HK1187364B - Apparatus and methods for integrated sample preparation, reaction and detection - Google Patents

Description

Apparatus and method for integrated sample preparation, reaction and detection

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims priority from U.S. provisional application serial No. 61/307,281, entitled "a facility and FOR INTEGRATED nuclear ACID and AMPLIFICATION", filed on 23/2/2010, which is incorporated herein by reference in its entirety.

This application is a continuation-in-part application of U.S. patent application Ser. No. 12/789,831 entitled "CASSETTE FOR PRS PREPARTATION" filed on 28.5.2010, which is a continuation of U.S. application Ser. No. 11/582,651 entitled "CASSETTE FOR PRS PREPARTATION" filed on 17.10.2006, which requires U.S. provisional application Ser. No. 60/728,569 entitled "METHOD APPPARATUSES FOR ISOLATING ACID", filed on 19.10.2005; U.S. provisional application No. 60/753,622 entitled "CASSETTE FOR SAMPLEPARATION" filed on 22.12.2005; and U.S. provisional application No. 60/753,618, entitled "CASSETTE FOR SAMPLE PREPARTATION," filed on 22/12/2005, each of which is incorporated herein by reference in its entirety. This application is also a continuation-in-part application of U.S. patent application Ser. No. 12/821,446 entitled "INSTRUMENT FOR CASSTEE FOR SAMPLE PREPARTATION" filed on 23.6.2010, which is a continuation of U.S. patent application Ser. No. 12/005,860 entitled "INSTRUMENT FOR CASSTEE FOR SAMPLE PREPARTATION" filed on 27.12.2007, which claims priority from U.S. provisional application Ser. No. 60/882,150 entitled "INSTRUMENT FOR CASSTEE FOR SAMPLE PREPARTATION" filed on 27.2006, 12.27.2006, each of which is incorporated herein by reference in its entirety.

Background

Embodiments described herein relate to apparatus and methods for sample preparation, reaction, and analysis. More particularly, embodiments described herein relate to cartridges and instruments within which nucleic acid isolation, amplification and analysis in an integrated process may be performed.

Some known diagnostic procedures include the isolation and analysis of nucleic acids, such as DNA or RNA. Known methods for isolating nucleic acids within a sample typically include several steps, such as: (1) removing proteins within the sample by adding a protease (e.g., proteinase K); (2) breaking down the remaining bulk sample to expose the nucleic acids contained therein (also known as cell lysis); (3) precipitating nucleic acids from the sample; and (4) washing and/or otherwise preparing the nucleic acid for further analysis.

In certain instances, amplification of the isolated nucleic acid (e.g., replication of the nucleic acid to increase its volume) is desirable for further analysis. Polymerase Chain Reaction (PCR) processes are known techniques for amplifying portions of nucleic acid molecules. In PCR, an input sample containing target DNA is mixed with reagents, including DNA polymerase (e.g., Taq polymerase). The input sample can be, for example, an isolated nucleic acid sample produced by the above-described procedure. The sample is then thermally cycled multiple times within the separation chamber to complete the reaction. The temperature and time period of the thermal cycle are carefully controlled to ensure accurate results. After the DNA sequence is sufficiently amplified, it can be analyzed using a variety of optical techniques.

Some known systems for performing nucleic acid separation and amplification comprise different portions (e.g., a separation portion and an amplification portion) between which the sample must be transferred using human intervention and/or processes that can compromise the integrity of the sample. Some known systems for performing nucleic acid isolation and amplification include complex control systems that require extensive preparation and/or calibration by experienced laboratory technicians. Accordingly, such known systems are not well suited for "bench top" applications, high volume diagnostic procedures, and/or use in a wide variety of laboratory settings.

In certain applications, multiple stages of the reaction may be required, with one or more later stages requiring the addition of reagents between stages of the reaction. For example, in reverse transcription PCR, the reverse transcription reaction is typically completed before performing the PCR process, which requires additional reagents. In some known systems, additional reagents required for later stages of the reaction are often transferred into the reaction chamber by human intervention and/or processes that can compromise the integrity of the sample. Accordingly, such known processes may induce errors and contamination, and may also be expensive and/or difficult to implement for high volume applications.

While some known systems include chambers containing reagents, such chambers are typically integrated into the cartridge and/or the reaction chamber. Accordingly, when such systems and/or cartridges are used in conjunction with different reactions and/or assays, it is common to use entirely different cartridges, cassettes, or other devices to facilitate the use of a particular combination of reagents to perform a desired reaction process. Thus, such known systems and/or cartridges are generally not interchangeable for different reaction procedures and/or assays.

While certain known systems include optical detection systems to detect one or more different reagents and/or targets within a test sample, such known systems typically include an excitation light source and/or an emission light detector in a portion of the device that is movable relative to the reaction chamber. For example, some known systems are configured to supply an excitation beam to a reaction chamber via a movable cover. Thus, such known systems are sensitive to detection variations, which may be due to variations in the position of the excitation and/or detection optical path.

Accordingly, there is a need for improved apparatus and methods for performing nucleic acid isolation and amplification.

SUMMARY

Cartridges and instruments for performing sample separation and downstream reactions are described herein. In certain embodiments, the instrument includes an isolation module, e.g., that can be used to isolate a nucleic acid sample, and a reaction module, e.g., that can be used to amplify a nucleic acid sample. The separation module includes a first housing and a second housing. The first housing defines a first chamber and a second chamber. At least the first chamber is configured to contain a sample, such as a sample containing nucleic acids. The second housing includes a sidewall and a penetrable member that collectively define a first volume configured to contain a first substance. The first substance may be, for example, a reagent to be added to the sample, a wash buffer solution, mineral oil, and/or any other substance. At least a portion of the second housing is configured to be disposed within the first housing such that the first volume is in liquid communication with the first chamber when a portion of the penetrable member is penetrated. The reaction module defines a reaction chamber and a second volume configured to contain a second substance. The reaction module is configured to be coupled to the separation module such that the reaction chamber and the second volume are each in fluid communication with the second chamber of the first housing.

In certain embodiments, PCR is performed in the cartridges and/or instruments provided herein. In a further embodiment, the reaction is monitored in real time by means of a fluorescent probe, e.g. a single stranded DNA molecule comprising a Minor Groove Binder (MGB) at the 5 'end and a fluorophore and a non-fluorescent quencher at its 3' end. In one embodiment, PCR is performed on multiple targets and the progress of the reaction is monitored in real time. In certain embodiments, the target is a gene sequence from one or more of the following viruses: influenza a, influenza b, Respiratory Syncytial Virus (RSV), herpes simplex virus type 1 (HSV 1), or herpes simplex virus type 2 (HSV 2). In certain embodiments, the reverse transcription reaction is performed in the cartridges and/or instruments provided herein prior to PCR.

Brief Description of Drawings

Fig. 1 and 2 are schematic views of a cartridge in a first configuration and a second configuration, respectively, according to an embodiment.

Fig. 3 is a schematic view of a cartridge having a first module, a second module, and a third module, according to one embodiment.

Fig. 4 is a schematic view of a cartridge having a first module, a second module, and a third module, according to one embodiment.

Fig. 5 is a schematic view of a cartridge having a first module and a second module, according to one embodiment.

Fig. 6 and 7 are schematic views of a portion of a cartridge in a first configuration and a second configuration, respectively, according to an embodiment.

Fig. 8 is a side perspective view of a cartridge according to one embodiment.

Fig. 9 is a top perspective view of the cartridge shown in fig. 8.

Fig. 10 is a side cross-sectional view of the cartridge shown in fig. 8.

Fig. 11 is a side exploded view of a portion of the cartridge shown in fig. 8.

Fig. 12 and 13 are perspective views of a reagent module of the cartridge shown in fig. 8.

Figure 14 is a perspective view of a portion of the reagent module shown in figures 12 and 13.

Fig. 15-18 are side cross-sectional views of the separation module of the cartridge shown in fig. 8 in a first configuration, a second configuration, a third configuration, and a fourth configuration, respectively.

Fig. 19 is a side cross-sectional view of a separate module of the cartridge shown in fig. 8.

FIG. 20 is taken along line X in FIG. 19₁-X₁A cross-sectional view of a portion of the shutter assembly of the separation module shown in figure 19 is obtained.

Fig. 21 is a perspective view of a portion of the shutter assembly of the separation module shown in fig. 19.

Fig. 22 is a perspective cross-sectional view of the cartridge shown in fig. 8.

FIG. 23 is a perspective view of a PCR module of the cartridge shown in FIG. 8.

Fig. 24 is a perspective cross-sectional view of the cartridge shown in fig. 8.

Fig. 25 is a side perspective view of a cartridge according to one embodiment.

Fig. 26 is a side perspective view of a separate module of the cartridge shown in fig. 25 in a first configuration.

Fig. 27 is a side cross-sectional view of the separation module shown in fig. 26 in a first configuration.

Fig. 28 is a side cross-sectional view of the separation module shown in fig. 26 in a second configuration.

FIG. 29 is a side perspective view of the PCR module of the cartridge shown in FIG. 25 in a first configuration.

FIG. 30 is a side cross-sectional view of the PCR module shown in FIG. 29 in a first configuration.

FIG. 31 is a side cross-sectional view of the PCR module shown in FIG. 29 in a second configuration.

Fig. 32 and 33 are side cross-sectional views of the cartridge shown in fig. 25 in a first configuration and a second configuration, respectively.

Fig. 34 is a schematic view of a portion of an apparatus according to an embodiment.

Fig. 35 is a perspective, cross-sectional schematic view of an apparatus according to one embodiment.

Fig. 36 is a perspective view of an instrument according to one embodiment.

Figure 37 is a perspective view of a first driver assembly of the instrument shown in figure 36.

Figure 38 is an exploded perspective view of the first actuator assembly shown in figure 37.

Figure 39 is a rear perspective view of the first actuator assembly shown in figure 37.

Figure 40 is a perspective view of a portion of the first actuator assembly shown in figure 37.

FIG. 41 is a top perspective view of the transfer drive assembly of the instrument shown in FIG. 36.

FIG. 42 is a bottom perspective view of the transfer drive assembly shown in FIG. 41.

FIG. 43 is a rear perspective view of the transfer drive assembly shown in FIG. 41.

FIG. 44 is a perspective view of a portion of the transfer actuator assembly shown in FIG. 41.

FIG. 45 is a perspective view of a portion of the transfer drive assembly shown in FIG. 41.

FIG. 46 is a perspective view of the worm drive shaft of the transfer drive assembly shown in FIG. 41.

Figure 47 is a top perspective view of a second actuator assembly of the instrument shown in figure 36.

Figure 48 is a side perspective view of the second actuator assembly shown in figure 47.

Figures 49-51 are perspective views of a portion of the second actuator assembly shown in figure 47.

Figure 52 is a side perspective view of the heater assembly of the instrument shown in figure 36.

FIG. 53 is a perspective view of a receiving block of the heater assembly shown in FIG. 52.

Fig. 54 and 55 are front and top views, respectively, of a receiving block of the heater assembly shown in fig. 52.

FIG. 56 is along line X in FIG. 54₂-X₂A cross-sectional view of the receiving block of the heater assembly shown in fig. 52 is obtained.

Fig. 57 is a perspective view of a clamp of the heater assembly shown in fig. 52.

FIG. 58 is a perspective view of a mounting block of the heater assembly shown in FIG. 52.

Fig. 59 is a perspective view of a heat sink of the heater module shown in fig. 52.

FIG. 60 is a perspective view of a mounting plate of the heater assembly shown in FIG. 52.

Fig. 61 and 62 are perspective views of a first insulating member and a second insulating member, respectively, of the heater assembly shown in fig. 52.

FIG. 63 is a perspective view of a heater block of the heater assembly shown in FIG. 52.

Fig. 64 and 66 are front and rear perspective views, respectively, of the optical assembly of the instrument shown in fig. 36.

Fig. 65 is an exploded perspective view of the optical assembly shown in fig. 64 and 66.

Fig. 67 is a perspective view of a mounting member of the optical assembly shown in fig. 64 and 66.

Fig. 68 is a perspective view of a slider of the optical assembly shown in fig. 64 and 66.

Fig. 69 is a perspective view of the sled of the optical assembly shown in fig. 64 and 66.

Fig. 70 is a perspective view of a portion of a fiber optic module of the optical assembly shown in fig. 64 and 66.

Fig. 71-73 are block diagrams of the electronic control system of the instrument shown in fig. 36.

Fig. 74-76 are schematic illustrations of an optical assembly in a first configuration, a second configuration, and a third configuration, respectively, according to an embodiment.

FIGS. 77-80 are flow diagrams of methods of detecting a target analyte in a nucleic acid-containing sample, according to embodiments.

FIG. 81 is a molecular marker generated by using a system and method according to one embodiment.

FIG. 82 is a cross-sectional perspective view of a portion of a separation module according to one embodiment configured to receive acoustic energy.

FIG. 83 is a cross-sectional perspective view of a portion of a separation module according to one embodiment configured to receive acoustic energy.

Fig. 84 is a cross-sectional perspective view of a portion of the cartridge and acoustic transducer shown in fig. 26.

Fig. 85 is a perspective view of a cartridge according to one embodiment.

Fig. 86 is a perspective view of the cartridge shown in fig. 85 without the cover.

FIG. 87 is a perspective view of the PCR module of the cartridge shown in FIG. 85.

FIG. 88 is a cross-sectional view of a PCR module according to one embodiment.

Fig. 89 is a perspective view of a cartridge according to one embodiment.

Fig. 90 is a perspective view of a cartridge according to one embodiment.

Fig. 91 is a perspective view of a cartridge according to one embodiment.

Fig. 92 is a perspective view of a cartridge according to one embodiment.

Fig. 93 is an exploded perspective view of the cartridge shown in fig. 92.

FIG. 94 is a perspective view of a cartridge with multiple PCR vials, according to one embodiment.

Detailed description of the invention

Cartridges and instruments for performing sample separation and reactions are described herein. In certain embodiments, the apparatus comprises an isolation module, e.g., that can be used to isolate a nucleic acid sample or an analyte sample, and a reaction module, e.g., that can be used to amplify a nucleic acid sample or test for binding of an analyte to other compounds. The separation module includes a first housing and a second housing. The first housing defines a first chamber and a second chamber. At least the first chamber is configured to contain a sample, such as a sample containing nucleic acids. The second housing includes a sidewall and a penetrable member that collectively define a first volume configured to contain a first substance. The first substance may be, for example, a reagent to be added to the sample, a wash buffer solution, mineral oil, and/or any other substance. At least a portion of the second housing is configured to be disposed within the first housing such that the first volume is in fluid communication with the first chamber when a portion of the penetrable member is penetrated. The reaction module defines a reaction chamber and a second volume configured to contain a second substance. The reaction module is configured to be coupled to the separation module such that the reaction chamber and the second volume are each in fluid communication with the second chamber of the first housing.

In certain embodiments, the apparatus includes a first module, a second module, and a third module. The first module defines a first chamber and a second chamber. At least a first chamber is configured to contain a sample. The second module defines a first volume configured to contain a first substance. The first substance may be, for example, a reagent to be added to the sample, a wash buffer solution, mineral oil, and/or any other substance. When the second module is coupled to the first module, a portion of the second module is configured to be disposed within the first chamber of the first module such that the first volume is configured to be selectively placed in fluid communication with the first chamber. The third module defines a reaction chamber and a second volume. The second volume is configured to contain a second substance. When the third module is coupled to the first module, a portion of the third module is disposed within the second chamber of the first module such that the reaction chamber and the second volume are each in fluid communication with the second chamber of the first module.

In certain embodiments, the apparatus includes a first module, a second module, and a third module. The first module defines a first chamber and a second chamber. The first module includes a first transfer mechanism configured to transfer the sample between the first chamber and the second chamber while maintaining fluidic separation between the first chamber and the second chamber. The second module defines a volume configured to contain a substance, such as a reagent or the like. When the second module is coupled to the first module, a portion of the second module is configured to be disposed within the first chamber of the first module such that the volume is configured to be selectively placed in fluid communication with the first chamber. The third module defines a reaction chamber. The third module is configured to be coupled to the first module such that the reaction chamber is in fluid communication with the second chamber. The third module includes a second transfer mechanism configured to transfer a portion of the sample between the second chamber and the reaction chamber.

In certain embodiments, the apparatus includes a first module and a second module. The first module includes a reaction vial, a substrate, and a first transfer mechanism. The reaction vial defines a reaction chamber and may be, for example, a PCR vial. The first transfer mechanism includes a plug movably disposed within the housing such that the housing and the plug define a first volume containing a first substance. The plug is movable between a first position and a second position. The first substance may be, for example, a reagent, mineral oil, or the like. The substrate defines at least a portion of the first flow path and the second flow path. The first flow path is configured to be in fluid communication with the reaction chamber, the first volume, and the separation chamber of the separation module. The second flow path is configured to be in fluid communication with the separation chamber. A portion of the plug is disposed within the first flow path such that the first volume is fluidly isolated from the reaction chamber when the plug is in the first position. A portion of the plug is positioned away from the first flow path such that the first volume is in fluid communication with the reaction chamber when the plug is in the second position. The plug is configured to create a vacuum within the reaction chamber to transfer the sample from the separation chamber to the reaction chamber when the plug is moved from the first position to the second position. The second module includes a second transfer mechanism and defines a second volume configured to contain a second substance. The second module is configured to be coupled to the first module such that the second volume can be selectively placed in fluid communication with the separation chamber via the second flow path. The second transfer mechanism is configured to transfer the second substance from the second volume to the separation chamber when the second transfer mechanism is actuated.

In certain embodiments, an instrument for manipulating and/or driving a cartridge containing a sample includes a block, a first optical member, a second optical member, and an optical assembly. The block defines a reaction volume configured to receive at least a portion of a reaction vessel. The patch may include and/or be attached to a mechanism for facilitating, generating, supporting and/or driving a reaction associated with the sample. In certain embodiments, for example, the block can be coupled to a heating element configured to thermally cycle the sample. A first optical member is at least partially disposed within the block such that the first optical member is in optical communication with the reaction volume. A second optical member is at least partially disposed within the block such that the second optical member is in optical communication with the reaction volume. The optical assembly includes an excitation module configured to generate a plurality of excitation light beams and a detection module configured to receive the plurality of emission light beams. The optical assembly is coupled to the first optical member and the second optical member such that each of the plurality of excitation light beams can be transmitted into the reaction volume and each of the plurality of emission light beams can be received by the reaction volume.

In certain embodiments, an instrument for operating and/or driving a cartridge includes a chassis, an acoustic transducer, and a drive mechanism. The chassis is configured to contain a cartridge having a housing defining a volume. The volume can receive a portion of a sample, such as a sample containing nucleic acids. The acoustic transducer is configured to generate acoustic energy. The drive mechanism is configured to move at least a portion of the acoustic transducer into contact with a portion of the cartridge. The drive mechanism is further configured to adjust a force applied by a portion of the acoustic transducer to a portion of the cartridge.

The term "beam of light" is used herein to describe any projection of electromagnetic energy, whether or not in the visible spectrum. For example, the light beam may include collimated projections of electromagnetic radiation in the visible spectrum produced by a laser, Light Emitting Diode (LED), flash lamp, or the like. The beam may be continuous for a desired period of time or discontinuous (e.g., pulsed or intermittent) for a desired period of time. In certain instances, the optical beam may include and/or be correlated with information (i.e., the optical beam may be an optical signal), such as the amount of analyte present in the sample.

The term "parallel" is used herein to describe a relationship between two geometric structures (e.g., two lines, two planes, a line and a plane, etc.), wherein the two geometric structures are substantially disjoint when they are substantially elongated to infinity. For example, as used herein, a first line is said to be parallel to a second line when the first and second lines do not intersect when they extend to infinity. Similarly, when a planar surface (i.e., a two-dimensional surface) is said to be parallel to a line, each point along the line is spaced away from the nearest portion of the surface by a substantially equal distance. When two geometries are nominally parallel to each other, for example when they are parallel to each other within a tolerance, they are described herein as being "parallel" or "substantially parallel" to each other. Such tolerances may include, for example, manufacturing tolerances, measurement tolerances, and the like.

The term "perpendicular" is used herein to describe a relationship between two geometric structures (e.g., two lines, two planes, a line and a plane, etc.) where the two geometric structures intersect at an angle of about 90 degrees in at least one plane. For example, as used herein, a first line is said to be perpendicular to a plane when the line and the plane intersect within the plane at an angle of about 90 degrees. When two geometries are nominally perpendicular to each other, for example when they are perpendicular to each other within a tolerance, they are described herein as being "perpendicular" or "substantially perpendicular" to each other. Such tolerances may include, for example, manufacturing tolerances, measurement tolerances, and the like.

Fig. 1 and 2 are schematic views of a cartridge 1001 in a first configuration and a second configuration, respectively, the cartridge 1001 including a separation module 1100 and a reaction module 1200, according to one embodiment. The separation module 1100 and the reaction module 1200 are coupled to each other such that the separation module 1100 and the reaction module 1200 can be placed in fluid communication with each other. As described herein, the separation module 1100 and the reaction module 1200 may be coupled together in any suitable manner. In certain embodiments, for example, the separation module 1100 and the reaction module 1200 may be separately constructed and coupled together to form the cartridge 1001. This arrangement between the separation module 1100 and the reaction module 1200 allows a variety of different configurations of the separation module 1100 to be used with a variety of different configurations of the reaction module 1200. Different configurations of the separation module 1100 and/or the reaction module 1200 can include different reagents and/or different structures within the separation module 1100 and/or the reaction module 1200.

Cartridge 1001 may be operated and/or driven by any of the instruments described herein. In certain embodiments, cartridge 1001 may be used to perform sample preparation, nucleic acid isolation, and/or Polymerase Chain Reaction (PCR) on a sample. In such embodiments, the separation module 1100 can separate a target nucleic acid from a sample contained therein. The isolated nucleic acids may then be amplified (e.g., using PCR) in the reaction module 1200, as described further below. The modular arrangement of the cartridge 1001 allows any number of different reaction modules 1200 to be used with the separation module 1100, the reaction modules 1200 each containing, for example, different reagents and/or each being configured to amplify a different type of sample, and vice versa.

The separation module 1100 includes a first housing 1110 and a second housing 1160. As described in greater detail herein, the second shell 1160 is coupled to the first shell 1110 such that the second shell 1160 may be placed in fluid communication with the first shell 1110. In certain embodiments, the first shell 1110 and the second shell 1160 are modular in arrangement such that different configurations of the first shell 1110 and the second shell 1160 may be used with each other. The different configurations of first housing 1110 and second housing 1160 may include, for example, different chemicals, reagents, samples, and/or different internal structures.

The first housing 1110 defines a first chamber 1114 and a second chamber 1190. At least one of the first chamber 1114 or the second chamber 1190 may contain a sample S. The sample S can be any biological sample, such as a biological sample containing one or more target nucleic acids, e.g., urine, blood, other materials containing tissue samples, and the like. The sample S may be introduced into the first chamber 1114 or the second chamber 1190 via any suitable mechanism, including, for example, by aspirating or injecting the sample S into the first chamber 1114 and/or the second chamber 1190 via a hole or a penetrable member in the first housing 1110 (not shown). Although the first chamber 1114 is shown in fluid communication with the second chamber 1190, in other embodiments, the first chamber 1114 may be selectively placed in fluid communication with the second chamber 1190. In other words, in certain embodiments, the first housing 1110 can include any suitable mechanism, such as a shutter (not shown in fig. 1 and 2), that can selectively place the first chamber 1114 in fluid communication with the second chamber 1190. Additionally, in other embodiments, the first housing 1110 can have any suitable flow control and/or transfer mechanism (not shown in fig. 1 and 2) to facilitate and/or control the transfer of a substance between the first chamber 1114 and the second chamber 1190, including, for example, a valve, a capillary flow control device, a pump, and the like. In still other embodiments, the first chamber 1114 can be fluidly separated from the second chamber 1190.

The second housing 1160 includes a sidewall 1147 and a penetrable member 1170. The sidewall 1147 and penetrable member 1170 define a first volume 1163. The first volume 1163 may be completely or partially filled with substance R1. The substance R1 may be any biological or chemical substance such as mineral oil, wash buffer, fluorescent dye, reagent, etc. As shown in fig. 1 and 2, a portion of the second housing 1160 is disposed within the first housing 1110 such that when the penetrable member 1170 is penetrated, ruptured, severed and/or ruptured, the first volume 1163 is in fluid communication with the first chamber 1114 as shown in fig. 2. Similarly stated, the separation module 1110 may be moved from the first configuration (fig. 1) to the second configuration (fig. 2) when the penetrable member 1170 is penetrated. When the first volume 1163 is in fluid communication with the first chamber 1114 as shown in fig. 2 (i.e., when the separation module is in the second configuration), the substance R1 can be transferred from the first volume 1163 to the first chamber 1114. The substance R1 can be transferred from the first volume 1163 into the first chamber 1114 by any suitable mechanism, such as by gravitational force, capillary force, or by some driving mechanism (not shown in fig. 1 and 2) acting on the first volume 1163.

The penetrable member 1170 may be constructed of a material that is substantially impermeable and/or substantially chemically inert to the substance R1. In this manner, the substance R1 can be stored within the first volume 1163 for an extended period of time without compromising the ability to use the second shell 1160 in any desired application, such as any of the embodiments described herein. Further, in certain embodiments, the penetrable member 1170 may be constructed of a material having a particular temperature characteristic such that the desired properties and integrity of the penetrable member 1170 are maintained through a particular temperature range. For example, in certain embodiments, it may be desirable to store the second housing 1160 containing the substance R1 in refrigerated conditions, or it may be desirable to manufacture the second housing 1160 by heat laminating the penetrable member 1170. In such embodiments, the permeable member 1170 may be selected such that the refrigerated and/or hot lamination conditions do not substantially degrade the desired properties and integrity of the permeable member 1170 for the intended application. In certain embodiments, the penetrable member 1170 may be constructed of a polymeric film, such as any form of polypropylene. In certain embodiments, the penetrable member 1170 may be constructed of Biaxially Oriented Polypropylene (BOP).

Although fig. 1-2 show at least a portion of the second shell 1160 disposed within the first shell 1110, in other embodiments, the first shell 1110 and the second shell 1160 may be coupled together by disposing at least a portion of the first shell 1110 within the second shell 1160,or by coupling or mating the first shell 1110 and the second shell 1160 together via an interface without seating within each other. Second shell 1160 may be coupled to first shell 1110 by any suitable mechanism, such as by adhesive bonding; welding a joint; snap-fit (e.g., an arrangement in which mating projections disposed on a first housing are received and/or retained within corresponding apertures defined by a second housing, or vice versa); interference fits in which the two parts are fastened together by friction after being pushed together (e.g. by friction)) (ii) a Screw-threaded couplings, including removable couplings, e.g.Or a flange coupling. The coupling between the first housing 1110 and the second housing 1160 may be fluid tight such that fluid transfer between the first volume 1163 and the first chamber 1114 does not result in leakage and/or contamination when the penetrable member 1170 has been breached or ruptured as shown in fig. 2. Fluid-tight coupling between first shell 1110 and second shell 1160 may be achieved through a tapered fit (threaded fit) using mating components, o-rings, gaskets, and the like.

The reaction module 1200 defines a reaction chamber 1262 and a second volume 1213. The second volume 1213 contains substance R2. Substance R2 may be any biological or chemical substance, such as mineral oil, wash buffers, reagents, etc., that participates in or otherwise supports a reaction within reaction chamber 1262 and/or other portions of cartridge 1001. The reaction module 1200 is coupled to the separation module 1100 such that the reaction chamber 1262 and the second volume 1213 can each be placed in liquid communication with the second chamber 1190 of the separation module 1100. The reaction module 1200 may be coupled to the separation module 1100 by any suitable mechanism, such as by adhesive bonding; welding a joint; snap-fit (e.g., an arrangement in which mating projections disposed on a first housing are received and/or retained within corresponding apertures defined by a second housing, or vice versa); interference fit of two partsFastening by friction after being pushed together (e.g. by friction)) (ii) a Screw-threaded couplings, including removable couplings, e.g.Or a flange coupling. The coupling between the first housing 1110 and the reaction module 1200 may be fluid tight such that fluid transfer between the separation module 1100 and the reaction module 1200 does not result in leakage and/or contamination. The fluid-tight coupling between the reaction module 1200 and the separation module 1100 may be achieved using a tapered fit of mating members, o-rings, gaskets, or the like. In certain embodiments, the coupling between the separation module 1100 and the reaction module 1200 is removable.

This arrangement allows the transfer of substances from the reaction chamber 1262 and/or the second volume 1213 to the second chamber 1190 or vice versa. For example, in use, a sample, reagent, and/or other support material, such as one or more of sample S, substance R1, or substance R2, can be transferred into or out of reaction chamber 1262 in conjunction with a desired reaction. Fluid transfer between second chamber 1190, reaction chamber 1262, and/or second volume 1213 can be achieved by gravitational forces, capillary forces, water pressure, and the like. In certain embodiments, the water pressure may be applied by a piston pump, a flapper pump, or any other suitable transfer mechanism. In certain embodiments, such fluid transfer mechanisms may be external to cartridge 1001 or internal to cartridge 1001 (e.g., disposed at least partially within separation module 1100 and/or reaction module 1200).

In certain embodiments, substance R1 and sample S or portions thereof can be transferred from first volume 1163 and first chamber 1114 to reaction chamber 1262 through second chamber 1190 in conjunction with a reverse transcription process to generate single-stranded complementary deoxyribonucleic acid (cDNA) from a ribonucleic acid (RNA) template using reverse transcriptase. After completion of the reverse transcription process, substance R2 may be transferred from second volume 1213 through second chamber 1190 to reaction chamber 1262 to perform a PCR process on newly synthesized cDNA or DNA present in sample S. In such embodiments, the substance R2 may include one or more PCR reagents, including Taq polymerase. In certain embodiments, substance R1 and/or substance R2 can include a DNA binding dye (e.g., Minor Groove Binder (MGB), MGB coupled to the 5' end of a DNA probe that specifically hybridizes to a target sequence, and a fluorophore), so the progress of the PCR process can be monitored in real time by detecting fluorescence from the fluorescent reporter in reaction chamber 1262 using any of the instruments and/or methods described herein.

In certain embodiments, cartridge 1001 (fig. 1 and 2) is used to isolate and amplify a nucleic acid sample. For example, the separation can occur in first chamber 1114 or second chamber 1190. In one embodiment, substance R1 includes reagents for nucleic acid isolation. DNA, RNA, and combinations thereof can be isolated by the cartridges provided herein. For example, in one embodiment, the substance R1 comprises magnetic beads derivatized with a reagent to isolate DNA or RNA.

Individual nucleic acids and total nucleic acids can be separated in the cartridges provided herein. For example, in one embodiment, substance R1 comprises a bead derived from a polyA sequence designed to isolate the total pool of messenger RNAs present in a sample. In another embodiment, the substance R1 comprises beads derived from specific nucleic acid sequences designed to isolate only a portion of the nucleic acids in a sample.

Once the nucleic acid is isolated, it can be amplified. In one embodiment, the amplification is by PCR. For the purposes of the present invention, reference to "PCR" on a nucleic acid sample includes reverse transcription-PCR (RT-PCR). In particular, when the nucleic acid sample is one or more target RNAs or RNA populations (e.g., total mRNA), RT-PCR will be performed on the target RNA. The PCR master mix (master mix) provided herein may therefore include reagents for reverse transcription. The reverse transcription step may occur in the same chamber or module as the PCR or in a different chamber or module. In one embodiment, reverse transcription and PCR are performed in the same chamber by providing a RT-PCR master mix. One of ordinary skill in the art will readily appreciate whether RT-PCR or PCR is required, based on the nucleic acid sample that was initially isolated. Any of the cartridges provided herein may be used to isolate DNA and/or RNA and perform RT-PCR and/or PCR.

For example, in one embodiment, if RNA is first isolated, a reverse transcriptase reaction is performed on the isolated sample, e.g., in second chamber 1190 or reaction chamber 1262. If DNA is isolated, it can be amplified, for example, by PCR in reaction chamber 1262. Similarly, if RNA is first isolated from sample S, it is subjected to a reverse transcription reaction, e.g., in reaction chamber 1262, and the product of this reaction is used in a downstream PCR reaction, e.g., in reaction chamber 1262. In certain embodiments, multiple target nucleic acids are amplified in a PCR, and the PCR reaction is monitored in real time. In one embodiment, amplification of multiple targets is monitored by employing individual DNA hybridization probes specific for each target, wherein each probe includes a fluorophore that emits light at a different wavelength or can be excited at a unique wavelength. In one embodiment, the DNA hybridization probe is provided in the second volume 1213 as substance R2 (or a portion thereof).

In one embodiment, the probe used to monitor the PCR is a DNA oligonucleotide that specifically hybridizes to a DNA target of interest and includes a non-fluorescent quencher at the 3 'end and a fluorophore at the 5' end. In addition, in this embodiment, the DNA oligonucleotide includes an MGB at the 5' end that is conjugated directly to the oligonucleotide or to a fluorophore (see Lukhtanov et al (2007). Nucleic Acids Research 35, p. e 30). DNA oligonucleotide probes fluoresce when bound to a target but not in solution. Thus, after product synthesis in PCR, more hybridization will occur and more fluorescence is generated. The amount of fluorescence is therefore proportional to the amount of target generated.

Real-time monitoring of the PCR reaction is not limited to the cartridges shown in FIGS. 1 and 2. Rather, any of the cartridges provided herein may employ real-time PCR, e.g., with DNA hybridization probes as described above.

In certain embodiments, cartridge 1001 may be operated by any of the instruments and/or methods described herein to facilitate a PCR process to occur within reaction chamber 1262. In such embodiments, reaction module 1200 can be coupled to and/or placed in contact with a thermal transfer instrument to allow the contents of reaction chamber 1262 to be thermally cycled in conjunction with a PCR process. In such embodiments, the reaction module 1200 may further be operably coupled to an optical instrument to allow real-time monitoring of the PCR process. In other embodiments, the reaction module 1200 and/or the separation module 1100 may be operably coupled to other energy sources, such as optical energy, ultrasonic energy, magnetic energy, hydraulic energy, and the like, to facilitate the reaction and/or separation processes occurring therein.

Although fig. 1-2 show reaction chamber 1262 and second volume 1213 in fluid communication with second chamber 1190, in other embodiments, fluid communication between reaction chamber 1262, second volume 1213, and/or second chamber 1190 of the separation module may be selective. In other words, in certain embodiments, the reaction module 1200 and/or the separation module 1100 can include a mechanism, such as a valve or a permeable membrane, that can selectively place the second chamber 1190 in fluid communication with the second volume 1213 and/or the reaction chamber 1262. Although the separation module 1100 is shown as defining a first volume 1163, in certain embodiments, the separation module 1100 may define any number of volumes and/or may contain any number of different substances. Similarly, although the reaction module 1200 is shown as defining one second volume 1213, in certain embodiments, the reaction module 1200 may define any number of volumes and may contain any number of different substances.

Fig. 3 is a schematic view of a cartridge 2001 according to one embodiment, the cartridge 2001 including a first module 2110, a second module 2160, and a third module 2200. The first module 2110 defines a first chamber 2114 and a second chamber 2190. The first chamber 2114 and/or the second chamber 2190 can contain any biological sample containing a target nucleic acid, e.g., urine, blood, other material containing a tissue sample, etc. Although the first chamber 2114 is shown in fluid communication with the second chamber 2190, in other embodiments, the first chamber 2114 can be selectively placed in fluid communication with the second chamber 2190. In other words, in certain embodiments, the first module 2110 can include any suitable mechanism, such as a shutter (not shown in fig. 3), that can selectively place the first chamber 2114 in fluid communication with the second chamber 2190. Additionally, in other embodiments, the first module 2110 can have any suitable flow control and/or transfer mechanism (not shown in fig. 3) to facilitate and/or control the transfer of a substance between the first chamber 2114 and the second chamber 2190, including, for example, a valve, a capillary flow control device, a pump, and the like.

The second module 2160 defines a first volume 2163 that may contain, in whole or in part, any biological or chemical substances. The substance may be, for example, mineral oil, wash buffer, reagents, etc., that may participate in or otherwise support a reaction within first chamber 2114 and/or any other portion of cartridge 2001. In one embodiment, the reaction in the first chamber 2114 is an isolation reaction, e.g., nucleic acid or peptide isolation. The second module 2160 may be coupled to the first module 2110 in any suitable manner as described herein. In certain embodiments, for example, the first module 2110 and the second module 2160 can be separately constructed and coupled together such that the first module 2110 and the second module 2160 are a modular arrangement. In such a module arrangement, a number of different configurations of the first module 2110 and the second module 2160 may be used with each other. Different configurations of the first module 2110 and/or the second module 2160 may include different reagents and/or different structures within the first module 2110 and/or the second module 2160. As shown in FIG. 3, a portion of the second module 2160 is disposed within the first chamber 2114 of the first module 2110 such that the first volume 2163 can be placed in fluid communication with the first chamber 2114. In other embodiments, the first volume 2163 can be selectively placed in fluid communication with the first chamber 2114. In certain embodiments, for example, the first module 2110 and/or the second module 2160 may include any suitable mechanism, such as a valve and/or any suitable fluid control and/or transfer mechanism as described herein, which may selectively place the first volume 2163 in fluid communication with the first chamber 2114 when the second module 2160 is coupled to the first module 2110. In certain embodiments, a substance and/or sample may be transferred between first volume 2163 and first chamber 2114 using any suitable fluid transfer mechanism as described herein. For example, in use, a sample, an isolated sample (e.g., isolated DNA, isolated RNA, isolated peptide, isolated protein), a reagent (e.g., an isolation reagent), and/or other support material can be transferred into and/or out of first chamber 2114 in conjunction with a desired reaction. In still other embodiments, the first volume 2163 can be fluidly separated from the first chamber 2114, such as by a valve, penetrable member, or selective transfer mechanism as described herein (not shown in fig. 3).

The third module 2200 defines a reaction chamber 2262 and a second volume 2213. Reaction chamber 2262 and/or second volume 2213 may contain, in whole or in part, one or more biological or chemical substances, such as mineral oil, wash buffers, one or more PCR reagents, etc., that participate in or otherwise support the reaction within reaction chamber 2262 and/or any other portion of cartridge 2001. The third module 2200 may be coupled to the first module 2110 in any suitable manner as described herein. In certain embodiments, the first module 2110 is an isolation module 2110, for example, to isolate one or more target nucleic acids from a biological sample. In certain embodiments, the first module 2110 is used for RNA isolation and first strand cDNA synthesis. In this embodiment, the first volume 2163 includes separation reagents and reagents for Reverse Transcription (RT) reactions. In certain embodiments, for example, the first module 2110 and the third module 2200 may be separately constructed and coupled together such that the first module 2110 and the third module 2200 are a modular arrangement. In such a modular arrangement, different configurations of the first and third modules 2110, 2200 may be used with each other. Different configurations of the first module 2110 and/or the third module 2200 may include different reagents and/or different structures within the first module 2110 and/or the third module 2200. As shown in fig. 3, a portion of the third module 2200 is disposed within the second chamber 2190 of the first module 2110 such that the reaction chamber 2262 and the second volume 2213 are each in fluid communication with the second chamber 2190. In other embodiments, the reaction chamber 2262 and/or the second volume 2213 may be selectively placed in fluid communication with the second chamber 2190. In other words, in certain embodiments, the first module 2110 and/or the third module 2200 may include any suitable mechanism, such as a valve and/or any suitable flow control and/or displacement mechanism as described herein, that may place the reaction chamber 2262 and/or the second volume 2213 in selective fluid communication with the second chamber 2190. In certain embodiments, substances and/or samples may be transferred between second chamber 2190, and reaction chamber 2262 and/or second volume 2213 using any suitable fluid transfer mechanism as described herein. For example, in use, samples, reagents and/or other support materials may be transferred into or out of reaction chamber 2262 in conjunction with a desired reaction. In still other embodiments, the reaction chamber 2262 and/or the second volume 2213 may be fluidly separated from the second chamber 2190, for example, by a penetrable member or selective transfer mechanism (not shown) as described herein.

In certain embodiments, cartridge 2001 may be used to perform sample preparation, nucleic acid isolation, and/or Polymerase Chain Reaction (PCRs) on a sample. In such embodiments, the target nucleic acid can be isolated from the sample in the first module 2110. The isolated nucleic acid can be RNA, DNA, or a combination thereof. As described above, if RNA is isolated, a reverse transcription reaction is performed in cartridge 2001, e.g., in first chamber 2114 or second chamber 2190, prior to PCR. The isolated nucleic acid (or newly synthesized cDNA if RNA is isolated) can then be amplified in the third module 2200 (e.g., using PCR), as described herein, e.g., real-time PCR with a DNA oligonucleotide probe comprising a fluorophore and MGB at the 5 'terminus and a non-fluorescent quencher at the 3' terminus. The modular arrangement of cartridge 2001 allows any number of different third modules 2200 to be used with the first module 2110, each of the third modules 2200 containing, for example, a different reagent and/or each being configured to amplify a different type of sample, or vice versa. In certain embodiments, cartridge 2001 may be operated by any of the instruments and/or methods described herein to facilitate the occurrence of a PCR process within reaction chamber 2262. In such embodiments, third module 2200 may be coupled to and/or placed in contact with a thermal transfer instrument to allow the contents of reaction chamber 2262 to be thermally cycled in conjunction with the PCR process. In such embodiments, the third module 2200 may be further operably coupled to an optical instrument to monitor the PCR process. In other embodiments, the third module 2200 and/or the first module 2110 can be operably coupled to other sources of energy, such as sources of light energy, ultrasonic energy, magnetic energy, hydraulic energy, and the like, to facilitate the reaction and/or separation processes occurring therein.

Although fig. 3 shows integrated cartridge 2001 as defining a first volume 2163 and a second volume 2213, in certain embodiments, integrated cartridge 2001 may define any number of first volumes 2163 and/or second volumes 2213 to contain any number of different substances and/or perform different functionalities. For example, the first volume 2163 and/or the second volume 2213 may contain separate wash buffers, elution buffers, reagents for reverse transcription reactions, PCR reagents, lysis buffers.

As noted above, in certain embodiments, any of the cartridges described herein may include one or more transfer mechanisms configured to transfer samples between different chambers defined within the cartridge. For example, fig. 4 is a schematic view of a cartridge 3001 according to one embodiment, said cartridge 3001 comprising a first module 3110, a second module 3160 and a third module 3200. The first module 3110 defines a first chamber 3114 and a second chamber 3190. In certain embodiments, the first module 3110 serves as an isolation module, e.g., to isolate one or more target nucleic acids, populations of nucleic acids (e.g., total RNA, total DNA, mRNA), or target peptides or proteins from a biological sample. First chamber 3114 and/or second chamber 3190 can contain any biological sample, such as a biological sample containing target nucleic acids, e.g., urine, blood, other materials containing tissue samples, etc. A first transfer mechanism 3140 is disposed between the first chamber 3114 and the second chamber 3190.

In certain embodiments, first transfer mechanism 3140 can be a selective transfer mechanism to selectively transfer samples and/or substances between first chamber 3114 and second chamber 3190. In such embodiments, for example, first transfer mechanism 3140 can transfer samples and/or substances having particular properties between first chamber 3114 and second chamber 3190 while limiting and/or preventing transfer of samples and/or substances having different properties between first chamber 3114 and/or second chamber 3190. In certain embodiments, first transfer mechanism 3140 may be an instrument that uses magnetic components to transfer samples and/or substances based on their magnetic properties. In other embodiments, the first transfer mechanism 3140 may transfer the sample and/or substance based on the surface charge of the sample and/or substance, for example, by using electrophoresis. In still other embodiments, first transfer mechanism 3140 can transfer samples and/or substances based on the size of molecules or ions within the samples and/or substances. In such embodiments, the first transfer mechanism 3140 may include a reverse osmosis mechanism for selectively transferring samples and/or substances. In other words, in certain embodiments, first transfer mechanism 3140 may rely on and/or generate a force, including, for example, a magnetic force, an electrostatic force, a pressure, or the like, to act on a target sample and/or substance and/or molecule and/or ion therein. First transfer mechanism 3140 may also include any suitable structure and/or multiple selective transfer mechanisms may be combined (e.g., to impart additional physical motion and/or provide additional selectivity). In certain embodiments, first transfer mechanism 3140 can selectively transfer particular molecules or ions between first chamber 3114 and second chamber 3190 while maintaining substantial fluidic separation between first chamber 3114 and second chamber 3190. In certain embodiments, the first transfer mechanism 3140 may be a magnetic shutter as disclosed in U.S. patent No. 7,727,473 entitled "CASSETTE FOR sample application" filed on 17.10.2006, which is incorporated herein by reference in its entirety. In still other embodiments, first transfer mechanism 3140 can non-selectively transfer substances and/or samples between first chamber 3114 and second chamber 3190.

The second module 3160 defines a first volume 3163 that may contain, in whole or in part, any biological or chemical substance, such as mineral oil, nucleic acid isolation reagents, reverse transcription reagents, elution buffers, lysis buffers, wash buffers, reagents, and the like, that may participate in or otherwise support a reaction within the first chamber 3114 and/or any other portion of the cartridge 3001. Second module 3160 may be coupled to first module 3110 in any suitable manner as described herein. In certain embodiments, for example, first module 3110 and second module 3160 may be separately constructed and coupled together such that first module 3110 and second module 3160 are in a modular arrangement. In such a modular arrangement, different configurations of the first module 3110 and the second module 3160 may be used with each other. Different configurations of the first module 3110 and/or the second module 3160 may include different reagents and/or different structures within the modules. As shown in fig. 4, a portion of the second module 3160 is disposed within the first compartment 3114 of the first module 3110 such that the first volume 3163 is in fluid communication with the first compartment 3114. In other embodiments, the first volume 3163 can be selectively placed in fluid communication with the first chamber 3114. In other words, in certain embodiments, first module 3110 and/or second module 3160 may include any suitable mechanism, such as a valve and/or any suitable fluid control and/or transfer mechanism as described herein, that may selectively place first volume 3163 in fluid communication with first chamber 3114. In certain embodiments, substances and/or samples can be transferred between first volume 3163 and first chamber 3114 using any suitable fluid transfer mechanism as described herein. For example, in use, samples, reagents, and/or other support materials can be transferred into or out of first chamber 3114 in conjunction with a desired reaction. In still other embodiments, the first volume 3163 can be fluidly separated from the first chamber 3114, such as by a penetrable member or a selective transfer mechanism (not shown) as described herein.

The third module 3200 defines a reaction chamber 3262. Reaction chamber 3262 may contain, in whole or in part, any biological or chemical substance such as mineral oil, reverse transcription reagents, elution buffers, lysis buffers, PCR reagents (e.g., Taq polymerase, primers, DNA oligonucleotide probes for monitoring the reaction, Mg²⁺) Wash buffers, reagents, etc., that participate in or otherwise support reactions within the reaction chamber 3262 and/or any other portion of the cartridge 3001. The third module 3200 may be coupled to the first module 3110 in any suitable manner as described herein. In certain embodiments, for example, the first module 3110 and the third module 3200 may be separately constructed and coupled together such that the first module 3110 and the third module 3200 are a modular arrangement. In such a modular arrangement, different configurations of the first module 3110 and the third module 3200 may be used with each other. Different configurations of the first module 3110 and/or the third module 3200 may include different reagents and/or different structures within the modules. As shown in fig. 4, a portion of third module 3200 is disposed within second chamber 3190 of first module 3110 such that reaction chambers 3262 can each be in fluid communication with second chamber 3190 to effect control of second transfer mechanism 3240.

Second transfer mechanism 3240 can transfer substances and/or reagents from second chamber 3190 to reaction chamber 3262 or vice versa. In certain embodiments, for example, a second transfer mechanism can transfer a predetermined volume of substance and/or reagent between second chamber 3190 and reaction chamber 3262. Similarly stated, in certain embodiments, second transfer mechanism 3240 can transfer a substance and/or reagent between second chamber 3190 and reaction chamber 3262 at a predetermined volumetric flow rate. In certain embodiments, for example, second transfer mechanism 3240 can be a pump configured to apply positive pressure or vacuum to second chamber 3190 and/or reaction chamber 3262. In such embodiments, second transfer mechanism 3240 can be a pump driven by the plug using any of the instruments and/or methods described herein. In certain embodiments, the second transfer mechanism 3240 can have a penetrable member as described herein such that the second transfer mechanism 3240 can penetrate, break, sever and/or rupture the penetrable member to transfer a substance and/or sample contained in the reaction chamber 3262 into the second chamber 3190 or vice versa. In other embodiments, for example, the second transfer mechanism 3240 can be a capillary flow control device. In still other embodiments, the second transfer mechanism 3240 can be any other selective or non-transfer mechanism as described herein.

In certain embodiments, cartridge 3001 can be used to perform sample preparation, nucleic acid isolation, reverse transcription (e.g., RNA is first isolated), and/or Polymerase Chain Reaction (PCR) on a sample. In such embodiments, the target nucleic acid can be isolated from the sample within the first module 3110. The isolated nucleic acids may then be amplified (e.g., using PCR) in a third module 3200, as described further below. As described herein, PCR on multiple targets can be monitored in real time with a cartridge of the present invention, such as cartridge 3001. In one embodiment, amplification of the multiplexed targets occurs with a DNA oligonucleotide probe disclosed by Lukhtanov et al (Nucleic Acids Research 35, p. e30, 2007). The modular arrangement of the cartridge 3001 allows any number of different third modules 3200 to be used with the first module 3110, said third modules 3200 each containing, for example, different reagents and/or each being configured to amplify a different type of sample, and vice versa. In certain embodiments, cartridge 3001 can be operated by any of the instruments and/or methods described herein to facilitate a PCR process to occur within reaction chamber 3262. In such embodiments, third module 3200 can be coupled to and/or placed in contact with a thermal transfer instrument to allow the contents of reaction chamber 3262 to be thermocycled in conjunction with a PCR process. In such embodiments, the third module 3200 may be further operably coupled to an optical instrument to monitor the PCR process. In other embodiments, the third module 3200 and/or the first module 3110 may be operably coupled to other energy sources, such as optical energy, ultrasonic energy, magnetic energy, hydraulic energy, etc., to facilitate the reaction and/or separation processes occurring therein.

Although in one embodiment, cartridge 3001 shown and described in connection with fig. 4 includes a first module, a second module, and a third module, in other embodiments, the cartridge may include two modules coupled together. For example, fig. 5 is a schematic view of a portion of a cartridge 4001 comprising a first module 4200 and a second module 4160, according to one embodiment. A portion of the cartridge 4001 may be coupled to a separation module 4110, as shown in fig. 5. The first module 4200 may include a reaction vial 4260, a base 4220, and a first transfer mechanism 4140. The reaction vial 4260 defines a reaction chamber 4262, which may wholly or partially contain any biological or chemical sample and/or substance containing a target nucleic acid, such as urine, blood, other materials containing tissue samples, etc., and/or mineral oil, wash buffers, lysis buffers, reverse transcription reagents, PCR reagents, etc., that participate in or otherwise support a reaction within the reaction chamber 4262 and/or any other portion of the cartridge 4001.

The reaction vial 4260 may be any suitable container for containing a sample, such as a nucleic acid sample, isolated or otherwise in a manner that allows a reaction to be performed in conjunction with the sample. In certain embodiments, the reaction vial 4260 may have a thin wall configured to accept and/or seat within a heating element and/or block (see, e.g., block 1710 described below). Reaction vial 4260 may be constructed of any suitable material having specific properties compatible with the desired reaction and/or process. In certain embodiments, the reaction vials 4260 may be constructed of a substantially thermally conductive material to allow thermal cycling of the substance and/or sample within the reaction vials 4260. In certain embodiments, the reaction vial 4260 may be constructed of a substantially mechanically robust material such that the sidewalls of the reaction vial 4260 substantially retain their shape and/or size when positive pressure or vacuum is applied to the volume within the reaction vial 4260. In certain embodiments, the reaction vial 4260 may be constructed of a material that is substantially chemically inert to the reaction within the reaction vial 4260 such that the material forming the reaction vial 4260 will not contaminate or otherwise affect the reaction within the reaction vial 4260.

The reaction vial 4260 may also be used in any suitable container containing a sample in a manner that allows for monitoring of such reactions (e.g., detection of analytes within the sample that are due to or bound to the reaction). In certain embodiments, for example, reaction vial 4260 may be a PCR vial, test tube, microcentrifuge tube, or the like. Further, in certain embodiments, at least a portion of the reaction vial 4260 may be substantially transparent to allow optical monitoring of the reaction occurring therein.

In certain embodiments, the reaction vial 4260 may be constructed entirely of the substrate 4220. In other embodiments, the reaction vial 4260 may be coupled to the substrate 4220 by any suitable mechanism as described herein.

The base 4220 defines at least a portion of a first flow path 4221 and a second flow path 4222. The first flow path 4221 is configured to be in fluid communication with the reaction chamber 4262 and the separation chamber 4114 of the separation module 4110. When first transfer mechanism 4140 is actuated, first transfer mechanism 4140 is configured to transfer sample S (or a portion thereof) from separation chamber 4114 to reaction chamber 4262 (as indicated by arrow AA). The substrate 4220 may define a portion of the first flow path 4221 and the second flow path 4222 using any suitable structure, material, and/or manufacturing process. In certain embodiments, the substrate 4220 may be a monolayer. In other embodiments, the substrate 4220 may be constructed of separate layers of material welded and coupled together to define a structure and flow path. In certain embodiments, the substrate 4220 may be constructed using processes including, for example, chemical etching, mechanical and/or ion milling, embossing, lamination, and/or silicon bonding. In certain embodiments, at least a portion of the substrate 4220 can have a heating element disposed thereon, disposed within and/or in contact with the heating element, such that, in use, a portion of the substrate defining the first flow path and/or the second flow path can be heated. For example, in certain embodiments, substrate 4220 can be disposed within any of the instruments disclosed herein, and first flow path 4221 and second flow path 4222 can be heated such that a substance contained therein (e.g., a portion of a sample to be transferred between separation chamber 4114 and reaction chamber 4262) can be heated and/or maintained at a temperature of about greater than 50 ℃. As described in more detail herein, this arrangement facilitates "hot start" transfer of substances and or reagents associated with the PCR process.

A first transfer mechanism 4140 is at least partially contained within the first module 4200 and is configured to facilitate transfer of sample S from separation chamber 4114 to a reaction chamber. In certain embodiments, the first transfer mechanism 4140 may facilitate transfer of the sample S while maintaining flow separation between the first flow path 4221 and the region outside of the first module 4200. For example, in certain embodiments, first transfer mechanism 4140 may be any mechanism that generates a force and/or facilitates transfer of sample S without the addition of a substance from a region outside of first module 4200 (e.g., without the addition of a compressed gas, etc.). This arrangement reduces potential contamination, improves process automation, and/or otherwise improves the speed and/or accuracy of sample S transfer. For example, the transfer of sample S may be programmed to occur at different time steps, with a different amount of sample S being transferred at each time step. Improving the transfer accuracy of sample S may also improve the quality of the PCR analysis. The first transfer mechanism may be any suitable mechanism as described herein. For example, in certain embodiments, first transfer mechanism 4140 can be a selective transfer mechanism to selectively transfer sample S between separation chamber 4114 and reaction chamber 4262. In certain embodiments, the first transfer mechanism 4140 may apply magnetic, electrostatic, and/or pressure to effect transfer of the sample S.

The first module 4200 may be coupled to the disconnect module 4110 in any suitable manner as described herein to allow liquid communication between the first module 4200 and the disconnect module 4110. In certain embodiments, for example, the first module 4200 and the split module 4110 may be separately constructed and coupled together such that the first module 4200 and the split module 4110 are a modular arrangement. In such a modular arrangement, different configurations of the first module 4200 and the split module 4110 may be used with each other. Different configurations of the first module 4200 and/or the separation module 4110 may include different reagents and/or different structures within the modules.

The second module 4160 includes a second transfer mechanism 4240 and defines a volume 4163 configured to contain a substance R1. As used herein, substance R1 and substance R2 may refer to one or more reagents. The substance R1 may be any biological or chemical substance, such as mineral oil, wash buffer, fluorescent dye, lysis buffer, wash buffer, elution buffer, reverse transcription reagent, PCR reagent (e.g. Taq polymerase, primers, DNA hybridization probes such as one or more of the probes described by Lukhtanov et al (2007), Nucleic Acids Research 35, page e 30), reagents, etc. Although fig. 5 shows the second module 4160 including one volume 4163, in other embodiments, the second module 4160 may include any number of volumes 4163 and/or containers within which multiple substances (including substance R1 and/or different substances) may be stored. The second module 4160 is configured to be coupled to the first module 4200 such that the volume 4163 may be selectively placed in flow communication with the reaction chamber 4262 via the second flow path 4222. When the second displacement mechanism 4240 is actuated, the second displacement mechanism 4240 is configured to displace at least a portion of the substance R1 from the volume 4163 to the reaction chamber 4262 (as indicated by arrow BB).

The second transfer mechanism 4240 can transfer substance R1 from the second volume 4163 to the reaction chamber 4262 or vice versa. In certain embodiments, for example, the second displacement mechanism can displace a predetermined volume of substance R1 between the second volume 4163 and the reaction chamber 4262. In certain embodiments, for example, the second transfer mechanism can transfer substance R1 between second chamber 4163 and reaction chamber 4262 at a predetermined volumetric flow rate. In certain embodiments, for example, the second displacement mechanism 4240 can be a pump configured to apply positive pressure or vacuum to the second volume 4163 and/or the reaction chamber 4262. In such embodiments, the second displacement mechanism 4240 may be a pump driven by the plug using any of the instruments and/or methods described herein. In certain embodiments, the second transfer mechanism 4240 can have a penetrable member as described herein such that, while in use, the second transfer mechanism 4240 can penetrate, break, sever and/or rupture the penetrable member and transfer the substance and/or sample contained in the reaction chamber 4262 into the second volume 4163 or vice versa. In certain other embodiments, for example, the second displacement mechanism 4240 can be a capillary flow control device. In still other embodiments, the second transfer mechanism 4240 can be any other selective or non-transfer mechanism as described herein.

In certain embodiments, cartridge 4001 can be used to perform sample preparation, nucleic acid isolation, and/or Polymerase Chain Reaction (PCR) on a sample or isolated portion thereof (e.g., an isolated nucleic acid sample). In such embodiments, the separation module 4110 can separate a target nucleic acid from a sample contained therein. The isolated nucleic acids can then be amplified (e.g., using PCR) in reaction chamber 4262, as described further below. Alternatively or additionally, if RNA is isolated, a reverse transcription reaction may be performed in reaction chamber 4262. In another embodiment, if RNA is isolated, an integrated reverse transcription-PCR reaction is performed in one of the reaction chambers, e.g., reaction chamber 4262. The modular arrangement of cartridge 4001 allows any number of different second modules 4160 to be used with first module 4200, said second modules 4160 each containing, for example, different reagents and/or each being configured to amplify a different type of sample, or to separate a different type of sample, and vice versa. In certain embodiments, cartridge 4001 can be operated by any of the instruments and/or methods described herein to facilitate an amplification process, such as a PCR process, to occur within reaction chamber 4262. In such embodiments, reaction vial 4260 may be coupled to and/or placed in contact with a thermal transfer device to allow the contents of reaction chamber 4262 to thermally cycle in conjunction with a PCR process. In such embodiments, the reaction vial 4260 may be further operably coupled to an optical instrument to monitor the PCR process. In other embodiments, the reaction vial 4260 and/or the separation module 4110 may be operably coupled to other energy sources, such as optical energy, ultrasonic energy, magnetic energy, hydraulic energy, etc., to facilitate the reaction and/or separation processes occurring therein.

Fig. 6 and 7 are schematic views of a portion of a cartridge 5001 in a first configuration and a second configuration, respectively, according to an embodiment. A portion of cartridge 5001 includes a first module 5200 and a second module 5100. The first module 5200 includes a reaction vial 5260, a substrate 5220, and a first transfer mechanism 5235. The reaction vial 5260 defines a reaction chamber 5262 that contains the sample in a manner that allows the reaction that binds to the sample S to occur. The reaction vial 5260 can have any suitable shape and/or size, and can be constructed using any suitable materials, as described herein. In certain embodiments, for example, the reaction vial 5260 may be a PCR vial, test tube, or the like.

The first transfer mechanism 5235 comprises a plug 5240 movably disposed within a housing 5230 such that the housing 5230 and the plug 5235 define a first volume 5213. The first volume 5213 contains a first substance R1. The first substance R1 can be, for example, a reagent (e.g., a PCR reagent, such as Taq polymerase, a primer, a DNA hybridization probe, such as the probes described above, or a combination thereof), a reverse transcription reagent, mineral oil, and the like. The plug 5240 can be actuated by any suitable mechanism, such as any of the instruments described herein.

The substrate 5220 defines at least a portion of a first flow path 5221 and a second flow path 5222. The first flow path 5221 is configured to be in fluid communication with the reaction chamber 5262, the first volume 5213, and the isolation chamber 5114 of the isolation module 5110 (shown in phantom in fig. 6). The second flow path 5222 is configured to be in flow communication with the separation chamber 5114. The separation chamber 5114 can be any suitable separation chamber and/or separation module of the type shown and described herein. Further, separation chamber 5114 may be coupled to first module 5200 in any suitable manner as described herein. In certain embodiments, the separation chamber 5114 can be coupled to the first module 5200 and the module arrangement as described herein. The removable coupling between the separation chamber 5114 and the first module 5200 can be fluid tight using any suitable mechanism as described herein.

The second module 5100 includes a second transfer mechanism 5150 and defines a second volume 5163 configured to contain a second substance R2. The second module 5100 is configured to be coupled to the first module 5200 such that the second volume 5163 can be selectively placed in fluid communication with the separation chamber 5114 via the second flow path 5222. The second module 5100 can include any mechanism and/or device configured to selectively place the second volume 5163 in fluid communication with the separation chamber 5114 and/or the second flow path 5222. For example, in certain embodiments, the second module 5100 can include a penetrable member that defines a portion of the boundary of the second volume 5163 and fluidly separates the second volume 5163 from the separation chamber 5114 and/or the second flow path 5222. In other embodiments, the second module 5100 can include a valve configured to place the second volume 5163 in fluid communication with the separation chamber 5114 and/or the second flow path 5222.

When the second transfer mechanism 5150 is actuated, the second transfer mechanism 5150 is configured to transfer at least a portion of the second substance R2 from the second volume 5163 into the separation chamber 5114. The second transfer mechanism 5150 can be any suitable transfer mechanism as described herein. For example, in certain embodiments, the second transfer mechanism 5150 can apply magnetic, electrostatic, and/or pressure to effect transfer of the substance R2 from the second volume 5163 to the separation chamber 5114. In certain embodiments, for example, the second transfer mechanism 5250 can be a pump driven by an embolus using any of the instruments and/or methods described herein. In certain other embodiments, for example, the second transfer mechanism 5250 can be a capillary flow control device.

Cartridge 5001 can be moved between at least a first configuration (fig. 6) and a second configuration (fig. 7) to facilitate reactions and/or assays involving sample S that is initially disposed within separation chamber 5114. When the cartridge 5001 is in the first configuration, the plug 5240 is in a first position within the housing 5230 such that a portion 5246 of the plug 5240 is disposed within the first flow path 5221. Thus, when the cartridge 5001 is in the first configuration, the first volume 5213 is fluidly isolated from the reaction chamber 5262. In this manner, when the cartridge 5001 is in the first configuration, the first substance R1 is maintained within the first volume 5213 and is prevented from passing into the reaction chamber 5262 (e.g., by leakage, gravity feed, capillary action, etc.). Further, when the cartridge 5001 is in the first configuration, the second volume 5163 is fluidly isolated from the second flow path 5222 and the separation chamber 5114. In this manner, when the cartridge 5001 is in the first configuration, the second substance R2 is maintained within the second volume 5163 and is prevented from transferring into the separation chamber 5114 (e.g., by leakage, gravity feed, capillary action, etc.).

By placing the second volume 5163 in fluid communication with the separation chamber 5114 via the second flow path 5222, the second transfer mechanism 5150 is actuated to transfer at least a portion of the second substance R2 into the separation chamber 5114 (as indicated by arrow CC in fig. 7), and the first transfer mechanism 5235 is actuated to move the cartridge 5001 to the second configuration (fig. 7). More specifically, the second volume 5163 can be placed in fluid communication with the separation chamber 5114 via the second flow path 5222 by any suitable mechanism, such as by penetrating a penetrable member, actuating a valve, or the like. In certain embodiments, the second volume 5163 can be placed in flow communication with the separation chamber 5114 by actuating the second transfer mechanism 5150. In this manner, the second volume 5163 can be placed in fluid communication with the separation chamber 5114 and a portion of the second substance R2 can be delivered into the separation chamber 5114 in one operation and/or in response to a single actuation event.

The first transfer mechanism 5235 is actuated by moving a plug 5240 within the housing 5230 as indicated by arrow DD in fig. 7. Similarly stated, when the first displacement mechanism 5235 is actuated, the plug 5240 is moved within the housing 5230 from a first position (as shown in fig. 6) to a second position (as shown in fig. 7). Thus, when the first displacement mechanism 5235 is actuated, a portion 5246 of the plug 5240 is at least partially removed from the first flow path 5221, thereby placing the first volume 5213 in fluid communication with the reaction chamber 5262 via the first flow path 5221. In this manner, a portion of the first substance R1 can be transferred from the first volume 5213 into the reaction chamber 5262, as indicated by arrow EE in fig. 7.

In addition, a vacuum is created within the reaction chamber 5262 as the plug 5240 is moved from the first position to the second position. This pressure differential within the cartridge 5001 (i.e., between the reaction chamber 5262 and the separation chamber 5114) causes at least a portion of the contents of the separation chamber 5114 (i.e., the sample S and/or the second substance R2) to be transferred into the reaction chamber 5262 via the first flow path 5221, as shown by arrows FF and GG in fig. 7. In this manner, substances and/or samples can be added, mixed, and/or transferred between the separation chamber 5114 and the reaction chamber 5262 by actuating the first transfer mechanism 5235 and/or the second transfer mechanism 5150. By performing mixing of the sample S and the substance R2 in the separation chamber 5114 instead of separately transferring the sample S and the substance R2 into the reaction chamber 5262, an additional transfer step may be eliminated. In addition, this arrangement and/or method can improve mixing of the sample S and the substance R2, thereby improving the accuracy and efficiency of the reaction in the reaction chamber 5262.

Although described as occurring in a particular order, in other embodiments, the operations associated with moving cartridge 5001 from the first configuration to the second configuration can occur in any order. Moreover, in other embodiments, cartridge 5001 can be placed in any number of different configurations involving any desired combination of operations.

In certain embodiments, cartridge 5001 can be used to perform a Polymerase Chain Reaction (PCR) on at least a portion of sample S (which can be, for example, one or more isolated target nucleic acids). In such embodiments, the isolated nucleic acids can be amplified (e.g., using PCR) in reaction chamber 5262, as described herein. In certain embodiments, cartridge 5001 can be operated by any of the instruments and/or methods described herein to facilitate the occurrence of a PCR process within reaction chamber 5262. In such embodiments, the reaction vials 5260 can be coupled to and/or placed in contact with a thermal transfer device to allow the contents of the reaction chambers 5262 to be thermally cycled in conjunction with a PCR process. In such embodiments, the reaction vial 5260 may be further operably coupled to an optical instrument to allow for real-time monitoring of the PCR process. In other embodiments, the reaction vial 5260 and/or the second module 5100 may be operably coupled to other energy sources, such as light energy, ultrasonic energy, magnetic energy, hydraulic energy, and the like, to facilitate the reaction and/or separation processes occurring therein.

In certain embodiments, the first substance R1 may include mineral oil, wax, or the like, such that the first substance R1 may form a layer on the surface of the fluid mixture (i.e., the sample S and the second substance R1) in the reaction chamber 5262 after the first substance R1 is transferred into the reaction chamber 5262. The surface layer of the first substance R1 can reduce evaporation of the fluid mixture in the reaction chamber 5262 during the course of the reaction (e.g., during thermal cycling), thereby improving the efficiency, accuracy, and/or control of the reaction therein. More specifically, by reducing evaporation of the fluid mixture in the reaction chamber 5262, the relative concentrations or proportions of the different constituent components in the reaction mixture can be more accurately controlled. In addition, reducing evaporation of the fluid mixture in the reaction chamber 5262 can also minimize condensation on the walls of the reaction vial 5260, thereby improving the accuracy of the optical monitoring or analysis of the reaction.

The mineral oil may be any mineral oil having suitable properties, such as desired physical properties, including, for example, density and/or surface tension. Mineral oil, etc. can also be selected such that it is chemically inert and physically stable when exposed to the conditions within the reaction chamber 5262.

Fig. 8-24 are various views of a cartridge 6001 according to one embodiment. In certain views, such as fig. 8 and 9, a portion of cartridge 6001 is shown as translucent so that components and/or features (features) within cartridge 6001 may be more clearly shown. Cartridge 6001 includes a sample preparation (or separation) module 6100 and an amplification (or PCR) module 6200, which may be coupled together to form an integrated cartridge 6001. One or more cartridges 6001 may be disposed within any suitable instrument of the type disclosed herein (see, e.g., instrument 3002 described below) that is configured to operate cartridge 6001, drive cartridge 6001, and/or interact with cartridge 6001 to perform nucleic acid separation, transcription, and/or amplification of a test sample contained within cartridge 6001. Cartridge 6001 allows for efficient and accurate diagnostic testing of samples by limiting sample throughput during and between isolation, transcription and/or PCR amplification processes. Furthermore, the modular arrangement of separation modules 6100 and amplification (or PCR) modules 6200 allows any number of different PCR modules 6200 to be used with any number of different separation modules 6100, the PCR modules 6200 each containing different reagents and/or being configured to amplify a different type of nucleic acid, the separation modules 6100 each containing different reagents and/or being configured to separate a different type of nucleic acid, and vice versa. This arrangement also allows the separation module 6100 and the amplification module 6200 to be stored separately. Separate storage may be useful, for example, if the reagents included within separation module 6100 have different storage requirements (e.g., expiration date, lyophilization requirements, storage temperature limits, etc.) than the reagents included within amplification module 6200.

As shown in fig. 11, the separation module 6100 includes a first (or separation) housing 6110 and a second (or reagent) housing 6160 coupled to the first housing 6110 and/or at least partially within the first housing 6110. For clarity purposes, the second housing 6160 is not shown in fig. 10 and 22. Fig. 11-14 show the second housing 6160 and the specific components contained therein, and fig. 15-18 show the second housing 6160 in a number of different stages of driving. The second housing 6160 includes a first end portion 6161 and a second end portion 6162 and defines a series of containment chambers 6163a, 6163b, 6163c, and 6163d that contain reagents and/or other substances used in the separation process. As described in more detail herein, the containment chamber can contain a protease (e.g., proteinase K), a lysis solution that dissolves a bulk material, a binding solution that magnetically charges the nucleic acid sample residing within the lysis chamber 6114, and a solution of magnetic beads that bind magnetically charged nucleic acids to aid in the transfer of nucleic acids within the separation module 6100 and/or the first housing 6110.

The housing chambers 6163a, 6163b, 6163c, and 6163d each include a drive 6166 (see, e.g., fig. 14) movably disposed therein. More specifically, as shown in fig. 18, the driver 6166a is disposed within the housing room 6163a, the driver 6166b is disposed within the housing room 6163b, the driver 6166c is disposed within the housing room 6163c, and the driver 6166d is disposed within the housing room 6163 d. As shown in fig. 15, the penetrable member 6170 is disposed about the second end portion 6162 of the second housing 6160 such that the interior portion of the second housing 6160, the penetrable member 6170, and the drivers 6166a, 6166b, 6166c, and 6166d collectively enclose and/or define the containment chambers 6163a, 6163b, 6163c, and 6163 d. Similarly stated, the inner portion of the second housing 6160, the penetrable member 6170, and the drivers 6166a, 6166b, 6166c, and 6166d collectively define fluid separation chambers 6163a, 6163b, 6163c, and 6163d within which reagents and/or substances may be stored. The penetrable member 6170 may be constructed of any suitable material of the types described herein, such as any form of polypropylene. In certain embodiments, the penetrable member 6170 may be constructed of Biaxially Oriented Polypropylene (BOP).

As shown in fig. 14, the drivers 6166 each include a plug portion 6167, a perforated portion 6168, and one or more driver holes 6169. The driver aperture 6169 is configured to accept a portion of a driver assembly to facilitate movement of the driver 6166 within a chamber (e.g., chamber 6163 a), as described herein. In particular, the driver aperture 6169 can receive a protrusion, such as protrusion 3446a of driver assembly 3400, as described below with respect to fig. 37-40. This arrangement allows the plug 6166 to be driven by the first end portion 6161 of the second housing 6160. In certain embodiments, the driver 6166 can include a retention mechanism (e.g., a protrusion, a snap ring, etc.) configured to retain a protrusion of a driver assembly (e.g., driver assembly 3400) to facilitate mutual movement of the driver 6166 through the driver assembly.

The plug portion 6167 of the actuator 6166 is configured to engage a portion of the second housing 6160 that defines a chamber (e.g., chamber 6163 a) within which the actuator 6166 is disposed such that the plug portion 6167 and a portion of the second housing 6160 form a substantially fluid-tight seal and/or a gas-tight seal. Thus, when the drive 6166 is positioned within a chamber (e.g., chamber 6163 a), leakage and/or transfer of material contained within the chamber is minimized and/or eliminated. In this manner, the end face of the plug portion 6167 defines a portion of the boundary of a chamber (e.g., chamber 6163 a). The plug portion 6167 is also configured such that when a force is applied to the driver 6166 (e.g., by the driver assembly 3400 shown and described below), the driver 6166 will move within the chamber (e.g., chamber 6163 a) to transfer the substance contained within the chamber into the lysis chamber 6114, as described below. In this manner, the drive 6166 can act as a transfer mechanism to transfer substances from a chamber (e.g., chamber 6163 a) of the separation module 6100 into another portion.

The perforated portion 6168 of the driver 6166 is configured to penetrate, break, sever, and/or rupture a portion of the penetrable member 6170 when the driver 6166 is moved within a chamber (e.g., chamber 6163 a) to place the chamber in liquid communication with an area outside the chamber. In this manner, each of the chambers 6163a, 6163b, 6163c, and 6163d can be selectively placed in fluid communication with another portion of the separation module 6100 (e.g., the lysis chamber 6114) to allow transfer of the substances contained within each of the chambers 6163a, 6163b, 6163c, and 6163d when the driver 6166a, 6166b, 6166c, and 6166d is driven, respectively, as described below.

The second housing 6160 includes a mixing pump 6181 that can be driven (e.g., by the driver assembly 3400 of the instrument 3002) to agitate, mix, and/or create turbulence within the sample, reagents, and/or other substances contained within a portion of the separation module 6100 (e.g., the lysis chamber 6114). As shown in fig. 12, pump 6181 includes a nozzle 6186 which may direct the flow, increase the pressure of the flow and/or increase turbulence within a portion of separation module 6100 to enhance mixing therein. Although the mixing pump 6181 is shown as a bellows-type pump, in other embodiments, the mixing pump 6181 can be any suitable mechanism for transferring energy to the solution within the lysis chamber 6114. Such mechanisms may include, for example, piston pumps, rotating members, and the like. In certain embodiments, the second housing 6160 can include any suitable mechanism for mixing the substances within the separation chamber 6114 to facilitate cell lysis of the sample contained therein and/or separation of the nucleic acids contained therein. In certain embodiments, the second housing 6160 can include an ultrasonic mixing mechanism, a thermal mixing mechanism, or the like.

As shown in fig. 11, the second housing 6160 is positioned within the aperture 6115 defined by the first end portion 6111 of the first housing 6110. Thus, a portion of the second housing 6160 defines at least a portion of the boundary of the lysis chamber 6114 when the second housing 6160 is positioned within the first housing 6110. More specifically, the penetrable member 6170 defines a portion of the boundary of the lysis chamber 6114 when the second housing 6160 is positioned within the first housing 6110. This arrangement allows the substance contained within the second housing 6160 to be transferred into the lysis chamber 6114 when a portion of the penetrable member 6170 is penetrated, broken, severed and/or ruptured (see, e.g., fig. 15). While at least a portion of the second housing 6160 is shown as being disposed within the first housing 6110 and/or the lysis chamber 6114, in other embodiments, the second housing 6160 can be coupled to the first housing 6110 without any portion of the second housing being disposed within the first housing. In still other embodiments, a portion of the first housing may be disposed within the second housing when the first housing and the second housing are coupled together.

As shown in fig. 12 and 13, the second housing 6160 includes a seal 6172 disposed about the second end portion 6162 such that when the second housing 6160 is coupled to the first housing 6110, the seal 6172 and a portion of the sidewall of the first housing 6110 collectively form a substantially fluid-tight seal and/or a gas-tight seal between the first housing 6110 and the second housing 6160. In other words, seal 6172 fluidly separates lysis chamber 6114 from the area outside cartridge 6001. In certain embodiments, the seal 6172 can also acoustically separate the second housing 6160 from the first housing 6110.

The first end portion 6161 of the second housing 6160 includes a projection 6171 configured to be received within a corresponding aperture 6119 (see, e.g., fig. 10) defined by the first housing 6110. Thus, when the second housing 6160 is positioned within the first housing 6110, the nub 6171 and aperture 6119 collectively retain the second housing 6160 within the first housing 6110. Similarly stated, the nub 6171 and aperture 6119 cooperate to limit movement of the second housing 6160 relative to the first housing 6110.

The modular arrangement of the first housing 6110 and the second housing 6160 allows any number of second housings 6160 (or reagent housings) each containing different reagents and/or substances to facilitate nucleic acid separation to be used with the first housing 6110 to form a separation module 6100. This arrangement also allows the first housing 6110 and the second housing 6160 to be stored separately. Separate storage can be useful, for example, if the reagents included within the second housing 6160 have different storage requirements (e.g., expiration date, lyophilization requirements, storage temperature limits, etc.) than the substance contained within the first housing 6110.

In use, the material contained within the second housing 6160 can be transferred into the first housing 6110 to facilitate the separation process. Figures 15-18 show cross-sectional views of a portion of the separation module 6100 during different stages of driving. For example, proteinase K can be stored in compartment 6163d and transferred into lysis compartment 6114, as shown in FIG. 15. More specifically, the driver 6166d can move within the chamber 6163d, as shown by arrow HH, when driven by any suitable external force, such as a force applied by the driver assembly 3400 of the instrument 3002 described herein. As the driver 6166d moves toward the lysis chamber 6114, the perforated portion 6168d contacts and penetrates a portion of the penetrable member 6170. In certain embodiments, the penetrable member 6170 may include perforations, stress riser tubes, or other structural discontinuities to ensure that the penetrable member 6170 easily penetrates the desired portion of the penetrable member 6170. In this manner, movement of the drive 6166d places the chamber 6163d in liquid communication with the lysis chamber 6114. The continuous movement of the drive 6166d transfers the contents of the chamber 6163d (e.g., proteinase K) into the lysis chamber 6114. In this manner, the driver 6166d acts as a shutter and transfer mechanism.

In another embodiment, the contents of chamber 6163d may include proteinase K (e.g., 10mg/mL, 15mg/mL, or 20mg/mL, mannitol, water, and bovine serum albumin. in a further embodiment, the beads are coated or derivatized with proteinase K. in another embodiment, the contents of chamber 6163d may include proteinase K, mannitol, water, and gelatin.

In another embodiment, the chamber 6163d also provides a positive control reagent. In one embodiment, the positive control reagent is a plurality of beads derived from an internal control nucleic acid sequence. In a further embodiment, the beads are provided in a solution of mannitol, BSA and water. In an even further embodiment, the beads and solution are provided as a lyophilized pellet, for example as a 50 μ L pellet.

Although described specifically for chamber 6163d, in other embodiments, proteinase K, a solution comprising proteinase K and/or a positive control reagent is present as substance R1 or R2.

In a similar manner, the lysis solution can be stored in compartment 6163c and transferred into the lysis compartment 6114, as shown in fig. 16. More specifically, the driver 6166c can move within the chamber 6163c, as shown by arrow II, when driven by any suitable external force, such as a force applied by the driver assembly 3400 of the instrument 3002 described herein. As the driver 6166c moves toward the lysis chamber 6114, the perforated portion 6168c contacts and penetrates a portion of the penetrable member 6170. In this way, the driver 61 66c places the chamber 6163c in fluid communication with the lysis chamber 6114. The continuous movement of the drive 6166c transfers the contents of the chamber 6163c (e.g., lysis solution) into the lysis chamber 6114. In this manner, the driver 6166c acts as a shutter and transfer mechanism. In one embodiment, the lysis solution stored in chamber 6163c or another chamber comprises guanidine HCl (e.g., 3M, 4M, 5M, 6M, 7M, or 8M), Tris HCl (e.g., 5mM, 10mM, 15mM, 20mM, 25mM, or 30 mM), triton-X-100 (e.g., 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, or 5%), NP-40 (e.g., 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, or 5%), Tween-20 (e.g., 5%, 10%, 15%, or 20%), CaCl₂(e.g., 1mM, 1.5mM, 2mM, 2.5mM, 3mM, 3.5mM, 4mM, 4.5mM, or 5 mM), molecular grade water. Although described specifically for chamber 6163c, in other embodiments, the lysis solution is present as species R1 or R2.

In a similar manner, the binding solution can be stored in chamber 6163b and transferred into lysis chamber 6114, as shown in fig. 17. More specifically, the driver 6166b can move within the chamber 6163b, as shown by arrow JJ, when driven by any suitable external force, such as a force applied by the driver assembly 3400 of the instrument 3002 described herein. As the driver 6166b moves toward the lysis chamber 6114, the perforated portion 6168b contacts and penetrates a portion of the penetrable member 6170. In this manner, movement of the drive 6166b places the chamber 6163b in liquid communication with the lysis chamber 6114. The continuous movement of the drive 6166b transfers the contents of the chamber 6163b (e.g., the binding solution) into the lysis chamber 6114. In this manner, the driver 6166b acts as a shutter and transfer mechanism. In one embodiment, the binding solution comprises isopropanol, e.g., 100% isopropanol, 90% isopropanol, 80% isopropanol, 70% isopropanol, in a volume of about 50 μ L, about 100 μ L, about 125 μ L, about 150 μ L, about 175 μ L, or about 200 μ L. Although described specifically for chamber 6163b, in other embodiments, the binding solution is present as species R1 or R2.

In a similar manner, a set of magnetic beads can be stored in the chamber 6163a and transferred into the lysis chamber 6114, as shown in fig. 18. More specifically, the driver 6166a can move within the chamber 6163a when driven by any suitable external force, such as a force applied by the driver assembly 3400 of the instrument 3002 described herein, as indicated by arrow KK. As the driver 6166a moves toward the lysis chamber 6114, the perforated portion 6168a contacts and penetrates a portion of the penetrable member 6170. In this manner, movement of the drive 6166a places the chamber 6163a in liquid communication with the lysis chamber 6114. The continuous movement of the drive 6166a transfers the contents of the chamber 6163a (e.g., magnetic beads) into the lysis chamber 6114. In this manner, the driver 6166a acts as a shutter and transfer mechanism. In one embodiment, the magnetic beads are paramagnetic. In one embodiment, the beads are magnetic silica beads and are provided at a concentration of 1.0mg/mL or 1.5mg/mL, 2.0mg/mL, 2.5mg/mL, 3.0mg/mL, or 3.5 mg/mL. In a further embodiment, the magnetic silica beads are stored in isopropanol, such as about 50% isopropanol, about 55% isopropanol, about 60% isopropanol, about 61% isopropanol, about 62% isopropanol, about 63% isopropanol, about 64% isopropanol, about 65% isopropanol, about 66% isopropanol, about 67% isopropanol, about 68% isopropanol, about 69% isopropanol, about 70% isopropanol, about 75% isopropanol, about 80% isopropanol, or about 85% isopropanol. In one embodiment, the beads are provided as a volume of about 50 μ L, about 100 μ L, about 125 μ L, about 150 μ L, about 175 μ L, or about 200 μ L. Although described specifically for chamber 6163a, in other embodiments, the beads are present as species R1 or R2.

As shown in fig. 10, the first housing 6110 includes a first end portion 6111 and a second end portion 6112 and defines a lysis chamber 6114, two wash chambers 6121 and 6122, three transfer assembly lumens 6123, 6124 and 6125, and an elution chamber 6190. The first housing 6110 also defines an aperture 6115 adjacent the separation chamber 6114. As shown in fig. 11 and described above, the second housing 6160 is positioned within the aperture 6115 such that a portion of the second housing 6160 (e.g., the penetrable member 6170) defines at least a portion of the boundary of the separation chamber 6114.

The first end portion 6111 also defines a fill aperture 6116 through which the lysis chamber 6114 can be placed in liquid communication with an area outside of the separation module 6100. As shown in fig. 8-10, the separation module 6100 includes a cover 6118 removably coupled around the fill hole 6116. In use, a sample containing the target nucleic acid, such as urine, blood, and/or other material containing a tissue sample, can be transferred into the lysis chamber 6114 via the fill hole 6116. The sample can be introduced into the lysis chamber 6114 via any suitable mechanism, including, for example, by aspirating or injecting the sample into the first chamber 6114 via the fill aperture 6116. In certain embodiments, the sample filter can be disposed within the fill aperture 6116 and/or the charging lid 6118. The filter may be, for example, a hydrophobic filter.

After the sample is disposed within the lysis chamber 6114, reagents and/or substances that promote cell lysis can be added to the lysis chamber 6114, as described above. In addition, the sample can be agitated and/or mixed via pump 6181 to facilitate the lysis process, as described above. In certain embodiments, the contents of the lysis chamber 6144 can be heated (e.g., by a third heating module 3780, as shown and described below with respect to the instrument 3002).

Separation module 6100 includes a series of transfer assemblies (also referred to as transfer mechanisms), shown in fig. 15-19 as transfer assembly 6140a, transfer assembly 6140b, and transfer assembly 6140 c. As described herein, the transfer assembly is configured to transfer substances (e.g., a portion of a sample comprising magnetically charged particles and isolated nucleic acids attached thereto) between the lysis chamber 6114, the wash chamber 6121, the wash chamber 6122, and the elution chamber 6190. More specifically, the transfer assembly 6140 is configured to transfer substances between the lysis chamber 6114, the wash chamber 6121, the wash chamber 6122, and the elution chamber 6190 while maintaining the separation chamber 6114, the wash chamber 6121, the wash chamber 6122, and the elution chamber 6190 substantially fluidically separated from other chambers (e.g., adjacent to the wash chamber) defined by the first housing 6110.

The transfer assembly 6140a is positioned within the transfer assembly lumen 6123 such that the transfer assembly 6140a is between the lysis chamber 6114 and the wash chamber 6121. Accordingly, the transfer assembly 6140a is configured to transfer substances between the lysis chamber 6114 and the wash chamber 6121.

Transfer assembly 6140b is positioned within transfer assembly lumen 6124 such that transfer assembly 6140b is between wash chamber 6121 and wash chamber 6122. Accordingly, transfer assembly 6140b is configured to transfer substances between the scrubbing chamber 6121 and the scrubbing chamber 6122.

Transfer assembly 6140c is positioned within transfer assembly lumen 6125 such that transfer assembly 6140c is between wash chamber 6122 and elution chamber 6190. Accordingly, transfer assembly 6140c is configured to transfer substances between wash chamber 6122 and elution chamber 6190.

The transfer assembly is described with respect to fig. 20 and 21, respectively, which fig. 20 and 21 show a representative transfer assembly 6140. The transfer assembly 6140 includes a housing 6141 and a movable member 6146, which is rotatably disposed within the housing 6141. The housing 6141 defines a first aperture 6142 and a second aperture 6143. When the transfer assembly 6140 is positioned within a transfer assembly lumen (e.g., transfer assembly lumen 6123), the housing 6141 is aligned such that a first aperture 6142 is aligned with and/or in fluid communication with a first chamber (e.g., lysis chamber 6114) and a second aperture 6143 is aligned with and/or in fluid communication with a second chamber (e.g., wash chamber 6121). Housing 6141 can be secured within a transfer assembly lumen (e.g., transfer assembly lumen 6123) by any suitable mechanism, such as by mechanical fasteners or retainers, chemical bonds or adhesives, interference fits, welded joints, etc. In addition, the housing 6141 can include one or more seals (not shown in fig. 20 and 21) such that the first chamber (e.g., the lysis chamber 6114) and the second chamber (e.g., the wash chamber 6121) are maintained in fluid separation from each other. Similarly stated, the housing 6141 and the first housing 6110 can collectively form a substantially fluid-tight seal and/or a gas-tight seal to eliminate and/or reduce leakage of materials between the first chamber (e.g., the lysis chamber 6114) and the second chamber (e.g., the washing chamber 6121).

The moveable member 6146 includes an outer surface 6147 that defines a groove or cavity 6148. The movable member 6146 is disposed within the housing 6141 such that the movable member 6146 can rotate as shown by arrow MM in fig. 20 and 21. For clarity, the outer surface 6147 of the movable member 6146 is shown spaced apart from the inner surface 6145 of the housing 6141 in fig. 20. The outer surface 6147 is in sliding contact with the inner surface 6145 of the housing 6141 such that the outer surface 6147 and the inner surface 6145 create a substantially fluid-tight seal and/or a gas-tight seal. In this manner, leakage of materials between the first chamber (e.g., the lysis chamber 6114) and the second chamber (e.g., the wash chamber 6121) via the interface between the housing 6141 and the movable member 6146 is eliminated and/or reduced.

The moveable member 6146 further defines a lumen 6149 configured to receive a portion of the driver 510. The driver 510 may be any suitable driver, such as the shaft 3510 of the transfer driver assembly 3500 of the instrument 3002 shown and described below with respect to fig. 41-46. As shown in fig. 20, the shape of the driver 510 can correspond to the shape of the lumen 6149 defined by the moveable member 6146, such that rotation of the driver 510 results in rotation of the moveable member 6146. Similarly stated, the driver 510 can be matingly positioned within the lumen 6149 such that relative rotational movement between the driver 510 and the moveable member 6146 is limited. In certain embodiments, driver 510 and lumen 6149 may have substantially similar hexagonal and/or octagonal shapes.

In use, the moveable member 6146 may be moved between a first position (not shown) and a second position (fig. 20) by rotating the moveable member 6146 as indicated by arrow MM. When the moveable member 6146 is in the first position, the groove or cavity 6148 is aligned with and/or in fluid communication with a first chamber (e.g., the lysis chamber 6114). When the moveable member 6146 is in the second position, the recess or cavity 6148 is aligned with and/or in fluid communication with a second chamber (e.g., the washing chamber 6121). Accordingly, one or more substances contained within a first chamber (e.g., the lysis chamber 6114) can be transferred to a second chamber (e.g., the wash chamber 6121) by capturing or disposing a portion of the substance within the cavity 6148 when the moveable member 6146 is in the first position, rotating the moveable member to the second position, and removing the substance from the cavity 6148.

In certain embodiments, the substance can be captured, disposed, and/or maintained within the cavity 6148 by magnetic force. For example, in certain embodiments, the driver 510 may include a magnetic portion. In use, driver 510 is aligned with desired transfer assembly 6140 and moved into lumen 6149, as shown by arrow LL in fig. 19. Because the shape of driver 510 can correspond to the shape of lumen 6149 as described above, an alignment operation can be performed in certain embodiments to ensure that driver 510 will fit within lumen 6149. When the magnetic portion of the driver 510 is within the lumen 6149, and when the moveable member 6146 is in the first position, the magnetic portion of the sample (e.g., magnetic beads and nucleic acids attached thereto) moves from the first chamber (e.g., the lysis chamber 6114) into the cavity 6148. The driver 510 is then rotated as shown by arrow MM in fig. 20 and 21. When the moveable member 6146 is in the second position, the driver 510 may be removed from the lumen 6149, thereby removing the magnetic force that retains the magnetic portion of the sample within the cavity 6148. Accordingly, a portion of the sample can then be removed from the cavity 6148 and into a second chamber (e.g., the washing chamber 6121). A portion of the sample can be removed from the cavity 6148 and into a second chamber (e.g., a wash chamber 6121) by any suitable mechanism, e.g., by gravity, fluid movement, etc. For example, as described below, in certain embodiments, the mixing mechanism 6130a can include a nozzle (e.g., nozzle 6131 a) to introduce a pressure jet into and/or adjacent to the cavity 6148 to move a portion of the sample out of the cavity 6148 and into a second chamber (e.g., wash chamber 6121).

The use of a transfer mechanism 6140 as described herein can eliminate the need for a separate waste chamber within the first housing 6110 and/or a flow path for conveying waste. Rather, as described above, the target portion of the sample moves between chambers (e.g., from wash chamber 6121 to wash chamber 6122), while other portions of the sample remain in a previous chamber (e.g., wash chamber 6122). Furthermore, because the transfer mechanism 6140 maintains a fluidic separation between the two chambers (e.g., the wash chamber 6121 and the wash chamber 6122), waste fluids are prevented from entering the chambers (e.g., the wash chamber 6122) along with the target portion of the sample. Thus, this arrangement also eliminates the need for a filtering mechanism within the first housing 6110, between the chambers described therein, and/or within the flow path defined by the separation module 6100.

The use of a transfer mechanism 6140 as described herein also allows a target portion of the sample to be transported within the separation module 6100 while maintaining the pressure within the separation module at or near ambient pressure. Similarly stated, the transfer mechanism 6140 as described herein transfers a target portion of the sample without creating a substantial pressure differential within the separation module 6100. Thus, this arrangement may reduce sample leakage from the separation module.

The separation module 6100 includes two mixing mechanisms 6130a and 6130b (also referred to as wash pumps). As described herein, the mixing mechanisms 6130a and 6130b are configured to generate fluid flow within the wash chamber 6121 and the wash chamber 6122, respectively, to facilitate washing and or mixing of a portion of the sample contained therein. Similarly stated, the mixing mechanisms 6130a and 6130b are configured to transfer energy into the washing chamber 6121 and the washing chamber 6122, respectively.

The mixing mechanism 6130a includes a driver 6132a and a nozzle 6131 a. The mixing mechanism 6130a is coupled to the first housing 6110 such that at least a portion of the nozzle 6131a is disposed within the washing chamber 6121. In particular, the mixing mechanism 6130a includes a coupling portion 6133a configured to couple to a corresponding coupling portion 6134a of the first housing 6110. Although the coupling portions 6133a and 6134a are shown as defining a threaded interface, in other embodiments, the mixing mechanism 6130a can be coupled to the first housing 6110 by any suitable method, such as by mechanical fasteners or retainers, chemical bonds or adhesives, interference fits, welded joints, or the like.

Similarly, the mixing mechanism 6130b includes a driver 6132b and a nozzle 6131 b. The mixing mechanism 6130b is coupled to the first housing 6110 such that at least a portion of the nozzle 6131b is disposed within the washing chamber 6122. In particular, the mixing mechanism 6130b includes a coupling portion 6133b configured to couple to a corresponding coupling portion 6134b of the first housing 6110. Although the coupling portions 6133b and 6134b are shown as defining a threaded interface, in other embodiments, the mixing mechanism 6130b can be coupled to the first housing 6110 by any suitable method, such as by mechanical fasteners or retainers, chemical bonds or adhesives, interference fits, welded joints, or the like.

The drivers 6132a and 6132b each include a top surface 6136a and 6136b, respectively, that are configured to be contacted and/or driven by a driver assembly of an instrument, such as the driver assembly 3600 of the instrument 3002 described herein. In use, the driver assembly can depress and/or move the top surface 6136a and 6136b of each driver 6132a and 6132b to create a pressure within each mixing mechanism 6130a and 6130 b. Pressure is transmitted into the wash chambers 6121 and 6122 to facilitate washing, mixing, and/or other interactions between and within the samples disposed therein. As described above, in certain embodiments, at least one of the nozzles (e.g., nozzle 6131 a) can include a tip portion that is angled, curved, and/or otherwise shaped to direct pressure energy and/or flow generated by the driver (e.g., driver 6132 a) to a particular region within the washing chamber (e.g., washing chamber 6121). For example, in certain embodiments, the nozzle 6131a can be shaped to direct the pressure energy and/or flow generated by the driver 6132a toward the cavity 6148 of the transfer mechanism 6140.

Although the drives 6132a and 6132b are each shown as a bellows-type pump, in other embodiments, the mixing mechanism 6130a and/or the mixing mechanism 6130b can include any suitable mechanism for generating and/or transferring energy into the washing chambers 6121 and 6122. Such mechanisms may include, for example, piston pumps, rotating members, and the like. In certain embodiments, the mixing mechanism may include an ultrasonic energy source, a thermal energy source, or the like.

Although the mixing mechanisms 6130a and 6130b are shown and described as generating energy and/or transferring energy into the scrubbing chambers 6121 and 6122, respectively, in other embodiments, the mixing mechanisms can also define a volume within which a substance fluidly separate from the scrubbing chambers (e.g., a scrubbing buffer solution) can be stored. Thus, when the mixing mechanism is actuated, the substance can be transferred into the washing chamber. In this manner, in certain embodiments, the mixing mechanism may also act as a transfer mechanism.

The amplification (or PCR) module includes a housing 6210 (having a first end portion 6211 and a second end portion 6212), a PCR vial 6260, and a transfer tube 6250. PCR vial 6260 is coupled to a first end portion 6211 of housing 6210 and defines a volume 6262 within which a sample can be disposed to facilitate a reaction that binds to the sample. PCR vial 6260 can be any suitable container for containing a sample in a manner that allows a reaction to be performed in conjunction with the sample. PCR vial 6260 may also be used in any suitable container containing a sample in a manner that allows for monitoring of such reactions (e.g., detecting analytes within the sample that are due to or bound to the reaction). In certain embodiments, at least a portion of the PCR vial 6260 can be substantially transparent to allow optical monitoring of the reaction occurring therein to be an optical system (e.g., the optical assembly 3800 of the instrument 3002 described herein).

As shown in fig. 8, 9, 10, and 22, the amplification module 6200 is coupled to the second end portion 6112 of the first housing 6110 of the separation module 6100 such that at least a portion of the transfer tube 6250 is disposed within the elution chamber 6190 of the separation module 6100. In this manner, the isolated nucleic acids, any substances, and/or any PCR reagents disposed within elution chamber 6190 can be transferred from elution chamber 6190 to PCR vial 6260 via transfer tube 6250 as described herein.

The housing 6210 defines a series of reagent chambers 6213a, 6213b, 6213c (see, e.g., fig. 22) and a pump cavity 6241. Reagent chambers 6213a, 6213b, 6213c may contain any suitable substance that combines with the reactions and/or processes occurring in PCR vial 6260. The reagent chambers 6213a, 6213b, 6213c may include, for example, elution fluids, a master mix, probes, and/or primers to facilitate the PCR process. As shown in fig. 24, housing 6210 defines a series of channels 6221a, 6221b, 6221c configured to place each of reagent chambers 6213a, 6213b, 6213c in liquid communication with elution chamber 6190 of separation module 6100. Although not shown in fig. 22, in certain embodiments, a penetrable member may be disposed within any of the reagent chambers 6213a, 6213b, 6213c and/or within any of the channels 6221a, 6221b, 6221c to fluidically separate the respective reagent chamber from the elution chamber 6190. In a manner similar to that described above with respect to penetrable member 6170, in such embodiments, the penetrable member may be perforated by a reagent plug to selectively place the reagent chamber in liquid communication with the elution chamber.

A reagent plug 6214a is movably disposed within the reagent chamber 6213a, a reagent plug 6214b is movably disposed within the reagent chamber 6213b, and a reagent plug 6214c is movably disposed within the reagent chamber 6213 c. In this manner, when the reagent plug (e.g., reagent plug 6214 a) is moved, as shown by arrow NN in fig. 22, the reagent plug transfers the contents of the reagent chamber (e.g., reagent chamber 6213 a) into the elution chamber 6190 via the bonded channel (e.g., channel 6221 a). In this way, the reagent plug acts as a transfer mechanism.

The agent plugs 6214a, 6214b, 6214c may be contacted and/or driven by an actuator assembly of an instrument, such as the actuator assembly 3600 of the instrument 3002 described herein. In certain embodiments, the reagent plugs 6214a, 6214b, 6214c can include a retention mechanism (e.g., a protrusion, snap ring, etc.) configured to retain a portion of the driver assembly (e.g., driver assembly 3400) to facilitate the mutual movement of the reagent plugs 6214a, 6214b, 6214c by the driver assembly.

The PCR module includes a transfer mechanism 6235 configured to transfer material from elution chamber 6190 of separation module 6100 and PCR vial 6260 of PCR module 6200 and/or between elution chamber 6190 of separation module 6100 and PCR vial 6260 of PCR module 6200. The transfer mechanism 6235 includes a transfer piston 6240 disposed within a pump cavity 6241. As transfer piston 6240 moves within pump cavity 6241, a vacuum and/or positive pressure is created within PCR volume 6262 as indicated by arrow OO in fig. 22. This pressure differential between PCR volume 6262 and elution chamber 6190 causes at least a portion of the contents of elution chamber 6190 to be transferred to (or from) PCR volume 6262 via transfer tube 6250 and channel 6222 (see, e.g., fig. 24). In this manner, substances and/or samples can be added, mixed, and/or transferred between elution chamber 6190 and PCR volume 6262 by driving transfer mechanism 6235. The transfer mechanism 6235 may be driven by any suitable mechanism, such as the driver assembly 3600 of the instrument 3002 described herein.

The transfer piston 6240 and the pump cavity 6241 may be in any suitable location within the PCR module 6200. For example, although transfer piston 6240 is shown as being disposed substantially on PCR vial 6260, in other embodiments, transfer piston 6240 can be disposed substantially on elution chamber 6190.

In certain embodiments, the housing 6210 defines one or more vent channels to fluidly couple the elution chamber 6190 and/or the PCR vial 6260 to the atmosphere. In certain embodiments, any of such vents can include a frit to minimize loss of sample and/or reagents from elution chamber 6190 and/or PCR vial 6260 and/or to prevent loss of sample and/or reagents from elution chamber 6190 and/or PCR vial 6260.

In use, after nucleic acid is separated and processed within separation module 6100, it is transferred via transfer assembly 6140c into elution chamber 6190, as described above. The magnetic beads are then removed (or "washed") from the nucleic acids by the elution buffer and removed from the elution chamber 6190. Thus, the elution chamber 6190 contains isolated and/or purified nucleic acids. In certain embodiments, the elution buffer is contained within the elution chamber 6190. In other embodiments, the elution buffer is contained in one of the reagent chambers of the PCR module 6200 (e.g., reagent chamber 6213 c) and transferred into the elution chamber 6190, as described above. In one embodiment, the elution buffer comprises a filtered solution of molecular grade water, tris HCl (e.g., about 10mM, about 15mM, about 20mM, about 25mM, about 30mM, about 35mM, or about 40 mM), magnesium chloride (e.g., about 1mM, about 2mM, about 3mM, about 4mM, about 5mM, about 6mM, about 7mM, about 8mM, about 9mM, about 10mM, or about 20 mM), glycerol (e.g., about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 12%, about 14%, about 16%, about 18%, about 20%, or about 25%). In one embodiment, the pH of the elution buffer is about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8.0, about 8.1, about 8.2, about 8.3, about 8.4, about 8.5, about 8.6, about 8.7, about 8.8, about 8.9, or about 9.0). In another embodiment, the elution buffer comprises a bactericidal agent, e.g., the elution buffers provided above further comprise a bactericidal agent. In one embodiment, the elution buffer also serves as a wash buffer. Although described specifically for the elution chamber 6190, in other embodiments, the elution buffer described above is present as species R1 or R2.

In certain embodiments, PCR reagents are subsequently transferred from the PCR module 6200 into the elution chamber 6190. More specifically, reagent plugs 6214a, 6214b, and/or 6214c are actuated (e.g., by instrument 3002) to introduce reagents into elution chamber 6190 via channels 6221a, 6221b, 6221 c. The PCR sample is then transferred from the elution chamber 6190 into the PCR vial 6260 via the transfer tube 6250 and the channel 6222. In particular, transfer piston 6240 can be driven to create a pressure differential within PCR module 6200 to transfer a PCR sample from elution chamber 6190 into PCR vial 6260, as described above. In this manner, PCR samples (isolated nucleic acids and PCR reagents) are prepared in the elution chamber 6190. By performing mixing of the reagents and nucleic acid sample within the elution chamber 642 (rather than transferring the isolated nucleic acids into and performing mixing within the PCR vial 6260), additional transfer of nucleic acids is avoided. This arrangement may result in improved accuracy of the post-PCR analysis, such that in some cases the analysis may be semi-quantitative in nature.

In other embodiments, however, PCR samples (isolated nucleic acids and PCR reagents) can be prepared in PCR vial 6260. In such embodiments, for example, PCR reagents can be stored in PCR vial 6260, e.g., in lyophilized form. The isolated nucleic acids can be transferred into PCR vial 6260 and mixed with the lyophilized PCR reagents to reconstitute the reagents within PCR vial 6260.

After the PCR sample is in PCR vial 6260, the PCR sample can be thermally cycled (e.g., via heater assembly 3700 of instrument 3002) to perform the desired amplification. After thermal cycling is complete and/or during thermal cycling, the PCR sample may optionally be analyzed (e.g., via the optical assembly 3800 of the instrument 3002) to analyze the sample. A description of the instrument 3002 is provided below.

Figures 25-33 are multiple views of a cartridge 7001 according to one embodiment. Certain parts of cartridge 7001 are similar to corresponding parts of cartridge 6001 and are therefore not described below. The discussion presented above with respect to cartridge 6001 is incorporated within the discussion of cartridge 7001 when applicable. For example, although the drive (e.g., drive 7163 a) within the second housing 7160 has a different size and/or shape than the drive (e.g., drive 6163 a) within the second housing 6160, many aspects of the structure and function of the drive within the second housing 6160 are similar to that of the drive within the housing 7160. Accordingly, the description presented above for a driver (e.g., driver 6160 a) is applicable to the driver (e.g., driver 7160 a) described below.

Cartridge 7001 comprises a sample preparation (or separation) module 7100 and an amplification (or PCR) module 7200, which are coupled together to form an integrated cartridge 7001. Boot 7005 is disposed around a portion of separation module 7100 and PCR module 7200. One or more cartridges 7001 may be disposed within any suitable instrument type disclosed herein (e.g., instrument 3002 described below) configured to manipulate cartridge 7001, actuate cartridge 7001, and/or interact with cartridge 7001 to perform nucleic acid separation, transcription, and/or amplification on a test sample contained within cartridge 7001.

As shown in fig. 26-28, the separation module 7100 includes a first (or separation) housing 7110 and a second (or reagent) housing 7160 coupled to the first housing 7110 and/or at least partially within the first housing 7110. The second housing 7160 defines a series of containment chambers 7163a, 7163b, 7163c and 7163d that contain reagents and/or other substances used in the separation process. As described herein, the containment chamber can contain a protease (e.g., proteinase K), a lysis solution that dissolves a substantial amount of material, a binding solution that magnetically charges the nucleic acid sample residing within the lysis chamber 7114, and a magnetic bead solution that binds magnetically charged nucleic acids to aid in nucleic acid transport within the isolation module 7100 and/or the first housing 7110. In one embodiment, the foregoing solutions provided above are used in the cartridges provided in fig. 26-28.

The containment compartments 7163a, 7163b, 7163c, and 7163d each include a drive movably disposed therein. More specifically, as shown in fig. 27 and 28, actuator 7166a is disposed within housing chamber 7163a, actuator 7166b is disposed within housing chamber 7163b, actuator 7166c is disposed within housing chamber 7163c, and actuator 7166d is disposed within housing chamber 7163 d. Drives 7166a, 7166b, 7166c, and 7166d are each similar to drive 6166 shown and described above (see, e.g., fig. 14). In particular, each of the drives 7166a, 7166b, 7166c, and 7166d may act as a transfer mechanism to transfer a substance from a chamber (e.g., chamber 7163 a) into another portion of the separation module 7100 when moved in the direction indicated by arrow PP in fig. 28.

As shown in fig. 27, the penetrable member 7170 is disposed about a portion of the second housing 7160 such that the interior portion of the second housing 7160, the penetrable member 7170, and the drivers 7166a, 7166b, 7166c, and 7166d collectively enclose and/or define containment chambers 7163a, 7163b, 7163c, and 7163 d. Similarly stated, the interior portion of the second housing 7160, the penetrable member 7170, and the drivers 7166a, 7166b, 7166c, and 7166d collectively define fluid separation chambers 7163a, 7163b, 7163c, and 7163d in which reagents and/or substances may be stored. The penetrable member 7170 may be constructed of any suitable material of the type described herein, such as any form of polypropylene. In certain embodiments, the penetrable member 7170 may be constructed of Biaxially Oriented Polypropylene (BOP).

The second housing 7160 includes a mixing pump 7181 that can be driven (e.g., by a driver assembly 3400 of the instrument 3002) to agitate, mix, and/or create turbulence within the sample, reagents, and/or other substances contained within a portion of the separation module 7100 (e.g., the lysis chamber 7114).

As shown in fig. 26-28, the second housing 7160 is positioned within an aperture defined by the first housing 7110. Thus, when the second housing 7160 is disposed within the first housing 7110, a portion of the second housing 7160 defines at least a portion of the boundary of the lysis chamber 7114. More specifically, the penetrable member 7170 defines a portion of the boundary of the lysis chamber 7114 when the second housing 7160 is disposed within the first housing 7110. This arrangement allows the substance contained within the second housing 7160 to be transferred into the lysis chamber 7114 when a portion of the penetrable member 7170 is penetrated, ruptured, severed and/or ruptured. In a similar manner as described above with respect to the separation module 6100, the substance contained within the second housing 7160 may be transferred into the first housing 7110 when the drives 7166a, 7166b, 7166c and 7166d are actuated.

As shown in fig. 27 and 28, the first housing 7110 comprises a first (or top) section 7112 and a second (or bottom) section 7111. In certain embodiments, the tip portion 7112 may be constructed separately from the bottom portion 7111 and may then be coupled to the bottom portion 7111 to form a first housing 7110. The first enclosure defines a lysis chamber 7114, two wash chambers 7121 and 7122, three transfer set lumens (not shown in fig. 27 and 28), and an elution chamber 7190. The first housing 7110 also defines an aperture adjacent to the separation chamber 7114 within which a portion of the second housing 7160 is disposed.

As shown in fig. 26-28, the separation module 7100 includes a cover 7118 that is removably coupled to the housing 7110. In use, after the lid 7118 is removed, a sample containing the target nucleic acid, such as urine, blood, and/or other material containing a tissue sample, can be transferred into the lysis chamber 7114 via the fill hole 7116. The sample may be introduced into the lysis chamber 7114 via any suitable mechanism, including, for example, by aspirating or injecting the sample into the first chamber 7114 via the fill aperture 7116.

After the sample is disposed within the lysis chamber 7114, reagents and/or substances that facilitate cell lysis may be added to the lysis chamber 7114, as described above. In addition, the sample can be agitated and/or mixed via pump 7181 to facilitate the lysis process, as described above. In certain embodiments, the contents of the lysis chamber 7144 can be heated (e.g., by a third heating module 3780, as shown and described below with respect to the instrument 3002). In addition, the second section 7111 of the first housing 7110 includes an acoustic coupling section 7182. Accordingly, in certain embodiments, at least a portion of an acoustic transducer (not shown) may be disposed in contact with acoustic coupling portion 7182. In this manner, acoustic and/or ultrasonic energy generated by the transducer may be transmitted through the acoustic coupling portion 7182 and the sidewall of the first housing 7110 and into the solution within the lysis chamber 7114.

Separation module 7100 includes a series of transfer assemblies (also referred to as transfer mechanisms) shown in fig. 26-28 as transfer assembly 7140a, transfer assembly 7140b, and transfer assembly 7140 c. As described herein, the transfer assembly is configured to transfer a substance (e.g., a portion of a sample comprising magnetically charged particles and isolated nucleic acids attached thereto) between the lysis chamber 7114, the wash chamber 7121, the wash chamber 7122, and the elution chamber 7190. More specifically, the transfer assembly 7140 is configured to transfer materials between the lysis chamber 7114, the wash chamber 7121, the wash chamber 7122, and the elution chamber 7190 while maintaining the separation chamber 7114, the wash chamber 7121, the wash chamber 7122, and the elution chamber 7190 in substantially fluid separation from other chambers (e.g., adjacent to the wash chamber) defined by the first housing 7110. Transfer assemblies 7140a, 7140b, and 7140c are similar in structure and function to transfer assembly 6140 shown and described above with respect to separation module 6100, and therefore are not described in detail below.

The separation module 7100 comprises two wash buffer modules 7130a and 7130b, each coupled to the upper portion 7112 of the first housing 7110. As described herein, each wash buffer module 7130a and 7130b contains a substance (e.g., a reagent to be added to the sample, a wash buffer solution, mineral oil, and/or any other substance) and is configured to transfer the substance into the wash chamber 7121 and the wash chamber 7122, respectively, when actuated. In addition, each wash buffer module 7130a and 7130b is configured to generate a fluid flow within the wash chamber 7121 and the wash chamber 7122, respectively, to facilitate washing and or mixing of the portions of the sample contained therein. Similarly stated, each wash buffer module 7130a and 7130b is configured to transfer energy into the wash chamber 7121 and the wash chamber 7122, respectively. In one embodiment, wash buffer modules 7130a and/or 7130b comprise a wash buffer comprising a filtered solution of molecular grade water, tris HCl (e.g., about 10mM, about 15mM, about 20mM, about 25mM, about 30mM, about 35mM, or about 40 mM), magnesium chloride (e.g., about 1mM, about 2mM, about 3mM, about 4mM, about 5mM, about 6mM, about 7mM, about 8mM, about 9mM, about 10mM, or about 20 mM), glycerol (e.g., about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 12%, about 14%, about 16%, about 18%, about 20%, or about 25%). In one embodiment, the pH of the wash buffer is about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8.0, about 8.1, about 8.2, about 8.3, about 8.4, about 8.5, about 8.6, about 8.7, about 8.8, about 8.9, or about 9.0). In another embodiment, the elution buffer comprises a bactericidal agent, e.g., the elution buffers provided above further comprise a bactericidal agent.

Although described specifically for chambers 7130a and/or 7130b, in other embodiments, the wash buffer described immediately above is present as species R1 and/or R2.

In another embodiment, the wash buffer modules 7130a and/or 7130b comprise a wash buffer comprising molecular grade water, guanidine HCl (e.g., about 0.7mM, about 0.8mM, about 0.81mM, about 0.82mM, about 0.83mM, about 0.84mM, about 0.85mM, about 0.9mM, about 1.0 mM), tris HCl (e.g., about 10mM, about 15mM, about 20mM, about 25mM, about 30mM, about 35mM, or about 40mM, and may have a pH of about 7.5, about 8, or about 8.5), triton-X-100 (e.g., about 0.25%, about 0.5%, about 0.75%, about 1%), Tween-20 (e.g., about 0.25%, about 0.5%, about 0.75%, about 1 EDTA (e.g., about 0.1mM, about 0.2mM, about 0.3mM, about 0.5mM, about 0.75mM, about 1mM, about 2mM, about 3mM, about 5mM, about 8mM, or about 8.5 mM), triton-100 mM, triton-1 mM, or about 0.5mM, or about 5mM, and/, Isopropanol (e.g., about 10%, about 20%, about 30%, about 40%, about 50%, about 60%). In one embodiment, the pH of the wash buffer is about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8.0, about 8.1, about 8.2, about 8.3, about 8.4, about 8.5, about 8.6, about 8.7, about 8.8, about 8.9, or about 9.0). Although described specifically for chambers 7130a and/or 7130b, in other embodiments, the wash buffer described immediately above is present as species R1 and/or R2.

The wash buffer module 7130a comprises a drive 7150a movably disposed within a housing 7137 a. The housing 7137a is coupled to the upper portion 7112 of the first housing 7110 such that the wash buffer module 7130a is substantially aligned with the wash chamber 7121. In particular, the housing 7137a includes a pair of projections 7133a configured to be positioned within respective apertures defined by the coupling portions 7134a of the upper section 7112 of a first housing 7110. Although the wash buffer module 7130a is shown coupled to the first housing 7110 by a "snap-fit," in other embodiments, the wash buffer module 7130a can be coupled to the first housing 7110 by any suitable method, such as by a threaded interface, a mechanical fastener or retainer, a chemical bond or adhesive, an interference fit, a welded joint, or the like.

The driver 7150a includes a plug portion 7151a, a perforated portion 7152a, and an engagement portion 7153 a. The engagement portion 7153a is configured to engage, removably couple to, and/or be received within a portion of the driver assembly to facilitate movement of the driver 7150a within the housing 7137a, as described herein. The driver 7150a may be operated and/or driven by any suitable instrument, such as the driver assembly 3600 described below with respect to fig. 47-51.

The plug portion 7151a of the driver 7150a is disposed within the housing 7137 a. The penetrable member 7135a is disposed about a distal portion of the housing 7137a such that the end face of the plug portion 7151a, the housing 7137a, and the penetrable member 7135a collectively define a volume within which the substance is disposed. The plug portion 7151a and the inner surface of the housing 7137a are configured to form a substantially fluid-tight seal and/or a gas-tight seal. In certain embodiments, the plug portion 7151a can comprise a sealing member, an o-ring, or the like.

When the driver 7150a is moved within the housing 7137a by the direction indicated by arrow QQ in fig. 28, the perforated portion 7152a of the driver 7150a is configured to penetrate, break, sever and/or rupture a portion of the penetrable member 7135 a. In this manner, movement of the drive 7150 places the chamber in fluid communication with the wash chamber 7121. Similarly stated, the wash buffer module 7130a can be selectively placed in fluid communication with the wash chamber 7121 when the driver 7150a is driven. After the substances within the wash buffer module 7130a are transferred into the wash chamber 7121, the driver 7150a may reciprocate within the housing 7137a to create pressure that is transferred into the wash chamber 7121 to facilitate washing, mixing, and/or other interactions between and within the samples disposed therein. The top portion 7112 of the first housing 7110 includes a nozzle 7131a configured to direct pressure energy and/or flow generated by an actuator 7150a to a particular region within the washing chamber 7121.

The wash buffer module 7130b comprises a drive 7150b movably disposed within a housing 7137 b. The housing 7137b is coupled to the upper portion 7112 of the first housing 7110 such that the wash buffer module 7130b is substantially aligned with the wash chamber 7122. In particular, the housing 7137b includes a pair of projections 7133b configured to seat within corresponding apertures defined by the coupling portions 7134b of the upper section 7112 of the first housing 7110. Although the wash buffer module 7130b is shown coupled to the first housing 7110 by a "snap-fit," in other embodiments, the wash buffer module 7130b may be coupled to the first housing 7110 by any suitable method, such as by a threaded interface, a mechanical fastener or retainer, a chemical bond or adhesive, an interference fit, a welded joint, or the like.

The driver 7150b includes a plug portion 7151b, a perforated portion 7152b, and an engagement portion 7153 b. The engagement portion 7153b is configured to engage, removably couple to, and/or be received within a portion of the driver assembly to facilitate movement of the driver 7150b within the housing 7137b, as described herein. The driver 7150b may be operated and/or driven by any suitable instrument, such as the driver assembly 3600 described below with respect to fig. 47-51.

The plug portion 7151b of the driver 7150b is disposed within the housing 7137 b. The penetrable member 7135b is disposed about the distal portion of the housing 7137b such that the end face of the plug portion 7151b, the housing 7137b, and the penetrable member 7135b collectively define a volume within which the substance is disposed. The plug portion 7151b and the inner surface of the housing 7137b are configured to form a substantially fluid-tight seal and/or a gas-tight seal. In certain embodiments, the plug portion 7151b can include a sealing member, an o-ring, or the like.

When the driver 7150b is moved within the housing 7137b, as indicated by the arrow QQ in fig. 28, the perforated portion 7152b of the driver 7150b is configured to penetrate, break, sever and/or rupture a portion of the penetrable member 7135 b. In this manner, movement of the drive 7150b places the chamber in fluid communication with the wash chamber 7122. Similarly stated, the wash buffer module 7130b can be selectively placed in fluid communication with the wash chamber 7122 when the driver 7150b is driven. After the substances within the wash buffer module 7130b are transferred into the wash chamber 7122, the driver 7150b may reciprocate within the housing 7137b to create pressure that is transferred into the wash chamber 7122 to facilitate washing, mixing, and/or other interactions between and within the samples disposed therein. The top portion 7112 of the first housing 7110 includes a nozzle 7131b configured to direct pressure energy and/or flow generated by an actuator 7150b to a particular region within the washing chamber 7122.

As shown in fig. 29-31, amplification (or PCR) module 7200 includes a substrate 7220 configured with a first (or upper) layer 7227 and a second (or lower) layer 7228. PCR module 7200 includes a PCR vial 7260 coupled to second layer 7228, a transfer mechanism 7235, a first reagent module 7270a, and a second reagent module 7270 b. PCR vial 7260 is coupled to first end portion 7211 of housing 7210 and defines a volume 7262 within which a sample can be disposed to facilitate a reaction that binds to the sample. PCR vial 7260 can be any suitable container for containing a sample in a manner that allows a reaction that binds to the sample to occur. PCR vial 7260 can also be used with any suitable container containing a sample in a manner that allows for monitoring of such reactions (e.g., detecting an analyte within the sample that is due to or bound to the reaction). In certain embodiments, at least a portion of PCR vial 7260 can be substantially transparent to allow optical monitoring of the reaction occurring therein to be an optical system (e.g., optical assembly 3800 of instrument 3002 described herein).

As shown in fig. 32 and 33, amplification module 7200 is coupled to a first housing 7110 of separation module 7100 such that at least a portion of transfer tube 7250 is disposed within elution chamber 7190 of separation module 7100. In this manner, the isolated nucleic acids, any substances, and/or any PCR reagents disposed within the elution chamber 7190 can be transferred from the elution chamber 7190 to the PCR vial 7260 via the transfer tube 7250 as described herein. More specifically, base 7220 defines flow channel 7222, which places PCR vial 7260 in liquid communication with elution chamber 7190 when PCR module 7200 is coupled to separation module 7100. As shown in fig. 30 and 31, a portion of the flow channel 7222 is defined in the transfer tube 7250 and the transfer port 7229 of the second layer 7228 of the substrate 7220. Although the flow channel 7222 is shown as being defined primarily by the second layer 7228 of the substrate 7220, in other embodiments, the flow channel 7222 can be defined by the first layer 7227 or in a portion of both the first layer 7227 and the second layer 7228.

The base 7220 also defines a flow channel 7223, a flow channel 7221a and a flow channel 7221 b. As described in greater detail herein, the flow channel 7223 is configured to place a volume 7237 defined within the transfer mechanism 7235 in fluid communication with the PCR vial 7260 via a transfer port 7229. The flow channel 7221a is configured to place the volume defined by the reagent module 7270a in liquid communication with the elution chamber 7190 via the transfer tube 7250. Flow channel 7221b is configured to place the volume defined by reagent module 7270b in liquid communication with PCR vial 7260 via transfer port 7229 and/or a portion of channel 7222. Any of the flow channels 7223, 7221a, and/or 7221b can be defined by the first layer 7227, the second layer 7228, or in a portion of the first layer 7227 and the second layer 7228.

PCR module 7200 includes two reagent modules 7270a and 7270b, each coupled to upper layer 7227 of substrate 7220. As described herein, each reagent module 7270a and 7270b contains a substance R1 and R2, respectively. Reagent module 7270a is configured to deliver substance R1 into elution chamber 7190 via flow channel 7221a as described herein. Reagent module 7270b is configured to transfer substance R2 into PCR vial 7260 via flow channel 7221b, as described herein. In this manner, each reagent module 7270a and 7270b acts as a reagent reservoir and transfer mechanism.

The substances R1 and R2 may be, for example, reagents to be added to the sample, elution buffer solutions, wash buffer solutions, mineral oil, and/or any other substance, as described herein. In certain embodiments, substance R1 may include an elution buffer and mineral oil. In certain embodiments, substance R2 may include reaction reagents that facilitate the PCR process within PCR vial 7260. In certain embodiments, the PCR master mix can be disposed within PCR vial 7260 in a lyophilized state such that the addition of substance R2 and/or the mixture of substance R1 and the target sample reconstitutes the lyophilized master mix to facilitate the PCR process.

For example, in one embodiment in which HSV is amplified via PCR, the master mixture is a lyophilized pellet comprising HSV1 and HSV2 primers specific for HSV1 and/or HSV2 sequences, a detection probe (e.g., a hybrid oligonucleotide probe comprising a fluorophore and MGB at the 5 'end and a non-fluorescent quencher at the 3' end), and an internal control primer and probe, KCl (e.g., about 40mM, about 50mM, about 60mM, about 70 mM), mannitol (e.g., about 70mM, about 80mM, about 90mM, about 100mM, about 110mM, about 120 mM), BSA (e.g., about 0.1mg/mL, about 0.5mg/mL, about 1 mg/mL), dNTPs (e.g., about 0.2mM, about 0.3mM, about 0.4mM, about 0.5mM, about 1 mM), Taq polymerase (e.g., about 0.1U/μ L, about 0.2U/μ L, about 0.3U/μ L).

In another embodiment, the master mix contains lyophilized reagents to perform multiplex PCR on the three targets and internal controls. In a further embodiment, the target nucleic acid is a nucleic acid specific for influenza a, a nucleic acid specific for influenza b, and a nucleic acid specific for RSV. In an even further embodiment, the multiplex reaction is monitored in real time by providing hybridization oligonucleotide probes specific for each target sequence, each probe comprising a fluorophore and MGB at the 5 'terminus and a non-fluorescent quencher at the 3' terminus.

In another embodiment, the lyophilized master mix contains reagents for PCR and reverse transcriptase reactions. For example, in one embodiment, the lyophilized master mix includes reverse transcriptase and Taq polymerase, dntps, rnase inhibitors, KCl, BSA, and primers to perform first strand cDNA synthesis and PCR.

The main mixture contains different primers and probes, depending on the target to be amplified. Each target will have a specific primer and probe set bound to it, and the primer and probe sets can be lyophilized with the other PCR reagents mentioned above to form a lyophilized master mix. The concentration of the components will also vary depending on the particular target to be amplified and whether multiple targets are amplified.

The reagent module 7270a includes a driver 7280a movably disposed within a housing 7277 a. Housing 7277a is coupled to upper portion 7227 of substrate 7220 such that reagent module 7270a is substantially aligned with channel 7221a, transfer tube 7250 and/or elution chamber 7190. As shown in fig. 29, housing 7277a includes a pair of protrusions 7273a configured to seat within respective apertures defined by coupling portions 7234a of upper portion 7227 of substrate 7220. Although reagent module 7270a is shown coupled to base 7220 by a "snap fit," in other embodiments reagent module 7270a may be coupled to base 7220 by any suitable method, such as by a threaded interface, a mechanical fastener or retainer ring, a chemical key or adhesive, an interference fit, a welded joint, or the like.

The driver 7280a includes a plug portion 7281a, a perforated portion 7282a, and an engagement portion 7283 a. The engagement portion 7283a is configured to engage, removably couple to, and/or accept within a portion of the driver assembly to facilitate movement of the driver 7280a within the housing 7277a, as described herein. Driver 7280a can be operated and/or driven by any suitable instrument, such as driver assembly 3600 described below with respect to fig. 47-51.

The plug portion 7281a of driver 7280a is disposed within housing 7277 a. The penetrable member 7175a is disposed about the distal end portion of the housing 7277a such that the end face of the plug portion 7281a, the housing 7277a and the penetrable member 7275a collectively define a volume within which the substance R1 is disposed. The plug portion 7281a and the inner surface of the housing 7277a are configured to form a substantially fluid-tight seal and/or a gas-tight seal. In certain embodiments, the plug portion 7281a can include a sealing member, an o-ring, or the like.

As the driver 7280a is moved within the housing 7277a by the direction indicated by arrow SS in fig. 31, the perforated portion 7282a of the driver 7280a is configured to pierce, break, sever and/or rupture a portion of the penetrable member 7275 a. In this manner, movement of the driver 7280a places the chambers in fluid communication with the channel 7221a and thus the elution chamber 7190. Similarly stated, reagent module 7270a may be selectively placed in liquid communication with elution chamber 7190 when driver 7280a is actuated.

The reagent module 7270b includes a driver 7280b movably disposed within a housing 7277 b. Housing 7277b is coupled to upper portion 7227 of substrate 7220 such that reagent module 7270b is substantially aligned with channel 7221 b. As shown in fig. 29, the housing 7277b includes a pair of protrusions 7273b configured to seat within respective apertures defined by the coupling portion 7234b of the upper portion 7227 of the substrate 7220. Although reagent module 7270b is shown coupled to base 7220 by a "snap fit," in other embodiments reagent module 7270b may be coupled to base 7220 by any suitable method, such as by a threaded interface, a mechanical fastener or retainer ring, a chemical key or adhesive, an interference fit, a welded joint, or the like.

The driver 7280b includes a plug portion 7281b, a perforated portion 7282b, and an engagement portion 7283 b. The engagement portion 7283b is configured to engage, removably couple to, and/or accept within a portion of the driver assembly to facilitate movement of the driver 7280b within the housing 7277b, as described herein. Driver 7280b can be operated and/or driven by any suitable instrument, such as driver assembly 3600 described below with respect to fig. 47-51.

The plug portion 7281b of driver 7280b is seated within housing 7277 b. The penetrable member 7275b is disposed about the distal end portion of the housing 7277b such that the end surface of the plug portion 7281b, the housing 7277b, and the penetrable member 7275b collectively define a volume within which the substance R2 is disposed. The plug portion 7281b and the inner surface of the housing 7277b are configured to form a substantially fluid-tight seal and/or a gas-tight seal. In certain embodiments, the plug portion 7281a can include a sealing member, an o-ring, or the like.

When the driver 7280b is moved within the housing 7277b by the direction shown by arrow SS in fig. 31, the perforated portion 7282b of the driver 7280b is configured to pierce, break, sever and/or rupture a portion of the penetrable member 7275 b. In this manner, movement of driver 7280b places the chamber in fluid communication with channel 7221b and thus PCR chamber 7260.

PCR module includes 7200 transfer mechanism 7235 configured to transfer material from elution chamber 7190 of separation module 7100 and PCR vial 7260 of PCR module 7200 and/or between elution chamber 7190 of separation module 7100 and PCR vial 7260 of PCR module 7200. Transfer mechanism 7235 is further configured to define a volume 7237 within which a substance can be contained, and selectively place volume 7237 in fluid communication with PCR vial 7260, as described herein. In this manner, the diversion mechanism 7235 also acts as a flow control mechanism.

The transfer mechanism 7235 includes a drive 7240 disposed within a housing 7236. The housing 7236 is coupled to the upper layer 7227 of the substrate 7220 and/or is part of the upper layer 7227 of the substrate 7220. Housing 7236 defines a volume 7237 in which a substance, such as mineral oil, may be stored. Although not shown as including a penetrable member, in other embodiments, a portion of the volume 7237 may be surrounded by and/or fluidly separated by a penetrable member, as described herein.

Actuator 7240 includes plug portion 7241, valve portion 7242, and engagement portion 7243. The engagement portion 7243 is configured to engage, removably couple to, and/or be received within a portion of the driver assembly to facilitate movement of the driver 7240 within the housing 7236, as described herein. Driver 7240 can be operated and/or driven by any suitable instrument, such as driver assembly 3600 described below with respect to fig. 47-51.

A plug portion 7241 of driver 7240 is disposed within housing 7236. The plug portion 7241 and the inner surface of the housing 7236 are configured to form a substantially fluid-tight seal and/or a gas-tight seal. In certain embodiments, plug portion 7241 can include a sealing member, an o-ring, or the like. In addition, a seal 7244 is disposed on a top end portion of the housing 7236.

The actuator 7240 is configured to move within the housing 7236 between a first position (fig. 30) and a second position (fig. 31). When the driver 7240 is in the first position, the valve portion 7242 of the driver 7240 is at least partially disposed within the channel 7223 such that the volume 7237 is substantially fluidically separated from the flow channel 7223 and/or the PCR vial 7260. Similarly stated, when the actuator 7240 is in the first position, a portion of the valve portion 7242 is in contact with the upper layer 7227 to create a substantially fluid-tight seal and/or a gas-tight seal. When the driver 7250 is moved within the housing 7236 by the direction shown by arrow RR in fig. 31, the valve portion 7242 is spaced from the upper layer 7227 and/or removed from the flow channel 7223, thereby placing the volume 7237 in fluid communication with the channel 7223 and thus the PCR chamber 7260. In this manner, as driver 7240 is moved, the substance within volume 7237 can be transferred into PCR volume 7262 defined by PCR vial 7260.

Further, as driver 7240 moves within housing 7236, a vacuum is created within PCR volume 7262 of PCR vial 7260 as indicated by arrow RR in fig. 31. This pressure differential between PCR volume 7262 and elution chamber 7190 causes at least a portion of the contents of elution chamber 7190 to be transferred into PCR volume 7262 via transfer tube 7250 and channel 7222 (see, e.g., fig. 24). In this manner, substances and/or samples can be added, mixed, and/or transported between the elution chamber 7190 and the PCR volume 7262 by driving the transfer mechanism 7235. The transfer mechanism 7235 can be actuated by any suitable mechanism, such as the actuator assembly 3600 of the instrument 3002 described herein.

In use, as described above, after one or more nucleic acids or nucleic acid populations are separated and processed within the separation module 7100, it is transferred into the elution chamber 7190 via the transfer assembly 7140 c. The reagent module 7270a may then be actuated to transfer the substance R1 into the elution chamber 7190. For example, in certain embodiments, the reagent module 7270a may be actuated to transfer a solution containing an elution buffer and mineral oil into the elution chamber 7190. The magnetic beads are then removed (or "washed") from the nucleic acids by the elution buffer and removed from the elution chamber 7190 (e.g., by the transfer module 7140 c). Thus, the elution chamber 7190 contains isolated and/or purified nucleic acid.

Reagent module 7270b may be actuated to transfer substance R2 into PCR volume 7262. For example, in certain embodiments, reagent module 7270b may be driven to transfer a solution containing multiple reaction reagents into PCR vial 7260. In certain embodiments, PCR vial 7260 can contain additional reagents and/or materials, such as a PCR master mix, in a lyophilized state. Preferably, when substance R2 is transferred into PCR vial 7260, the lyophilized contents can be reconstituted in preparation for reaction.

Target sample S can be transferred (before or after actuation of reagent module 7270b described above) from elution chamber 7190 into PCR vial 7260 via transfer tube 7250 and channel 7222. In particular, driver 7240 of transfer mechanism 7235 can be driven to create a pressure differential within PCR module 7200 to transfer a PCR sample from elution chamber 7190 into PCR vial 7260 via channel 7222, as described above. In this manner, the PCR sample (isolated nucleic acids and PCR reagents) can be prepared in part in the elution chamber 7190. In addition, when transfer mechanism 7235 is actuated, volume 7237 defined therein is placed in fluid communication with PCR volume 7262 via channel 7223, as described above. Thus, in certain embodiments, additional substances (e.g., mineral oil) may be added to the PCR vial via the same procedure as the sample transfer procedure.

After the PCR sample is in the PCR vial 7260, at least a portion of the PCR sample S can be thermally cycled (e.g., via the heater assembly 3700 of the instrument 3002) to perform the desired amplification. After thermal cycling is complete and/or during thermal cycling, the PCR sample may optionally be analyzed (e.g., via the optical assembly 3800 of the instrument 3002) to analyze the sample. Alternatively, as described throughout, the PCR sample may optionally be analyzed during PCR, for example with DNA hybridization probes each conjugated to an MGB and a fluorophore. A description of the instrument 3002 and other suitable instruments for operating the cartridge is provided below.

Any of the cartridges described herein may be operated and/or driven by any suitable instrument to perform a separation process and/or reaction on a sample contained within the cartridge. For example, in certain embodiments, any of the cartridges described herein may be operated and/or driven by an instrument to perform real-time nucleic acid separation and amplification on a test sample within the cartridge. In this way, the system (e.g., cartridge or series of cartridges and instruments) can be used for rapid detection of many different assays, such as influenza a (flu), influenza b, and Respiratory Syncytial Virus (RSV) from nasopharyngeal specimens.

In certain embodiments, the instrument may be configured to facilitate, generate, support and/or drive a reaction in a sample contained in a reaction chamber defined by a cartridge of the type shown and described herein. Such instruments may also include optical components to detect one or more different substances and/or analytes within the sample before, during, and/or after the reaction. For example, fig. 34 is a schematic illustration of an instrument 1002 according to one embodiment. The instrument 1002 includes a block 1710, a first optical member 1831, a second optical member 1832, and an optical assembly 1800. Block 1710 defines a reaction volume 1713 configured to accept at least a portion 261 of the reaction vessel 260 containing the sample S. The reaction vessel 260 may be any suitable vessel for containing the sample S in a manner that allows a reaction that binds to the sample S to occur. The reaction vessel 260 may also be any suitable vessel for containing the sample S in a manner that allows for monitoring of such reactions (e.g., detection of analytes within the sample S that result from or bind to the reactions). In certain embodiments, for example, the reaction vessel 260 may be a PCR vial, test tube, or the like. Further, in certain embodiments, at least a portion 261 of the reaction vessel 260 may be substantially transparent to allow optical monitoring of the reaction occurring therein.

Block 1710 may be any suitable structure and/or may be coupled to any suitable mechanism for facilitating, generating, supporting, and/or driving a reaction that binds to sample S in reaction vessel 260. For example, in certain embodiments, block 1710 can be coupled to and/or can include a mechanism for cyclically heating sample S in reaction vessel 260. In this manner, block 1710 can generate a thermally induced reaction of sample S, such as a PCR process. In other embodiments, block 1710 may be coupled to and/or may include a mechanism for introducing one or more substances into the reaction vessel 260 to produce a chemical reaction that binds to the sample S.

Reaction volume 1713 can have any suitable size and/or shape for containing portion 261 of reaction chamber 260. In certain embodiments, for example, the shape of reaction volume 1713 may substantially correspond to the shape of portion 261 of reaction chamber 260 (e.g., as shown in fig. 34). However, in other embodiments, the shape of reaction volume 1713 may be different than the shape of portion 261 of reaction chamber 260. Although the portion 261 of the reaction chamber 260 is shown in fig. 34 as being spaced apart from the sidewalls of the block 1710 that define the reaction volume 1713, in other embodiments, the portion 261 of the reaction chamber 260 may be in contact with a portion of the block 1710. In still other embodiments, the reaction volume 1713 may contain a substance (e.g., a saline solution, a thermally conductive gel, etc.) disposed between the portion 261 of the reaction chamber 260 and a portion (e.g., a sidewall) of the block 1710.

Although block 1710 is shown in fig. 34 as containing only a portion 261 of the reaction chamber 260 within the reaction volume 1713, in other embodiments, block 1710 may be configured such that the entire reaction chamber 260 is received within the reaction volume 1713. In certain embodiments, for example, block 1710 can include a hood or other mechanism (not shown in fig. 34) that retains substantially the entire reaction chamber 260 within the reaction volume 1713. Furthermore, in certain embodiments, the block 1710 may surround substantially the entire reaction chamber 260. In other embodiments, the block 1710 may substantially surround a portion 261 of the reaction chamber 260 disposed within the reaction volume 1713.

As shown in fig. 34, the first optical member 1831 is at least partially disposed within the block 1710 such that the first optical member 1831 is in optical communication with the reaction volume 1713. In this manner, an optical beam (and/or optical signal) may be transmitted between the reaction volume 1713 and the area outside the block 1710 via the first optical member 1831. The first optical member 1831 may be any suitable structure, device, and/or mechanism through or from which a light beam may be transmitted. In some embodiments, the first optical member 1831 may be any suitable optical fiber that transmits a light beam, such as a multimode fiber or a single mode fiber. In other embodiments, the first optical member 1831 may include a mechanism configured to modify and/or transform the light beam, such as an optical amplifier, an optical signal converter, a lens, a filter, and the like. In still other embodiments, the second optical member 1832 may comprise a Light Emitting Diode (LED), a laser, or other device configured to generate a light beam.

The second optical member 1832 is at least partially disposed within the block 1710 such that the second optical member 1832 is in optical communication with the reaction volume 1713. In this manner, an optical beam (and/or optical signal) may be transmitted between the reaction volume 1713 and the area outside the block 1710 via the second optical member 1832. The second optical member 1832 may be any suitable structure, device, and/or mechanism through or from which a light beam may be transmitted. In some embodiments, the second optical member 1832 may be any suitable optical fiber that transmits a light beam, such as a multimode fiber or a single mode fiber. In other embodiments, the second optical member 1832 may include a mechanism configured to modify and/or transform the light beam, such as an optical amplifier, an optical signal converter, a lens, a filter, and the like. In still other embodiments, the second optical member 1832 may comprise a photodiode or other device configured to receive and/or detect a light beam.

Optical assembly 1800 includes an excitation module 1860 and a detection module 1850. Excitation module 1860 is configured to generate a series of excitation light beams (and/or optical signals, not shown in fig. 34). Accordingly, excitation module 1860 may include any suitable device and/or mechanism for generating a series of excitation light beams, such as a laser, one or more Light Emitting Diodes (LEDs), flash lamps, or the like. In certain embodiments, each of the light beams generated by excitation module 1860 may have substantially the same characteristics (e.g., wavelength, amplitude, and/or energy) as each of the other light beams generated by excitation module 1860. However, in other embodiments, the first beam of light generated by excitation module 1860 may have different characteristics (e.g., wavelength, amplitude, and/or energy) than one of the other beams of light generated by excitation module 1860. In certain embodiments, for example, the excitation module 1860 may include a series of LEDs, each configured to produce a light beam having a different wavelength than the light beams produced by the other LEDs.

The detection module 1850 is configured to receive a series of emitted light beams (and/or light signals, not shown in fig. 34). Accordingly, detection module 1850 can comprise any suitable light detector, such as an optical detector, a photoresistor, a photovoltaic cell, a photodiode, a photocell, a CCD camera, or the like. The emitted beam may be generated by any suitable source, such as by excitation of the constituent components of the sample S. In some embodiments, the detection module 1850 may be configured to selectively accept each emitted light beam regardless of whether each light beam has the same respective characteristics (e.g., wavelength, amplitude, and/or energy) as the other emitted light beams. However, in other embodiments, detection module 1850 may be configured to selectively accept each emitted light beam based on a particular characteristic (e.g., wavelength, amplitude, and/or energy) of the light beam. In certain embodiments, for example, detection module 1850 can comprise a series of photodetectors, each configured to receive a light beam having a different wavelength than light beams received by the other photodetectors.

As shown in fig. 34, a first optical member 1831 and a second optical member 1832 are coupled to the optical assembly 1800. In this manner, a series of excitation light beams may each be transmitted into the reaction volume 1713 and/or the portion 261 of the reaction vessel 260, and a series of emission light beams may each be received by the reaction volume 1713 and/or the portion 261 of the reaction vessel 260. More specifically, the first optical member 1831 is coupled to the excitation module 1860 such that a series of excitation light beams generated by the excitation module 1860 may be transmitted into the reaction volume 1713 and/or the portion 261 of the reaction vessel 260. Similarly, the second optical member 1832 is coupled to the detection module 1850 such that the plurality of emitted light beams are each receivable by the reaction volume 1713 and/or the portion 261 of the reaction vessel 260.

The series of light beams generated by the excitation module 1860 pass through the first optical member 1831 and are transmitted along the first optical path 1806 into the reaction volume 1713 and/or the portion 261 of the reaction vessel 260. Thus, the series of light beams generated by the excitation module 1860 are each delivered at a substantially constant location into the reaction volume 1713 and/or the portion 261 of the reaction vessel 260. Similarly, a series of light beams received by detection module 1850 pass through second optical component 1832 and are received by reaction volume 1713 and/or portion 261 of reaction vessel 260 along second optical path 1807. Thus, the series of light beams received by the detection module 1850 are each received by the reaction volume 1713 and/or the portion 261 of the reaction vessel 260 at a substantially constant position. By transmitting and receiving the excitation light beam and the emission light beam, respectively, at constant locations within the reaction volume 1713, detection variations within a multi-channel analysis combined with transmitting the excitation light beam and/or receiving the emission light beam from a plurality of different locations may be reduced.

In addition, by including the first optical member 1831 and the second optical member 1832 within the block 1710, the position of the first optical member 1831 (and the first optical path 1806) and/or the position of the second optical member 1832 (and the second optical path 1807) is constant relative to the reaction volume 1713. This arrangement may also reduce inter-test detection variations associated with optical path and/or optical components by minimizing and/or eliminating relative movement between the first optical member 1831, the second optical member 1832, and/or the reaction volume 1713.

In some embodiments, a series of excitation light beams may be sequentially delivered into the reaction volume 1713, and a series of emission light beams may be sequentially received by the reaction volume 1713. For example, in some embodiments, excitation module 1860 may generate a series of light beams each having a different wavelength in a sequential (or time-programmed) manner. Each light beam is transmitted into the reaction volume 1713 where it can, for example, excite the sample S contained in the reaction vessel 260. Similarly, in such embodiments, the emitted light beams are generated in a sequential (or time-course) manner (due to excitation of particular analytes and/or targets within the sample S). Thus, detection module 1850 can receive a series of light beams each having a different wavelength in a sequential (or time-course) manner. In this manner, the instrument 1802 can be used to detect multiple different analytes and/or targets within a sample S.

Although a portion of the first optical member 1831 disposed within the block 1710 and a portion of the second optical member 1832 disposed within the block 1710 are shown in fig. 34 as being substantially parallel and/or in the same plane, in other embodiments, the block may include the first optical member in any position and/or orientation relative to the second optical member. Similarly stated, although the first optical path 1806 is shown in fig. 34 as being substantially parallel and/or in the same plane as the second optical path 1807, in other embodiments, the instrument may be configured to produce a first optical path at any position and/or orientation relative to the second optical path.

For example, fig. 35 shows a partial schematic cross-sectional view of a portion of an apparatus 2002 according to one embodiment. The instrument 2002 includes a slug 2710, a first optical member 2831, a second optical member 2832, and an optical assembly (not shown in FIG. 35). The slug 2710 defines a reaction volume 2713 configured to accept at least a portion 261 of the reaction vessel 260 containing the sample S. The reaction vessel 260 can be any suitable vessel for containing the sample S in a manner that allows reactions that bind to the sample S to occur and allows such reactions to be monitored, as described herein. In certain embodiments, for example, the reaction vessel 260 may be a PCR vial, test tube, or the like. Further, in certain embodiments, at least a portion 261 of the reaction vessel 260 may be substantially transparent to allow optical monitoring of the reaction occurring therein.

Slug 2710 may be any suitable structure and/or may be coupled to any suitable mechanism for facilitating, generating, supporting, and/or facilitating a reaction that binds to sample S in reaction vessel 260. For example, in certain embodiments, slug 2710 may be coupled to and/or may include a mechanism for cyclically heating sample S in reaction vessel 260. In this manner, block 2710 can generate a thermally induced reaction of sample S, such as a PCR process. In other embodiments, slug 2710 may be coupled to and/or may include a mechanism for introducing one or more substances into reaction vessel 260 to produce a chemical reaction that binds to sample S.

Reaction volume 2713 can be of any suitable size and/or shape for containing portion 261 of reaction chamber 260. As shown in fig. 35, when portion 261 is disposed within reaction volume 2713, reaction volume 2713 defines a longitudinal axis L_AAnd substantially surrounds a portion 261 of the reaction chamber 260. In this manner, any stimulus (e.g., heating or cooling) provided to sample S by slug 2710 or a mechanism attached thereto may be provided in a substantially spatially consistent manner.

As shown in fig. 35, the first optical member 2831 is at least partially disposed within the slug 2710 such that the first optical member 2831 defines a first optical path 2806 and is in optical communication with the reaction volume 2713. In this manner, a light beam (and/or light signal) may be transmitted between the reaction volume 2713 and the region outside the slug 2710 via the first optical member 2831. The first optical member 2831 can be any suitable structure, device, and/or mechanism of the type shown and described herein through which or from which a light beam can be transmitted. In certain embodiments, the first optical member 2831 can be any suitable optical fiber that transmits a light beam, such as a multimode fiber or a single mode fiber.

A second optical member 2832 is at least partially disposed within slug 2710 such that second optical member 2832 defines a second optical path 2807 and is in optical communication with reaction volume 2713. In this manner, light beams (and/or optical signals) may be transmitted between the reaction volume 2713 and the region outside the slug 2710 via the second optical member 2832. The second optical member 2832 can be any suitable structure, device, and/or mechanism of the type shown and described herein through which or from which a light beam can be transmitted. In certain embodiments, the second optical member 2832 may be any suitable optical fiber that transmits a light beam, such as a multimode fiber or a single mode fiber.

As described above, the first optical member 2831 and the second optical member 2832 are coupled to an optical assembly (not shown in fig. 35). The optical assembly may generate one or more excitation light beams and may detect one or more emission light beams. Thus, one or more excitation light beams may be transmitted into the reaction volume 2713 and/or the reaction vessel 260, and one or more emission light beams may be received by the reaction volume 2713 and/or the portion 261 of the reaction vessel 260. More specifically, the first optical member 2831 may transmit an excitation beam from the optical assembly into the reaction volume 2713 to excite a portion of the sample S contained in the reaction vessel 260. Similarly, the second optical member 2832 can transmit an emission beam generated by an analyte or other target within the sample S from the reaction volume 2713 to the optical assembly. In this manner, the optical assembly can monitor the reactions occurring within the reaction vessel 260.

As shown in fig. 35, the first lightA portion of the optical member 2831 and the first optical path 2806 are disposed substantially in the first plane P_XYAnd (4) the following steps. First plane P_XYLongitudinal axis L with reaction volume 2713_ASubstantially parallel and/or including the longitudinal axis L of the reaction volume 2713 _A. However, in other embodiments, the first plane P_XYNeed not be aligned with the longitudinal axis L of the reaction volume 2713_ASubstantially parallel and/or including the longitudinal axis L of the reaction volume 2713_A. A portion of the second optical member 2832 and the second optical path 2807 are disposed substantially in the second plane P_YZAnd (4) the following steps. Second plane P_YZLongitudinal axis L with reaction volume 2713_ASubstantially parallel and/or including the longitudinal axis L of the reaction volume 2713_A. However, in other embodiments, the second plane P_YZNeed not be aligned with the longitudinal axis L of the reaction volume 2713_ASubstantially parallel and/or including the longitudinal axis L of the reaction volume 2713_A. Further, as shown in fig. 35, first optical path 2806 and second optical path 2807 define an offset angle Θ that is greater than about 75 degrees. More specifically, the longitudinal axis L of the reaction volume 2713_AIn a substantially parallel direction (i.e. in a direction parallel to the first plane P)_XYAnd a second plane P_YZIn a substantially perpendicular plane), first optical path 2806 and second optical path 2807 define an offset angle Θ that is greater than about 75 degrees. In a similar manner, first optic member 2831 and second optic member 2832 define an offset angle Θ that is greater than about 75 degrees. This arrangement minimizes the amount of excitation beam received by the second optical member 2832 (i.e., the "detection" optical member), thereby improving the accuracy and/or sensitivity of optical detection and/or monitoring.

In certain embodiments, a portion of instrument 2002 can create first optical path 2806 and second optical path 2807 within reaction volume 2713 such that skew angle Θ is between about 75 degrees and about 105 degrees. In certain embodiments, a portion of instrument 2002 can create a first optical path 2806 and a second optical path 2807 within reaction volume 2713 such that the skew angle Θ is about 90 degrees.

Although a portion of instrument 2002 is shown as generating a first lightPath 2806 and second optical path 2807, which are substantially parallel and intersect at point PT in reaction volume 2713, but in other embodiments, piece 2713, first optical member 2831, and/or second optical member 2832 can be configured such that first optical path 2806 is not parallel and/or does not intersect second optical path 2807. For example, in certain embodiments, first optical path 2806 and/or first optical member 2831 may be parallel to and offset (i.e., tilted) from second optical path 2807 and/or second optical member 2831. Similarly stated, in certain embodiments, the first optical member 2831 and the second optical member 1832 may each pass through the distance Y₁And Y₂Spaced apart from a reference plane defined by a block 2710, where Y ₁Is different from Y₂. Thus, along the longitudinal axis L_AOn which the first optical member 2831 and/or the first optical path 2806 intersect the reaction volume 2713 at a location other than along the longitudinal axis L_AWhere the second optical member 2832 and/or the second optical path 2807 intersect the reaction volume 2713. In this manner, first optical path 2806 and/or first optical member 2831 may be tilted with respect to second optical path 2807 and/or second optical member 2831.

In other embodiments, the longitudinal axis L_AAnd first optical path 2806 and/or first optical member 2831₁May be different from the longitudinal axis L_AAnd angle γ defined by second optical path 2807 and/or second optical member 2832₂(i.e., first optical path 2806 may not be parallel to second optical path 2807). In still other embodiments, slug 2713, first optical member 2831, and/or second optical member 2832 may be configured such that first optical path 2806 intersects second optical path 2807 at a location outside of reaction volume 2713.

Distance Y₁And a distance Y₂May be any suitable distance such that the first optical member 2831 and the second optical member 1832 are configured to create and/or define a first optical path 2806 and a second optical path 2807, respectively, in a desired portion of the reaction vessel 260. For example, in certain embodiments Distance Y₁This may be such that when reaction vessel 260 is positioned within block 2710, first optical member 2831 and/or first optical path 2806 enter and/or intersect reaction volume 2713 at a location below the location of fill line FL of sample S. In this way, the excitation beam delivered by the first optical member 2831 will enter the sample S below the fill line. This arrangement may improve optical detection of analytes within the sample by reducing the excitation beam attenuation that may occur by transmitting the excitation beam through the headspace of the reaction vessel (i.e., the portion of the reaction vessel 260 on the fill line LF that is substantially devoid of sample S). However, in other embodiments, distance Y₁This may be such that when reaction vessel 260 is positioned within block 2710, first optical member 2831 and/or first optical path 2806 enter reaction volume 2713 at a location above the location of fill line FL for sample S.

Similarly, in certain embodiments, distance Y₂This may be such that when reaction vessel 260 is positioned within block 2710, second optical member 2832 and/or second optical path 2807 enter and/or intersect reaction volume 2713 at a location below the location of fill line FL for sample S. In this manner, the emitted beam received by the second optical member 2832 will exit the sample S below the fill line. This arrangement may improve optical detection of analytes within the sample by reducing attenuation of the emitted light beam that may occur by receiving the emitted light beam through the headspace of the reaction vessel. However, in other embodiments, distance Y ₂This may be such that when reaction vessel 260 is positioned within block 2710, second optical member 2832 and/or second optical path 2807 enter and/or intersect reaction volume 2713 at a location that is above the location of fill line FL for sample S.

Fig. 36-70 show various views of an instrument 3002 and/or a portion of an instrument configured to operate a series of cartridges, drive and/or interact with the series of cartridges, to perform nucleic acid separation and amplification processes on a test sample within the cartridges. The cartridge may include any of the cartridges shown and described herein, such as cartridge 6001. Such a system can be used for the rapid detection of many different assays, such as influenza a (flu), influenza b and Respiratory Syncytial Virus (RSV) from nasopharyngeal specimens. The instrument 3002 is shown without the housing 3002 and/or specific portions of the instrument 3002 to more clearly show the components therein. For example, fig. 47 shows the instrument 3002 without the optical assembly 3800.

As shown in fig. 36, instrument 3002 includes a chassis and/or frame 3300, a first driver assembly 3400, a sample transfer assembly 3500, a second driver assembly 3600, a heater assembly 3700, and an optics assembly 3800. The frame 3300 is configured to house, contain, and/or provide mounting for each component and/or assembly of the instrument 3002, as described herein. The first driver assembly 3400 is configured to drive a driver or transfer mechanism (e.g., driver or transfer mechanism 6166) of a separation module (separation module 6100) of a cartridge to deliver one or more reagents and/or substances into a lysis chamber within the separation module. Transfer drive assembly 3500 is configured to drive a transfer assembly (e.g., transfer assembly 6140 a) to transfer a portion of a sample between multiple chambers and/or volumes within a separation module (e.g., separation module 7100). Second driver assembly 3600 is configured to drive a separation module (e.g., separation module 6100) and/or a mixing mechanism (e.g., mixing mechanism 6130 a) of a PCR module (e.g., PCR module 6200) and/or a wash buffer module (e.g., wash buffer module 7130 a) to deliver to and/or mix one or more reagents and/or substances within a chamber within the separation module and/or PCR module. The heater assembly 3700 is configured to heat one or more portions of the cartridge (e.g., PCR vial 7260, substrate 7220, and/or the region of housing 7110 adjacent to lysis chamber 7114) to push and/or facilitate a process within the cartridge (e.g., to push, facilitate, and/or generate a "hot start" process, a thermal lysis process, and/or a PCR process). The optical assembly 3800 is configured to monitor reactions occurring within the cartridge. More specifically, optical assembly 3800 is configured to detect one or more different analytes and/or targets within a test sample in a cartridge. Each of these components is discussed separately below, followed by a description of various methods that may be performed by the instrument 3002.

As shown in fig. 36, the frame 3300 includes a base frame 3310, a front member 3312, two side members 3314, and a rear member 3320. The base member 3310 supports the functional components described herein and includes six mounting or support brackets. In certain embodiments, the support brackets may be adjustable to allow the instrument 3302 to be level when installed and/or mounted on a laboratory bench. The rear member 3320 is coupled to the base member 3310 and is configured to support and or retain the power component 3361. The rear member 3320 may also provide a mounting bracket for any other components related to the operation of the instrument 3302, such as a processor, control elements (e.g., motor controllers, heating system controllers, etc.), communication interfaces, a cooling system, and the like. Fig. 71-73 are block diagrams of the control and computer system of the instrument 3002.

The side members 3314 each include an upper portion 3316 and a lower portion 3315. A front member 3312 is coupled to each side member 3314 and defines a bore within which a cartridge 3350 containing a plurality of assay cartridges may be positioned for machining. In certain embodiments, cartridge 3350 may be configured to contain six cartridges of the type shown and described herein (shown, for example, as cartridge 6001 in fig. 36). In use, a cartridge 3350 containing a plurality of cartridges is disposed within the instrument 3002 and maintained in a fixed position relative to the chassis 3300 during the separation and/or amplification process. Thus, the cartridge containing the sample is not moved between the multiple stations for analysis. Rather, as described herein, samples, reagents, and/or other substances are transported, processed, and/or manipulated within portions of the cartridge by instrument 3002, as described herein. Although the instrument 3002 is shown as being configured to accept a cartridge 3350 containing six cartridges, in other embodiments, the instrument may be configured to accept any number of cartridges 3350 containing any number of cartridges.

Fig. 37-40 show various views of the first driver assembly 3400 of the instrument 3002. The first driver assembly 3400 is configured to drive and/or operate a transfer mechanism and/or a reagent driver (e.g., reagent drivers 6166a, 6166b, 6166c, and 6166 d) of a separation module (e.g., separation module 6100) of a cartridge to deliver one or more reagents and/or substances into a lysis chamber within the separation module. In particular, a first driver assembly 3400 may drive a first one of the reagent drivers (e.g., reagent driver 6166 d) from a respective cartridge disposed within the cartridge 3350 and then, at a different time, drive a second one of the reagent drivers (e.g., reagent driver 6166 c) from the respective cartridge.

The first drive assembly includes an engagement rod 3445, a first (or x-axis) motor 3440, and a second (or y-axis) motor 3441 supported by the frame assembly 3410. As shown in fig. 38 and 40, the engagement bar 3445 includes a series of projections 3346a, 3346b, 3346c, 3346d, 3346e, and 3346 f. The projections are each configured to engage one or more reagent drivers (e.g., reagent driver 6166 a) of a separation module (e.g., separation module 6100), disposed within one or more reagent drivers (e.g., reagent driver 6166 a) of a separation module (e.g., separation module 6100), and/or drive one or more reagent drivers (e.g., reagent driver 6166 a) of a separation module (e.g., separation module 6100), disposed within instrument 3002. In certain embodiments, the engagement bar 3445 and/or the protrusion (e.g., protrusion 3346 a) can comprise a retention mechanism (e.g., a protrusion, a snap ring, etc.) configured to retain the protrusion and/or aperture of a driver (e.g., reagent driver 6166 a) to facilitate mutual movement of the reagent drivers within the separation module.

Frame assembly 3410 includes a first axis (or x-axis) mounting frame 3420 removably coupled to a second axis (or y-axis) mounting frame 3430. In particular, first axle mounting frame 3420 can move along the y-axis relative to second axle mounting frame 3430 as indicated by arrow AAA in fig. 37. Similarly stated, a first one of the axle mounting frames 3420 may be moved relative to a second one of the axle mounting frames 3430 in an "alignment direction" (i.e., along the y-axis) to facilitate alignment of the engagement rods 3445 and/or projections (e.g., projection 3346 a) with a desired series of drives and/or transfer mechanisms.

The first axis mount frame 3420 provides support for a first axis (or x-axis) motor 3440 that is configured to move the engagement bar 3445 and/or projection (e.g., projection 3346 a) along the x-axis, as shown by arrow BBB in fig. 37. Similarly stated, a first spindle motor 3440 is coupled to the first spindle mounting frame 3420 and is configured to move the engagement rod 3445 and/or projections (e.g., projection 3346 a) in a "drive direction" (i.e., along the x-axis) to drive the desired series of drives and/or transfer mechanisms. The movement of the articulation bar 3445 is guided by two x-axis guide shafts 3421, each movably disposed within a respective bearing 3422. The bearing 3422 is positioned relative to the first shaft mounting frame 3420 and/or the first motor 3440 by a bearing mounting member 3423.

The second axle mounting frame 3430 is coupled to the frame assembly 3300 and between the two side frame members 3314 of the frame assembly 3300. The second axis mounting frame 3430 provides support for a second (or y-axis) motor 3441 and a first axis mounting frame 3420. The second motor 3441 is configured to move the first shaft mounting frame 3420, and thus the engaging lever 3445, along the y-axis (or in an aligned direction), as indicated by arrow BBB in fig. 37. In this manner, the engagement bar 3445 and/or projections (e.g., projection 3346 a) can be aligned with a desired series of drivers and/or transfer mechanisms prior to actuation of the drivers and/or transfer mechanisms. The first shaft mounting frame 3420 is coupled to the second shaft mounting frame 3430 by a pair of bearing blocks 3432 that are slidably disposed about a corresponding pair of y-axis guide shafts 3431.

In use, a first driver assembly 3400 may sequentially drive a series of transfer mechanisms and/or reagent drivers (e.g., drivers 6166a, 6166b, 6166c, and 6166 d) of a set of cartridges (e.g., cartridge 6001) disposed within instrument 3001. First, the engagement bar 3445 can be aligned with a desired transfer mechanism and/or reagent drive (e.g., drive 6166 d) by moving the first axis mount frame 3420 in an alignment direction (i.e., along the y-axis). The engagement rod 3445 may then be moved in a drive direction (i.e., along the x-axis) to drive the desired transfer mechanism and/or reagent driver (e.g., driver 6166 d) from each cartridge. In this manner, the first driver assembly 3400 may drive and/or operate respective reagent drivers from cartridges disposed in a parallel (or simultaneous) manner within the instrument 3002. However, in other embodiments, the driver assembly 3400 and/or the engagement rod 3445 may be configured to sequentially drive respective reagent drivers from cartridges disposed in a sequential (or series) fashion within the instrument 3002.

The first driver assembly 3400 may drive the desired transfer mechanism and/or reagent driver by moving the engagement rod 3445 in a first direction along the x-axis. However, in other embodiments, the first driver assembly 3400 may drive the desired transfer mechanism and/or reagent driver by reciprocating the engagement bar 3445 along the x-axis (i.e., alternatively moving the engagement bar 3445 in a first direction and a second direction). When the desired transfer mechanism and/or reagent driver has been actuated, the first driver assembly 3400 may drive another transfer mechanism and/or reagent driver (e.g., driver 6166 c) in a manner similar to that described above.

Although first driver assembly 3400 is shown and described as driving a transfer mechanism and/or a reagent driver, in other embodiments, first driver assembly 3400 may drive any suitable portion of any cartridge described herein. For example, in certain embodiments, the first driver assembly 3400 may drive, operate, and or move the ultrasonic transducer to facilitate ultrasonic lysis.

Fig. 41-46 show various views of a transfer driver assembly 3500 of instrument 3002. Transfer drive assembly 3500 is configured to drive and/or operate a transfer assembly or mechanism, such as transfer assembly 6140 shown and described above with respect to fig. 20 and 21. In particular, transfer driver assembly 3500 may drive a first one of the transfer assemblies (e.g., transfer assembly 6140 a) from a respective cartridge disposed within cartridge 3350 and subsequently, at a different time, drive a second one of the transfer assemblies (e.g., transfer assembly 6140 b) from the respective cartridge.

Transfer drive assembly 3500 includes a series of drive shafts 3510. Although the transfer drive assembly 3500 includes six drive shafts, only one is identified in FIGS. 41-46. Driver shafts 3510 are each configured to engage one or more transfer assemblies (e.g., transfer assembly 6140 a) of a separation module (e.g., separation module 6100), to be disposed within one or more transfer assemblies (e.g., transfer assembly 6140 a) of a separation module (e.g., separation module 6100), and/or to drive one or more transfer assemblies (e.g., transfer assembly 6140 a) of a separation module (e.g., separation module 6100) disposed within instrument 3002. As shown in fig. 44, each driver shaft 3510 has a first end portion 3511 and a second end portion 3512. The first end portion 3511 is coupled to a drive gear 3513 (see fig. 41-42), which in turn is driven by a worm drive shaft 3541. As shown in fig. 41 and 42, a rotational position indicator 3542 is coupled to a first end portion 3511 of one of the driver shafts 3510. The rotational position indicator 3542 defines a slot and/or aperture 3543 whose rotational position can be sensed (e.g., via an optical sensing mechanism) to provide feedback regarding the rotational position of the driver shaft 3510.

Second end portion 3512 of each shaft 3510 includes an engagement portion 3514 configured to receive within and/or engage a transfer assembly (e.g., transfer assembly 6140 a) of a cartridge (e.g., cartridge 6001) disposed within instrument 3002 (e.g., transfer assembly 6140 a). In this manner, engagement portion 3514 can operate and/or actuate the transfer assembly to facilitate transfer of a portion of a sample within a cartridge, as described above. The engagement portion 3514 has a shape that corresponds to the shape of a portion of the transfer set (e.g., lumen 6149 defined by movable member 6146) such that rotation of the driver shaft 3510 results in rotation of a portion of the transfer set. In particular, as shown in fig. 44, the engagement portion has an octagonal shape. In certain embodiments, engagement portion 3514 can include a retention mechanism (e.g., a protrusion, a snap ring, etc.) configured to retain a protrusion and/or a hole of the transfer set to facilitate reciprocal movement of a portion of the transfer set within the separation module.

The engagement portion 3514 defines a lumen 3515 within which a magnet (not shown) can be disposed. In this manner, the driver shaft 3510 may generate and/or apply a force (i.e., a magnetic force) to a portion (i.e., a magnetic bead) of the contents disposed within a cartridge (e.g., cartridge 6001) to facilitate transfer of a portion of a sample via a transfer assembly, as described above.

The driver shaft 3510 is moved by a first (or x-axis) motor 3580, a second (or y-axis) motor 3560, and a third (or rotary) motor 3540. As described in more detail below, the x-axis motor 3580 is supported by a support frame 3571, the y-axis motor 3560 is supported by an engagement frame assembly 3550, and the rotation motor 3540 is supported by a rotation frame assembly 3530.

The rotating frame assembly 3530 provides support for a rotating motor 3540 that is configured to rotate the driver shaft 3510 about the y-axis, as indicated by arrow CCC in fig. 41. Similarly stated, a rotary motor 3540 is coupled to the rotary frame assembly 3530 and is configured to rotate the driver shaft 3510 in a "drive direction" (i.e., about the y-axis) to drive the desired series of transfer assemblies. The rotating frame assembly 3530 includes a rotating plate 3531, a pair of worm gear drive bearing housings 3533, and a worm gear drive shaft 3541. The worm drive shaft 3541 is coupled to the rotary motor 3540 by a pulley assembly and is supported by two worm drive bearing mounts 3533. The worm drive shaft 3541 engages a drive gear 3513 of each drive shaft 3510. Accordingly, as the worm drive shaft 3541 rotates in a first direction (i.e., about the z-axis), each drive shaft 3510 rotates in a second direction (i.e., about the y-axis, as indicated by arrow CCC in fig. 41).

The rotating frame assembly 3530 also includes a y-axis position indicator 3534 that may be slidably disposed within a pair of corresponding slide members 3553 on the engagement frame assembly 3550. In this manner, as the rotating frame assembly 3530 translates along the y-axis (e.g., in an "engagement direction"), as shown by arrow DDD in fig. 41, the y-axis position indicator 3534 and corresponding slide member 3553 may guide linear movement and/or provide feedback regarding the position of the rotating frame assembly 3530.

The engagement frame assembly 3550 provides support for a y-axis motor 3560 that is configured to move the rotation frame assembly 3530, and thus the drive shaft 3510, along the y-axis, as shown by arrow DDD in fig. 41. Similarly stated, a y-axis motor 3560 is coupled to the engagement frame assembly 3550 and is configured to move the driver shaft 3510 in an "engagement direction" (i.e., along the y-axis) to drive the desired series of transfer mechanisms. The engagement frame assembly 3550 includes a support frame 3551 that provides support with respect to a drive link 3561 (which translates rotational motion of the y-axis motor into linear motion of the rotating frame assembly 3530). Movement of the engagement frame assembly 3530 is guided by two y-axis guide shafts 3552, each movably disposed within a respective bearing 3554. Bearing 3554 is coupled to rotating plate 3531, as shown in fig. 43.

The support frame 3571 is coupled to the frame assembly 3300 and between the lower end portions 3315 of the two side frame members 3314 of the frame assembly 3300. The support frame 3571 provides support for the x-axis motor 3580 and the engagement frame assembly 3550. The x-axis motor 3580 is configured to move the engagement frame assembly 3550, and thus the drive shaft 3510, along the x-axis (or in an aligned direction), as indicated by the arrow EEE in fig. 41. In this manner, the driver shaft 3510 can be aligned with a desired series of transfer mechanisms before the transfer mechanisms are driven. The support frame 3571 is coupled to the adapter frame assembly 3550 by a pair of bearing blocks 3573 that are slidably disposed about a respective pair of x-axis guide shafts 3572.

In use, the transfer driver assembly 3500 may sequentially drive a series of transfer mechanisms (e.g., transfer mechanisms 6140a, 6140b, and 6166 c) of a set of cartridges (e.g., cartridge 6001) disposed within the instrument 3001. First, by moving the engagement frame assembly 3550 in an alignment direction (i.e., along the x-axis), the actuator shaft 3510 can be aligned with the desired transfer mechanism. The driver shaft 3510 may then be moved in an engaging direction (i.e., along the y-axis) to engage a desired transfer mechanism (e.g., transfer mechanism 6140 a) from each cartridge. The driver shaft 3510 may then be moved in a driving direction (i.e., rotated about the y-axis) to drive a desired transfer mechanism (e.g., transfer mechanism 6140 a) from each cartridge. In this manner, transfer driver assembly 3500 can drive and/or operate respective transfer mechanisms from cartridges disposed in parallel (or simultaneously) within instrument 3002. However, in other embodiments, transfer driver assembly 3500 and/or driver shaft 3510 can be configured to sequentially drive respective transfer mechanisms from cartridges disposed in a sequential (or serial) manner within instrument 3002.

Fig. 47-51 show various views of a second driver assembly 3600 of the instrument 3002. Second driver assembly 3600 is configured to drive and/or operate a transfer mechanism (e.g., transfer mechanism 7235), a wash buffer module (e.g., wash buffer module 7130 a), a mixing mechanism (e.g., mixing mechanism 6130 a), and/or a reagent module (e.g., reagent module 7270 a) of any cartridge shown or described herein. In particular, the second driver assembly 3600 can drive a first one (e.g., the mixing mechanism 6130 a) from a respective transfer mechanism, mixing mechanism, etc., of cartridges disposed within the cartridge 3350, and subsequently drive a second one (e.g., the mixing mechanism 6130 b) from the respective transfer mechanism, mixing mechanism, etc., of cartridges at a different time.

Second drive assembly 3600 includes an engagement rod 3645 supported by a frame assembly 3610, a first (or x-axis) motor 3640 and a second (or y-axis) motor 3641. As shown in fig. 48, the adapter bar 3645 includes a series of projections 3346. Although the engagement rod 3645 includes six protrusions (one for each cartridge in the cartridge 3350), only one protrusion 3346 is labeled. The projections are each configured to engage one or more transfer mechanisms (e.g., transfer mechanism 7235), wash buffer modules (e.g., wash buffer module 7130 a), mixing mechanisms (e.g., mixing mechanism 6130 a), and/or reagent modules (e.g., reagent module 7270 a) of the cartridge, be disposed within one or more transfer mechanisms (e.g., transfer mechanism 7235), wash buffer modules (e.g., wash buffer module 7130 a), mixing mechanisms (e.g., mixing mechanism 6130 a), and/or reagent modules (e.g., reagent module 7270 a) of the cartridge, operate one or more transfer mechanisms (e.g., transfer mechanism 7235), wash buffer modules (e.g., wash buffer module 7130 a), mixing mechanisms (e.g., mixing mechanism 6130 a), and/or reagent modules (e.g., reagent module 7270 a) of the cartridge, and/or drive one or more transfer mechanisms (e.g., transfer mechanism 7235), of the cartridge, A wash buffer module (e.g., wash buffer module 7130 a), a mixing mechanism (e.g., mixing mechanism 6130 a), and/or a reagent module (e.g., reagent module 7270 a), disposed within the instrument 3002. In certain embodiments, the engagement rods 3645 and/or the projections 3346 may include a retention mechanism (e.g., a projection, a snap ring, etc.) configured to retain a portion of the driver (e.g., the engagement portion 7153a of the driver 7150a, shown and described above with respect to fig. 27 and 28) to facilitate mutual movement of the driver within a portion of the cartridge.

The frame assembly 3610 includes a second axis (or y-axis) mounting frame 3630 that is removably coupled to the first axis (or x-axis) mounting frame 3620. In particular, the second shaft mounting frame 3630 may be movable along the x-axis relative to the first shaft mounting frame 3620, as indicated by arrows GGG in fig. 37. Similarly stated, the second shaft mounting frame 3630 can be moved relative to the first shaft mounting frame 3620 in an "alignment direction" (i.e., along the x-axis) to facilitate alignment of the engagement rods 3645 and/or the protrusions 3346 with a desired series of transfer mechanisms, mixing mechanisms, reagent modules, and the like.

Second axis mounting frame 3620 provides support for second axis (or y-axis) motor 3641, which is configured to move engagement bar 3645 and/or projection 3346 along the y-axis, as shown by arrow FFF in fig. 37. Similarly stated, a second shaft motor 3641 is coupled to the second shaft mounting frame 3620 and is configured to move the engagement rods 3645 and/or the projections 3346 in a "drive direction" (i.e., along the y-axis) to drive the desired series of transfer mechanisms, mixing mechanisms, reagent modules, and the like. The movement of the engaging bar 3645 is guided by two y-axis guide shafts 3631, each movably disposed within a respective bearing coupled to the second shaft mounting frame 3620.

A first one of the axle mounting frames 3630 is coupled to the frame assembly 3300 and between the lower portions 3316 of the two side frame members 3314 of the frame assembly 3300. A first shaft mounting frame 3630 provides support for a first (or x-axis) motor 3640 and a second shaft mounting frame 3620. The first motor 3640 is configured to move the second shaft mounting frame 3620 and, thus, the engagement rod 3645 along the x-axis (or in an aligned direction), as indicated by the arrow GGG in fig. 47. In this manner, the engagement rods 3645 and/or protrusions 3346a can be aligned with a desired series of transfer mechanisms, mixing mechanisms, reagent modules, etc., prior to actuation of such mechanisms. A second shaft mounting frame 3620 is coupled to the first shaft mounting frame 3630 by a pair of bearing housings 3622 that are slidably disposed about a corresponding pair of x-axis guide shafts 3631. A first (or x-axis) motor 3640 is coupled to a second axis mounting frame 3620 via a mounting member 3624 (see, e.g., fig. 51).

In use, the second driver assembly 3600 may sequentially drive a series of transfer mechanisms (e.g., transfer mechanism 7235), wash buffer modules (e.g., wash buffer module 7130 a), mixing mechanisms (e.g., mixing mechanism 6130 a), and/or reagent modules (e.g., reagent module 7270 a) of a set of cartridges (e.g., cartridge 6001) disposed within the instrument 3001. First, the docking bar 3645 may be aligned with a desired mechanism (e.g., the mixing mechanism 6130 a) by moving the second axle mounting frame 3630 in an alignment direction (i.e., along the x-axis). The adapter rod 3645 may then be moved in a driving direction (i.e., along the y-axis) to drive the desired mechanism (e.g., the mixing mechanism 6130 a) from each cartridge. In this manner, second driver assembly 3600 can drive and/or operate the respective transfer mechanisms, wash buffer modules, mixing mechanisms, and/or reagent modules from cartridges disposed in parallel (or simultaneously) within instrument 3002. However, in other embodiments, the second driver assembly 3600 and/or the adapter rod 3645 can be configured to sequentially drive respective mechanisms from cartridges disposed in a sequential (or series) manner within the instrument 3002.

The second actuator assembly 3600 may actuate the desired mechanism by moving the engagement rod 3645 in a first direction along the y-axis. However, in other embodiments, the second driver assembly 3600 may drive the desired transfer mechanism and/or reagent driver by reciprocating the docking rod 3645 along the y-axis (i.e., alternatively moving the docking rod 3645 in a second direction and a second direction). When the desired mechanism has been actuated, the second actuator assembly 3600 may actuate another mechanism and/or actuator (e.g., the mixing mechanism 6130 b) in a manner similar to that described above.

Although second actuator assembly 3600 is shown and described as driving a transfer mechanism and/or a reagent actuator, in other embodiments, second actuator assembly 3600 may drive any suitable portion of any cartridge described herein. For example, in certain embodiments, the second driver assembly 3600 may drive, operate, and or move the ultrasonic transducer to facilitate the transmission of acoustic energy into a portion of the cartridge.

Fig. 52-63 show various views of a heater assembly 3700 of the instrument 3002. The heater assembly 3700 is configured to heat one or more portions of the cartridge (e.g., PCR vial 7260, substrate 7220, and/or the region of housing 7110 adjacent to lysis chamber 7114) to push and/or facilitate processes within the cartridge (e.g., to push, facilitate, and/or generate a "hot start" process, a thermal lysis process, and/or a thermal cycling process for PCR). In particular, the heater assembly 3700 can drive and/or heat a respective first portion of a cartridge (e.g., PCR vial 6260) disposed within the cartridge 3350, and subsequently, at a different time, drive and/or heat a respective second portion of the cartridge (e.g., a portion of the separation module 6100 adjacent to the lysis chamber 6114).

The heater assembly 3700 includes a series of receiving modules 3710 (one for each cartridge in the cassette 3350), a positioning assembly 3770, a first heating module 3730, a second heating module 3750, and a third heating module 3780. Receiving block 3710 is configured to receive a drugAt least a portion of the reaction chamber of the cartridge, such as PCR vial 6260 of cartridge 6001. As shown in fig. 53-56, the receiving block 3710 includes a mounting surface 3714 and defines a reaction volume 3713. Reaction volume 3713 has a size and/or shape that substantially corresponds to the size and/or shape of PCR vial 6260 of cartridge 6001. As shown in fig. 54 and 56, when PCR vial 6260 is disposed within reaction volume 3713, reaction volume 3713 defines a longitudinal axis L_AAnd substantially surrounds a portion of PCR vial 6260. In this manner, any stimulation (e.g., heating or cooling) provided by the heater assembly 3700 on the sample within the PCR vial 6260 can be provided in a substantially spatially consistent manner. Further, as shown in fig. 56, the sidewalls defining a portion of the receiving block 3710 of the reaction volume 3713 have a substantially uniform wall thickness. This arrangement allows heat transfer between the reaction volume 3713 and the remainder of the heater assembly 3700 to occur in a substantially spatially consistent manner.

The receiving block 3710 is coupled to the mounting block 3734 (see, e.g., fig. 58) by a clamp block 3733 (see, e.g., fig. 57), such that the thermoelectric device 3731 is in contact with the mounting surface 3714. In this manner, the reaction volume 3713 and the sample contained therein can be cyclically heated to produce a thermally induced reaction of the sample S, such as a PCR process.

Each receiving block 3710 defines a first (or firing) lumen 3711, a second (or firing) lumen 3712, and a third (or temperature monitoring) lumen 3715. A thermocouple or other suitable temperature measuring device may be positioned adjacent the PCR vial via the third lumen 3715. As shown in fig. 52, the excitation fiber 3831 is at least partially disposed within the first lumen 3711 such that the excitation fiber 3831 and/or the first lumen 3711 define a first optical path 3806 and are in optical communication with the reaction volume 3713. In this manner, a light beam (and/or light signal) may be transmitted between the reaction volume 3713 and the region outside the block 3710 via the excitation fiber 3831 and/or the first lumen 3711. The excitation fiber 3831 may be any suitable structure, device, and/or mechanism of the type shown and described herein through which or from which a light beam may be transmitted. In certain embodiments, the excitation fiber 3831 may be any suitable optical fiber that transmits a light beam, such as a multimode fiber or a single mode fiber.

The detection fiber 3832 is at least partially disposed within the second lumen 3712 such that the detection fiber 3832 and/or the second lumen 3712 defines a second optical path 3807 and is in optical communication with the reaction volume 3713. In this manner, a light beam (and/or light signal) may be transmitted between the reaction volume 3713 and the region outside the block 3710 via the detection fiber 3832 and/or the second lumen 3712. Sensing fiber 3832 may be any suitable structure, device, and/or mechanism of the type shown and described herein through which or from which a light beam may be transmitted. In certain embodiments, the detection fiber 3832 may be any suitable optical fiber that transmits a light beam, such as a multimode fiber or a single mode fiber.

The excitation fiber 3831 and the detection fiber 3832 are coupled to the optical assembly 3800 as described below. The optical assembly 3800 may produce one or more excitation light beams and may detect the one or more emission light beams. Accordingly, the excitation fiber 3831 may transmit an excitation beam from the optical assembly into the reaction volume 3713 to excite the portion of the sample S contained within the PCR vial 6260. Similarly, the detection fiber 3832 may transmit an emission beam generated by an analyte or other target within the sample S from the PCR vial 6260 to the optical assembly 3800.

As shown in fig. 55, the first lumen 3711 and the second lumen 3712 define an offset angle Θ of about 90 degrees. Similarly stated, the first optical path 3806 and the second optical path 3807 define an offset angle Θ of about 90 degrees. More specifically, the longitudinal axis L of the reaction volume 3713_AViewed in a substantially parallel orientation, the first optical path 3806 and the second optical path 3807 define an offset angle Θ of about 90 degrees. In a similar manner, the excitation fibers 3831 and the detection fibers 3832 disposed within the first and second lumens 3711 and 3712, respectively, define a skew angle Θ of about 90 degrees. This arrangement minimizes the amount of excitation beam received by the detection fiber 3832, thereby improving the accuracy and/or sensitivity of optical detection and/or monitoring.

In certain embodiments, the first lumen 3711 and the second lumen 3712 can be positioned such that the offset angle Θ is greater than about 75 degrees. In other embodiments, the first lumen 3711 and the second lumen 3712 can be positioned such that the offset angle Θ is between about 75 degrees and about 105 degrees.

As shown in fig. 54, the centerline of the first lumen 3711 is substantially parallel to and offset (i.e., tilted) from the centerline of the second lumen 3712. Similarly stated, the excitation fiber 3831 (and thus the first optical path 3806) is inclined from the detection fiber 3832 (and thus the second optical path 3807). In other words, the first lumen 3711 (and/or excitation fiber 3831) and the second lumen 3712 (and/or detection fiber 3832) each pass through the distance Y ₁And Y₂Spaced apart from a reference plane defined by the receiving block 3710, where Y₁Is different from Y₂. Thus, along the longitudinal axis L_AAt which the excitation fiber 3831 and/or the first optical path 3806 intersects the reaction volume 3713 at a location other than along the longitudinal axis L_AWhere the fiber 3832 and/or the second optical path 3807 intersects the reaction volume 3713 is detected. In this manner, the first optical path 3806 and/or the excitation fiber 3831 may be tilted from the second optical path 3807 and/or the second optical member 3831.

Distance Y₁And a distance Y₂May be any suitable distance such that the excitation fiber 3831 and the detection fiber 3832 are configured to create and/or define the first optical path 3806 and the second optical path 3807, respectively, in desired portions of the PCR vial 6260. For example, in certain embodiments, distance Y₁This may be such that the first lumen 3711, excitation fiber 3831, and/or first optical path 3806 enters and/or intersects the reaction volume 3713 at a location below the location of the fill line of the sample within the PCR vial 6260 disposed within the receiving block 3710. In this way, the excitation beam delivered by the excitation fiber 3831 will enter the sample below the fill line. However, in other embodiments, distance Y ₁May be such that the first lumen 3711, excitation fiber 3831, and/or first optical path 3806 are at the location of the fill line of the sample within the PCR vial 6260The upper position enters the reaction volume 3713.

Similarly, in certain embodiments, distance Y₂This may be such that the second lumen 3712, detection fiber 3832, and/or second optical path 3807 enters and/or intersects the reaction volume 3713 at a location below the location of the fill line of the sample within the PCR vial 6260 disposed within the receiving block 3710. However, in other embodiments, distance Y₂This may be such that the second lumen 3712, detection fiber 3832, and/or second optical path 3807 enter and/or intersect the reaction volume 3713 at a location on the fill line of the sample within the PCR vial 6260.

The first heating module 3730 includes a series of thermoelectric devices 3731 (one for each cartridge and/or each receiving module 3710), a mounting module 3734, a series of clamp modules 3733, and heat sinks 3732. As shown in fig. 58, the mounting block 3734 includes a first portion 3735 and a second portion 3737. The first portion 3735 includes an angled surface 3736 to which the thermoelectric devices 3731 are each coupled. In this manner, each receiving block 3710 is coupled to the mounting block 3734 by a respective clamp block 3733 such that the thermoelectric device 3731 is in contact with the mounting surface 3714 of the receiving block 3710.

A second portion 3737 of the mounting block 3734 is coupled to the heat sink 3732. The heat sink (see, e.g., fig. 59) can be any suitable means for facilitating heat transfer between receiving block 3710 and an area external to instrument 3002. In certain embodiments, heat sink 3732 can include devices and/or mechanisms to actively cool mounting block 3734 (i.e., remove heat from mounting block 3734).

Positioning assembly 3770 is coupled to heat sink 3732 and a portion of frame assembly 3300 and is configured to move heater assembly 3700 linearly in a direction along the y-axis. Thus, when actuated, positioning assembly 3770 may move heater assembly 3700 relative to cartridge 3350 and/or the cartridges therein such that PCR vials (e.g., PCR vials 6260) are seated within receiving block 3710, as described above. Positioning assembly 3770 includes a motor 3771 and linkage assembly 3772 configured to convert rotational motion of motor 3771 to linear motion. Movement of the heater assembly 3700 is guided by the y-axis guide shaft 3773.

In use, the first heating module 3730 may cyclically heat the respective PCR vials of the cartridges disposed within the instrument 3001 to facilitate the PCR process and/or the mixing of the contents contained therein. Furthermore, because the cartridges are each heated by a separate thermoelectric device 3731 via a separate receiving block 3710, in certain embodiments, the thermal cycling of a first cartridge may occur at a different time than the thermal cycling of a second cartridge. Further, because each cartridge may be thermally cycled independently of the other cartridges in the instrument, in certain embodiments, the thermal cycling profile (e.g., time and temperature of the thermal cycling event) for a first cartridge may be different than the thermal cycling profile for a second cartridge. In certain embodiments, the first heating module 3730 is not used for thermocycling, but is maintained at a constant temperature, e.g., the temperature at which reverse transcription of an RNA sample is performed.

The second heating module 3750 includes a series of resistive heaters 3751 (one for each cartridge and/or each receiving module 3710), a mounting plate 3754, a first insulating component 3752, and a second insulating component 3753. As shown in fig. 60, the mounting plate 3754 includes a first portion 3755 and a second portion 3760. The first portion 3755 provides mounting support for the respective resistive heaters 3751. Similarly stated, the resistive heaters 3731 are each coupled to a mounting plate 3754.

The mounting plate 3754 is coupled to the mounting block 3734 of the first heating module 3730 such that the first insulating component 3752 is disposed between the mounting block 3734 and the first portion 3755 of the mounting plate 3754 and the second insulating component 3753 is disposed between the mounting block 3734 and the second portion 3760 of the mounting plate 3754. In this manner, the second heating module 3750 may function substantially independently of the first heating module 3730. Similarly stated, this arrangement reduces and/or limits heat transfer between the mounting plate 3754 and the mounting blocks 3734.

A first portion 3755 of mounting plate 3754 includes a top surface 3758 and defines a recess 3756 and a series of lumens 3757 (one for each cartridge in cartridge 3350). In use, as the heater assembly 3700 is moved into position around each cartridge within the instrument 3002, each PCR vial is seated through the respective lumen 3757 and into the reaction volume 3713 defined by the respective receiving block 3710. Thus, in certain embodiments, when the heater assembly 3700 is placed around each cartridge, the sidewalls of the mounting plate 3754 defining the lumen 3757 are placed around a portion of each PCR vial 6260 and/or substantially around a portion of each PCR vial 6260. However, in other embodiments, the PCR vial 6260 may be separate from the lumen 3757 and/or not reside within the lumen 3757. For example, in certain embodiments, only a transfer port (e.g., transfer port 7229 of PCR module 7200, shown and described above with respect to fig. 30 and 31) may be disposed within lumen 3737 of mounting plate 3754 when heater assembly 3700 is placed around each cartridge.

As shown in fig. 60, the second portion 3760 of the mounting plate 3754 defines a series of recesses and/or cavities 3761 (one for each cartridge in the box 3350). In use, as the heater assembly 3700 is moved into position around each cartridge within the instrument 3002, a portion of the cartridge seats within a corresponding recess 3761 of the mounting plate 3754. More specifically, as shown in fig. 52, a portion of a separation module (e.g., separation module 6100) corresponding to elution chamber 6190 (not identified in fig. 52) is disposed within a respective recess 3761. Thus, when the heater assembly 3700 is placed around each cartridge, the sidewall of the second portion 3760 of the mounting plate 3754 defining the recess 3761 is placed around a portion of the elution chamber 6190 and/or substantially around a portion of the elution chamber 6190. In this manner, the second heating module 3750 can heat and/or thermally cycle a portion of the sample contained within the elution chamber 6190 of each cartridge.

In use, the second heating module 3750 may heat a portion of each cartridge disposed within the instrument 3001 to facilitate, improve, and/or facilitate a reaction process occurring within the cartridge. For example, in certain embodiments, the second heating module 3750 can heat a portion of a substrate of a PCR module (e.g., the substrate 7220 of the PCR module 7200 shown and described above with respect to fig. 29-31). In one embodiment, heating by the second heating module 3750 is accomplished to facilitate the reverse transcription reaction, or for "hot start" PCR.

More specifically, in certain embodiments, the second heating module 3750 can facilitate a "hot start" method associated with the PCR process. The hot start method involves the use of a "hot start enzyme" (polymerase) to reduce non-specific priming of nucleic acids in the amplification reaction. More specifically, when the enzyme is maintained at ambient temperature (e.g., below about 50 ℃), non-specific hybridization can occur, which can result in non-specific priming in the presence of the polymerase. Thus, a hot start enzyme is an enzyme that is inactivated at ambient temperature and does not become active until heated to a predetermined temperature. Such predetermined temperatures may be temperatures in excess of about 40 ℃, 50 ℃, 70 ℃, or 95 ℃. To facilitate the "hot start" method, the second heating module 3750 can heat the elution chamber (e.g., elution chamber 7190) to maintain the eluted nucleic acid sample at an elevated temperature (e.g., at a temperature greater than about 40 ℃, 50 ℃, 70 ℃, or 95 ℃) prior to addition of the master mix to the amplification reaction within the PCR vial (e.g., PCR vial 7260). In certain embodiments, for example, the second heating module 3750 may maintain the temperature of the sample within the elution chamber 7190 at a temperature between about 50 ℃ and about 95 ℃. By heating the eluted nucleic acids in this manner, non-specific hybridization and/or false priming in the presence of a polymerase can be eliminated and/or reduced.

The reaction reagents (e.g., substance R2 contained within reagent module 7270b as shown in fig. 30 and 31 above) can then be added to a PCR vial (e.g., PCR vial 7260) to the lyophilized master mix contained therein. The heated nucleic acid sample from the elution chamber (e.g., elution chamber 7190) can then be transferred into a PCR vial, as described above. In addition, the second heating module 7250 can also heat the flow path (e.g., channel 7222) between the elution chamber and the PCR vial so that the contents therein (e.g., an eluted nucleic acid sample transferred from the elution chamber to the PCR vial) can be maintained at a high temperature (e.g., at a temperature in excess of about 40 ℃, 50 ℃, 70 ℃, or 95 ℃). In certain embodiments, for example, the second heating module 3750 can maintain the temperature of the sample within the flow path at a temperature between about 50 ℃ and about 95 ℃. After the heated eluted sample is transferred into the PCR vial, the solution is mixed by temperature cycling (generated by the first heating module 3730) and then the PCR reaction is initiated.

The third heater assembly 3780 includes at least one heater (not shown) and a heater block 3784. As shown in fig. 63, the heat block 3784 defines a series of recesses and/or cavities 3786a, 3786b, 3786c, 3786d, 3786e, 3786f, one for each cartridge in the cassette 3350). In use, when the heater assembly 3700 is moved into position around each cartridge within the instrument 3002, a portion of the cartridge seats within a corresponding recess (e.g., recess 3786 a) of the heating block 3784. More specifically, as shown in fig. 52, a portion of a separation module (e.g., separation module 6100) corresponding to lysis chamber 6114 (not identified in fig. 52) is disposed within a respective recess. Thus, when the heater assembly 3700 is placed around each cartridge, the sidewalls of the heating block 3784 defining the recess 3786 are placed around a portion of the lysis chamber 6114 and/or substantially around a portion of the lysis chamber 6114. In this manner, the third heater assembly 3780 can heat and/or thermally cycle a portion of the sample contained within the lysis chamber 6114 of each cartridge. In one embodiment, heating by third heater assembly 3780 occurs during the reverse transcription and/or PCR reaction.

Fig. 64-70 show various views of the optical assembly 3800 of the instrument 3002. The optical assembly 3800 is configured to monitor a reaction occurring with respect to a cartridge disposed within the instrument 3002. More specifically, optical assembly 3800 is configured to detect one or more different analytes and/or targets within a test sample before, during, and/or after a PCR reaction occurring within a PCR vial (e.g., PCR vial 6260) of a cartridge. As described herein, the optical assembly 3800 can analyze the sample in a sequential and/or time-course manner and/or in real-time. Optical assembly 3800 includes an excitation module 3860, a detection module 3850, a slide assembly 3870, and a fiber optic assembly 3830.

For example, in one embodiment, the optical assembly is used to monitor a nucleic acid amplification reaction in real time. In a further embodiment, the amplification reaction is PCR. In another embodiment, the optical assembly is used to measure results from a binding assay, such as binding between an enzyme and a substrate or ligand and receptor.

The fiber optic assembly 3830 includes a series of excitation optical fibers (identified in fig. 64 as excitation fibers 3831a, 3831b, 3831c, 3831d, 3831e, 3831f, 3831 g). The excitation fibers 3831a, 3831b, 3831c, 3831d, 3831e, and 3831f are each configured to transmit light beams and/or light signals from the excitation module 3860 to a respective receiving block 3710. Accordingly, a first end portion of each excitation fiber 3831a, 3831b, 3831c, 3831d, 3831e, and 3831f is positioned within the lumen 3711 of the receiving block 3710, as described above. The excitation fiber 3831g is a calibration fiber and is configured to transmit light beams and/or light signals from the excitation module 3860 to an optical calibration module (not shown). The excitation optical fiber 3831 may be any suitable optical fiber that transmits a light beam, such as a multimode fiber or a single mode fiber.

The optical fiber assembly 3830 included a series of detection optical fibers (identified in fig. 64 as detection fibers 3832a, 3832b, 3832c, 3832d, 3832e, 3832f, 3832 g). Sensing fibers 3832a, 3832b, 3832c, 3832d, 3832e, and 3832f are each configured to transmit light beams and/or light signals from receiving block 3710 to sensing module 3850. Accordingly, a first end portion of each of the sensing fibers 3832a, 3832b, 3832c, 3832d, 3832e, and 3832f is disposed within the lumen 3712 of the receiving block 3710, as described above. The detection fiber 3832g is a calibration fiber and is configured to receive a light beam and/or a light signal from an optical calibration module (not shown). The detection optical fiber 3832 may be any suitable optical fiber that transmits a light beam, such as a multimode fiber or a single mode fiber.

The fiber optic assembly 3830 also includes a fiber mounting block 3820. As shown in FIG. 70, the fiber mounting block 3820 defines a series of lumens 3825a-3825g and a series of lumens 3824a-3824 g. Lumens 3824 are each configured to receive a second end portion of a respective excitation optical fiber (e.g., excitation fiber 3831a as identified in fig. 65). Similarly, the lumens 3825 are each configured to receive a second end portion of a respective detection optical fiber (e.g., a detection fiber 3832a as identified in fig. 65). Fiber mounting block 3820 is coupled to slide rail 3890 of slide assembly 3870 to optically couple excitation fiber 3831 to excitation module 3860 and to optically couple detection fiber 3832 to detection module 3850, as described in more detail below.

As shown in fig. 65, the fiber optic assembly 3830 includes a series of spacers, lenses, and sealing members to facilitate the optical coupling described herein and/or to modify, condition, and/or transform the light beam transmitted by the fiber optic assembly 3830. More specifically, the fiber optic assembly 3830 includes a series of excitation spacers 3833 and detection spacers 3834 configured to be positioned within the fiber mounting block 3820 and/or sled 3890. The fiber optic assembly 3830 also includes a series of excitation lenses 3835 and detection lenses 3836 configured to be disposed within the fiber mounting block 3820 and/or sled 3890. The fiber optic assembly 3830 also includes a series of excitation seal members 3837 and detection seal members 3838 configured to be disposed within the fiber mounting block 3820 and/or sled 3890. The excitation sealing member 3837 and the detection sealing member 3838 are configured to seal and/or prevent contamination from entering the optical path defined by the optical assembly 3800.

As shown in fig. 64-66, the optical assembly 3800 includes an excitation module 3860 configured to generate a series of excitation light beams (and/or optical signals, not shown). The excitation module 3860 includes an excitation circuit board 3861 on which a series of excitation light sources 3862 are mounted. The light source 3862 may be any suitable device and/or mechanism for generating a series of excitation light beams, such as a laser, a Light Emitting Diode (LED), a flash lamp, and the like. In certain embodiments, the light beams generated by each light source 3862 may have substantially the same characteristics (e.g., wavelength, amplitude, and/or energy) as the light beams generated by the other light sources 3862. However, in other embodiments, the first light source 3862 may generate a light beam having a first set of characteristics (e.g., wavelengths combined with a red light beam), and the second light source 3862 may generate a light beam having a second, different set of characteristics (e.g., wavelengths combined with a green light beam). This arrangement allows each different beam (i.e., a beam having different characteristics) to be delivered to each of the receiving blocks 3710 in a sequential manner, as described in more detail herein. As shown in fig. 65, the excitation module 3860 includes a series of spacers 3863, filters 3864, and lenses 3865 to facilitate the optical coupling described herein, and/or to modify, condition, and/or transform the light beams generated by the excitation module 3860 and transmitted by the excitation fibers 3831.

As shown in fig. 64-66, the optical assembly 3800 includes a detection module 3850 configured to accept and/or detect a series of emitted light beams (and/or light signals, not shown). The detection module 3850 includes a series of detection circuit boards 3851 on which emission light detectors 3852 are mounted. The emitted light detector 3852 may be any suitable device and/or mechanism for detecting a series of emitted light beams, such as an optical detector, a photoresistor, a photovoltaic cell, a photodiode, a photocell, a CCD camera, or the like. In some embodiments, each detector 3852 may be configured to selectively accept the emitted light beam regardless of the characteristics (e.g., wavelength, amplitude, and/or energy) of the emitted light beam. However, in other embodiments, the detector 3852 may be configured (or "tuned") to correspond to an emitted light beam having a particular set of characteristics (e.g., wavelengths combined with a red light beam). In certain embodiments, for example, each of the detectors 3852 may be configured to receive emitted light resulting from excitation of a portion of the sample when excited by a corresponding light source 3862 of the excitation module 3860. This arrangement allows each different emitted beam (i.e., a beam having different characteristics) to be received by each receiving block 3710 in a sequential manner, as described in more detail herein. As shown in fig. 65, the detection module 3850 includes a series of spacers 3853, filters 3854, and lenses 3855 to facilitate the optical coupling described herein, and/or to modify, condition, and/or convert the emitted light beams received by the detection module 3850.

Slide assembly 3870 includes a mounting member 3840, a slider 3880, and a slide track 3890. The slider 3880 is coupled to the mounting member 3840 and is slidably mounted to the slide rail 3890. As described in more detail below, in use, the drive screw 3872, which is rotated by the stepper motor 3873, may be rotated within a portion of the slider 3880 to cause the slider 3880 (and thus the mounting member 3840) to move relative to the slide track 3890, as shown by arrow HHH in fig. 64 and 66. In this manner, the mounting member 3840 may be moved relative to the track 3890 to sequentially move each of the excitation light sources 3862 and emission light detectors 3852 into optical communication with the second ends of each of the excitation fibers 3831 and emission fibers 3832, respectively. Further details of the operation of slide assembly 3870 and optical assembly 3800 are provided below.

As shown in FIG. 67, the mounting member 3840 defines a series of firing lumens 3844a-3844f and a series of launching lumens 3845a-3845 f. As shown in fig. 65, each excitation light source 3862 is disposed within a respective excitation lumen 3844, and each emission light detector 3852 is disposed within a respective emission lumen 3845. The mounting member 3840 is coupled to the slider 3880 such that movement of the slider 3880 causes movement of the mounting member 3840 (and thus the excitation light source 3862 and the emission light detector 3852).

As shown in fig. 68, the slider 3880 includes a first portion 3881 and a second portion 3882. The first portion 3881 includes a guide projection 3886 and defines a series of firing lumens 3884a-3884f and a series of firing lumens 3855a-3855 f. When the sliders 3880 are coupled to the mounting member 3840, the firing lumens 3884 of the sliders 3880 are each aligned with a corresponding firing lumen 3844 of the mounting member 3840. Similarly, the launching lumens 3885 of the sliders 3880 are each aligned with a corresponding launching lumen 3845 of the mounting member 3840. The guide projections are configured to slidably seat within corresponding channels 3896 on the slide track 3890.

The second portion 3882 of the slide 3880 defines a pair of guide tube lumens 3887 and lead screw lumens 3888. In use, the drive screw 3872 rotates within the lead screw lumen 3888 to move the sled 3880 relative to the sled 3890. The movement of the sliders 3880 is guided by guide rails 3871, which guide rails 3871 are slidably disposed within respective guide tube chambers 3887.

As shown in fig. 69, the glide track 3890 defines seven excitation wells 3894a, 3894b, 3894c, 3894d, 3894e, 3894f, and 3894g, and seven detection wells 3895a, 3895b, 3895c, 3895d, 3895e, 3895f, and 3895 g. The fiber mounting block 3820 is coupled to the sled 3890 such that the excitation fibers 3831 are in optical communication with each respective excitation aperture and the detection fibers 3832 are in optical communication with each respective excitation aperture. In this manner, as the slider 3880 and the mounting member 3840 are moved together relative to the track 3890, the firing and sensing apertures of the slider 3880 and the mounting member 3840 are each sequentially aligned with the firing and sensing apertures 3894 and 3895, respectively, of the track 3890.

In use, the slide assembly 3870 may controllably move the slide block 3880 during or after the amplification process, such that each light source 3862 and optical detector 3852 pair passes each pair of excitation fibers 3831 and detection fibers 3832 in sequence. In this manner, optical assembly 3800 can analyze samples within each of the six PCR vials (e.g., PCR vial 6260) in a time-programmed and/or multiplexed format.

Fig. 71-73 are schematic block diagrams of an electronic control and computer system for the instrument 3002.

Although the optical assembly 3800 is shown as including a detection module 3850 adjacent to the excitation module 3860, in other embodiments, the optical assembly of the instrument may include a detection module located in a position relative to the excitation module. For example, fig. 74-76 are schematic diagrams of an optical assembly 4800 configured to perform time-lapse optical detection of a series of samples, as described above with respect to optical assembly 3800. The optical assembly 4800 is part of an instrument (e.g., any of the instruments shown and described herein) configured to contain six reaction vials 260. The optical assembly 4800 includes an excitation module 4860, a detection module 4850, and a fiber assembly 4830. The excitation module 4860 includes four excitation light sources 4862a, 4862b, 4862c, and 4862 d. The excitation light sources are each configured to produce an excitation light beam having a different wavelength. For example, light source 4862a is configured to produce a light beam having color #1 (e.g., red), light source 4862b is configured to produce a light beam having color #2 (e.g., green), light source 4862c is configured to produce a light beam having color #3 (e.g., blue), and light source 4862d is configured to produce a light beam having color #4 (e.g., yellow).

Detection module 4850 includes four detectors 4852a, 4852b, 4852c, and 4865 d. The detectors are each configured to accept an emission beam having a different wavelength. For example, detector 4852a is configured to receive a light beam resulting from excitation of an analyte having excitation color #1, detector 4852b is configured to receive a light beam resulting from excitation of an analyte having excitation color #2, detector 4852cv is configured to receive a light beam resulting from excitation of an analyte having excitation color #3, and detector 4852d is configured to receive a light beam resulting from excitation of an analyte having excitation color # 4.

The fibre assembly 4830 comprises a series of excitation fibres 4831 and a series of detection fibres 4832. In particular, one excitation fiber is used to optically couple each reaction vial 260 to the excitation module 4860 and one detection fiber 4832 is used to optically couple each reaction vial 260 to the detection module 4850. The excitation module 4860 and detection module 4850 are configured to move relative to the fiber assembly 4830. In this manner, the light source and its corresponding detector (e.g., light source 4862a and detector 4852 a) may each be sequentially aligned with the excitation and detection fibers for a particular reaction vial 260.

In use, when the optical assembly 4800 is in a first configuration, as shown in fig. 74, the light source 4862a and detector 4852a are in optical communication with the first reaction vial 260. Thus, the sample contained in the first reaction vial can be analyzed with excitation light having color # 1. Excitation module 4860 and detection module 4850 are then moved as indicated by arrow III in fig. 75 to place the optical assembly in the second configuration. When the optical assembly 4800 is in the second configuration, as shown in fig. 75, the light source 4862a and detector 4852a are in optical communication with the second reaction vial 260, and the light source 4862b and detector 4852b are in optical communication with the first reaction vial 260. Thus, the sample contained within the first reaction vial can be analyzed with excitation light having color #2, and the sample contained within the second reaction vial can be analyzed with excitation light having color # 1. Excitation module 4860 and detection module 4850 are then moved as indicated by arrow JJJ in fig. 76 to place the optical assembly in a third configuration. When the optical assembly 4800 is in the third configuration, as shown in fig. 76, the light source 4862a and detector 4852a are in optical communication with the third reaction vial 260, the light source 4862b and detector 4852b are in optical communication with the second reaction vial 260, and the light source 4862c and detector 4852c are in optical communication with the first reaction vial 260. Thus, a sample contained in a first reaction vial can be analyzed with excitation light having color #3, a sample contained in a second reaction vial can be analyzed with excitation light having color #2, and a sample contained in a third reaction vial can be analyzed with excitation light having color # 1.

FIG. 75 is a flow diagram of a method 100 for detecting nucleic acids in a biological sample, according to one embodiment. In particular, the illustrated method is a "single-stage target detection" method, which can be performed using any of the cartridges shown and described herein, as well as any of the instruments shown and described herein. More specifically, the operations of method 100 described below may be performed in a cartridge without opening the cartridge and/or otherwise exposing the sample, reagents, and/or PCR mixture to external conditions. Similarly stated, the operations of method 100 described below may be performed in a cartridge without human intervention to transfer samples and/or reagents. For purposes of description, method 100 is described as being performed with separation module 7100 and PCR module 7200 of cartridge 7001 shown and described above with respect to fig. 25-33.

The method includes eluting the nucleic acid from the magnetic capture bead in an elution chamber, 102. This process may occur, for example, within the elution chamber 7190 of the separation module 7100. More specifically, referring to fig. 29-31, elution buffer may be stored within the reagent module 7270a and may be transferred into the elution chamber 7190 as described above to complete the elution operation. The elution buffer can be any suitable elution buffer described herein and/or one that is compatible with nucleic acid amplification (e.g., via PCR and reverse transcription).

The eluted nucleic acid is then transferred from the elution chamber to the PCR chamber, 104. The PCR chamber can be, for example, PCR vial 7260 shown in fig. 29-31. Although the elution chamber 7190 and PCR vial 7260 are shown above as being within different modules and/or housings, in other embodiments, the elution chamber and PCR chamber may be located within a housing or mechanism of unitary construction. As described above, in certain embodiments, the PCR chamber may include lyophilized amplification reagents such that the reagents are reconstituted after nucleic acid transfer. The eluted nucleic acids are then transferred into PCR vial 7260 as described above using transfer mechanism 7235 or any suitable mechanism.

The PCR mixture is then thermally cycled and/or heated in a PCR chamber, 106. The PCR mixture can be cycled between any suitable temperature range using the instrument 3002, as indicated above. In certain embodiments, the PCR mixture may be raised to a constant temperature to activate the enzymes for amplification.

The amplification reaction is monitored in real time, 108. In certain embodiments, the amplification reaction may be monitored by binding the product (i.e., amplification) with a fluorescently labeled Minor Groove Binder (MGB) and/or any other affinity-based hybridization interaction). Monitoring may be performed using the optical assembly 3800 of the instrument 3002 shown and described above.

After amplification is complete, the detection probe (e.g., MGB) can bind to the target amplicon, 110. This provides endpoint detection.

In certain embodiments, the method includes performing melting analysis and/or annealing analysis, 112. This operation can be performed to identify or confirm molecular targets of specific or mismatched sequences.

FIG. 76 is a flow diagram of a method 200 of detecting nucleic acids in a biological sample, according to one embodiment. In particular, the illustrated method is a "two-stage target detection" method, which can be performed using any of the cartridges shown and described herein and any of the instruments shown and described herein. More specifically, the operations of method 200 described below may be performed in a cartridge without opening the cartridge and/or otherwise exposing the sample, reagents, and/or PCR mixture to external conditions. Similarly stated, the operations of method 200 described below may be performed in a cartridge without human intervention to transfer samples and/or reagents. For purposes of description, the method 200 is described as being performed with the separation module 6100 and PCR module 6200 shown and described above with respect to fig. 8-24.

The method includes eluting nucleic acids from magnetic capture beads in an elution chamber, 202. This process may occur, for example, within the elution chamber 6190 of the separation module 6100. More specifically, referring to fig. 8-10, elution buffer can be stored within reagent chamber 6213c and can be transferred into the elution chamber as described above to complete the elution operation. The elution buffer can be any suitable elution buffer described herein and/or one that is compatible with nucleic acid amplification (e.g., via PCR and reverse transcription).

The eluted nucleic acid is then transferred from the elution chamber to the PCR chamber, 204. The PCR chamber can be, for example, a PCR vial 6260 as shown in fig. 8. As described above, in certain embodiments, the PCR chamber may include lyophilized amplification reagents such that the reagents are reconstituted after nucleic acid transfer. The eluted nucleic acids are then transferred using transfer mechanism 6235 or any suitable mechanism, as described above.

The PCR mixture is then thermally cycled and/or heated in a PCR chamber, 206. The PCR mixture can be cycled between any suitable temperature range using the instrument 3002, as indicated above. In certain embodiments, the PCR mixture may be raised to a constant temperature to activate the enzymes for amplification.

The amplification reaction is monitored in real time, 208. In certain embodiments, the amplification reaction may be monitored by binding the product (i.e., amplification) with a fluorescently labeled Minor Groove Binder (MGB) and/or any other affinity-based hybridization interaction). Monitoring may be performed using the optical assembly 3800 of the instrument 3002 shown and described above.

After amplification is complete, the detection probe (e.g., MGB) can bind to the target amplicon, 210. This provides endpoint detection. The method includes performing melting analysis and/or annealing analysis, 212. This operation can be performed to identify or confirm molecular targets of specific or mismatched sequences. As used herein, MGBs may be used as probes by themselves, or may be conjugated to another molecule and used as probes. For example, in one embodiment, the MGB is conjugated to the 5' end of a specific DNA oligonucleotide probe along with a fluorescent dye. In this embodiment, the probe comprises a non-fluorescent quencher at the 3' end. When the probe is in solution, the fluorescence of the 5' fluorescent dye is quenched. However, when the probe binds to its complement, the fluorescence is no longer quenched. Accordingly, the amount of fluorescence generated by the probe is directly proportional to the amount of target generated. By conjugating different fluorochromes (i.e., each fluorochrome will emit light at a different wavelength when excited, or may be excited at a unique wavelength) to each probe, the probes may be "multiplexed" in the reaction.

A second set of probes is then delivered to the PCR chamber, 214. In certain embodiments, the second set of probes may include a second set of MGB probes or other generic probes formulated to bind specific or mismatched target sequences that melt (dissociation energy to disrupt affinity) at temperatures greater than about 70 degrees celsius. In certain embodiments, the second set of MGB probes is formulated to bind to a specific or mismatched target sequence that melts at a temperature greater than about 75 degrees celsius. In other embodiments, the second set of MGB probes is formulated to bind to a specific or mismatched target sequence that melts at a temperature greater than about 80 degrees celsius. In yet other embodiments, the second set of MGB probes is formulated to bind to a specific or mismatched target sequence that melts at a temperature greater than about 85 degrees celsius.

In certain embodiments, a second set of probes can be stored within reagent chamber 6213b and can be transferred into PCR vial 6260, either directly or via elution chamber 6190, as described above. In this manner, the second set of probes can be added to the PCR mixture without opening the cartridge or PCR vial or otherwise exposing the PCR mixture to contaminants.

The method then includes performing a second melting analysis and/or annealing analysis, 216. This operation can be performed to identify or confirm molecular targets of specific or mismatched sequences.

FIG. 77 is a flow diagram of a method 300 of detecting nucleic acids in a biological sample, according to one embodiment. In particular, the illustrated method is a "two-step reverse transcription PCR (RT-PCR), with single-stage target detection" method, which can be performed using any of the cartridges shown and described herein, as well as any of the instruments shown and described herein. More specifically, the operations of method 300 described below may be performed in a cartridge without opening the cartridge and/or otherwise exposing the sample, reagents, and/or PCR mixture to external conditions. Similarly stated, the operations of method 300 described below may be performed in a cartridge without human intervention to transfer samples and/or reagents. For purposes of description, the method 200 is described as being performed with the separation module 6100 and PCR module 6200 shown and described above with respect to fig. 8-24.

The method includes eluting nucleic acids from magnetic capture beads in an elution chamber, 302. This process may occur, for example, within the elution chamber 6190 of the separation module 600. More specifically, referring to fig. 8-10, elution buffer can be stored within reagent chamber 6213c and can be transferred into the elution chamber as described above to complete the elution operation. The elution buffer can be any suitable elution buffer described herein and/or one that is compatible with nucleic acid amplification (e.g., via PCR and reverse transcription).

The eluted nucleic acid is then transferred from the elution chamber to the PCR chamber, 304. The PCR chamber can be, for example, a PCR vial 6260 as shown in fig. 8. As described above, in certain embodiments, the PCR chamber may include lyophilized amplification reagents such that the reagents are reconstituted after nucleic acid transfer. The eluted nucleic acids are then transferred using a syringe pump or any suitable mechanism, as described above.

The mixture is then heated to a substantially constant temperature within the PCR chamber, 306. In this way, the enzyme for reverse transcription can be activated.

After reverse transcription is complete, PCR reagents are delivered to the PCR chamber, 308. PCR reagents can be stored in reagent chambers 6213b and/or 6213a and can be transferred into PCR vial 6260 directly or via elution chamber 6190, as described above. In this manner, PCR reagents can be added to the PCR mixture after reverse transcription is complete without opening the cartridge or PCR vial or otherwise exposing the PCR mixture to contaminants.

The amplification reaction is monitored in real time, 310. In certain embodiments, the amplification reaction may be monitored by binding the product (i.e., amplification) with a fluorescently labeled Minor Groove Binder (MGB) and/or any other affinity-based hybridization interaction). However, any DNA binding reagent can be used to monitor the PCR reaction in real time. Monitoring may be performed using the optical assembly 3800 of the instrument 3002 shown and described above.

As used herein, "DNA binding agent" refers to any detectable molecule capable of binding double-stranded or single-stranded DNA, e.g., detectable by fluorescence. In one embodiment, the DNA binding agent is a fluorescent dye or other chromophore, enzyme or agent capable of directly or indirectly generating a signal when binding double-stranded or single-stranded DNA. The agent may be indirectly bound, i.e., the DNA binding agent may be attached to another agent that directly binds DNA. It is only necessary that the agent is capable of producing a detectable signal when bound to double stranded nucleic acid or single stranded DNA that is distinguishable from the signal produced when the same agent is in solution.

In one embodiment, the DNA binding agent is an intercalating agent. Intercalating agents such as ethidium bromide and SYBR green fluoresce more strongly when inserted into double-stranded DNA than when bound to single-stranded DNA, RNA or in solution. Other intercalating agents exhibit a change in fluorescence spectrum when bound to double-stranded DNA. For example, actinomycin D fluoresces red when bound to single stranded nucleic acids and green when bound to double stranded templates. Any intercalating agent that provides a distinguishable detectable signal when the agent binds to double stranded DNA or is unbound, is suitable for practicing the invention, whether the detectable signal increases, decreases or transitions, as is the case with actinomycin D.

In another embodiment, the DNA binding agent is an exonuclease probe using fluorescence resonance energy transfer. For example, in one embodiment, the DNA binding reagent is an oligonucleotide probe having a reporter and a quencher dye at the 5 'and 3' ends, respectively, and specifically binds to the target nucleic acid molecule. In solution and when intact, the fluorescence of the reporter dye is quenched. However, exonuclease activity of certain Taq polymerases acts to cleave probes during PCR, and the reporter is no longer quenched. Thus, the fluorescence emission is directly proportional to the amount of target generated.

In another embodiment, the DNA binding agent employs an MGB conjugated to the 5' end of the oligonucleotide probe. In addition to the MGB at the 5' end, a reporter dye is conjugated to the 5' end of the probe and a quencher dye is placed at the 3' end. For example, in one embodiment, a DNA probe described by Lukhtanov (Lukhtavon (2007). Nucleic acids research 35, p. e 30) is used. In one embodiment, the MGB is directly conjugated to an oligonucleotide probe. In another embodiment, the MGB is conjugated to a reporter dye. When the probe is in solution, the fluorescence of the 5' fluorescent dye is quenched. However, when the probe binds to its complement, the fluorescence is no longer quenched. Accordingly, the amount of fluorescence generated by the probe is directly proportional to the amount of target generated. By conjugating different fluorochromes (i.e., each fluorochrome will emit light at a different wavelength when excited, or may be excited at a unique wavelength) to each probe, the probes may be "multiplexed" in the reaction.

In yet other embodiments, the minor groove binder is used to monitor the PCR reaction in real time. For example, Hoechst 33258 (Searle & Embrey, 1990, Nuc. acids Res.18 (13): 3753-3762) shows fluorescence that changes with increasing target amounts. Other MGBs for use with the present invention include distamycin and fusin.

According to embodiments described herein, the DNA binding reagent produces a detectable signal, either directly or indirectly. The signal is directly detectable, e.g., by fluorescence or absorbance, or indirectly detectable via a surrogate label moiety or a binding ligand attached to the DNA binding reagent.

According to embodiments described herein, the DNA binding reagent produces a detectable signal, either directly or indirectly. The signal is directly detectable, e.g., by fluorescence or absorbance, or indirectly detectable via a surrogate label moiety or a binding ligand attached to the DNA binding reagent. For example, in one embodiment, a DNA probe conjugated to a fluorescent reporter dye is employed. The DNA probe has a quencher dye on the opposite end of the reporter dye and fluoresces only when bound to its complementary sequence. In a further embodiment, the DNA probe has an MGB at the 5' end and a fluorescent dye.

Other non-limiting DNA binding reagents for use with the present invention include, but are not limited to, Molecular Beacons, Scorpion and FRET probes.

After amplification is complete, the detection probe (e.g., MGB) may bind to the target amplicon, 312. This provides endpoint detection. The method includes performing melting analysis and/or annealing analysis, 314. This operation can be performed to identify or confirm molecular targets of specific or mismatched sequences.

FIG. 78 is a flow diagram of a method 400 of detecting nucleic acids in a biological sample, according to one embodiment. In particular, the illustrated method is an alternative "single-stage target detection" method with respect to the method 100 shown and described above. The method 400 may be performed using any of the cartridges shown and described herein and any of the instruments shown and described herein. More specifically, the operations of method 400 described below may be performed in a cartridge without opening the cartridge and/or otherwise exposing the sample, reagents, and/or PCR mixture to external conditions. Similarly stated, the operations of method 400 described below may be performed in a cartridge without human intervention to transfer samples and/or reagents. For descriptive purposes, the method 400 is described as being performed by the separation module 10100 and the PCR module 10200 shown and described herein with respect to fig. 85-87.

Method 400 differs from method 100 in that the elution buffer is stored in the elution chamber of the housing instead of in reagent chamber 6213c as described for method 100. Thus, the method includes eluting the nucleic acid from the magnetic capture bead in an elution chamber, 402. This process occurs within the elution chamber of separation module 10100. The elution buffer can be any suitable elution buffer that is compatible with nucleic acid amplification (e.g., via PCR and reverse transcription).

The eluted nucleic acid is then transferred from the elution chamber to the PCR chamber, 404. The PCR chamber can be, for example, a PCR vial 10260 as shown in fig. 85-87. Although the elution chamber 10190 and the PCR vial 10260 are shown within different modules and/or housings, in other embodiments, the elution chamber and the PCR chamber may be located within a housing or mechanism that is integrally constructed. As described above, in certain embodiments, the PCR chamber may include lyophilized amplification reagents such that the reagents are reconstituted after nucleic acid transfer. The eluted nucleic acids are then transferred using a syringe pump or any suitable mechanism, as described above.

The PCR mixture is then thermally cycled and/or heated in a PCR chamber, 406. The PCR mixture can be cycled between any suitable temperature range using the instrument 3002, as indicated above. In certain embodiments, the PCR mixture may be raised to a constant temperature to activate the enzymes for amplification.

The amplification reaction is monitored in real time, 408. In certain embodiments, the amplification reaction may be monitored by binding the product (i.e., amplification) with a fluorescently labeled Minor Groove Binder (MGB) and/or any other affinity-based hybridization interaction). Monitoring may be performed using the optical assembly 3800 of the instrument 3002 shown and described above.

After amplification is complete, the detection probe (e.g., MGB) can bind to the target amplicon, 410. This provides endpoint detection. In certain embodiments, the method comprises performing melting analysis and/or annealing analysis, 412. This operation can be performed to identify or confirm molecular targets of specific or mismatched sequences.

Data generated using the systems and methods described herein can be analyzed using any number of different methods. For example, the data can be analyzed for sequence identification of amplified nucleic acids via melting or annealing analysis using affinity probes. One or more specific genetic status profiles are indicated/generated using melting/annealing profiling-molecular profiling (consisting of modified bases and MGB-fluorescence-affinity constant-Kd with an affinity directed to binding to a target nucleic acid) of a unique "affinity probe" or molecular target. For example, fig. 81 is a graph indicating molecular markers generated from a set of probes that bind to amplified nucleic acids derived from a biological sample. Molecular markers represent disease states (or the presence of unique nucleic acid sequences) associated with recovery of a biological sample. The molecular marker or profile depends on the specific interaction of the molecular target with the target nucleic acid, which can only be generated from the molecular target within the cartridge. In other words, the spectrum is a fingerprint trace (i.e., a unique sequence of peaks or "spectral responses" indicative of one or more diseased states (oncology, infectious disease) or genetic states).

Multiplex within spectrum-more than one diseased state- (multiplex label) -multiplex for temperature and time (within a specific wavelength), unique "probes" or multiple probes (unique molecular entities-molecular reactants, indicators, markers).

The multichannel method comprises the following steps: more than one fingerprint trace (fingerprint group) may be used in the authentication process. Multi-panel fingerprints-a spectral array of fingerprints can be used to determine the results. The variables that generate the multichannel or array data are the wavelength-differential fluorescence used, the temperature range for annealing or dissociation (melting), and the data acquisition rate (data-dependent domain).

Heating and cooling controls of the affinity probes and amplification targets can be used to obtain the desired identification of diseased fingerprints. For data generation (annealing and melting), the temperature range may be in the range of 70-100 degrees celsius.

Although separation module 6001 above is shown to include separation module 6100 with mixing pump 6181 for facilitating the lysis process, in other embodiments, any suitable mechanism for transferring energy into solution to push and/or enhance cell lysis may be used. For example, in certain embodiments, sonic energy may be used.

For example, fig. 82 shows a second housing 8160 of a separation module according to one embodiment configured to deliver ultrasonic energy into a sample contained within a separation chamber (not shown) of the separation module (e.g., separation module 6100, separation module 7100, etc.) to facilitate cell lysis and/or separation of nucleic acids contained therein. The second housing 8160 may be coupled to and/or disposed within a corresponding first housing (not shown in fig. 82) in a manner similar to that described above with respect to fig. 11. More specifically, the second housing 8160 includes a seal (not shown) similar to seal 6172 shown and described above, which substantially acoustically separates the second housing 8160 from the first housing.

Second housing 8160 defines a series of containment chambers 8163a, 8163b, 8163c, and 8163d that contain reagents and/or other substances used in the separation process. In particular, the containment chamber can contain a protease (e.g., proteinase K), a lysis solution that dissolves a bulk material, a binding solution for nucleic acids, and a solution of magnetic beads that bind magnetically-charged nucleic acids to aid in the transport of nucleic acids within the separation module and/or the first housing.

The second housing 8160 also defines a bore 8185 within which a portion of the ultrasound transducer 8195 may be disposed. An acoustic coupling member 8182 is coupled to a portion of the sidewall of the second housing 8160 within the aperture 8185. Accordingly, in use, at least a portion of the acoustic transducer 8195 can be disposed within the aperture 8185 and in contact with the acoustic coupling member 8182. In this manner, acoustic and/or ultrasonic energy generated by the transducer 8195 can be transmitted through the acoustic coupling member 8182 and the side wall of the second housing 8160 and into the solution within the separation chamber. Acoustic transducer 8195 may be any suitable acoustic transducer and may be configured to resonate between 20kHz and 300 kHz.

The ultrasound transducer 8195 may be moved into the bore 8185 by an instrument, such as the driver of the instrument 3002 described herein. Such drivers may include, for example, stepper motors configured to move the ultrasonic transducer 8195 through a predetermined distance into contact with the acoustic coupling member 8182. In certain embodiments, for example, the instrument can include a driver assembly similar to the first driver assembly 3400 shown and described above with respect to fig. 37-40. In such embodiments, a first driver assembly may include a series of ultrasonic transducers that move into the bore via a coupling rod similar to coupling rod 3445.

In certain embodiments, the driver may be configured to vary the force applied by the ultrasound transducer 8195 to the acoustic coupling member 8182. This may be done, for example, by moving the ultrasound transducer 8195 relative to the coupling member 8182 as the ultrasound transducer is driven. Such an arrangement may allow for dynamic adjustment of the ultrasonic energy transfer through acoustic coupling member 8182 and/or the heat generated by the ultrasonic energy transfer through acoustic coupling member 8182.

In certain embodiments, acoustic coupling member 8182 is constructed from a thermally conductive material. In this manner, heat transfer from the acoustic coupling member 8182 to the adjacent side wall of the second housing 8160 may be minimized. This arrangement may minimize deformation and/or melting of the side walls of second housing 8160 and/or prevent deformation and/or melting of the side walls of second housing 8160 when in contact with the side walls when acoustic transducer 8195 is driven. Additionally, in certain embodiments, acoustic coupling member 8182 may be configured and/or constructed with acoustic impedance to facilitate the transfer of ultrasonic energy through acoustic coupling member 8182 and into the separation chamber.

Fig. 83 shows a second housing 9160 of a separation module according to one embodiment configured to deliver ultrasonic energy into a sample contained within a separation chamber (not shown) of the separation module to facilitate cell lysis and/or separation of nucleic acids contained therein. The second housing 9160 can be coupled to and/or disposed within a corresponding first housing (not shown in fig. 83) in a similar manner as described above. More specifically, the second housing 9160 includes a seal (not shown) similar to the seal 6172 shown and described above, which substantially acoustically separates the second housing 9160 from the first housing.

The second housing 9160 defines a series of containment chambers 9163a, 9163b, 9163c, and 9163d, which contain reagents and/or other substances used in the separation process. The second housing 9160 also defines an aperture 9185 within which a portion of the ultrasound transducer 9195 can be positioned. In contrast to the aperture 8185 described above, the aperture 9185 can be in fluid communication with the separation chamber via an aperture in a sidewall of the second housing 9160.

The acoustic coupling member 9183 is disposed within the aperture 9185 and through a portion of a sidewall of the second housing 9160. More specifically, the acoustic coupling member 9183 is coupled to the sidewall such that a first portion 9186 of the acoustic coupling member 9183 is within the aperture 9185 and a second portion 9187 of the acoustic coupling member 9183 is within the separation chamber. The seal 9184 is disposed between a sidewall of the second housing 9160 and the acoustic coupling member 9183 to substantially fluidly separate the separation chamber and/or the acoustic coupling member 9183 from the second housing.

In use, at least a portion of the acoustic transducer 8195 can be disposed within the aperture 9185 and in contact with the first portion 9186 of the acoustic coupling member 9183. In this manner, the acoustic and/or ultrasonic energy generated by the transducer 9195 may be transmitted through the acoustic coupling member 9183 and into the solution within the separation chamber.

The ultrasound transducer 8195 may be moved into the bore 9185 by an instrument, such as a driver of the instrument 3002 described herein. Such drivers may include, for example, stepper motors configured to move the ultrasonic transducer 9195 into contact with the acoustic coupling member 9183 by a predetermined distance. In certain embodiments, for example, the instrument can include a driver assembly similar to the first driver assembly 3400 shown and described above with respect to fig. 37-40. In such embodiments, a first driver assembly may include a series of ultrasonic transducers that move into the bore via a coupling rod similar to coupling rod 3445.

In certain embodiments, the driver can be configured to vary the force applied by the ultrasound transducer 5195 to the acoustic coupling member 5183. This can be done, for example, by moving the ultrasound transducer 8195 relative to the coupling member 9183 upon actuation of the ultrasound transducer. Such an arrangement may allow for dynamic adjustment of the ultrasonic energy transfer through the acoustic coupling members 9183 and/or the heat generated by the ultrasonic energy transfer through the acoustic coupling members 9183.

As described above, in certain embodiments, the acoustic coupling member 5183 can be configured and/or constructed to have an acoustic impedance to facilitate the transfer of ultrasonic energy through the acoustic coupling member 9183 and into the separation chamber.

Although fig. 82 and 83 show a second housing of the separation module configured to transmit ultrasonic energy into the sample contained within the separation module, in other embodiments, any portion of the cartridge may be configured to transmit ultrasonic energy into the sample. For example, fig. 84 shows a separation module 7100 (see, e.g., fig. 26-28) and an ultrasound transducer 7195. In particular, as described above, the housing 7110 includes an acoustic coupling portion 7182. In use, at least a portion of the ultrasonic transducer 7195 may be disposed in contact with the acoustic coupling portion 7182. In this manner, acoustic and/or ultrasonic energy generated by the transducer may be transmitted through the acoustic coupling portion 7182 and the sidewall of the first housing 7110 and into the solution within the separation chamber 7114.

The ultrasonic transducer 7195 may be moved into contact with the acoustic coupling portion 7182 by an instrument, such as a driver of the instrument 3002 described herein. Such drivers may include, for example, stepper motors configured to move the ultrasonic transducer 7195 through a predetermined distance into contact with the acoustic coupling portion 7182. In certain embodiments, for example, the instrument can include a driver assembly similar to the first driver assembly 3400 shown and described above with respect to fig. 37-40. In such embodiments, a first driver assembly may include a series of ultrasonic transducers that move into contact with acoustic coupling portion 7182 via a coupling rod similar to coupling rod 3445.

In certain embodiments, the driver may be configured to vary the force applied by the ultrasonic transducer 7195 to the acoustic coupling portion 7182. This may be done, for example, by moving the ultrasonic transducer 7195 relative to the acoustic coupling portion 7182 as the ultrasonic transducer is driven. This arrangement may allow dynamic adjustment of the ultrasonic energy transfer through the acoustic coupling portion 7182 and/or the heat generated by the ultrasonic energy transfer through the acoustic coupling portion 7182. As shown in fig. 83, the ultrasound transducer 7195 may include a spring 7196 or other biasing member configured to maintain and/or bias the ultrasound transducer relative to a driver assembly of the instrument.

Although PCR module 6200 is shown and described above as comprising three reagent chambers 6213a, 6213b, and 1213c within which PCR reagents, elution buffers, and the like may be stored, in other embodiments, the PCR module may comprise any number of reagent chambers. In certain embodiments, the PCR module may be devoid of any reagent chambers. For example, fig. 85-87 show a cartridge 10001 according to one embodiment. The cartridge 10001 comprises a nucleic acid separation module 10100 and an amplification (or PCR) module 10200 that are coupled together to form an integrated cartridge 10001. Integrated cartridge 10001 is similar in many respects to cartridge 6001 and/or cartridge 7001 shown and described above, and therefore is not described in detail herein. As shown in fig. 86, which shows the cartridge without the cover 10005, the PCR module 10200 comprises a housing 10210, a PCR vial 10260 and a transfer tube 10250. The amplification module 10200 is coupled to the separation module 10100 such that at least a portion of the transfer tube is disposed within the elution chamber of the separation module 10100.

Housing 10210 includes transfer port 10270. Transfer port 10270 defines one or more lumens and/or channels through which isolated nucleic acids and/or other substances or reagents can be transferred into PCR vial 10260. Housing 10210 and/or transfer port 10270 may define one or more vent passages to fluidly couple the elution chamber and/or PCR vial 10260 to the atmosphere. In certain embodiments, any of such vents may include a frit, a valve, and/or other suitable mechanism to minimize loss of sample and/or reagents from the elution chamber and/or PCR vial 10260, and/or to prevent loss of sample and/or reagents from the elution chamber and/or PCR vial 10260.

First end portion 10271 of transfer port 10270 is disposed outside of PCR vial 10260 and second end portion 10272 of transfer port 10270 is disposed within the PCR vial. More specifically, second end portion 10272 is disposed within PCR vial 10260 such that a volume V of PCR vial 10260 within which a sample can be disposed does not exceed a predetermined magnitude. In this manner, because there is a limited "head space" on the sample within the PCR vial 10260, condensation that may form on the walls of the PCR vial 10260 during thermal cycling may be minimized and/or eliminated.

The PCR module 10200 includes a transfer piston 10240 configured to generate pressure and/or vacuum within the elution chamber and/or the PCR vial 10260 to transfer at least a portion of the sample and/or reagents within the elution chamber into the PCR vial 10260, as described above.

The elution buffer used for cartridge 10001 is stored in the elution chamber (not shown in fig. 85-87) of separation module 10100. The PCR reagents are stored in lyophilized form in PCR vial 10260 as described above. In use, the isolated nucleic acid is eluted from the capture bead in the elution chamber. The eluted nucleic acids are then transferred into the PCR vial 10260 as described above and mixed with the PCR reagents within the PCR vial 10260.

Although the PCR module 6200 is shown and described as including three reagent chambers 6213a, 6213b, and 6213c disposed adjacent a first end portion 6211 of the housing 6210 (see, e.g., fig. 8), in other embodiments, the PCR module can include any number of reagent chambers or modules disposed in any position and/or orientation. Further, in certain embodiments, the agent plug (e.g., plug 6214 a) and/or any of the transfer mechanisms described herein can be biased. For example, fig. 88 is a cross-sectional view of PCR module 11200 coupled to separation module 6100'. PCR module 11200 includes a housing 11210 defining three reagent chambers 11213 in which substances and/or reagents of the type described herein may be stored. A plug 11214 and a spring 11215 (only one shown and labeled in fig. 88) are disposed within each reagent chamber 11213. In this manner, the plug (or transfer mechanism) is biased in the non-actuated position. However, in other embodiments, the plug may be biased in the actuated position and may be held in place by a locking tab or the like. In this way, the driving of the plug may be assisted by the spring force.

The PCR module also includes a mixing mechanism (or transfer mechanism) 11130 that is in fluid communication with the elution chamber 6190' via nozzle 11131. Pipette 11250 places elution chamber 6190 in fluid communication with PCR vial 11260.

In certain embodiments, the PCR module may include a PCR vial or reaction chamber disposed adjacent to the elution chamber of the separation module. For example, fig. 89 shows cartridge 12001 with separation module 6100' coupled to PCR module 12200. PCR module 12200 includes a PCR chamber 12260 adjacent to elution chamber 6190'. Similarly stated, when PCR module 12200 is coupled to separation module 6100 ', PCR vial 12260 is disposed between PCR chamber 12231 and separation module 6100'.

Although the cartridges shown and described herein include a separation module that includes an elution chamber (e.g., elution chamber 7190) coupled to a PCR module such that, in use, a portion of the separated sample is transferred into a PCR vial (e.g., PCR vial 7260), in other embodiments, the PCR module need not include a PCR vial. For example, in certain embodiments, the cartridge may include an elution chamber that is also configured as a reaction volume in which PCR occurs. For example, fig. 90 shows cartridge 13001 according to one embodiment, which includes separation module 6100' and PCR module 13200. The PCR module 13200 includes a substrate 13220 and a series of reagent modules 13270. In use, reagent module 13270 is configured to transfer one or more reagents and/or substances of the type shown and described herein via flow tube 13229 into elution chamber 6190 'of separation module 6100'. In this manner, PCR can occur in the elution chamber 6190'. In such embodiments, an instrument similar to instrument 3002 can be configured as a thermocycling elution chamber 6190' to facilitate PCR. In addition, the instrument can include an optical assembly configured to optically monitor the reaction within the elution chamber 6190'. In certain embodiments, the housing 6110 'can include an excitation optic (not shown) and/or a detection optic (not shown) disposed therein in a location adjacent to the elution chamber 6190'.

Although the cartridges shown and described herein generally include a PCR module coupled in series with a separation module, in other embodiments, the cartridge may include a PCR module coupled to a separation module in any orientation, position, and/or position. Similarly stated, although the cartridge is shown and described herein as including a PCR module coupled to an end portion of the separation module, in other embodiments, the PCR module can be integrated and/or coupled to the separation module in any manner. For example, fig. 91 shows a cartridge 14001 that includes a separation module 14100 and a PCR module 14200. The separation module 14100 includes a series of washing mechanisms 14130 similar to those described above. The PCR module includes a series of reagent modules 14270. Reagent module 14270 is positioned adjacent to washing mechanisms 14130 and/or between washing mechanisms 14130.

In use, reagent module 14270 is configured to transfer one or more reagents and/or substances of the type shown and described herein via flow tube 14229 into elution chamber 14190 of separation module 14100. In this manner, PCR can occur in the elution chamber 14190.

Fig. 92 and 93 show another embodiment in which reagent module 15270 of PCR module 15200 is positioned adjacent to washing mechanism 15130 of separation module 15100 and/or between washing mechanisms 15130 of separation module 15100. Cartridge 15001 differs from cartridge 14001 in that the substance contained within reagent module 15270 is transferred into PCR vial 15260 via a series of internal flow paths 15228. The PCR module includes a transfer mechanism 15235 to transfer a portion of the separated sample from the elution chamber 15190 into the PCR vial 15260.

Although the PCR module shown and described herein includes a single PCR vial, in other embodiments, the PCR module may include any number of PCR vials. One example is shown in fig. 94, which shows a PCR module 16200 with four PCR vials 16260.

While various embodiments have been described above, it should be understood that they have been presented by way of example only, and not limitation. When the above-described methods and/or diagrams indicate particular events and/or flow patterns occurring in a particular order, the ordering of the particular events and/or flow patterns may be modified. Additional specific events may be performed concurrently in a parallel process, as well as performed sequentially, as may be possible. While the embodiments have been particularly shown and described, it will be understood that various changes in form and detail may be made.

Although many of the chambers described herein, such as the chamber 6163a, the wash buffer module 7130a, and the reagent module 7270a, are described as containing a substance, sample, and/or reagent that is maintained in fluid separation by the penetrable members (e.g., the penetrable member 6170, the penetrable member 7135a, and the penetrable member 7275), in certain embodiments, any of the chambers herein may be only partially filled with the desired substance, sample, and/or reagent. More specifically, any of the chambers described herein can include a first volume of a desired substance (which is generally in a liquid state) and a second volume of a gas, such as air, oxygen, nitrogen, and the like. This arrangement reduces the force used to move the transfer mechanism or perforating member (e.g., the perforating portion 6168 of the drive 6166) within the chamber prior to rupturing the penetrable member. More specifically, by including a portion of the chamber volume as a gas, the gas compresses to reduce the volume of the chamber as the transfer mechanism moves within the chamber, thereby allowing the piercing member to contact the penetrable member. In certain embodiments, any of the chambers described herein can include about 10% of its volume as a gas.

Although separation module 6100 is shown and described above as including transfer assembly 6140a configured to transfer substances between lysis chamber 6114 and wash chamber 6121 while maintaining substantial fluidic separation of lysis chamber 6114 from wash chamber 6121, in other embodiments, any of the modules described herein can include a transfer mechanism that transfers substances between chambers while allowing liquid communication between the chambers. For example, in certain embodiments, a module may include a transfer mechanism configured to selectively control the flow of a substance between a first chamber and a second chamber. Such a transfer mechanism may include, for example, a trap door.

Although the cartridge is shown and described herein as including a plurality of modules (e.g., a separation module and a reaction module) that are coupled together prior to placement within an instrument that operates the cartridge, in other embodiments, the cartridge may include a plurality of modules, at least one of which is configured to be coupled to another module within and/or by the instrument. Similarly, in certain embodiments, the instrument may be configured to couple one module (e.g., a reagent module) to another module (e.g., a reaction module, a separation module, etc.) as part of the cartridge process.

Although a transfer mechanism, such as transfer mechanism 6140, is shown and described herein as using magnetic forces to facilitate movement of the target portion of the sample within the cartridge, in other embodiments, any of the transfer mechanisms shown and described herein may employ any suitable type of force to facilitate movement of the target portion of the sample within the cartridge. For example, in certain embodiments, the transfer mechanism may comprise a pump. In other embodiments, the transfer mechanism can produce peristaltic movement of the target portion of the sample.

Although the cartridge and/or portions thereof have been described primarily for use with nucleic acid separation and amplification reactions, and for use with the particular instruments described herein, the cartridge is not so limited. Although the instrument and/or portions thereof have been described primarily for use with nucleic acid separation and amplification reactions, and for use with the particular cartridges described herein, the instrument is not so limited.

For example, the cartridges, instruments, and/or portions thereof provided herein can be used in a Next Generation Sequencing (NGS) platform. NGS technology has been reported to generate three to four orders of magnitude more sequences than the Sanger method, and runs are less expensive (harismenday et al (2009) Genome Biology 10, pages R32.1-R32.13). NGS applications include, but are not limited to, genomic shotgun sequencing, Bacterial Artificial Chromosome (BAC) end sequencing, single nucleotide polymorphism discovery and resequencing, other mutation discovery, chromatin immunoprecipitation (ChIP), micro RNA discovery, large-scale expressed sequence tag sequencing, primer walking, or Serial Analysis of Gene Expression (SAGE).

In one embodiment, the module is used to adapt the cartridge of the present invention to an NGS platform instrument for nucleic acid sequence analysis. Alternatively, a sample transfer module (e.g., an automated liquid handling instrument) can transfer the nucleic acid amplification products to a flow cell of the NGS instrument.

In one embodiment, a module is provided such that the cartridge of the present invention is compatible for use with one of the following NGS platforms: roche 454 GS-FLX platform, Illumina Sequencing Platforms (e.g., HiSeq 2000, HiSeq 1000, MiSeq, Genome Analyzer IIx), Illumina Solexa IG Genome Analyzer, Applied Biosystems 3730xl platform, ABI SOLID^TM(e.g., 5500xl or 5500SOLIDTM System). The module may be adapted to one of the above-described devices, or may be a sample transfer module that transfers the product of a nucleic acid amplification reaction to an NGS instrument.

In one embodiment, the cartridge of the invention is used for genomic shotgun sequencing, Bacterial Artificial Chromosome (BAC) end sequencing, single nucleotide polymorphism discovery and resequencing, other mutation discovery, chromatin immunoprecipitation (ChIP), microrna discovery, large-scale expressed sequence tag sequencing, primer walking, or gene expression Sequencing (SAGE).

In one embodiment, nucleic acid isolation and/or amplification (e.g., PCR) is performed in the cartridges and instruments of the invention as described herein. In a further embodiment, upon completion of the amplification reaction, the sample transfer module transfers the amplification products to the flow cell of the respective NGS instrument for library preparation and subsequent sequencing.

In another embodiment, nucleic acid isolation and/or amplification (e.g., PCR) is performed in the cartridge and/or instrument of the invention as described herein. In a further embodiment, after completion of the amplification reaction, the cartridge is transferred to a module that is amenable for use with one of the NGS instruments provided above. The nucleic acid amplification products are then transferred to the flow cell of the respective NGS instrument for library preparation and subsequent sequencing.

In certain embodiments, the apparatus includes a first module, a second module, and a third module. The first module defines a first chamber and a second chamber, at least the first chamber configured to contain a sample. The second module defines a first volume configured to contain a first substance. When the second module is coupled to the first module, a portion of the second module is configured to be disposed within the first chamber of the first module such that the first volume is configured to be selectively placed in fluid communication with the first chamber. The third module defines a reaction chamber and a second volume configured to contain a second substance. When the third module is coupled to the first module, a portion of the third module is disposed within the second chamber of the first module such that the reaction chamber and the second volume are each in fluid communication with the second chamber of the first module.

In certain embodiments, any of the modules described herein may include an acoustic coupling member configured to transmit acoustic energy to a chamber defined by the module.

In certain embodiments, any of the modules described herein can include a transfer mechanism configured to transfer a sample between a first chamber within the module and a second chamber within the module. Such transfer mechanisms may use any suitable mechanism for transferring substances, including flow of solutions, magnetic forces, and the like.

In certain embodiments, any of the modules described herein can include a valve configured to transfer a sample between a first chamber within the module and a second chamber within the module. In certain embodiments, such a valve may be configured to maintain fluid separation between the first chamber and the second chamber.

In certain embodiments, the apparatus includes a first module, a second module, and a third module. The first module defines a first chamber and a second chamber. The first module includes a first transfer mechanism configured to transfer the sample between the first chamber and the second chamber while maintaining fluidic separation between the first chamber and the second chamber. The second module defines a volume configured to contain a substance. When the second module is coupled to the first module, a portion of the second module is configured to be disposed within the first chamber of the first module such that the volume is configured to be selectively placed in fluid communication with the first chamber. A third module defines a reaction chamber, the third module configured to be coupled to the first module such that the reaction chamber is in fluid communication with the second chamber. The third module includes a second transfer mechanism configured to transfer a portion of the sample between the second chamber and the reaction chamber.

In certain embodiments, the apparatus includes a first module and a second module. The first module includes a reaction vial, a substrate, and a first transfer mechanism. The reaction vial defines a reaction chamber. A first transfer mechanism includes a plug movably disposed within a housing such that the housing and the plug define a first volume containing a first substance. The substrate defines at least a portion of the first flow path and the second flow path. The first flow path is configured to be in fluid communication with the reaction chamber. The first volume of the separation module and the separation chamber, the second flow path are configured to be in fluid communication with the separation chamber. A portion of the plug is disposed in the first flow path such that the first volume is fluidly isolated from the reaction chamber when the plug is in a first position within the housing. A portion of the plug is positioned away from the first flow path such that the first volume is in fluid communication with the reaction chamber when the plug is in the second position within the housing. The plug is configured to create a vacuum within the reaction chamber to transfer the sample from the separation chamber to the reaction chamber when the plug is moved from the first position to the second position. The second module includes a second transfer mechanism and defines a second volume configured to contain a second substance. The second module is configured to be coupled to the first module such that the second volume can be selectively placed in fluid communication with the separation chamber via the second flow path. The second transfer mechanism is configured to transfer the second substance from the second volume to the separation chamber when the second transfer mechanism is actuated.

In certain embodiments, an instrument includes a block, a first optical component, a second optical component, and an optical assembly. The block defines a reaction volume configured to receive at least a portion of a reaction vessel. A first optical member is at least partially disposed within the block such that the first optical member defines a first optical path and is in optical communication with the reaction volume. A second optical member is at least partially disposed within the block such that the second optical member defines a second optical path and is in optical communication with the reaction volume. A first plane including the first optical path and a second plane including the second optical path define an angle greater than about 75 degrees. An optical assembly is coupled to the first and second optical members such that the excitation light beam can be transmitted into the reaction volume and the emission light beam can be received by the reaction volume.

Although various embodiments have been described as having particular combinations of parts and/or features, other embodiments having any combination of parts and/or features from any of the embodiments as discussed above are possible.

Claims (31)

1. An instrument, comprising:

an apparatus, the apparatus comprising:

a separation module comprising a first housing and a second housing, the first housing defining a first chamber and a second chamber, at least the first chamber configured to contain a sample, the second housing comprising a sidewall and a penetrable member that together define a first volume configured to contain a first substance, a portion of the second housing configured to be disposed within the first housing such that the first volume is in fluid communication with the first chamber when a portion of the penetrable member is penetrated; and

a reaction module defining a reaction chamber and a second volume configured to contain a second substance, the reaction module configured to be coupled to the separation module such that the reaction chamber and the second volume are each in fluid communication with the second chamber of the first housing, wherein the separation module comprises a transfer mechanism configured to transfer a sample between the first chamber and the second chamber,

wherein the separation module comprises a shutter disposed at least partially within the first housing, the shutter configured to transfer sample between the first chamber and the second chamber while maintaining fluidic separation between the first chamber and the second chamber,

Wherein the reaction module comprises a plug disposed at least partially within the second volume, the plug configured to exert a force on the second substance to transfer the second substance from the second volume into at least one of the reaction chamber or the second chamber when the plug is moved within the second volume;

a frame configured to receive, contain and/or provide mounting for each component and/or assembly of the instrument;

a first driver component;

a sample transfer assembly;

a second driver assembly;

a heater assembly configured to heat one or more portions of the instrument to facilitate and/or facilitate a procedure within the instrument; and

an optical assembly configured to monitor a reaction occurring within the instrument.

2. The apparatus of claim 1, wherein the reaction module is configured to be removably coupled to the separation module.

3. The apparatus of claim 1, wherein a portion of the reaction module is disposed within the second chamber of the first housing when the reaction module is coupled to the separation module.

4. The apparatus of claim 1, wherein:

the reaction module includes a substrate at least partially defining a first flow path configured to be in fluid communication with a reaction chamber and the second chamber of the first housing and a second flow path configured to be in fluid communication with a second volume and the second chamber of the first housing.

5. The apparatus of claim 1, wherein the separation module comprises a plug disposed at least partially within the first volume of the second housing, the plug configured to penetrate the portion of the penetrable member when moved within the first volume.

6. The apparatus of claim 1, wherein the separation module comprises an acoustic coupling member configured to transmit ultrasonic energy into the first chamber.

7. The apparatus of claim 1, wherein the first volume contains a first substance comprising a lysis buffer, a nucleic acid isolation reagent, or a combination thereof.

8. The apparatus of claim 1, wherein the second volume contains a second substance comprising reagents for performing PCR.

9. The apparatus of claim 8, wherein the second substance further comprises reverse transcriptase.

10. The apparatus of claim 1, wherein the reaction chamber contains a lyophilized PCR master mix.

11. The apparatus of claim 10, wherein the reaction chamber further comprises a reverse transcriptase.

12. An instrument, comprising:

an apparatus, the apparatus comprising:

a first module defining a first chamber and a second chamber, the first module comprising a first transfer mechanism configured to transfer a sample between the first chamber and the second chamber while maintaining fluidic separation between the first chamber and the second chamber;

A second module defining a volume configured to contain a substance, a portion of the second module configured to be disposed within a first chamber of the first module when the second module is coupled to the first module such that the volume is configured to be selectively placed in fluid communication with the first chamber; and

a third module defining a reaction chamber, the third module configured to be coupled to the first module such that the reaction chamber is in fluid communication with the second chamber, the third module comprising a second transfer mechanism configured to transfer a portion of a sample between the second chamber and the reaction chamber;

a frame configured to receive, contain and/or provide mounting for each component and/or assembly of the instrument;

a first driver component;

a sample transfer assembly;

a second driver assembly;

a heater assembly configured to heat one or more portions of the instrument to facilitate and/or facilitate a procedure within the instrument; and

an optical assembly configured to monitor a reaction occurring within the instrument.

13. The apparatus of claim 12, wherein a portion of the third module is disposed within the second chamber of the first module when the third module is coupled to the first module.

14. The apparatus of claim 12, wherein:

the volume is a first volume;

the substance is a first substance; and

the third module defines a second volume configured to contain a second substance, the third module configured to be coupled to the first module such that the second volume is in fluid communication with the second chamber, the third module including a third transfer mechanism configured to transfer a portion of the second substance between the second volume and the second chamber.

15. The apparatus of claim 12, wherein the second module includes a penetrable member at least partially defining the volume, the penetrable member further defining a portion of a boundary of a first chamber of the first module when the second module is coupled to the first module.

16. The apparatus of claim 12, wherein said second module comprises a third transfer mechanism disposed at least partially within said volume, said third transfer mechanism configured to transfer said first substance from said volume into said first chamber when said third transfer mechanism is actuated.

17. The apparatus of claim 12, wherein:

said first module defining a third chamber between said first chamber and said second chamber; and

the first transfer mechanism includes a valve housing defining a first aperture in fluid communication with the first chamber and a second aperture in fluid communication with the third chamber, and a valve member defining a cavity in fluid communication with the first aperture when the valve member is in a first position and the second aperture when the valve member is in a second position.

18. The apparatus of claim 12, wherein the second transfer mechanism comprises a plunger disposed within a cylinder in fluid communication with the reaction chamber, the plunger configured to create a vacuum within the reaction chamber when the plunger is moved within the cylinder.

19. An instrument, comprising:

an apparatus, the apparatus comprising:

a first module comprising a reaction vial defining a reaction chamber, a substrate defining at least a portion of a first flow path and a second flow path, the first flow path configured to be in fluid communication with the reaction chamber and a separation chamber of the separation module, and a first transfer mechanism configured to transfer a sample from the separation chamber to the reaction chamber when the first transfer mechanism is actuated; and

A second module comprising a second transfer mechanism and defining a volume configured to contain a substance, the second module configured to be coupled to the first module such that the volume is selectively placed in fluid communication with the reaction chamber via the second flow path, the second transfer mechanism configured to transfer the substance from the volume to the reaction chamber when the second transfer mechanism is actuated,

wherein the first transfer mechanism is configured to create a vacuum in the reaction chamber to create a flow of sample from the separation chamber to the reaction chamber;

a frame configured to receive, contain and/or provide mounting for each component and/or assembly of the instrument;

a first driver component;

a sample transfer assembly;

a second driver assembly;

a heater assembly configured to heat one or more portions of the instrument to facilitate and/or facilitate a procedure within the instrument; and

an optical assembly configured to monitor a reaction occurring within the instrument.

20. The apparatus of claim 19, wherein:

a portion of the first module is disposed within the separation chamber of the separation module when the first module is coupled to the separation module.

21. The apparatus of claim 19, wherein the first transfer mechanism comprises a plunger disposed within a housing in fluid communication with the reaction chamber, the plunger configured to create a vacuum within the reaction chamber when the plunger is moved within the housing.

22. The apparatus of claim 19, wherein:

the volume is a first volume;

the substance is a first substance; and

said first transfer mechanism comprising a plug removably disposed within a housing such that said housing defines a second volume, said second volume containing a second substance, a portion of said plug disposed within a first flow path such that said second volume is fluidly isolated from said reaction chamber when said plug is in a first position within said housing, a portion of said plug disposed away from said first flow path such that said second volume is in fluid communication with said reaction chamber when said plug is in a second position within said housing,

the plug is configured to create a vacuum within the reaction chamber when the plug is moved from a first position to a second position.

23. The instrument of claim 19, wherein the second module comprises a penetrable member at least partially defining the volume, the volume being placed in fluid communication with the reaction chamber when a portion of the penetrable member is penetrated.

24. The apparatus of claim 19, wherein the second transfer mechanism comprises a plug disposed within the volume of the second module, the plug configured to exert a force on the substance to transfer the substance from the volume into the reaction chamber when the plug is moved within the volume.

25. The apparatus of any one of claims 1, 12 and 19, wherein the heater assembly comprises a series of receiving modules, positioning modules, a first heating module, a second heating module, and a third heating module.

26. The apparatus of any one of claims 1, 12 and 19, wherein the optical assembly is configured to detect one or more different analytes and/or targets within a test sample in the instrument.

27. The apparatus of any one of claims 1, 12 and 19, wherein the optical assembly comprises an excitation module, a detection module, a sliding assembly, and a fiber optic assembly.

28. The apparatus of any one of claims 1, 12 and 19, further comprising an acoustic transducer configured to generate acoustic energy and a drive mechanism configured to move at least a portion of the acoustic transducer into contact with a portion of the instrument.

29. The instrument of any one of claims 1, 12 and 19, wherein the first driver assembly is configured to drive a driver or transfer mechanism of a separation module of the instrument to deliver one or more reagents and/or substances into a lysis chamber within the separation module.

30. The instrument of any one of claims 1, 12 and 19, wherein the sample transfer assembly is configured to actuate a transfer assembly to transfer a portion of a sample between a plurality of chambers and/or volumes within the separation module.

31. The instrument of any one of claims 1, 12 and 19, wherein the second driver assembly is configured to drive the mixing mechanism of the separation module and/or PCR module and/or wash buffer module to transfer and/or mix one or more reagents and/or substances within a chamber within the separation module and/or PCR module.