HK1235344B - Systems and methods for biochemical analysis including a base instrument and a removable cartridge - Google Patents

Description

System and method for biochemical analysis comprising a basic instrument and a removable cartridge

Related applications

This application claims priority to U.S. Application No. 62/003,264, filed May 27, 2014, which is hereby incorporated by reference in its entirety.

background

Embodiments of the present invention generally relate to systems and methods for performing biochemical reactions, and more particularly to systems and methods in which a base instrument interacts with a removable cartridge to perform reactions for at least one of sample preparation or biochemical analysis.

Various biochemical protocols involve performing a large number of controlled reactions on a support surface or in a designated reaction chamber. Controlled reactions can be performed to analyze biological samples or prepare biological samples for subsequent analysis. Analysis can identify or reveal the characteristics of the chemicals involved in the reaction. For example, in a cycle array sequencing assay (e.g., synthesis sequencing (SBS)), a dense array of DNA features (e.g., template nucleic acids) is sequenced through iterative cycles of enzyme manipulation. After each cycle, an image can be captured and subsequently analyzed with other images to determine a series of DNA features. In another biochemical assay, an unknown analyte with an identifiable label (e.g., a fluorescent label) can be exposed to an array of known probes having a predetermined address within the array. Observing the chemical reaction that occurs between the probe and the unknown analyte can help identify or reveal the characteristics of the analyte.

There is a general demand for systems that automatically perform assays, such as those described above, where the system requires less work or involvement from the user. Currently, most platforms require the user to prepare the biological sample individually before loading the biological sample into the system for analysis. It may be desirable for the user to load one or more biological samples into the system, select the assay to be performed by the system, and have results from the analysis within a predetermined period of time, such as one day or less. At least some systems used today are unable to perform certain protocols, such as whole genome sequencing, that provide data with a sufficient level of quality and within a certain cost range.

Brief Description

In an embodiment, a system is provided that includes a removable cartridge having a cartridge housing. The removable cartridge further includes a fluid network disposed within the cartridge housing. The fluid network is configured to receive and fluidically direct a biological sample for at least one of sample analysis or sample preparation. The removable cartridge further includes a flow control valve that is operably coupled to the fluid network and movable relative to the fluid network to control the flow of the biological sample therethrough. The cartridge housing includes a housing side that defines an exterior of the removable cartridge and allows access to the flow control valve. The system further includes a base instrument having a control side configured to detachably engage the housing side of the removable cartridge. The housing side and the control side together define a system interface. The base instrument includes a valve actuator that engages the flow control valve via the system interface. The removable cartridge further includes a detection assembly held by at least one of the removable cartridge or the base instrument. The detection assembly includes an imaging detector and a reaction chamber in fluid communication with the fluid network. The imaging detector is configured to detect a specified reaction within the reaction chamber.

In some embodiments, the control side and the housing side are generally planar and face each other, wherein the system interface is a single-sided interface in which the base instrument and the removable box are operably coupled to each other only through the housing side and the control side.

In some embodiments, the base instrument and the removable cartridge are operably coupled such that the base instrument and the removable cartridge are secured to each other at the system interface using at least one of a fluid coupling, an electrical coupling, or a thermal coupling established through the system interface.

In some embodiments, the control side represents the top of the base instrument relative to gravity, such that the removable cartridge rests on and is supported by the base instrument.

In some embodiments, the valve actuator includes an elongated actuator body extending through the housing side and into the cartridge housing.

In some embodiments, the flow control valve includes an elongated actuator body extending through the control side and into the base instrument.

In some embodiments, the base instrument has an instrument side facing in an opposite direction relative to the control side, the base instrument has an instrument size extending between the control side and the instrument side, and the base instrument and the removable box have a combined size that is larger than the instrument size.

In some embodiments, the removable cartridge and the base instrument each include a contact array of electrical contacts that are electrically coupled to each other at the system interface.

In some embodiments, the housing side is a first housing side and the cartridge housing further comprises a second housing side, the first housing side and the second housing side facing different directions, wherein the system interface is a multi-sided interface in which the base instrument and the removable cartridge are operably coupled to each other along each of the first housing side and the second housing side.

In some embodiments, the first housing side and the second housing side are generally perpendicular to each other, the base instrument has an instrument housing including a first control side and a second control side, the first control side and the second control side face perpendicularly to each other and form an open side recess of the base instrument, and the removable box is arranged in the open side recess so that the first housing side and the second housing side engage the first control side and the second control side.

In some embodiments, the valve actuator includes an elongated body extending through the system interface between the first housing side and the first control side, and the second housing side and the second control side include corresponding contact arrays of electrical contacts, and the contact arrays are electrically coupled to each other along the system interface.

In some embodiments, the first housing side and the second housing side face in generally opposite directions, the base instrument has an instrument side and a cartridge receiving slot open to the instrument side, and the removable cartridge is disposed within the cartridge receiving slot.

In some embodiments, the removable cartridge and the base instrument are fluidly coupled along the first housing side and electrically coupled along the second housing side.

In some embodiments, the base instrument includes a locking mechanism that engages at least one of the first housing side or the second housing side to retain the removable cartridge within the base instrument.

In some embodiments, the removable cartridge and the base instrument each include flow ports that are fluidly coupled to each other at the system interface.

In some embodiments, the system further comprises a locking mechanism attached to at least one of the removable cartridge or the base instrument, the locking mechanism configured to removably secure the cartridge housing to the base instrument.

In some embodiments, the imaging detector is held by the base instrument and the reaction chamber is held by the removable cartridge.

In some embodiments, the flow control valve includes a flexible membrane configured to control the flow of the biological sample through the fluidic network, the flexible membrane being bent by the valve actuator between a first condition and a second condition.

In some embodiments, the housing side of the cartridge housing includes an access opening therethrough that receives the valve actuator.

In some embodiments, the flow control valve comprises a rotatable valve configured to control the flow of fluid through the fluid network, the rotatable valve being rotated by the valve actuator.

In some embodiments, the base instrument includes a thermal block, and the fluid network of the cartridge housing includes a sample channel in which a designated reaction to the biological sample occurs, the housing side including an access opening extending along the sample channel and configured to receive the thermal block for changing the temperature of the sample channel.

In some embodiments, the fluidic network comprises a plurality of channels and a storage module comprising a plurality of reservoirs for storing reagents for at least one of sample preparation or sample analysis.

In some embodiments, the base instrument includes a system controller having a valve control module configured to control operation of the valve actuator to control flow of the biological sample through the fluidic network.

In some embodiments, the valve control module is configured to control operation of the valve actuator to perform a sequencing-by-synthesis (SBS) protocol.

In an embodiment, a method for sequencing nucleic acids is provided. The method includes providing a removable cartridge having a cartridge housing, a fluid network disposed within the cartridge housing, and a flow control valve operably coupled to the fluid network and movable relative to the fluid network. The cartridge housing includes a housing side defining the exterior of the removable cartridge. The method also includes contacting the removable cartridge with a base instrument. The housing side of the removable cartridge detachably engages a control side of the base instrument to jointly define a system interface. The base instrument includes a valve actuator that engages the flow control valve through the system interface. The method also includes fluidically directing a biological sample to flow through the fluid network of the cartridge to perform at least one of sample analysis or sample preparation in the cartridge. The biological sample is directed to flow into a reaction chamber, wherein the flow of the biological sample is controlled by the action of the valve actuator on the flow control valve. The method also includes detecting the biological sample using an imaging detector directed to the reaction chamber, wherein the detection assembly is held by at least one of the removable cartridge or the base instrument.

In some embodiments, the method further comprises removing the removable cartridge from the base instrument.

In some embodiments, the method further comprises contacting a second removable cartridge with the base instrument, wherein the housing side of the second removable cartridge detachably engages the control side of the base instrument to collectively define the system interface.

In some embodiments, fluidically directing the biological sample and imaging the biological sample are repeated sequentially a plurality of times.

In some embodiments, the method further comprises sealing the biological sample within a sample preparation zone of the fluidic network, and amplifying the biological sample while the biological sample is sealed within the sample preparation zone.

In some embodiments, the flow control valve includes a movable valve having at least one flow channel extending between valve ports, and the valve actuator is configured to move the movable valve between different positions.

In some embodiments, when the biological sample flows through the flow channel and is directed into the reaction chamber, the movable valve is in a sample position, and the method further includes moving the movable valve to a component position and allowing a reagent to flow through the flow channel into the reaction chamber, the reagent reacting with the biological sample in the reaction chamber.

In some embodiments, the component position includes a plurality of component positions, and the method further comprises moving the movable valve between the component positions according to a predetermined sequence to allow different reagents to flow into the reaction chamber.

In some embodiments, the biological sample comprises nucleic acid, and the predetermined sequence is according to a sequencing-by-synthesis (SBS) protocol.

In some embodiments, the flow cell includes the reaction chamber, and the biological sample is fixed to one or more surfaces of the flow cell. In an embodiment, a removable box is provided, the removable box including a box housing having a sample port, the sample port being open to the outside of the box housing and configured to receive the biological sample. The box housing has an array of electrical contacts and a mechanical interface device exposed to the outside. The box housing is configured to be removably coupled to a basic instrument. The removable box may also include a fluid network having multiple channels, a reaction chamber and a storage module. The storage module includes multiple reservoirs for storing reagents. The fluid network is configured to guide the reagents from the reservoirs to the reaction chamber, wherein the mechanical interface device is movable relative to the fluid network to control the flow of fluid through the fluid network. The system also includes an imaging device arranged in the box housing and positioned to detect a specified reaction in the reaction chamber. The imaging device is electrically coupled to the array of electrical contacts for communicating with the basic instrument. The mechanical interface device can be configured to be moved by the basic instrument when the removable box is coupled to the basic instrument.

In some embodiments, the mechanical interface device includes a channel valve configured to control the flow of fluid through one of the channels of the fluid network.

In some embodiments, the cartridge housing includes an access opening allowing access to the mechanical interface device.

In some embodiments, the mechanical interface device comprises a rotatable valve.

In some embodiments, the cartridge housing includes an inlet opening exposed to the exterior, and the channel includes a sample channel in fluid communication with the sample port, the inlet opening extending along the sample channel and configured to receive a heat block for controlling the temperature of the sample channel.

In some embodiments, the cartridge housing includes a fluid coupling port exposed to the exterior and in fluid communication with the fluid network, the fluid coupling port being configured to engage an instrument port to receive fluid therethrough.

In some embodiments, the cartridge housing includes a first housing side and a second housing side facing in opposite directions, the first housing side including the array of electrical contacts, and the second housing side including the mechanical interface device.

In some embodiments, the removable cartridge further comprises a locking mechanism attached to the cartridge housing, the locking mechanism configured to removably secure the cartridge housing to the base instrument.

In some embodiments, the storage module includes reagents for performing a sequencing-by-synthesis (SBS) protocol. In an embodiment, a removable cartridge is provided, comprising a cartridge housing having a sample port, the sample port being open to the exterior of the cartridge housing and configured to receive a biological sample. The removable cartridge may further include a rotatable valve disposed within the cartridge housing. The rotatable valve has a fluid side and a plurality of valve ports open at the fluid side. The rotatable valve has at least one flow channel extending between the valve ports, wherein the rotatable valve is rotatable between different rotational positions. The removable cartridge may further include a microfluidic body having a body side slidably coupled to the fluid side of the rotatable valve. The microfluidic body may at least partially define a fluid network including a sample channel in fluid communication with the sample port. The sample channel has a network port open to the body side of the microfluidic body. The fluid network may further include a reservoir configured to hold a reagent. The reservoir is in fluid communication with the reservoir port open to the fluid side of the microfluidic body. The fluid network also includes a feed channel in fluid communication with a reaction chamber of the fluid network. The feed channel has a feed port open to a main body side of the microfluidic body. The rotatable valve is configured to rotate between first and second rotational positions. When the rotatable valve is in the first rotational position, the network port is fluidically coupled to the feed port via the rotatable valve. When the rotatable valve is in the second rotational position, the reservoir port is fluidically coupled to the feed port via the rotatable valve.

In some embodiments, the cartridge housing has an exterior side configured to engage an underlying instrument, the rotatable valve comprising a mechanical interface device accessible at the exterior side and configured to engage the underlying instrument.

In some embodiments, the storage module includes reagents for performing a sequencing-by-synthesis (SBS) protocol.

In some embodiments, the rotatable valve in the first rotational position is configured to receive the sample liquid when the force on the fluid moves the sample liquid toward the feed port, wherein the rotatable valve in the second rotational position is configured to allow the sample liquid to move into the reservoir when the moving force pushes the sample liquid away from the feed port into the reservoir.

In some embodiments, the rotatable valve rotates about an axis, and the feed port is aligned with the axis.

In an embodiment, a removable cartridge is provided, comprising a cartridge housing having a sample port that is open to the exterior of the cartridge housing and configured to receive a biological sample. The cartridge housing may include a matching side configured to face and be removably coupled to a basic instrument. The removable cartridge also includes a fluid network disposed within the housing. The fluid network includes a sample channel fluidically connected to the sample port. The removable cartridge also includes a channel valve having a flexible member configured to move between a first and a second position. The flexible member blocks flow through the sample channel when in the first position and allows flow through the sample channel when in the second position. The matching side of the cartridge housing includes an access opening that exposes the channel valve to the exterior of the cartridge housing. The access opening is configured to receive a valve actuator of the basic instrument for moving the flexible member between the first and second positions.

In some embodiments, the flexible member comprises a flexible layer covering an interior lumen of the fluid network, the flexible layer being configured to be pushed into the lumen to prevent flow therethrough.

In some embodiments, the removable cartridge further comprises a rotatable valve disposed within the cartridge housing, the rotatable valve configured to rotate between different positions to change a flow path of the fluid network, the rotatable valve comprising a mechanical interface device accessible along the mating side.

In some embodiments, the fluid network includes a network port connected to the sample channel fluid, a feed port connected to the reaction chamber fluid, and a reservoir port connected to a reservoir fluid configured to store reagents, and the detachable box also includes a rotatable valve arranged in the box housing, the rotatable valve fluidly coupling the feed port and the network port when in a first rotational position and fluidly coupling the feed port and the reservoir port when in a second rotational position.

In some embodiments, the mating side is a first mating side and the removable cartridge includes a second mating side, the first mating side and the second mating side facing in opposite directions, the second mating side configured to mechanically, fluidly, or thermally engage the instrument.

In an embodiment, a base instrument is provided that includes a system housing having a mating side configured to engage a removable cartridge. The base instrument also includes a rotary motor configured to engage a rotatable valve of the removable cartridge. The base instrument also includes a valve actuator configured to engage a channel valve of the removable cartridge and an array of electrical contacts configured to be electrically coupled to the removable cartridge. The base instrument also includes a system controller configured to control the rotary motor and the valve actuator to execute an assay protocol within the removable cartridge. The system controller is configured to receive imaging data from the removable cartridge via the array of electrical contacts. Optionally, the base instrument includes a heat block for heating a portion of the removable cartridge.

In some embodiments, the base instrument further comprises a heat block for heating a portion of the removable cartridge.

In an embodiment, a removable cartridge is provided, comprising a cartridge housing having a sample port, the sample port being open to the exterior of the cartridge housing and configured to receive a biological sample. The cartridge housing includes a mating side configured to face and removably couple to a primary instrument. The removable cartridge also includes a microfluidic body disposed within the cartridge housing. The microfluidic body has a body side and includes a fluid network. The fluid network has a plurality of discrete channels and corresponding ports open on the body side at a valve receiving area. The removable cartridge also includes a rotatable valve disposed within the cartridge housing. The rotatable valve has a fluid side and at least one flow channel extending between the plurality of valve ports. The valve ports are open to the fluid side. The fluid side is rotatably coupled to the valve receiving area on the body side of the microfluidic body, wherein the rotatable valve is movable between different rotational positions to fluidically couple the discrete channels. The rotatable valve has a mechanical interface device accessible along the mating side and configured to engage the primary instrument such that the rotatable valve is controlled by the primary instrument.

In some embodiments, the rotatable valve rotates about an axis, and the valve port includes an inlet port through which the axis extends.

In an embodiment, a removable cartridge is provided, comprising a cartridge housing having a sample port that is open to the exterior of the cartridge housing and configured to receive a biological sample. The cartridge housing includes a mating side configured to be removably coupled to a base instrument. The removable cartridge also includes a microfluidic structure disposed within the cartridge housing and comprising a plurality of stacked printed circuit board (PCB) layers. The PCB layers include a fluidic layer that defines channels and reaction chambers when the PCB layers are stacked. The PCB layers also include a wiring layer. The removable cartridge also includes a CMOS imager configured to be mounted to the microfluidic structure and electrically coupled to the conductive wiring layer. The CMOS imager is oriented to detect a specified reaction within the reaction chamber.

In some embodiments, the removable cartridge further includes input/output (I/O) contacts exposed to the exterior of the cartridge housing, the I/O contacts being electrically coupled to the imager.

In some embodiments, the microfluidic structure includes a channel valve, wherein at least a portion of the channel valve is defined by the PCB layer, the channel valve being configured to be actuated to prevent and allow flow through one of the channels.

Brief Description of the Drawings

1A is a schematic diagram of a system formed in accordance with an embodiment configured to perform at least one of biochemical analysis or sample preparation.

FIG. 1B is a flow chart illustrating a method of performing a specified reaction for at least one of sample preparation or sample analysis.

2 is a schematic diagram of a system formed in accordance with an embodiment configured to perform at least one of biochemical analysis or sample preparation.

3 is a side view of a system formed in accordance with an embodiment including a base instrument and a removable cartridge.

4 is a top view of a system formed in accordance with an embodiment including a base instrument and a removable cartridge.

5 is a cross-sectional view of a portion of a system formed in accordance with an embodiment showing a flow control valve having a first position.

6 is a cross-sectional view of a portion of the system of FIG. 5 showing the flow control valve having a second position.

7 is a cross-sectional view of a portion of a system formed in accordance with an embodiment showing a flow control valve having a first position.

8 is a cross-sectional view of a portion of the system of FIG. 5 showing the flow control valve having a second position.

9 is a cross-sectional view of a portion of a system formed in accordance with an embodiment showing a flow control valve having a first position.

10 is a cross-sectional view of a portion of the system of FIG. 5 showing the flow control valve having a second position.

11 is a perspective view of an exposed portion of a removable case formed in accordance with an embodiment.

12 is a cross-section of a rotatable valve that may be used with the removable cartridge of FIG. 11 .

FIG. 13 shows an arrangement of ports that may be fluidly interconnected using a rotatable valve.

14 shows a flow chart of an example of a method for using a flexible printed circuit board (PCB) and roll-to-roll (R2R) printed electronics for monolithic integration of CMOS technology and interdigitated fluidic devices.

FIG. 15 shows an exploded view of an example of a fluid stack having certain layers that can be laminated and bonded together using the method of FIG. 16 .

16 shows a perspective view of an example of a CMOS device that can be integrated into the fluidics layer of a microfluidic cartridge using the method of FIG. 14 .

17A , 17B, 18 , 19 , and 20 illustrate side views of the structure and illustrate an example of a process for attaching a CMOS device to a flexible PCB using the method of FIG. 14 .

21 shows a side view of an example of a structure formed using the method of FIG. 14 , in which the fluidics layer and CMOS device are integrated together in a microfluidics cartridge.

22A and 22B illustrate perspective views of examples of diaphragm valves that may be integrated into the fluidics layer.

23A and 23B show cross-sectional views of a diaphragm valve in an open and closed state, respectively.

FIG. 24 shows a schematic diagram of an example of a microfluidic cartridge including integrated CMOS technology and finger-like fluidics.

25 and 26 illustrate perspective views of a microfluidic cartridge assembly, which is a physical illustration of the integrated microfluidic cartridge shown in FIG. 24 .

27A and 27B illustrate perspective views of examples of fluidic assemblies installed in the microfluidic cartridge assembly shown in FIGs. 25 and 26 .

28A and 28B illustrate plan and cross-sectional views, respectively, of examples of heater traces that may be installed in the fluidic assemblies shown in FIGs. 27A and 27B.

29, 30, 31, 32, 33A and 33B illustrate various other views of the microfluidic cartridge assembly of FIG. 25 showing further details therein.

34 through 42 illustrate the process of disassembly of the microfluidic cartridge assembly of FIG. 25 as a means of revealing the internal components therein.

43 illustrates a transparent perspective view of a portion of the microfluidic cartridge assembly of FIG. 25 and showing the various reagent fluid reservoirs and sample loading ports therein.

44 illustrates another transparent perspective view of a portion of the microfluidic cartridge assembly of FIG. 25 and further illustrating the fluid channels therein.

Figure 45 shows a cross-sectional view of the microfluidic cartridge assembly of Figure 25 showing further details therein.

46A, 46B, 47A, 47B, and 48 illustrate various views of the housing of the microfluidic cartridge assembly of FIG. 25 showing further details therein.

Figures 49, 50, 51A, 51B and 52 show various views of the bottom plate of the microfluidic cartridge assembly of Figure 25 showing more details therein.

Figures 53A and 53B illustrate additional perspective views of the fluidics assembly of the microfluidic cartridge assembly showing more detail therein.

Figures 54A, 54B and 54C show other views illustrating more details of the flexible PCB heater of the fluidics assembly of the microfluidic cartridge assembly.

55A and 55B show perspective and plan views, respectively, of the inlet/outlet layer of the fluidics layer shown in FIG. 15 and FIG. 27 .

56A and 56B illustrate a perspective view and a plan view of the fluidic channel layer of the fluidic layer shown in FIG. 15 and FIG. 27 , respectively.

57A and 57B illustrate perspective and plan views, respectively, of the flexible PCB layer of the fluidic layer shown in FIG. 15 and FIG. 27 .

58A and 58B illustrate perspective and plan views, respectively, of the bottom layer of the sequencing chamber of the fluidics layer shown in FIG. 15 and FIG. 27 .

59A and 59B illustrate perspective and plan views of the sequencing chamber layer of the fluidics layer shown in FIG. 15 and FIG. 27 , respectively.

60A and 60B show perspective and plan views of the top layer of the sequencing chamber of the membrane layer and fluidic layer shown in FIG. 15 and FIG. 27 , respectively.

61A and 61B illustrate a flow diagram of an example of a method for using a microfluidic cartridge assembly to perform multiplex PCR and downstream mixing required for sequencing.

62 shows a side view of an example of a CMOS flow cell in which up to approximately 100% of the biosensor active area is accessible for reagent delivery and illumination.

FIG63 shows an exploded view of an illustrative example of the CMOS flow cell shown in FIG49.

64 and 65 show perspective and side views, respectively, of the CMOS flow cell shown in FIG. 63 when fully assembled.

Figure 66 shows a perspective view of an example of a flow cell cover of the CMOS flow cell shown in Figures 63, 64 and 65.

Figures 67, 68, 69 and 70 illustrate examples of processes for providing an extended planar surface in a CMOS flow cell onto which a flow cell cover can be mounted.

71A, 71B, 71C and 71D illustrate another example of a process for providing an extended planar surface in a CMOS flow cell onto which a flow cell cover may be mounted.

72, 73, 74 and 75 illustrate yet another example of a process for providing an extended planar surface in a CMOS flow cell onto which a flow cell cover may be mounted.

Detailed description

The embodiment set forth herein can be used for performing a specified reaction for sample preparation and/or biochemical analysis. The term "biochemical analysis" can include at least one of a biological analysis or a chemical analysis. Figure 1A is a schematic diagram of a system 100 configured to perform biochemical analysis and/or sample preparation. System 100 includes a basic instrument 102 and a detachable cartridge 104 configured to detachably engage the basic instrument 102. Basic instrument 102 and detachable cartridge 104 can be configured to interact with each other to transport a biological sample to different locations within the system 100, perform a specified reaction including the biological sample so that the biological sample is prepared for subsequent analysis, and optionally detect one or more events using the biological sample. An event can indicate a specified reaction with a biological sample. In some embodiments, detachable cartridge 104 is similar to integrated microfluidic cartridge 1100 (shown in Figure 24) or microfluidic cartridge assembly 1200 (shown in Figures 25 and 26).

Although reference is made below to the base instrument 102 and removable cartridge 104 shown in FIG. 1A , it should be understood that the base instrument 102 and removable cartridge 104 illustrate only one exemplary embodiment of the system 100 and that other embodiments exist. For example, the base instrument 102 and removable cartridge 104 include various components and features that collectively perform a plurality of operations for preparing and/or analyzing biological samples. In the illustrated embodiment, each of the base instrument 102 and removable cartridge 104 is capable of performing certain functions. However, it should be understood that the base instrument 102 and removable cartridge 104 may perform different functions and/or may share such functions. For example, in the illustrated embodiment, the removable cartridge 104 is configured to detect a specified reaction using an imaging device. In an alternative embodiment, the base instrument 102 may include an imaging device. As another example, in the illustrated embodiment, the base instrument 102 is a "dry" instrument that does not provide, receive, or exchange liquids with the removable cartridge 104. In alternative embodiments, the base instrument 102 may provide the removable cartridge 104 with reagents or other liquids, for example, that are subsequently consumed by the removable cartridge 104 (eg, used in a designated reaction).

As used herein, a biological sample may include one or more biological or chemical substances, such as nucleosides, nucleic acids, polynucleotides, oligonucleotides, proteins, enzymes, polypeptides, antibodies, antigens, ligands, receptors, polysaccharides, carbohydrates, polyphosphates, nanopores, organelles, lipid layers, cells, tissues, organisms, and/or biologically active chemical compounds, such as analogs or mimetics of the aforementioned substances. In some examples, a biological sample may include whole blood, lymph, serum, plasma, sweat, tears, saliva, sputum, cerebrospinal fluid, amniotic fluid, semen, vaginal secretions, serous fluid, joint fluid, pericardial fluid, peritoneal fluid, pleural fluid, transudate, exudate, cystic fluid, bile, urine, gastric juice, intestinal fluid, fecal samples, liquids containing single or multiple cells, liquids containing organelles, fluidized tissues, fluidized organisms, liquids containing multicellular organisms, biological swabs, and biological washes.

In some embodiments, the biological sample can include added materials such as water, deionized water, saline solutions, acid solutions, alkaline solutions, detergent solutions, and/or pH buffers. The added materials can also include reagents used to perform biochemical reactions during a given assay protocol. For example, the added liquid can include materials to perform multiple polymerase chain reaction (PCR) cycles with the biological sample.

In some embodiments, the biological sample of the present invention can be in a form or state different from the biological sample in the system 100. For example, the biological sample in the system 100 can include whole blood or saliva that is processed subsequently (for example, via separation or amplification process) to provide prepared nucleic acid. Prepared nucleic acid can then be analyzed (for example, quantified by PCR or by SBS sequencing) by system 100. Accordingly, when term " biological sample " is used when describing the first operation such as PCR and when describing the second operation subsequently such as sequencing, it should be understood that the biological sample in the second operation can be modified before or during the first operation about the biological sample. For example, amplicon nucleic acid produced from the template nucleic acid amplified in the previous amplification step (for example PCR) can be performed sequencing step (for example SBS). In this case, amplicon is a copy of template, and amplicon exists with the quantity higher than the quantity of template.

In some embodiments, the system 100 can automatically prepare a sample for biochemical analysis based on a substance provided by the user (e.g., whole blood or saliva). However, in other embodiments, the system 100 can analyze a biological sample that has been partially or pre-prepared for analysis by the user. For example, the user can provide a solution that includes nucleic acids that have been isolated and/or amplified from whole blood.

As used herein, a "specified reaction" includes a change in at least one of the chemical, electrical, physical, or optical properties (or mass) of an analyte of interest. In a particular embodiment, a specified reaction is a concomitant binding event (e.g., the merging of a fluorescently labeled biomolecule with an analyte of interest). A specified reaction may be a separate binding event (e.g., the release of a fluorescently labeled biomolecule from an analyte of interest). A specified reaction may be a chemical transformation, a chemical change, or a chemical interaction. A specified reaction may also be a change in an electrical property. For example, a specified reaction may be a change in ion concentration within a solution. Exemplary reactions include, but are not limited to, chemical reactions such as reduction, oxidation, addition, elimination, rearrangement, esterification, amidation, etherification, cyclization, or displacement; binding interactions in which a first chemical is bound to a second chemical; dissociation reactions in which two or more chemicals are separated from one another; fluorescence; luminescence; bioluminescence; chemiluminescence; and biological reactions such as nucleic acid replication, nucleic acid amplification, nucleic acid hybridization, nucleic acid complex formation, phosphorylation, enzymatic action, receptor binding, or ligand binding. A specified reaction may also be the addition or elimination of a proton, such as one that can be detected as a change in the pH of the surrounding solution or environment. An additional designated reaction may be the detection of the flow of ions across a membrane (e.g., a natural or synthetic bilayer membrane), e.g., when ions flow through the membrane, the current is interrupted and the interruption can be detected. Field sensing of charged tags may also be used as thermal sensing and other analytical sensing techniques known in the art.

In a specific embodiment, the specified reaction includes the merging of fluorescently labeled molecules into analyte. The analyte can be an oligonucleotide, and the fluorescently labeled molecules can be nucleotides. When the excitation light is directed to the oligonucleotide with the labeled nucleotide and the fluorophore emits a detectable fluorescent signal, the specified reaction can be detected. In an optional embodiment, the fluorescence detected is the result of chemiluminescence or bioluminescence. The specified reaction can also be, for example, by increasing fluorescence (or) resonance energy transfer (FRET) by bringing the donor fluorophore very close to the acceptor fluorophore, reducing FRET by separating the donor and acceptor fluorophores, increasing fluorescence by separating a quencher and a fluorophore, or reducing fluorescence by making a quencher and a fluorophore be located in one place.

As used herein, "reaction component" includes any substance that can be used to obtain a specified reaction. For example, reaction component includes reagents, catalysts such as enzymes, reactants for reaction, samples, products of reaction, other biomolecules, salts, metal cofactors, chelating agents and pH buffer solutions (such as hydrogenation buffer). Reaction component can be transported to various positions in a fluid network in solution or in combination in one or more mixtures. For example, reaction component can be transported to a reaction chamber where the biological sample is fixed. Reaction component can interact directly or indirectly with the biological sample. In some embodiments, detachable box 104 is pre-installed with one or more reaction components necessary for performing a specified assay protocol. Pre-loading can occur at a location (such as a manufacturing facility) before box 104 is received by a user (such as at a consumer's facility).

In some embodiments, the base instrument 102 may be configured to interact with the removable cartridge 104 per activity period. After the activity period, the removable cartridge 104 may be replaced with another removable cartridge 104. In other embodiments, the base instrument 102 may be configured to interact with one removable cartridge 104 per activity period. As used herein, the term "activity period" includes performing at least one of a sample preparation and/or a biochemical analysis protocol. Sample preparation may include separating, isolating, modifying, and/or amplifying one or more components of a biological sample so that the prepared biological sample is suitable for analysis. In some embodiments, an activity period may include continuous activity, wherein multiple controlled reactions are performed until (a) a specified number of reactions are performed, (b) a specified number of events are detected, (c) a specified period of system time elapses, (d) the signal-to-noise ratio drops to a specified threshold; (e) the target component is identified; (f) a system failure or malfunction is detected; and/or (g) one or more resources used to perform the reactions are exhausted. Alternatively, the activity period may include suspending system activity for a period of time (eg, minutes, hours, days, weeks), and thereafter completing the activity period until at least one of (a)-(g) occurs.

The assay protocol may include a sequence of operations for performing a specified reaction, detecting a specified reaction, and/or analyzing a specified reaction. Together, the removable cartridge 104 and the basic instrument 102 may include components necessary for performing different operations. The operations of the assay protocol may include fluid manipulation, thermal control operations, detection operations, and/or mechanical operations. Fluid manipulation includes controlling the flow of a fluid (e.g., liquid or gas) passing through the system 100, which may be activated by the basic instrument 102 and/or by the removable cartridge 104. For example, fluid manipulation may include controlling a pump to introduce a flow of a biological sample or reaction component into a detection zone. Thermal control operations may include controlling the temperature of a specified portion of the system 100. As an example, thermal control operations may include raising or lowering the temperature of a polymerase chain reaction (PCR) zone in which a liquid containing a biological sample is stored. Detection operations may include controlling the activation of a detector or monitoring the activity of a detector to detect predetermined characteristics, quantity, or features of a biological sample. As an example, detection operations may include capturing an image of a specified area containing a biological sample to detect fluorescent emission from the specified area. Detection operations may include controlling a light source to illuminate the biological sample or controlling a detector to observe the biological sample. Mechanical operations may include controlling the motion or position of a specified component. For example, mechanical operation may include controlling a motor to move a valve control component in base instrument 102, which is operably engaged with a rotatable valve in removable cartridge 104. In some cases, a combination of different operations may occur simultaneously. For example, while a pump controls the flow of fluid through a detection zone, a detector may capture an image of the detection zone. In some cases, different operations for different biological samples may occur simultaneously. For example, a first biological sample may undergo amplification (e.g., PCR) while a second biological sample may undergo detection.

As used herein, "liquid" is a relatively incompressible substance and has the ability to flow and conform to the shape of a container or channel holding the substance. The liquid can be based on water and include polar molecules that exhibit surface tension that holds the liquid together. The liquid can also include non-polar molecules, such as in oil-based or non-aqueous substances. It should be understood that reference to a liquid in this application can include a liquid formed by a combination of two or more liquids. For example, separate reagent solutions can be combined later to carry out a specified reaction.

The removable cartridge 104 is configured to be detachably engaged or removably coupled to the base instrument 102. As used herein, when the term "detachably engaged" or "removably coupled" (or similar terms) is used to describe the relationship between the removable cartridge and the base instrument, the term is intended to mean that the connection between the removable cartridge and the base instrument is easily separable without damaging the base instrument. Accordingly, the removable cartridge can be electrically detachably engaged to the base instrument such that the electrical contacts of the base instrument are not damaged. The removable cartridge can be mechanically detachably engaged to the base instrument such that features of the base instrument that retain the removable cartridge are not damaged. The removable cartridge can be fluidically detachably engaged to the base instrument such that the ports of the base instrument are not damaged. For example, the base instrument is not considered "destroyable" if only a simple adjustment (or realignment) of the component or a simple replacement (e.g., replacing a nozzle) is required. Components (e.g., the removable cartridge 104 and the base instrument 102) are readily separable when the components can be separated from each other without expending undue effort or a significant amount of time in separating the components. In some embodiments, the removable cartridge 104 and the base instrument 102 are easily separable without destroying the removable cartridge 104 or the base instrument 102 .

In some embodiments, the removable cartridge 104 can be permanently modified or partially damaged during an active period with the base instrument 102. For example, a container holding a liquid can include a foil cover that is pierced to allow the liquid to flow through the system 100. In such an embodiment, the foil cover can be damaged, making it necessary to replace the damaged container with another container. In a particular embodiment, the removable cartridge 104 is a disposable cartridge so that the removable cartridge 104 can be replaced and optionally discarded after a single use.

In other embodiments, the removable cartridge 104 can be used during more than one active session while engaged with the base instrument 102, and/or can be removed from the base instrument 102, refilled with reagents, and reattached to the base instrument 102 to perform additional designated reactions. Accordingly, the removable cartridge 104 can, in some cases, be refurbished so that the same removable cartridge 104 can be used with different consumables (e.g., reaction components and biological samples). Refurbishment can be performed at a manufacturing facility after the cartridge is removed from the base instrument at the customer's facility.

As shown in Figure 1A, detachable box 104 includes fluid network 106, which can maintain and guide the fluid (such as liquid or gas) passing through it. Fluid network 106 includes a plurality of interconnected fluid elements that can store fluid and/or allow fluid to flow through it. The non-limiting example of fluid element includes the reservoir, reaction chamber, waste reservoir, detection chamber, the multipurpose room for reaction and detection of the port, cavity, storage module, storage module of passage, passage. Fluid element can be coupled to each other with a specified mode fluidly so that system 100 can perform sample preparation and/or analysis.

As used herein, the term "fluidically coupled" (or similar terms) refers to two spatial regions connected together so that a liquid or gas can be guided between the two spatial regions. In some cases, fluid coupling allows a fluid to be guided back and forth between the two spatial regions. In other cases, fluid coupling is unidirectional so that there is only one direction of flow between the two spatial regions. For example, an assay reservoir can be coupled with channel fluids so that liquid can be transported to the channel from the assay reservoir. However, in some embodiments, it may not be possible to guide the fluid in the channel back to the assay reservoir. In a specific embodiment, the fluid network 106 is configured to receive a biological sample and guide the biological sample through sample preparation and/or sample analysis. The fluid network 106 can guide the biological sample and other reaction components to a waste reservoir.

One or more embodiments may include retaining a biological sample (such as template nucleic acid) at a designated location, wherein the biological sample is analyzed. As used herein, the term "retain" includes attaching the biological sample to a surface or limiting the biological sample to a designated space when being used with respect to the biological sample. As used herein, the term "fixed" includes attaching the biological sample to a surface in or on a solid support when being used with respect to the biological sample. Fixing may include attaching the biological sample to a surface at the molecular level. For example, the biological sample may be fixed to the surface of the substrate using absorption techniques and covalent bonding techniques (wherein functional groups or cross-linking agents are convenient for attaching the biological sample to the surface) including non-covalent interactions (such as dehydration of electrostatic force, van der Waals force, and hydrophobic interface). The biological sample may be fixed to the surface of the substrate based on the characteristics of the surface of the substrate, the liquid medium carrying the biological sample, and the characteristics of the biological sample itself. In some cases, the substrate surface may be functionalized (such as chemically or physically modified) so that the biological sample is fixed to the substrate surface. The substrate surface may first be modified to have a functional group that is bound to the surface. The functional group can then be bound to the biological sample to immobilize the biological sample thereon. In some cases, the biological sample can be immobilized to the surface via a gel, for example, as described in U.S. Patent Publication Nos. 2011/0059865 A1 and 2014/0079923 A1, each of which is incorporated herein by reference in its entirety.

In some embodiments, nucleic acid can be fixed to surface and amplified using bridge amplification.For example, in U.S. Patent number 5,641,658, WO 07/010251, U.S. Patent number 6,090,592, U.S. Patent Publication No. 2002/0055100 A1, U.S. Patent number 7,115,400, U.S. Patent Publication No. 2004/0096853 A1, U.S. Patent Publication No. 2004/0002090A1, U.S. Patent Publication No. 2007/0128624 A1 and U.S. Patent Publication No. 2008/0009420 A1, useful bridge amplification methods are described, and each file is fully incorporated herein. Another useful method for amplifying nucleic acid on the surface is for example using the rolling circle amplification (RCA) of the method elaborating in more detail below. In some embodiments, nucleic acid can be attached to surface and amplified using one or more primers. For example, one of primers can be in solution, and another primer can be fixed on the surface (for example 5 '-attachment). As an example, nucleic acid molecules can be hybridized to one of the primers on the surface, followed by an extension of the fixed primer to produce the first copy of nucleic acid. The primer in the solution is then hybridized to the first copy of nucleic acid, and nucleic acid can extend using the first copy of nucleic acid as a template. Alternatively, after the first copy of nucleic acid is produced, the organic nucleic acid molecule can be hybridized to the second fixed primer on the surface, and can extend after the primer in the solution is extended simultaneously or. In any embodiment, multiple copies of nucleic acid are provided using repeated cycles of the extension (such as amplification) of the fixed primer and the primer in the solution. In some embodiments, biological sample can be confined in a predetermined space together with the reaction component configured to use during the amplification (such as PCR) of biological sample.

In the illustrated embodiment, the removable cartridge 104 includes a cartridge housing 110 having a plurality of housing sides 111-114. The housing sides 111-114 include non-mating sides 111-113 and a mating side 114. The mating side 114 is configured to engage the base instrument 102. In the illustrated embodiment, the cartridge housing 110 forms a substantially unitary structure. In alternative embodiments, the cartridge housing 110 may be constructed from one or more sub-components, which are assembled by a user of the system 100. The sub-components may be assembled before the removable cartridge 104 is detachably attached to the base instrument 102 or after one of the sub-components is detachably attached to the base instrument 102. For example, the storage module 150 may be held by a first sub-housing (not shown), while the remaining components of the removable cartridge 104 (e.g., the fluidics network and imaging device) may comprise a second sub-housing (not shown). The first and second sub-housings may be combined to form the cartridge housing 110.

The fluid network 106 is maintained by the cartridge housing 110 and includes a plurality of sample ports 116 open to the non-matching side 112. In an optional embodiment, the sample ports 116 may be located along the non-matching side 111 or 113 or may be located along the matching side 114. Each sample port 116 is configured to receive a biological sample. By way of example only, the biological sample may be whole blood or saliva. In some embodiments, the biological sample may be nucleic acid and other materials (e.g., reagents, buffers, etc.) for performing PCR. Although three sample ports 116 are shown in FIG1A , embodiments may include only one, two, or more than three sample ports.

The fluidic network 106 also includes a fluidic coupling port 118 that is open to the mating side 114 and exposed to the exterior of the cartridge housing 110. The fluidic coupling port 118 is configured to be fluidically coupled to a system pump 119 of the base instrument 102. The fluidic coupling port 118 is in fluidic communication with a pump channel 133, which is part of the fluidic network 106. During operation of the system 100, the system pump 119 is configured to provide a negative pressure that causes fluid to flow through the pump channel 133 and through the remainder of the fluidic network 106. For example, the system pump 119 can cause a biological sample to flow from the sample port 116 to the sample preparation area 132, where the biological sample can be prepared for subsequent analysis. The system pump 119 can cause a biological sample to flow from the sample preparation area 132 to the reaction chamber 126, where a detection operation is performed to obtain data (e.g., imaging data) about the biological sample. The system pump 119 can also cause a fluid to flow from the reservoirs 151, 152 of the storage module 150 to the reaction chamber 126. After the testing operation is performed, the system pump 119 may cause the flow of fluid into the waste reservoir 128 .

In addition to the fluid network 106, the removable cartridge 104 may also include one or more mechanical interface devices 117 that can be controlled by the base instrument 102. For example, the removable cartridge 104 may include a valve assembly 120 having a plurality of flow control valves 121-123 that are operably coupled to the fluid network 106. Each flow control valve 121-123 may represent a mechanical interface device 117 that is controlled by the base instrument 102. For example, the flow control valves 121-123 may be selectively activated or controlled by the base instrument 102 in conjunction with the selective activation of the system pump 119 to control the flow of fluid within the fluid network 106.

For example, in the illustrated embodiment, the fluid network 106 includes a sample channel 131 immediately downstream of the sample port 116 and in fluid communication with the sample port 116. Only a single sample channel 131 is shown in Figure 1A, but alternative embodiments may include multiple sample channels 131. The sample channel 131 may include a sample preparation area 132. The valve assembly 120 includes a pair of channel valves 121, 122. The channel valves 121, 122 can be selectively activated by the base instrument 102 to obstruct or prevent fluid from flowing through the sample channel 131. In particular embodiments, the channel valves 121, 122 can be activated to form a seal that retains a specified volume of liquid within the sample preparation area 132 of the sample channel 131. The specified volume within the sample preparation area 132 may include a biological sample.

The valve assembly 120 may also include a movable valve 123. The movable valve 123 may be similar to the rotatable valve assembly 1410 (shown in Figures 27A and 27B). The movable valve 123 has a valve body 138 that may include at least one flow channel 140 extending between corresponding ports. The valve body 138 can be moved between different positions to align the ports with the corresponding ports of the fluid network 106. For example, the position of the movable valve 123 can determine the type of fluid flowing into the reaction chamber 126. In the first position, the movable valve 123 can align with the corresponding port of the sample channel 131 to provide a biological sample to the reaction chamber 126. In the second position, the movable valve 123 can align with one or more corresponding ports of the reservoir channels 161 and 162, which are respectively in fluid communication with the reservoirs 151 and 152 of the storage module 150. Each reservoir 151 and 152 is configured to store reaction components that can be used to perform a specified reaction. Reservoir channels 161, 162 are located downstream of and in fluid communication with reservoirs 151, 152, respectively. In some embodiments, movable valves 123 can be individually moved to different positions to align with corresponding ports of the reservoir channels.

In the illustrated embodiment, the movable valve 123 is a rotatable valve configured to rotate about an axis 142. Accordingly, the movable valve 123 is hereinafter referred to as the rotatable valve 123. However, it should be understood that alternative embodiments may include a movable valve that does not rotate to different positions. In such embodiments, the movable valve can slide in one or more linear directions to align with the corresponding ports. The rotatable valve and linear motion valve described herein may be similar to the device described in International Application No. PCT/US2013/032309, filed on March 15, 2013, which is incorporated herein by reference in its entirety.

In some embodiments, the biological sample is illuminated by a light source 158 of the base instrument 102. Alternatively, the light source 158 may be incorporated into the removable cartridge 104. For example, the biological sample may include one or more fluorophores that emit light when excited by light of an appropriate wavelength. In the illustrated embodiment, the removable cartridge 104 includes an optical path 154. The optical path 154 is configured to allow illumination light 156 from the light source 158 of the base instrument 102 to be incident on the biological sample within the reaction chamber 126. Accordingly, the reaction chamber may have one or more optically transparent sides or windows. The optical path 154 may include one or more optical elements, such as lenses, reflectors, fiber optic lines, etc., that actively direct the illumination light 156 into the reaction chamber 126. In an exemplary embodiment, the light source 158 may be a light emitting diode (LED). However, in alternative embodiments, the light source 158 may include other types of light-generating devices, such as a laser or a lamp.

In some embodiments, the detection component 108 includes an imaging detector 109 and a reaction chamber 126. The imaging detector 109 is configured to detect a specified reaction within the reaction chamber 126. The imaging detector 109 may be similar to a CMOS image sensor 262 (shown in Figure 40). In some embodiments, the imaging detector 109 may be positioned relative to the reaction chamber 126 to detect a light signal (e.g., absorption, reflection/refraction, or light emission) from the reaction chamber 126. The imaging detector 109 may include one or more imaging devices, such as a charge coupled device (CCD) camera or a complementary metal oxide semiconductor (CMOS) imager. In some embodiments, the imaging detector 109 may detect a light signal emitted from a chemiluminescent device. In yet other embodiments, the detection component 108 may not be limited to imaging applications. For example, the detection component 108 may be one or more electrodes that detect the electrical properties of a liquid.

As described herein, the base instrument 102 is configured to operably engage the removable cartridge 104 and control various operations within the removable cartridge 104 to perform a designated reaction and/or obtain data from a biological sample. To this end, the mating side 114 is configured to allow or permit the base instrument 102 to control the operation of one or more components of the removable cartridge 104. For example, the mating side 114 may include a plurality of access openings 171-173 that allow the valves 121-123 to be controlled by the base instrument 102. The mating side 114 may also include an access opening 174 configured to receive the thermal block 206 of the base instrument 102. The access opening 174 extends along the sample channel 131. As shown, the access openings 171-174 are open to the mating side 114.

The base instrument 102 has a control side 202 configured to detachably engage the mating side 114 of the removable case 104. The mating side 114 of the removable case 104 and the control side 202 of the base instrument 102 collectively define a system interface 204. The system interface 204 represents a common boundary between the removable case 104 and the base instrument 102, through which the base instrument 102 and the removable case 104 are operably engaged. More specifically, the base instrument 102 and the removable case 104 are operably engaged along the system interface 204, such that the base instrument 102 can control various features of the removable case 104 via the mating side 114. For example, the base instrument 102 may have one or more controllable components that control corresponding components of the removable case 104.

In some embodiments, the base instrument 102 and the removable cartridge 104 are operably coupled such that the base instrument 102 and the removable cartridge 104 are secured to each other at the system interface 204 using at least one of an electrical coupling, a thermal coupling, an optical coupling, a valve coupling, or a fluidic coupling established via the system interface 204. In the illustrated embodiment, the base instrument 102 and the removable cartridge 104 are configured to have electrical coupling, thermal coupling, valve coupling, and optical coupling. More specifically, the base instrument 102 and the removable cartridge 104 can transfer data and/or electrical power via the electrical coupling. The base instrument 102 and the removable cartridge 104 can transfer thermal energy to and/or from each other via the thermal coupling, and the base instrument 102 and the removable cartridge 104 can transfer optical signals (e.g., illumination light) via the optical coupling.

In the illustrated embodiment, the system interface 204 is a single-sided interface 204. For example, the control side 202 and the housing side 114 are generally planar and face in opposite directions. The system interface 204 is single-sided, such that the removable case 104 and the base instrument 102 are operably coupled to each other only via the mating side 114 and the control side 202. In alternative embodiments, the system interface can be a multi-sided interface. For example, at least two, three, four, or five sides of the removable case can be mating sides configured to couple with the base instrument. The multiple sides can be planar and can be arranged orthogonal or opposite to each other (e.g., surrounding all or part of a rectangular volume).

To control the operation of the removable cartridge 104, the base instrument 102 may include valve actuators 211-213 configured to operably engage the flow control valves 121-123, a thermal block 206 configured to provide and/or remove thermal energy from the sample preparation area 132, and a contact array 208 of electrical contacts 209. The base instrument 102 may also include a light source 158 positioned along the control side 202. The base instrument 102 may also include a system pump 119 having a control port 210 positioned along the control side 202.

The system 100 may also include a locking mechanism 176. In the illustrated embodiment, the locking mechanism 176 includes a rotatable snap lock 177 configured to engage a snap lock engagement element 178 of the removable cartridge 104. Alternatively, the removable cartridge 104 may include the rotatable snap lock 177, and the base instrument 102 may include the snap lock engagement element 178. When the removable cartridge 104 is mounted to the base instrument 102, the snap lock 177 may rotate and engage the locking engagement element 176. The camming effect created by the locking mechanism 176 may push or drive the removable cartridge 104 toward the base instrument 102 to secure the removable cartridge 104 thereto.

Base instrument 102 may include a user interface 125 configured to receive user input for performing a specified assay protocol and/or to communicate information about the assay to the user. User interface 125 may be integrated with base instrument 102. For example, user interface 125 may include a touchscreen attached to the housing of base instrument 102 and configured to recognize a touch from the user and the location of the touch relative to information displayed on the touchscreen. Alternatively, user interface 125 may be remotely located relative to base instrument 102.

The base instrument 102 may also include a system controller 220 configured to control the operation of at least one of the valve actuators 211-213, the thermal block 206, the contact array 208, the light source 158, or the system pump 119. The system controller 220 is conceptually shown as a series of circuit modules, but may be implemented using any combination of dedicated hardware boards, DSPs, processors, and the like. Alternatively, the system controller 220 may be implemented using an off-the-shelf PC having a single processor or multiple processors, with functional operations distributed among the processors. As another option, the circuit modules described below may be implemented using a hybrid configuration, where some modular functions are performed using dedicated hardware, while other modular functions are performed using an off-the-shelf PC, etc.

The system controller 220 may include a plurality of circuit modules 221-224 configured to control the operation of certain components of the base instrument 102 and/or the removable cartridge 104. For example, the circuit module 221 may be a flow control module 221 configured to control the flow of fluid through the fluid network 106. The flow control module 221 may be operably coupled to the valve actuators 211-213 and the system pump 119. The flow control module 221 may selectively activate the valve actuators 211-213 and the system pump 119 to cause the flow of fluid through one or more pathways and/or prevent the flow of fluid through one or more pathways.

As an example only, the valve actuator 213 can be rotatably engaged with the rotatable valve 123. The valve actuator 213 may include a rotary motor 214 configured to drive (e.g., rotate) the valve actuator 213. The flow control valve 221 can activate the valve actuator 213 to move the rotatable valve 123 to a first rotational position. When the rotatable valve 123 is in the first rotational position, the flow control module 221 can activate the system pump 219 to remove the biological sample from the sample preparation area 132 and place it in the reaction chamber 126. The flow control module 221 can then activate the valve actuator 213 to move the rotatable valve 123 to a second rotational position. When the rotatable valve 123 is in the second rotational position, the flow control module 221 can activate the system pump 219 to remove one or more of the reaction components from the corresponding reservoir and place them in the reaction chamber 126. In some embodiments, the system pump 219 can be configured to provide positive pressure so that the fluid is actively pumped in the opposite direction. Such an operation can be used to add multiple liquids into a common reservoir, thereby mixing the liquids in the reservoirs. Accordingly, the fluid coupling port 118 can allow fluid (eg, gas) to exit the cartridge housing 110 or can receive fluid into the cartridge housing 110 .

The system controller 220 may also include a thermal control module 222. The thermal control module 222 may control the thermal block 206 to provide and/or remove thermal energy from the sample preparation area 132. In a specific example, the thermal block 206 may increase and/or decrease the temperature experienced by the biological sample in the sample channel 131 according to a PCR protocol. Although not shown, the system 100 may include additional thermal devices positioned adjacent to the sample preparation area 132. For example, the removable cartridge 104 may include a thermal device similar to the flexible PCB heater 1412 (shown in Figures 27A and 27B).

The system controller 220 may also include a detection module 223 configured to control the detection assembly 108 to obtain data about the biological sample. The detection module 223 may control the operation of the detection assembly 108 via the contact array 208. For example, the detection assembly 108 may be communicatively coupled to the contact array 194 of electrical contacts 196 along the mating side 114. In some embodiments, the electrical contacts 196 may be flexible contacts (e.g., pogo contacts or contact beams) that can be repositioned to and from the mating side 114. The electrical contacts 196 are exposed to the exterior of the cartridge housing and electrically coupled to the detection assembly 108. The electrical contacts 196 may be referred to as input/output (I/O) contacts. When the base instrument 102 and the removable cartridge 104 are operably engaged, the detection module 223 may control the detection assembly 108 to obtain data at predetermined times or during predetermined time periods. As an example, when a biological sample has a fluorophore attached thereto, detection module 223 can control detection assembly 108 to capture an image of reaction chamber 126. Multiple images can be obtained.

Optionally, system controller 220 includes an analysis module 224 configured to analyze data to provide at least partial results to a user of system 100. For example, analysis module 224 may analyze imaging data provided by imaging detector 109. The analysis may include identifying sequences of nucleic acids of the biological sample.

The system controller 220 and/or circuit modules 221-224 may include one or more logic-based devices, including one or more microcontrollers, processors, reduced instruction set computers (RISC), application specific integrated circuits (ASICs), field programmable gate arrays (FPGAs), logic circuits, and other circuits capable of performing the functions described herein. In an exemplary embodiment, the system controller 220 and/or circuit modules 221-224 execute a set of instructions stored therein to perform one or more assay protocols. The storage element may be in the form of an information source or physical memory element in a basic instrument 102 and/or a removable cartridge 104. The protocol performed by the assay system 100 may be for example quantitative analysis of DNA or RNA, protein analysis, DNA sequencing (e.g., sequencing by synthesis (SBS)), sample preparation, and/or the preparation of a fragment library for sequencing.

The set of instructions may include various commands that instruct the system 100 to perform specific operations, such as the methods and processes of the various embodiments described herein. The set of instructions may be in the form of a software program. As used herein, the terms "software" and "firmware" are interchangeable and include any computer program stored in a memory, including RAM memory, ROM memory, EPROM memory, EEPROM memory, and non-volatile RAM (NVRAM) memory, for execution by a computer. The above memory types are merely exemplary and are therefore not intended to limit the types of memory that may be used for storage of computer programs.

The software may be in various forms such as system software or application software. In addition, the software may be in the form of a collection of separate programs or a program module or a portion of a program module within a larger program. The software may also include modular programming in the form of object-oriented programming. After the detection data is obtained, the detection data may be processed automatically by the system 100, processed in response to user input, or processed in response to a request made by another processing machine (e.g., a remote request via a communication link).

The system controller 220 can be connected to other components or subsystems of the system 100 via a communication link, which can be hardwired or wireless. The system controller 220 can also be communicatively connected to an off-device system or server. The system controller 220 can receive user input or commands from a user interface (not shown). The user interface can include a keyboard, a mouse, a touch screen panel, and/or a voice recognition system, among others.

The system controller 220 can be used to provide processing capabilities, such as storing, interpreting and/or executing software instructions and controlling the overall operation of the system 100. The system controller 220 can be configured and/or programmed to control data and/or power aspects of various components. Although the system controller 220 is shown as a single structure in FIG1A, it should be understood that the system controller 220 can include multiple individual components (e.g., processors) distributed at different locations throughout the system 100. In some embodiments, one or more components can be integrated with the base instrument, and one or more components can be remotely located relative to the base instrument.

Fig. 1 B is a flow chart illustrating a method 180 for carrying out a specified reaction for at least one of sample preparation or sample analysis. In a specific embodiment, method 180 may include sequencing nucleic acid. Method 180 may use the structure or aspect of the various embodiments (e.g., system and/or method) discussed herein. In various embodiments, some steps may be omitted or added, and some steps may be combined, and some steps may be performed simultaneously, and some steps may be performed in parallel, and some steps may be divided into a plurality of steps, and some steps may be performed in different orders, or some steps or the series of steps may be re-performed in an iterative manner.

For example, method 180 may include providing a removable cartridge having a cartridge housing at 182. The removable cartridge may include a fluid network disposed within the cartridge housing. The removable cartridge may also include a flow control valve operably coupled to and movable relative to the fluid network. The flow control valve may be, for example, a channel valve or a movable valve, such as a rotatable valve. The cartridge housing may include a housing side defining an exterior of the removable cartridge.

Method 180 may also include, at 184, attaching (e.g., contacting) the removable cartridge to the base instrument. The housing side of the removable cartridge may be detachably coupled to the control side of the base instrument to jointly define a system interface. The base instrument includes a valve actuator that engages the flow control valve through the system interface. For example, the valve actuator may include an elongated body extending from the control side and inserted into an opening along the housing side of the removable cartridge. Alternatively, the valve actuator directly engages a portion of the flow control valve.

At 186, one or more biological samples may be received by the removable cartridge. For example, a user may use a pipette to add the biological sample to a sample port in fluid communication with the fluid network. The receiving at 186 may occur before or after the contacting at 184. Method 180 may include, at 188, fluidically directing the biological sample to flow through the fluid network of the removable cartridge to perform at least one of sample analysis or sample preparation in the cartridge. For example, the biological sample may be directed to a sample preparation area of the fluid network, wherein the flow of the biological sample is controlled by the action of a valve actuator on a flow control valve. The biological sample may undergo an amplification process, such as PCR, while the biological sample is sealed within the sample preparation area. As another example, the biological sample may be directed to flow into a reaction chamber, wherein the flow of the biological sample is controlled by the action of a valve actuator on the flow control valve.

Optionally, at 190, method 180 includes detecting the biological sample introduced into the reaction chamber using an imaging detector. The detection assembly may be held by at least one of a removable cartridge or a base instrument. For example, the detection assembly may be incorporated into the removable cartridge. The base instrument may be electrically coupled to the detection assembly to control its operation. Optionally, fluidically introducing the biological sample at 186 and/or imaging the biological sample at 190 may be repeated multiple times according to a predetermined schedule or sequence.

In some embodiments, method 180 includes removing a removable cartridge from a base instrument at 192. After the assay protocol is completed, the removable cartridge can be removed from the base instrument. In some cases, the removable cartridge can be refilled or refurbished. For example, the removable cartridge can be disinfected and/or sterilized, and the used storage module can be replaced with a new storage module. Method 1800 can then return to 182, where another removable cartridge is provided and installed relative to the same base module at 184. In a manner similar to the first removable cartridge, the housing side of the second removable cartridge can be detachably engaged with the control side of the base instrument to jointly define a system interface.

FIG2 is a schematic diagram of a system 300 configured to perform at least one of a biochemical analysis or sample preparation. System 300 may include features identical or similar to those of system 100 ( FIG1A ). For example, system 300 includes a base instrument 302 and a removable cartridge 304 configured to detachably engage base instrument 302. Base instrument 302 and removable cartridge 304 may have features similar to base instrument 102 and removable cartridge 104 (shown in FIG1A ), respectively. As shown in FIG2 , base instrument 302 has an instrument housing 303 comprising an instrument side 306 and a cartridge receiving slot 308 open to instrument side 306. In some embodiments, instrument side 306 may represent the top of base instrument 302 relative to gravity and partially form the exterior of instrument housing 303. In the illustrated embodiment, cartridge receiving slot 308 is defined by interior docking side or control sides 311-313 of instrument housing 303. The control sides 311 and 313 are opposite to each other, and the control side 312 extends between the control sides 311, 313. The control side 312 may face an opening 316 of the cartridge receiving slot 308.

Removable cartridge 304 is sized and shaped to fit within cartridge receiving slot 308 and operably engage base instrument 302. As shown, removable cartridge 304 includes a cartridge housing 320 having housing sides 321-324. Housing sides 321-323 are configured to operably engage docking or control sides 311-313, thereby establishing at least one of an electrical, thermal, optical, and/or fluidic coupling between base instrument 302 and removable cartridge 304. Therefore, housing sides 321-323 are hereinafter referred to as mating sides 321-323. Housing side 324 does not operably engage base instrument 302. Accordingly, housing side 324 may be referred to as non-mating side 324.

Similar to the removable cartridge 104 ( FIG. 1A ), the removable cartridge 304 includes a number of features and components for controlling operations within the removable cartridge 304 to perform a specified reaction. For example, the removable cartridge 304 has a sample port 330 that is open to the non-mating side 324 and configured to receive one or more biological samples. Alternatively, the sample port 330 can be open to one of the mating sides 321-323. In such an embodiment, the biological sample can be placed in the sample port 330 before the removable cartridge 304 is loaded into the cartridge receiving slot 308.

The removable cartridge 304 may also include a fluidic network 332 having a sample preparation area 334. The fluidic network 332 may include or fluidically interconnect various other components of the removable cartridge 304, such as a storage module 336, a removable valve 338, a detection assembly 340 having an imaging detector 342, and a waste reservoir 344. Optionally, the removable cartridge 304 may also include an optical path 346 and a contact array 348. The components of the removable cartridge 304 may be similar to those described above with respect to the removable cartridge 304.

The basic instrument 302 may include components that operably engage the removable cartridge 304 to perform a designated reaction. For example, the basic instrument 302 may include a heat block 350, a valve actuator 352, a light source 356, a contact array 358, and a system pump 360. When the removable cartridge 304 is loaded into the cartridge receiving slot 308, or after the removable cartridge 304 is loaded into the cartridge receiving slot 308, the removable cartridge 304 and the basic instrument 302 may engage with each other. More specifically, when the removable cartridge 304 is operably loaded into the basic instrument 302, the heat block 350 may be positioned proximate to the sample preparation area 334, the valve actuator 352 may operably engage the movable valve 338, the light source 356 may be communicatively coupled to the optical path 346, the contact array 358 may be electrically engaged with the contact array 348, and the system pump 360 may be communicatively engaged with the fluidic network 332. Accordingly, the removable cartridge 304 may be controlled by the base instrument 302 in a similar manner as the removable cartridge 104 is controlled by the base instrument 102 .

The base instrument 302 can be configured to allow the removable cartridge 304 to be freely inserted into the cartridge receiving slot 308 without damaging the components, based on either the control sides 311-313 or the mating sides 321-323. For example, one or more components of the base instrument 302 may be biased toward or move toward the removable cartridge 304. In some embodiments, the thermal block 350 and the valve actuator 352 are secured to a component support frame 362. After the removable cartridge 304 is positioned within the cartridge receiving slot 308, the component support frame 362 may be biased toward or move toward the mating side 321. Similarly, the system pump 360 may be secured to a component support frame 364. After the removable cartridge 304 is positioned within the cartridge receiving slot 308, the component support frame 364 may be biased toward or move toward the mating side 323.

The component supports 362, 364 can be automatically activated by the system controller 370. For example, the system controller 370 can determine that the removable cartridge 304 is loaded or has been loaded into the cartridge receiving slot 308. The system controller 370 can then activate a drive mechanism or mechanisms to drive the component supports 362, 364 toward the mating sides 321, 323. Alternatively, the component supports 362, 364 can be operably linked to one or more operator-controlled mechanisms that, upon activation by a user of the system 300, can drive the component supports 362, 364 toward the mating sides 321, 323, respectively. Accordingly, the base instrument 302 can be configured to allow the removable cartridge 304 to freely advance (e.g., without substantial snagging or kicking) into the cartridge receiving slot 308.

The embodiments described herein include systems in which the removable cartridge and the base instrument can form a multi-sided system interface. For example, each mating side 321-323 can operably engage a corresponding control side that defines the cartridge receiving slot 308. The mating sides 321-323 and the corresponding control sides 311-313 together define a system interface, which can be referred to as a multi-sided interface. Such embodiments can be desirable to balance the forces experienced by the removable cartridge 304. For example, the heat block 350 and the valve actuator 352 can apply a force 374 in a first direction (as indicated by the arrow). The system pump 360 applies a force 376 in an opposite second direction (as indicated by the arrow). The interaction between the contact arrays 348, 358 can also provide a portion of the force 376.

In some embodiments, at least one force 374, 376 facilitates providing intimate contact between corresponding components. For example, force 374 can provide intimate contact between thermal block 350 and sample preparation area 334 to enable thermal control of sample preparation area 334. Similarly, force 374 can allow valve actuator 352 and movable valve 338 to properly engage each other so that valve actuator 352 can selectively control movable valve 338. Force 376 can achieve intimate contact between corresponding electrical contacts of contact arrays 348, 358.

Figures 3 and 4 illustrate various systems including corresponding base instruments and removable cartridges, and in particular, illustrate various multi-sided interfaces that may be utilized by one or more embodiments. For example, Figure 3 is an end view of system 400 including base instrument 402 and removable cartridge 404. Base instrument 402 includes an open side recess 406 sized and shaped to receive removable cartridge 404. As shown, open side recess 406 is formed by first and second control sides 411, 412 oriented perpendicularly relative to each other. More specifically, first and second control sides 411, 412 form an L-shaped recess. First and second control sides 411, 412 operably engage first and second mating sides 413, 414, respectively, of removable cartridge 404. A multi-sided interface 415 is formed between first control side 411 and first mating side 413 and second control side 412 and second mating side 414. More specifically, at least one of a valve coupling, a fluid coupling, an electrical coupling, an optical coupling, or a thermal coupling may be established along each of the first and second mating sides 413 , 414 .

Fig. 4 is a top-down view of a system 420 comprising a basic instrument 422 and a removable cassette 424. Basic instrument 422 includes a cassette receiving slot 426, which may be similar or identical to cassette receiving slot 308 (Fig. 2). Cassette receiving slot 426 is sized and shaped to accommodate removable cassette 424. As shown, cassette receiving slot 426 is formed by control sides 431-434. Control sides 431, 433 are opposed to each other, and control sides 432, 434 are opposed to each other. Control sides 431-434 are respectively operatively engaged with corresponding sides 441-444 of removable cassette 424. A multi-side interface 427 is formed between the respective sides of removable cassette 424 and basic instrument 422.

Figures 5-12 illustrate different valve control mechanisms by which a base instrument can control (e.g., regulate) the flow of a fluid network passing through a removable cartridge. Each of Figures 5-12 illustrates a cross-section of a system in which a valve coupling is established between the base instrument and the removable cartridge via a system interface. Each of Figures 5-12 illustrates a channel valve, which the base instrument can activate to open and close a corresponding channel. For example, Figures 5 and 6 illustrate a portion of a system 500 that can be similar to the systems described above, such as systems 100 (Figure 1A), 300 (Figure 2), 400 (Figure 3), and 420 (Figure 4).

Fig. 5 and 6 illustrate a cross section of a part of a system 500 with a basic instrument 502 and a detachable cartridge 504 that are operably engaged along a system interface 506. As shown, the detachable cartridge 504 has a cartridge housing 508 and a microfluidic body 510 maintained by the cartridge housing 508. In the illustrated embodiment, the microfluidic body 510 comprises a plurality of layers 521-523 stacked side by side. The layers 521-523 can be printed circuit board (PCB) layers, such as those described below with respect to Figure 14-75. One or more layers 521-523 can be etched so that when the layers 5212-523 are stacked side by side, the microfluidic body 510 forms a sample channel 526. The sample channel 526 is a part of a fluid network such as a fluid network 106 (Fig. 1A) and comprises a valve or inner chamber 528.

The removable cartridge 504 includes a passageway valve 530 configured to regulate the flow of fluid through the sample passageway 526. For example, the passageway valve 530 may allow maximum clearance so that the fluid can flow unimpeded. The passageway valve 530 may also hinder the flow of fluid through it. As used herein, the term "impede" may include slowing the flow of the fluid or completely preventing the flow of the fluid. As shown, the sample passageway 530 includes first and second ports 532 and 534 in fluid communication with the valve chamber 528. The fluid is configured to flow into the valve chamber 528 through the first port 532 and out of the valve chamber 528 through the second port 534. In the illustrated embodiment, the passageway valve 530 comprises a flexible membrane that can bend between first and second conditions. The flexible membrane is in the first condition in FIG. 5 and in the second condition in FIG. 6. In a specific embodiment, the flexible membrane is a flexible layer, such as membrane layer 918 (shown in FIG. 23A and 23B). The flexible layer is configured to be pushed into the valve chamber 528 to prevent the flow of fluid through it. In alternative embodiments, the channel valve 530 may be a physical element that can be moved between different conditions or positions to regulate the flow of the fluid.

Also shown, base instrument 502 includes a valve actuator 540 configured to activate access valve 530. For example, valve actuator 540 can bend a flexible membrane between a first and a second condition. Valve actuator 540 includes an elongated body 542, such as a rod or rod, extending through system interface 506. More specifically, elongated body 542 extends from a control side 544 of base instrument 502. Removable cartridge 504 includes an access opening 546 for receiving valve actuator 540. Access opening 546 opens to a mating side 548 of removable cartridge 504. As shown, elongated body 542 protrudes away from control side 544 and into access opening 546 on mating side 548. Access opening 546 allows valve actuator 540 to directly engage access valve 530, which is a flexible membrane in the illustrated embodiment. In FIG. 5 , valve actuator 540 is in a first state or position. In FIG. 6 , valve actuator 540 is in a second state or position. In the second position, the valve actuator 540 moves a distance toward the access valve 530 and engages with the access valve 530. The valve actuator 540 can deform the access valve 530 so that the access valve 530 covers the first port 532. Therefore, fluid flow through the first port 532 is blocked by the access valve 530.

In some embodiments, the system 500 can have first and second access valves similar or identical to the access valve 530 shown in Figures 5 and 6, wherein the first access valve is upstream of a sample preparation area (not shown) of the fluid network and the second access valve is downstream of the sample preparation area. Thus, the first and second access valves can effectively seal the fluid, which may contain a biological sample, within the sample preparation area. The fluid containing the biological sample can then be heated to subject the fluid to an amplification protocol, such as a PCR protocol.

Figures 7 and 8 illustrate a cross-section of a portion of a system 550 having a base instrument 552 and a removable cartridge 554 operably coupled along a system interface 556. Base instrument 552 and removable cartridge 554 can be similar to base instrument 502 and removable cartridge 504, respectively, shown in Figures 5 and 6 . Base instrument 552 includes a valve actuator 590 with an elongated body 592, such as a nozzle, that extends outward from a control side 594 of base instrument 552 and is inserted into an access opening 596 on a mating side 598 of removable cartridge 554. Valve actuator 590 extends through system interface 556. Optionally, base instrument 552 can include a sealing member 595, such as an O-ring, surrounding elongated body 592 and sealing access opening 596 to provide a closed chamber. In an exemplary embodiment, removable cartridge 554 includes a passage valve 580, which can be a flexible membrane, pneumatically activated by valve actuator 590. More specifically, valve actuator 590 is configured to provide a fluid (e.g., air) to increase pressure within the closed chamber, thereby deforming channel valve 580. When channel valve 580 deforms, it can cover first port 582 of sample channel 576, thereby preventing flow through sample channel 576.

9-10 illustrate a system 600 similar to systems 500 and 550. More specifically, FIG9-10 illustrate system 600 having a base instrument 602 and a removable cartridge 604 operably engaged along a system interface 606. Removable cartridge 604 includes a movable valve 630 that is rotatably engaged by a valve actuator 640 of base instrument 602. Movable valve 630 is a planar body that is shaped to allow flow through sample channel 626 when in a first rotational position (shown in FIG9 ) and to prevent flow through sample channel 626 when in a second rotational position (shown in FIG10 ). More specifically, movable valve 630 can cover port 632 when in the second rotational position.

FIG11 is a perspective view of an exposed portion of a removable cartridge 700 having a microfluidic body 702 and a rotatable valve 704. The removable cartridge 700 may be similar to the removable cartridge 104 ( FIG1 ) and other removable cartridges described herein. The rotatable valve 704 may be similar to the removable valve 123 ( FIG1 ). The rotatable valve 704 is configured to be rotatably mounted to a body side or surface 706 of the microfluidic body 702. The rotatable valve 704 has a fluid side 708 configured to slidably engage the body side 706 when rotated about an axis 710. The fluid body 702 may include a fluid network 760 having a plurality of sample channels 763, 764, a plurality of reservoir channels 765, and a feed channel 766. Channels 763-766 are discrete channels. For example, channels 763-766 can be disconnected based on the rotational position of the rotatable valve 704.

Channels 763-766 have corresponding ports that open to the main body side 706. In the illustrated embodiment, four sample channels 763 are in fluidic communication with a single sample channel 764. Thus, the sample channels 763 can be referred to as channel sections, and the sample channel 764 can be referred to as a common sample channel. Each sample channel 763 is operably coupled to a pair of channel valves 761, 762. The channel valves 761, 762 can be similar to the channel valves described herein, such as the channel valve 530. When in their respective closed positions, the channel valves 761, 762 can seal a liquid containing a corresponding biological sample. In some embodiments, the sample channels 763 extend adjacent to the thermal control region 770. When the biological sample is sealed in the corresponding sample channel 763, a heating element (not shown) and a heat block (not shown) can be positioned adjacent to the thermal control region 770. The heating element and the heat block can coordinate to increase and/or decrease the temperature experienced by the biological sample within the sample channel 763. In such an embodiment, the sample channel 763 can constitute a sample preparation zone.

Feed channel 766 is in fluid communication with reaction chamber 716, and reservoir channel 765 can be in fluid communication with the corresponding reservoir (not shown) of storage module (not shown). Sample channel 764 has network port 721, feed channel 766 has feed port 722, and reservoir channel 765 has corresponding reservoir port 723. Network port 721, feed port 722 and reservoir port 723 are open to main body side 706. Reservoir port 723 is in fluid communication with corresponding module port 724 by corresponding reservoir channel 765. As shown, module port 724 can be located at various positions away from feed port 722 or axis 710 along main body side 706. Module port 724 is configured to be coupled to reservoir (not shown) fluidically. Module port 724 can have a position based on the size of reservoir.

In the illustrated embodiment, the microfluidic body 702 has a total of 15 channels that are directly interconnected to the rotatable valve 704. More specifically, there is only one sample channel 764 and only one feed channel 766, but 13 reservoir channels 765 that are directly interconnected (fluidically) to the rotatable valve 704. In other embodiments, the microfluidic body 702 may include multiple sample channels 764 and/or multiple feed channels 766 that are directly interconnected to the rotatable valve 704. Each sample channel 763 can be fluidically coupled to a corresponding sample port (not shown) configured to receive a biological sample from a user.

The fluid side 708 is configured to slidably engage the body side 706 at the valve receiving area 728. The rotatable valve 704 is sized and shaped so that the fluid side 708 covers the valve receiving area 728 and one or more ports 721-723 along the body side 706. The rotatable valve 704 includes a flow channel 744 (shown in FIG. 12 ) configured to fluidly interconnect the feed port 722 to the one or more ports 721, 723. The rotatable valve 704 can prevent flow through one or more ports and allow flow through one or more other ports based on the position and configuration of the rotatable valve 704.

FIG12 illustrates a cross-section of a rotatable valve 704 operably engageable with a valve actuator 730. More specifically, the rotatable valve 704 includes a valve body 732 having a fluid side 708 and an operating side 734. The operating side 734 may include a mechanical interface device 736 configured to engage the valve actuator 730. In the illustrated embodiment, the mechanical interface device 736 comprises a planar body or fin that coincides with the axis 710. The valve actuator 730 includes a slot 738 configured to receive the mechanical interface device 736, enabling the valve actuator 730 to operably engage the rotatable valve 704. More specifically, the valve actuator 730 may engage the rotatable valve 704 such that the valve actuator 730 can rotate the rotatable valve 704 about the axis 710.

The fluid side 708 includes a plurality of valve ports 740, 742 and flow channels 744 extending between the valve ports 740, 742. The fluid side 708 is slidably coupled to the body surface 706 at the valve receiving area 728. In the illustrated embodiment, the rotatable valve 704 includes only two valve ports 740, 742 and only one flow channel 744. In other embodiments, the rotatable valve 704 may include more than two valve ports and/or more than one flow channel.

As shown in FIG12 , feed port 722 is fluidly aligned and coupled to valve port 740, and valve port 742 is fluidly aligned and coupled to network port 721. Depending on the rotational position of rotatable valve 704, valve port 742 may also be fluidly coupled to one of component ports 723. As mentioned above, rotatable valve 704 is configured to rotate about axis 710. In some embodiments, feed port 722 and valve port 740 are positioned such that they are aligned with axis 710. More specifically, axis 710 extends through each of feed port 722 and valve port 740.

When the valve actuator 730 is operably engaged to the rotatable valve 704, the valve actuator 730 can apply an actuator force 748 in a direction against the body side 706. In such an embodiment, the actuator force 748 can be sufficient to seal the flow passage 744 between the valve ports 740, 742 and to seal the reservoir port 723 and/or the network port 721.

Accordingly, the rotatable valve 704 can fluidly couple the feed port 722 and the network port 721 in a first rotational position, and fluidly couple the feed port 722 and the corresponding reservoir port 723 in a second rotational position. When the rotatable valve 704 is rotated between different rotational positions, the rotatable valve 704 effectively changes the flow path of the fluid network.

Fluid can flow in either direction through flow channel 744. For example, a system pump (not shown), such as system pump 119 ( FIG. 1 ), can be in fluid communication with feed port 722. The system pump can generate suction that pulls the fluid through network port 721 (or corresponding reservoir port 723) and then into flow channel 744 and then through feed port 722. Alternatively, the system pump can provide positive pressure that moves the fluid within flow channel 744, causing the fluid to flow through feed port 722 and then into flow channel 744 and then through network port 721 (or corresponding reservoir port 723).

Figure 13 is the top-down view of the main body side 706 illustrating network port 721, feed port 722 and reservoir port 723. Represent flow channel 744 on two different rotational positions.Reservoir port 723 can comprise reservoir port 723A-723D.Each reservoir port 723A-723D is coupled to corresponding reservoir fluidically by corresponding reservoir channel 765 (Figure 10).More specifically, reservoir port 723A is coupled to hydrogenation buffer fluidically, and reservoir port 723B is coupled to nucleotide solution fluidically, and reservoir port 723C is coupled to washing solution fluidically, and reservoir port 723D is coupled to lysate fluidically.As mentioned above, based on the rotational position of rotatable valve 704 (Figure 11), flow channel 744 can be coupled to sample channel 763,764 or corresponding reservoir fluidically with feed port 722.

Table 1 shows various states of the synthesis sequencing (SBS) protocol, but it should be understood that other determination protocols can be implemented. In stage 1, the flow channel 744 has a rotational position that fluidically couples the network port 721 and the feed port 722. In stage 1, the channel valve (not shown) can be selectively activated to seal the second, third and fourth biological samples in the corresponding sample preparation area, but allows the first biological sample to flow through the network port 721. Accordingly, in stage 1, the system pump can apply a suction force that pulls the first biological sample into the flow channel 744. In stage 2, the rotatable valve 704 is rotated to the second rotational position, and the first biological sample is stored in the flow channel 744, so that the flow channel 744 fluidically couples the reservoir port 723A and the feed port 722. In the second rotational position, the system pump can provide a positive displacement force that pushes the first biological sample through the reservoir port 723A and into the hydrogenation buffer reservoir.

In stage 3, rotatable valve 704 is rotated back to the first rotational position, and the channel valve is selectively activated, allowing the second biological sample to be drawn into flow channel 744. In stage 4, rotatable valve 704 is rotated back to the second rotational position, while the first biological sample is stored in flow channel 744, and the second biological sample is added to the hydrogenation buffer with the first biological sample. During stages 5-8, the third and fourth biological samples are removed from the corresponding sample preparation areas and added to the hydrogenation buffer. Accordingly, four biological samples can be stored in a single reservoir with the hydrogenation buffer. Reactions can occur on the biological samples and the hydrogenation buffer, which prepares the biological samples for SBS sequencing.

In stage 9, the combined biological sample/hydrogenation buffer is drawn out through reservoir port 723A, through flow channel 744, through feed port 722 and into a reaction chamber (not shown). The biological sample can be fixed to the surface defining the reaction chamber. For example, a cluster comprising the biological sample can be formed. Stages 10-13 represent sequencing cycles. In stage 10, the rotatable valve 704 can be at the third rotational position so that the nucleotide solution can be drawn out through flow channel 744 and into the reaction chamber. At this time, bases can be incorporated into the corresponding biological sample (e.g., template nucleic acid). In stage 11, the rotatable valve 704 can be at the fourth rotational position so that the washing solution can flow through the reaction chamber and transmit the nucleotide solution away from the reaction chamber. After stage 11, the reaction chamber can be imaged by an imaging detector. The color of the light emitted from the cluster can be used to identify the bases merged by the cluster. In stage 12, the rotatable valve 704 can be at the fourth rotational position so that the lysate can flow through the reaction chamber, and the fluorophore (and if present, the reversible terminator portion) can be removed from the cluster. In stage 13, the rotatable valve 704 can again be in the third rotational position and a wash solution can flow through the reaction chamber to remove the lysate. Stages 10-13 can be repeated until sequencing is complete and/or until the reagents are exhausted.

Table 1

The above-mentioned embodiments can be used in conjunction with the subject matter of U.S. Provisional Patent Application No. 61/951,462 (Attorney Docket No. IP-1210-PRV 296PRV2) (hereinafter "the '462 application"), which is incorporated herein by reference in its entirety. At least a portion of the '462 application is provided below.

Can be used in conjunction with various nucleic acid sequencing techniques the method described herein. Particularly applicable technologies are those in which nucleic acids are attached to fixed positions in the array so that their relative positions do not change, and wherein the array is repeatedly detected or imaged. The following embodiment is particularly applicable: images are obtained in different color channels, for example, overlapping with different markers for distinguishing a nucleotide base type from another nucleotide base type. In some embodiments, the process for determining the nucleotide sequence of the target nucleic acid can be an automated process. Preferred embodiments include sequencing by synthesis ("SBS") technology.

"Sequencing by synthesis ("SBS") techniques" generally involve the enzymatic extension of a nascent nucleic acid chain by the repeated addition of nucleotides to a reference template chain. In traditional methods of SBS, a single nucleomonomer can be provided to a target nucleic acid in each delivery in the presence of a polymerase. However, in the methods described herein, more than one type of nucleomonomer can be provided to a target nucleic acid in the presence of a polymerase in a delivery.

SBS can utilize the nucleotide monomer with terminator part or lack the nucleotide monomer of any terminator part.Utilize the method for the nucleotide monomer that lacks terminator to comprise for example pyrophosphate sequencing and use the order-checking of the Nucleotide of gamma-phosphate labeling, as set forth in more detail below.In the method for the nucleotide monomer that lacks terminator, the quantity of the Nucleotide that adds in each circulation is normally variable and depends on the pattern that template sequence and Nucleotide are transported.For utilizing the SBS technology of the nucleotide monomer with terminator part, terminator can actually be irreversible under the order-checking condition used, as the traditional Sanger order-checking situation of utilizing dideoxynucleotide is just like this, or terminator can be reversible, as the order-checking method situation of being developed by Solexa (present Illumina Co., Ltd.) is just like this.

In some embodiments, the SBS technique can utilize nucleotide monomers with a labeling portion or lack nucleotide monomers with a labeling portion. Accordingly, the incorporation event can be detected by the release of a proton or pyrocarbonate, etc., of the incorporation of a characteristic such as the fluorescence of the labeling, the characteristic of the nucleotide monomer such as the molecular weight or the charge, the nucleotide. In an embodiment in which two or more different nucleotides are present in a sequencing reagent, the different nucleotides can be distinguished from each other, or alternatively, two or more different labels can be indistinguishable under the detection technology employed. For example, the different nucleotides present in a sequencing reagent can have different labels, and they can be distinguished using the appropriate optical device exemplified by the sequencing method developed by Solexa (present Illumina Co., Ltd.).

In another exemplary type of SBS, cycle sequencing is accomplished by the gradual addition of reversible terminator nucleotides comprising, for example, cleavable or photobleachable dye labels, as described, for example, in International Patent Publication No. WO 04/018497 and U.S. Patent No. 7,057,026, the disclosures of which are incorporated herein by reference. This method is commercialized by Illumina Co., Ltd. and is also described in International Patent Publication No. WO 91/06678 and International Patent Publication No. WO 07/123,744, each of which is incorporated herein by reference. The availability of fluorescently labeled terminators (wherein termination can be reversible and the fluorescent label can be cleaved) facilitates effective cyclic reversible termination (CRT) sequencing. Polymerases can also be co-designed to effectively merge these modified nucleotides and extend from these modified nucleotides.

Preferably, in sequencing embodiments based on reversible terminators, the label does not substantially inhibit extension under SBS reaction conditions. However, the detection label may be removable, for example, by cleavage or degradation. Images may be captured after the label is incorporated into the arrayed nucleic acid feature. In a specific embodiment, each cycle involves the simultaneous delivery of four different nucleotide types to the array, and each nucleotide type has a spectrally different label. Four images may then be obtained, each using a detection channel selected for one of the four different labels. Alternatively, different nucleotide types may be added sequentially, and an image of the array may be obtained between each addition step. In such an embodiment, each image will show a nucleic acid feature incorporating a specific type of nucleotide. Due to the different sequence content of each feature, different features may be present or absent in different images. However, the relative position of the features will remain unchanged in the image. Images obtained from such a reversible terminator-SBS method may be stored, processed, and/or analyzed as described herein. After the image capture step, the label may be removed and the reversible terminator portion may be removed for subsequent cycles of nucleotide addition and detection. Removal of labels after they are detected in a particular cycle and prior to subsequent cycles may offer the advantage of reducing background signal and cross-talk between cycles. Examples of useful labeling and removal methods are set forth below.

In a specific embodiment, some or all of the nucleotide monomers may include a reversible terminator. In such an embodiment, a reversible terminator/cleavable fluorophor may include a fluorophor linked to a nucleic acid portion via a 3' ester bond (Metzker, Genome Res. 15: 1767-1776 (2005), which is incorporated herein by reference). Other methods separate terminator chemicals from the cracking of fluorescent labels (Ruparel et al., Proc Natl Acad Sci USA 102: 5932-7 (2005), which is incorporated herein by reference in its entirety). Ruparel et al. describe the development of reversible terminators that use small 3' allyl groups to prevent extension but can easily be unblocked using a palladium catalyst by brief processing. Fluorophores are attached to bases via photocleavable cross-linkers, which can easily be cracked by being exposed to 30 seconds of long wavelength UV light. Therefore, disulfide reduction or photocleavage can be used as cleavable cross-linkers. Another method of reversible termination is the use of the natural terminator that guarantees after a large amount of dyes are placed on dNTP.The existence of a large amount of charged dyes on dNTP can serve as effective terminator by spatial and/or electrostatic hindrance.The existence of an incorporation event prevents further incorporation unless the dye is removed.The cracking of the dye has removed the fluorophor and effectively makes the termination reversal. In United States Patent (USP) 7,427,673 and United States Patent (USP) 7,057,026, the example of the Nucleotide of modification is described, and the disclosure of these patents is fully incorporated into this paper by reference.

Additional exemplary SBS systems and methods that may be utilized with the methods and systems described herein are described in U.S. Patent Publication No. 2007/0166705, U.S. Patent Publication No. 2006/0188901, U.S. Patent 7,057,026, U.S. Patent Publication No. 2006/0240439, U.S. Patent Publication No. 2006/0281109, International Patent Publication No. WO 05/065814, U.S. Patent Publication No. 2005/0100900, International Patent Publication No. WO 06/064199, International Patent Publication No. WO 07/010,251, U.S. Patent Publication No. 2012/0270305, and U.S. Patent Publication No. 2013/0260372.

Some embodiments can utilize the detection of four different nucleotides using less than four different labels.For example, the method and system described in the combined materials of U.S. Patent No. 2013/0079232 can be utilized to perform SBS.As a first example, a pair of nucleotide types can be detected at the same wavelength, but based on the difference in intensity compared with another member of this pair or based on the change of this pair of members (such as via chemical modification, photochemical modification or physical modification) and be distinguished, compared with the signal detected by the other member of this pair, this change makes the apparent signal appear or disappear.As a second example, three of the four different nucleotide types can be detected under specific conditions, and the fourth " dark state " nucleotide type lacks the label that can be detected under those conditions or is minimally detected under those conditions (such as due to the minimum detection of background fluorescence, etc.).The incorporation of the first three nucleotide types into nucleic acid can be determined based on the existence of their corresponding signals, and the incorporation of the fourth nucleotide type into nucleic acid can be determined based on the lack of any signal or the minimum detection.As a third example, a nucleotide type can include the label detected in two different channels, and other nucleotide types are detected in no more than one channel. The three exemplary configurations mentioned above are not considered mutually exclusive and can be used in various combinations. An exemplary embodiment combining all three examples is a fluorescence-based SBS method using a first nucleotide type detected in a first channel (e.g., labeled dATP that is detected in the first channel when excited by a first excitation wavelength), a second nucleotide type detected in a second channel (e.g., labeled dCTP that is detected in the second channel when excited by a second excitation wavelength), a third nucleotide type detected in both the first and second channels (e.g., labeled dTTP that is detected in both channels when excited by the first and/or second excitation wavelengths), and a fourth nucleotide type that is not or minimally detected in either channel (e.g., unlabeled dGTP).

In addition, as described in the incorporated materials of U.S. Patent Publication No. 2013/0079232, a single channel can be used to obtain sequencing data. In such a so-called single-dye sequencing method, the first nucleotide type is labeled, but the label is removed after the first image is generated, and the second nucleotide type is labeled only after the first image is generated. The third nucleotide type retains its label in the first and second images, and the fourth nucleotide type retains its label in both images.

Some embodiments can utilize sequencing by complex formation. Such technology utilizes DNA ligase to incorporate oligonucleotides and identify the incorporation of such oligonucleotides. Oligonucleotides generally have different labels associated with the identity of specific nucleotides in the sequence to which the oligonucleotides hybridize. Like other SBS methods, images can be obtained using labeled sequencing reagents after processing the array of nucleic acid features. Each image will show nucleic acid features with a specific type of incorporation label. Due to the different sequence content of each feature, different features may be present or absent in different images, but the relative position of the features will remain unchanged in the image. The images obtained from the sequencing method based on complex formation can be stored, processed and/or analyzed as described herein. Exemplary sequencing systems and methods that can be utilized together with the methods and systems described herein are described in U.S. Patents 6,969,488, U.S. Patents 6,172,218 and U.S. Patents 6,306,597.

Some embodiments can utilize nanopore sequencing (Deamer, D.W. & Akeson, M. "Nanopores and nucleic acids: prospects for ultrarapid sequencing." Trends Biotechnol. 18, 147-151 (2000); Deamer, D. and D. Branton, "Characterization of nucleic acids by nanopore analysis." Acc. Chem. Res. 35: 817-825 (2002); Li, J, M. Gershow, D. Stein, E. Brandin and J. A. Golovchenko, "DNA molecules and configurations in a solid-state nanopore microscope" Nat. Mater. 2: 611-615 (2003), the disclosures of which are incorporated herein by reference in their entirety). In such embodiments, the target nucleic acid passes through a nanopore. The nanopore can be a synthetic pore or a biological membrane protein, such as α-hemolysin. As a target nucleic acid passes through a nanopore, each base pair can be identified by measuring fluctuations in the pore's electrical conductivity. (U.S. Patent 7,001,792; Soni, G.V. & Meller, "A. Progress toward ultrafast DNA sequencing using solid-state nanopores." Clin. Chem. 53, 1996-2001 (2007); Healy, K. "Nanopore-based single-molecule DNA analysis." Nanomed. 2, 459-481 (2007); Cockroft, S.L., Chu, J., Amorin, M. & Ghadiri, M.R. "A single-molecule nanopore device detects DNA polymerase activity with single-nucleotide resolution." J. Am. Chem. Soc. 130, 818-820 (2008), the disclosures of which are incorporated herein by reference in their entireties). In other embodiments, an endonuclease can be coupled to a nanopore so that nucleotides sequentially released from the ends of the nucleic acid by the endonuclease are detected as they pass through the nanopore. Each nucleotide can be distinguished based on a different base moiety or based on an added moiety. The data obtained from nanopore sequencing can be stored, processed, and/or analyzed as described herein. In particular, the data can be processed into images according to the exemplary processing of optical images and other images described herein.

Some embodiments can utilize the method for real-time monitoring related to DNA polymerase activity.Can detect nucleotide incorporation by fluorescence resonance energy transfer (FRET) interaction between the polymerase carried by fluorophore and the nucleotide of gamma phosphate labeling as described in, for example, U.S. Patent No. 7,329,492 and U.S. Patent No. 7,211,414 (wherein each patent is incorporated herein by reference), or can use zero-mode waveguide as described in, for example, U.S. Patent No. 7,315,019 (which is incorporated herein by reference) and use fluorescent nucleotide analogues and engineering polymerase as described in, for example, U.S. Patent No. 7,405,281 and U.S. Patent Publication No. 2008/0108082 (wherein each patent is incorporated herein by reference) to detect nucleotide incorporation. Illumination can be confined to a scaled volume around the surface-bound polymerase, allowing incorporation of fluorescently labeled nucleotides to be observed with low background (Levene, M.J. et al. "Zero-mode waveguides for single-molecule analysis at high concentrations." Science 299, 682-686 (2003); Lundquist, P.M. et al. "Parallel confocal detection of single molecules in realtime." Opt. Lett. 33, 1026-1028 (2008); Korlach, J. et al. "Selective aluminum passivation for targeted immobilization of single DNA polymerase molecules in zero-mode waveguide nanostructures." Proc. Natl. Acad. Sci. USA 105, 1176-1181 (2008), the disclosures of each of which are incorporated herein by reference in their entirety). Images resulting from such methods can be stored, processed, and analyzed as described herein.

Some SBS embodiments include detection of protons released when nucleotides are incorporated into extension products. For example, sequencing based on detection of released protons can use electrical detectors and related technologies commercially available from Ion Torrent (Guilford, CT, a Life Technologies subsidiary) or the sequencing methods and systems described in U.S. Patent Publication No. 2009/0026082, U.S. Patent Publication No. 2009/0127589, U.S. Patent Publication No. 2010/0137143, or U.S. Patent Publication No. 2010/0282617, each of which is incorporated herein by reference.

Can advantageously carry out above-mentioned SBS method in many formats so that a plurality of different target nucleic acids are manipulated simultaneously.In a specific embodiment, different target nucleic acids can be processed in common reaction vessels or on the surface of specific substrate.This allows the convenient transportation of sequencing reagents, the removal of unreacted reagent and the detection of incorporation event in a multiplexed manner.In the embodiment using surface-bound target nucleic acid, target nucleic acid can be in array format.In array format, target nucleic acid can generally be attached to surface in a spatially distinguishable manner.Can be by direct covalent bonding, be attached to pearl or other particle or be attached to polymerase or be attached to other molecules on surface and combine target nucleic acid.Array can comprise the single copy of the target nucleic acid at each position (also referred to as feature), or have multiple copies of identical sequence and can be present in each position or feature.Can produce multiple copies by amplification method such as bridge amplification or emulsion PCR as described further below.

The methods described herein can use arrays having features at any of a variety of densities, including at least about 10 features/cm ² , 100 features/cm ² , 500 features/cm ² , 1,000 features/cm ² , 5,000 features/cm ² , 10,000 features/cm ² , 50,000 features/cm ² , 100,000 features/cm ² , 1,000,000 features/cm ² , 5,000,000 features/cm ² , or more. The methods and apparatus described herein can include detection components or devices having a resolution at least sufficient to resolve individual features at one or more of these exemplified densities.

The advantage of the method of setting forth herein is that they provide the parallel quick and effective detection of multiple target nucleic acids. Accordingly, the disclosure provides an integrated system that can use technology known in the art such as the technology illustrated above to prepare and detect nucleic acid. Therefore, the integrated system of the disclosure may include a fluid component that can transport amplification reagent and/or sequencing reagent to one or more fixed DNA fragments, and this system includes components such as pumps, valves, reservoirs, fluid circuits, etc. Can configure and/or use flow cell in the integrated system for the detection of target nucleic acid. For example, exemplary flow cell has been described in U.S. Patent Publication No. 2010/0111768A1 and U.S. Patent Application No. 13/273,666, each patent is incorporated herein by reference. As illustrated to flow cell, one or more fluid components of integrated system can be used for amplification method and for detection method. Taking nucleic acid sequencing embodiment as an example, one or more fluid components of integrated system can be used for the amplification method set forth herein and for the conveying of sequencing reagent in sequencing method such as illustrated above. Alternatively, integrated system can include independent fluid system to carry out amplification method and carry out detection method. Examples of integrated sequencing systems capable of creating amplified nucleic acids and also determining the sequence of the nucleic acids include, but are not limited to, the MiSeq ^™ or NextSeq ^™ platforms (Illumina, Inc., San Diego, CA) or the devices described in U.S. Patent Application Publication Nos. 2012/0270305 A1 or 2013/0260372 A1, each of which is incorporated herein by reference.

"Activity detector" means any device or component capable of detecting an activity indicating a specific reaction or process. An activity detector may be capable of detecting a predetermined event, characteristic, quantity, or characteristic within a predetermined volume or region. For example, an activity detector may be capable of capturing an image of a predetermined volume or region. An activity detector may be capable of detecting ion concentrations within a predetermined volume of a solution or along a predetermined region. Exemplary activity detectors include: a charge coupled device (CCD) (e.g., a CCD camera); a photomultiplier tube (PMT); a molecular characterization device or detector, such as those used with a nanopore; a microcircuit arrangement, such as those described in U.S. Patent No. 7,595,883, which is incorporated herein by reference in its entirety; and a sensor manufactured by CMOS with a field effect transistor (FET), including a chemically sensitive field effect transistor (chemFET), an ion-sensitive field effect transistor (ISFET), and/or a metal oxide semiconductor field effect transistor (MOSFET). Exemplary activity detectors are described, for example, in International Patent Publication No. WO2012/058095.

The term "biosensor" includes any structure with multiple reaction sites. The biosensor may include a solid-state imaging device (such as a CCD or CMOS imager) and a flow cell optionally mounted thereto. The flow cell may include at least one flow channel in fluid communication with the reaction site. As a specific example, the biosensor is configured to be fluidically and electrically coupled to a bioassay system. The bioassay system may transport reactants to the reaction site and perform multiple imaging events according to a predetermined protocol (such as sequencing by synthesis). For example, the bioassay system may guide the solution to flow along the reaction site. At least one solution may include four types of nucleotides with the same or different fluorescent labels. Nucleotide may be incorporated into the corresponding oligonucleotides located at the reaction site. The bioassay system may then use an excitation light source (such as a solid-state light source, such as a light emitting diode or LED) to illuminate the reaction site. The excitation light may have one or more predetermined wavelengths, including a certain range of wavelengths. The excited fluorescent label provides an emission signal that can be detected by a light detector.

In one aspect, the solid-state imager comprises a CMOS image sensor comprising an array of photodetectors configured to detect emission signals. In some embodiments, each photodetector has a single pixel, and the ratio of pixels to detection paths defined by the filter walls can be substantially one to one. Exemplary biosensors are described, for example, in U.S. Patent Application No. 13/833,619.

"Detection surface" means any surface including a photodetector. The detector can be based on any appropriate technology, such as those including a charge coupled device (CCD) or a complementary metal oxide semiconductor (CMOS). In a specific embodiment, a CMOS imager with a single proton avalanche diode (CMOS-SPAD) can be used, for example, to distinguish fluorophores using fluorescence lifetime imaging (FLIM). Exemplary CMOS-based systems that can be used for FLIM are described in U.S. Patent Publication No. 2008/0037008 A1, "Biomedical Optics Express 1:1302-1308 (2010)" by Giraud et al., or "IEEE European Solid-State Device Conference (ESSCIRC), Athens, Greece, IEEE, pp. 204-207 (2009)" by Stoppa et al., each of which is incorporated herein by reference in its entirety. Other useful detection devices that can be used include, for example, those described in U.S. Patent 7,329,860 and U.S. Patent Publication No. 2010/0111768, each of which is incorporated herein by reference in its entirety.

In addition, it will be appreciated that other signal detection devices as known in the art can be used to detect the signal produced in the method set forth herein. For example, it is particularly useful to detect the detector for detecting pyrophosphate or proton. Detectors can be used, for example, to detect pyrophosphate release from 454Life Sciences (Branford, Conn., a Roche Company) available on the market or as described in U.S. Patent Publication No. 2005/0244870, which is incorporated herein by reference in its entirety. Exemplary systems for detecting primer extension based on proton release are included in systems available on the market from IonTorrent (Guilford, Conn., a ThermoFisher subsidiary) or as described in U.S. Patent Publication No. 2009/0026082, 2009/0127589, 2010/0137143 and 2010/0282617, each of which is incorporated herein by reference in its entirety. Exemplary detection surfaces and detectors are described, for example, in US Patent Publication No. 2013/0116128A1, which is incorporated herein by reference in its entirety.

"Sequencing module" means a CMOS chip suitable for sequencing applications. The module may include a surface comprising a substrate comprising a hydrophilic region for nucleic acid attachment and amplification surrounded by a hydrophobic region. For example, a dynamic pad having a hydrophilic patch, such as the dynamic pad described above, may be used. Alternatively or in addition, a stack of dynamic pads comprising some pads in a hydrophilic state while surrounding pads in a hydrophobic state may form a hydrophilic region surrounded by a hydrophobic region. The surface for nucleic acid attachment may optionally include multiple isolation regions, such that each isolation region contains multiple nucleic acid molecules, preferably obtained from one nucleic acid molecule for sequencing. For example, the hydrophilic region may comprise a gel. The hydrophilic region may be smooth, textured, porous, non-porous, etc. The hydrophobic region is preferably located between the hydrophilic regions. The reagents are moved across the surface by any number of forces.

The subject matter described herein includes, in one or more embodiments, a disposable microfluidic box and a method for making and using the same. The method for making a disposable integrated microfluidic box optionally utilizes a flexible printed circuit board (PCB) and roll-to-roll (R2R) printed electronics for monolithic integration of CMOS technology and finger-shaped fluidic devices. That is, the disposable integrated microfluidic box includes a stack of fluid layers, wherein the CMOS sensor is integrated, all of which are installed in a housing. Accordingly, conventional injection molding fluidics devices can be integrated with flexible PCB technology. The fluid layer is formed using materials suitable for use in R2R printed electronics processes. In addition, the fluid layer includes a polymerase chain reaction (PCR) zone and a reagent mixing and distribution zone. The fluid layer also includes a set of diaphragm valves, and the PCR zone can be completely blocked by the diaphragm valves.

The method uses a disposable integrated microfluidics cartridge to perform multiplex PCR and downstream mixing required for sequencing.

Embodiments described herein include CMOS flow cells in which a majority, or up to approximately 100%, of the biosensor active area is accessible for reagent delivery and illumination.

FIG14 illustrates a flow chart illustrating an example of a method 100 for monolithic integration of CMOS technology and flexible printed circuit boards (PCBs) and roll-to-roll (R2R) printed electronics using finger-shaped fluidic devices. Specifically, using method 100, multi-layer laminated fluidic devices can be integrated with flexible PCB technology (see FIG15 ). Furthermore, using structures formed using method 100, conventional injection molded fluidics can be integrated with flexible PCB technology (see FIGS26 through 45 ). Method 100 may include, but is not limited to, the following steps.

At step 110, fluidic layers are formed and then laminated and bonded together. For example, FIG15 shows an exploded view of a set of fluidic layers 200 that can be laminated and bonded together in this step. In this example, fluidic layers 200 include, in order, an inlet/outlet layer 210, a fluidic channel layer 220, a flexible PCB layer 260, a bottom sequencing chamber layer 280, a sequencing chamber layer 250, and a membrane layer 240 coplanar with a top sequencing chamber layer 290. The inlet/outlet layer 210, fluidic channel layer 220, flexible PCB layer 260, bottom sequencing chamber layer 280, sequencing chamber layer 250, membrane layer 240, and top sequencing chamber layer 290 are suitable for formation using a R2R printed electronics process.

The inlet/outlet layer 210 can be formed from, for example, polycarbonate, polymethyl methacrylate (PMMA), cyclic olefin copolymer (COC), and/or polyimide. The inlet/outlet layer 210 can be from about 25 μm to about 1000 μm thick in one example, or about 250 μm thick in another example. An arrangement of openings (or holes) is provided in the inlet/outlet layer 210. The openings (or holes) provide fluid paths that can be used, for example, as feed ports and/or discharge ports for various liquid supply reservoirs (not shown). More details of the inlet/outlet layer 210 are shown and described herein below with reference to Figures 55A and 55B.

The fluid channel layer 220 can be formed from, for example, polycarbonate, PMMA, COC, and/or polyimide. In one example, the fluid channel layer 220 can be from approximately 25 μm to approximately 1000 μm thick, or in another example, approximately 250 μm thick. An arrangement of fluid channels is provided within the fluid channel layer 220. The fluid channels provide fluid pathways along the fluid channel layer 220 from one destination to another. Because the fluid channel layer 220 is sandwiched between the inlet/outlet layer 210 and the flexible PCB layer 260, fluids can be confined within the fluid channels by the inlet/outlet layer 210 on the bottom and the flexible PCB layer 260 on the top. In one example, the fluid channel layer 220 is used to perform PCR and downstream mixing required for sequencing. Further details of the fluid channel layer 220 are shown and described herein below with reference to Figures 56A and 56B.

Flexible PCB layer 260 can be formed from, for example, polycarbonate, PMMA, COC, and/or polyimide. In one example, flexible PCB layer 260 can be from about 30 μm to about 300 μm thick, or in another example, about 200 μm thick. An arrangement of openings (or holes) is provided in flexible PCB layer 260. The openings (or holes) provide fluid paths that can serve as inlets and/or outlets for diaphragm valves for controlling the flow of liquid in the fluid channels of fluid channel layer 220. Further details of flexible PCB layer 260 are shown and described herein below with reference to Figures 57A and 57B.

Sequencing chamber bottom layer 280 can be formed from, for example, polycarbonate, PMMA, COC, and/or polyimide. Sequencing chamber bottom layer 280 can be from about 25 μm to about 1000 μm thick in one example, or about 250 μm thick in another example. An arrangement of openings is provided in sequencing chamber bottom layer 280 for forming diaphragm valves within the stack of layers of fluidics layer 200. Sequencing chamber bottom layer 280 also includes CMOS devices, such as CMOS image sensors 262, positioned proximate to the sequencing chambers of sequencing chamber layer 250. Sequencing chamber bottom layer 280 is coplanar with the CMOS devices and serves as a fluidic connection layer to the inlets/outlets of the sequencing chambers of sequencing chamber layer 250. Further details of sequencing chamber bottom layer 280 are shown and described herein below with reference to Figures 58A and 58B.

Sequencing chamber layer 250 can be formed from, for example, polycarbonate, PMMA, COC, and/or polyimide. Sequencing chamber layer 250 can be from about 50 μm to about 300 μm thick in one example, or about 100 μm thick in another example. An arrangement of openings is provided in sequencing chamber layer 250 for forming a diaphragm valve within the stack of fluidics layer 200. Sequencing chamber layer 250 also includes a sequencing chamber. Further details of sequencing chamber layer 250 are shown and described herein below with reference to Figures 59A and 59B.

Diaphragm layer 240 can be formed from, for example, a silicone elastomer. Diaphragm layer 240 can be from about 25 μm to about 1000 μm thick in one example, or about 250 μm thick in another example. Diaphragm layer 240 serves as an elastic membrane for opening and closing a diaphragm valve within the stack of fluidic layer 200, wherein the diaphragm valve is sequentially created by the combination of flexible PCB layer 260, sequencing chamber bottom layer 280, sequencing chamber layer 250, and diaphragm layer 240. Further details of the diaphragm valve are shown and described herein below with reference to Figures 22A, 22B, 23A, and 23B. Further details of diaphragm layer 240 are shown and described herein below with reference to Figures 60A and 60B.

Sequencing chamber top layer 290 can be formed from a low-autofluorescence material with good optical properties, such as COC. Sequencing chamber top layer 290 can be from about 25 μm to about 1000 μm thick in one example, or about 250 μm thick in another example. Sequencing chamber top layer 290 is used to cover the sequencing chambers in sequencing chamber layer 250. Further details of sequencing chamber top layer 290 are shown and described herein below with reference to Figures 60A and 60B.

Referring now again to FIG. 14 , at step 115 , a CMOS device is attached to the flexible PCB. For example, a CMOS image sensor 262 (see FIG. 15 ) is attached to the sequencing chamber bottom layer 280 of the fluidics layer 200 . FIG. 16 shows a perspective view of an example of a CMOS image sensor 262 . In one example, the CMOS image sensor 262 is approximately 9,200 μm long, approximately 8,000 μm wide, and approximately 800-1,000 μm thick; and may have approximately 50 I/O pads. The CMOS image sensor 262 may include a pixel array. In one example, the pixel array is 4,384 x 3,292 pixels, with an overall size of approximately 7,272 μm x 5,761 μm. It will be appreciated that CMOS wafers can have a wide range of sizes and I/O pad counts. For example, rectangular wafers (e.g., non-square dimensions that appear long and thin) can be used with finger-shaped fluidics devices to utilize only a portion of the wafer in any given analysis protocol.

Continuing with step 115, Figures 17A, 17B, 18, 19, and 20 illustrate side views of structure 400, illustrating an example process for attaching a CMOS device to a flexible PCB. Structure 400 is a multi-layer structure. Referring now to Figure 17A, the initial formation of structure 400 begins with a flexible PCB. For example, the flexible PCB includes, in order, a polyimide layer 410, a PCB heater layer 412, a polyimide layer 414, a PCB routing layer 416, and a polyimide layer 418. In other words, Figure 17 illustrates a flexible PCB with a PCB heater layer and a PCB routing layer—also known as a rolled foil.

17B , a low temperature isotropic conductive adhesive (low temperature ICA) 420 is dispensed atop the polyimide layer 418 .

18 , a CMOS device, such as a CMOS image sensor 262, is placed on the reel; that is, atop the cryogenic ICA 420. In one example, the CMOS image sensor 262 is placed atop the cryogenic ICA 420 using a well-known pick and place process. FIG18 shows the I/O pads 422 of the CMOS image sensor 262 in contact with the cryogenic ICA 420 and thereby electrically connected to the PCB wiring layer 416. Other attachment options exist, including but not limited to controlled collapse/flip chip soldering, wire bonding, etc. FIG18 also shows that the CMOS image sensor 262 includes a bio-layer 424 facing away from the polyimide layer 418. A protective film 426 can be placed atop the bio-layer 424 until ready for use.

Next and now referring to Figure 19, a set of fluidic layers 428 are provided on top of the polyimide layer 418 of the flexible PCB. That is, a laminated polycarbonate film is provided coplanar with the CMOS surface. An example of the fluidic layer 428 is the fluidic layer 200 shown in Figure 15.

20 , flip chip bonding of the CMOS image sensor 262 on the foil roll is completed by dispensing an underfill epoxy adhesive 430 around the CMOS image sensor 262 .

Referring now again to FIG. 14 , at step 120 , final assembly of the microfluidic cartridge, including the integrated fluidics layer and CMOS device, is performed. For example, FIG. 21 shows a side view of an example of a microfluidic cartridge 800 . The microfluidic cartridge 800 includes a fluidics portion 810 and a CMOS portion 812 , which are based on the structure 400 shown in FIG. The final assembly steps may include, for example, dispensing (printing) an underfill epoxy adhesive 430 , removing the protective film 426 , laminating a low-temperature non-conductive adhesive 814 (e.g., a UV or thermal non-conductive adhesive) to the CMOS portion 812 , laminating a low-autofluorescence cyclic olefin copolymer (COC) layer 816 to the CMOS portion 812 of the microfluidic cartridge 800 , and laminating a flexible PCB heater 818 to both sides of the fluidics portion 810 . During the formation of the microfluidic cartridge 800 , it is important to use a self-aligned process flow so that the surfaces of the CMOS device and the fluidics layer are flush with each other.

A fluid path is formed through the microfluidic cartridge 800. That is, a sample inlet 820 is provided at the input end of the fluidics portion 810, and an outlet 822 is provided downstream of the CMOS portion 812. The sample inlet 820 supplies a PCR chamber 824. The PCR chamber 824 then supplies a reagent dispensing area 826. The reagent dispensing area 826 then supplies a sequencing chamber 828. The biolayer 424 of the CMOS image sensor 262 is oriented toward the sequencing chamber 828. The sequencing chamber 828 then supplies the outlet 822. In addition, the microfluidic cartridge 800 includes certain diaphragm valves 830 that control the flow of liquid into and out of the PCR chamber 824.

22A and 22B show perspective views of an example of a diaphragm valve 830, wherein the diaphragm valve can be integrated into, for example, the fluid layer 200. Referring now to FIG22A, which is a perspective view of the diaphragm valve 830. In this example, the diaphragm valve 830 comprises, in order, a bottom layer 910, a fluid channel layer 912, and a reservoir layer 914. The bottom layer 910, the fluid channel layer 912, and the reservoir layer 914 can be formed from, for example, polycarbonate, PMMA, COC, and/or polyimide. The reservoir layer 914 has a recessed area with a small reservoir 916 created therein. A diaphragm layer 918 is stretched across the reservoir 916. The reservoir 916 has an inlet 920 and an outlet 922, which provide a flow path to a corresponding fluid channel 924. 22B shows the diaphragm valve 830 without the diaphragm layer 918 covering the reservoir 916 in order to better illustrate the features of the reservoir 916 and the inlet 920 and outlet 922. The diaphragm layer 918 is formed of a flexible and stretchable elastomeric film material, such as a silicone elastomer.

Figures 23A and 23B each show a cross-sectional view of diaphragm valve 830 taken along line A-A of Figure 22A. An actuator, such as actuator 1010, can be used to open and close diaphragm valve 830. For example, Figure 23A shows diaphragm valve 830 in an open state, wherein actuator 1010 is not engaged with diaphragm layer 918. In contrast, Figure 23B shows diaphragm valve 830 in a closed state, wherein actuator 1010 is engaged with diaphragm layer 918. In other words, the top end of actuator 1010 is used to push the center portion of diaphragm layer 918 against outlet 922 and thereby prevent liquid from flowing therethrough. Diaphragm valve 830 (i.e., diaphragm valves 242, 244, and 246) can be actuated using, for example, mechanical or air actuation, such as a solenoid or a pneumatic pump.

FIG24 shows a schematic diagram of an example of a microfluidic cartridge 1100 that includes integrated CMOS technology and finger-shaped fluidics. Specifically, the microfluidic cartridge 1100 includes a fluidics layer 200 that is fluidically and operatively connected to four sample supplies 1110 (e.g., sample supplies 1110a, 1110b, 1110c, 1110d), thirteen reagent supplies 1112 (e.g., reagent supplies 1112a-1112m), and an outlet pump 1114. The fluidics layer 200 includes a PCR zone 270 and a reagent mixing and dispensing zone 275. The PCR zone 270 includes, for example, four PCR channels 222 (e.g., PCR channels 222a, 222b, 222c, and 222d). The inlets of the PCR channels 222a, 222b, 222c, and 222d are fed by the sample supplies 1110a, 1110b, 1110c, and 1110d, respectively. Because the microfluidic cartridge 1100 includes four PCR channels 222 fed by four sample supplies 1110, the microfluidic cartridge 1100 is configured for 4X sample multiplexing.

Four diaphragm valves 242 are used to control the inputs of the four PCR channels 222. That is, diaphragm valves 242a, 242b, 242c, and 242d are used to control the inputs of PCR channels 222a, 222b, 222c, and 222d, respectively. Similarly, four diaphragm valves 244 are used to control the outputs of the four PCR channels 222. That is, diaphragm valves 244a, 244b, 244c, and 244d are used to control the outputs of PCR channels 222a, 222b, 222c, and 222d, respectively. The outputs of the four PCR channels 222 are fed into a common PCR output channel 224, which in turn feeds the reagent mixing and dispensing area 275. The presence of diaphragm valves 242 and 244 in the fluidics layer 200 allows the PCR zone 270 to be completely sealed.

Reagent mixing and distribution area 275 includes the arrangement of 13 reagent channels 226 (e.g., reagent channels 226a-226m). In addition, 13 reagent channels 226a-226m are fed via 13 reagent supplies 1112a-1112m respectively. A rotatable valve assembly (not shown) is used to fluidically connect a certain PCR channel 222 to a certain reagent supply 1112. In doing so, a certain PCR mixing can be created. A rotatable valve assembly (not shown) is also used to fluidically connect a certain PCR mixing to a sequencing feed channel 228, which feeds the inlet of the sequencing chamber 258. In addition, a CMOS image sensor 262 is located at the sequencing chamber 258.

A sequencing outlet channel 230 is provided at the outlet of the sequencing chamber 258. An outlet pump 1114 is fluidically and operatively connected to the sequencing outlet channel 230. The outlet pump 1114 is used to provide positive or negative pressure to move liquid in any direction along the flow path of the fluidic layer 200. In addition, a series of three diaphragm valves 246 are provided along the length of the sequencing outlet channel 230. The diaphragm valves 242, 244, and 246 can be implemented based on the diaphragm valve 830 shown and described in Figures 22A, 22B, 23A, and 23B.

The three diaphragm valves 246 at the sequencing outlet channel 230 can be used as pumps instead of or in combination with the outlet pump 1114. Therefore, in one embodiment, the microfluidic cartridge 1100 includes only the outlet pump 1114, and the three diaphragm valves 246 are omitted. In another embodiment, the microfluidic cartridge 1100 includes only the three diaphragm valves 246, and the outlet pump 1114 is omitted. In yet another embodiment, the microfluidic cartridge 1100 includes the outlet pump 1114 and the three diaphragm valves 246. In yet another embodiment, the microfluidic cartridge 1100 includes any other type of pumping mechanism instead of the outlet pump 1114 and/or the three diaphragm valves 246. More details of an example of implementing the microfluidic cartridge 1100 are shown and described herein below with reference to Figures 25 to 60B.

Figures 25 and 26 show perspective views of a microfluidic cartridge assembly 1200, which is an example of a physical representation of the integrated microfluidic cartridge 1100 shown in Figure 24 . Microfluidic cartridge assembly 1200 is an example of a conventional injection-molded fluidic device integrated with flexible PCB technology. In this example, microfluidic cartridge assembly 1200 is a multi-compartment microfluidic cartridge comprising a housing 1210 secured atop a base plate 1212. Housing 1210 and base plate 1212 may, for example, be formed from a molded plastic and secured together via screws (see Figure 32 ). The overall height of microfluidic cartridge assembly 1200 may, for example, range from approximately 12 mm to approximately 100 mm. The overall length of microfluidic cartridge assembly 1200 may, for example, range from approximately 100 mm to approximately 200 mm. The overall width of microfluidic cartridge assembly 1200 may, for example, range from approximately 100 mm to approximately 200 mm.

Inside the housing 1210 is the fluidic assembly 1400 shown in Figures 27A and 27B. That is, Figures 27A and 27B show perspective views of an example of the fluidic assembly 1400 installed in the microfluidic cartridge assembly 1200 shown in Figures 25 and 28. The fluidic assembly 1400 is based on the integrated microfluidic cartridge 1100 shown in Figure 24. That is, the fluidic assembly 1400 includes the fluidic layer 200 shown and described in Figures 15 and 24. The fluidic assembly 1400 also includes a rotatable valve assembly 1410 arranged relative to the thirteen reagent channels 226a-226m in the reagent mixing and dispensing area 275 of the fluidic layer 200. The length of the fluidic layer 200 can be, for example, from about 100 mm to about 200 mm. The width of the fluidic layer 200 can be, for example, from about 100 mm to about 200 mm.

Additionally, fluidics assembly 1400 includes a flexible PCB heater 1412 wrapped around both sides of PCR zone 270 of fluidics layer 200. Two independently controlled heater traces are provided in flexible PCB heater 1412, such that there is one heater trace on one side of PCB zone 270 and another heater trace on the other side of PCB zone 270. Flexible PCB heater 1412 is an example of flexible PCB heater 818 of microfluidic cartridge 800 shown in FIG. 21 . Examples of heater traces are shown and described herein in further detail below with reference to FIG. 28A and 28B . Examples of flexible PCB heater 1412 are shown and described herein in further detail below with reference to FIG. 54A, 54B, and 54C .

25 and 26, the housing 1210 of the microfluidic cartridge assembly 1200 further includes four sample loading ports 1214 (e.g., sample loading ports 1214a, 1214b, 1214c, 1214d) that are substantially aligned with the four PCR channels 222 (e.g., PCR channels 222a, 222b, 222c, 222d) of the fluidic layer 200. The housing 1210 of the microfluidic cartridge assembly 1200 further includes 13 reagent reservoirs 1216 that feed the 13 reagent channels 226 (e.g., reagent channels 226a-226m) of the fluidic layer 200. The 13 reagent reservoirs 1216 can be of the same size or different sizes. For example, the reagent reservoirs 1216 can hold a volume of liquid ranging from approximately 0.001 ml to approximately 0.150 ml.

The housing 1210 of the microfluidic cartridge assembly 1200 also includes a waste reservoir 1218 fed by the sequencing outlet channel 230. The waste reservoir 1218 can hold, for example, a volume of liquid ranging from about 25 ml to about 100 ml. FIG26 shows that the reagent reservoir 1216 and the waste reservoir 1218 can be covered and sealed using, for example, a foil seal 1220.

Figures 28A and 28B respectively illustrate a plan view and a cross-sectional view of an example heater trace 1500 that can be installed in the fluidic assembly 1400 shown in Figures 27A and 27B. Specifically, Figure 28A illustrates a plan view of an example heater trace 1500 having a serpentine-type layout. Figure 28B illustrates a cross-sectional view of one side of a flexible PCB heater 1412 of the fluidic assembly 1400 that includes the heater trace 1500. Flexible PCB heater 1412 is a multilayer structure that sequentially includes, for example, a single-sided flexible copper layer 1510, an adhesive layer 1512, a dielectric layer 1514, a copper heater layer 1516 (in which the heater trace 1500 is patterned), and a layer 1518. Copper heater layer 1516 illustrates a cross-section of heater trace 1500 taken along line A-A in Figure 28A.

Figures 29, 30, 31, 32, 33A, and 33B show various other views of the microfluidic cartridge assembly 1200 of Figure 25, illustrating further details thereof. Specifically, Figure 29 shows a perspective view of the housing 1210 side of the microfluidic cartridge assembly 1200, while Figure 30 shows a plan view, both of which show further details of the arrangement of the 13 reagent reservoirs 1216 and the waste reservoir 1218. Figure 31 shows a plan view of the housing 1210 side of the microfluidic cartridge assembly 1200 with the foil seal 1220 installed. The foil seal 1220 has an opening that leaves the four sample loading ports 1214 exposed and accessible.

FIG32 shows a perspective view of the housing 1212 side of the microfluidic cartridge assembly 1200. FIG33A shows a plan view of the base plate 1212 side of the microfluidic cartridge assembly 1200. FIG33B shows a side view of the microfluidic cartridge assembly 1200. FIG32, 33A, and 33B show further details of the base plate 1212. Specifically, the base plate 1212 includes an opening 1222 and an opening 1224 for exposing a portion of the PCR region 270 of the fluidics layer 200 of the fluidics assembly 1400. Identified through the opening 1224 are a set of I/O pads 1226 for contacting the flexible PCB heater 1412 of the fluidics assembly 1400.

Along one edge of opening 1222 are four openings 1228 for accessing and activating the four diaphragm valves 242 of fluidic layer 200 of fluidic assembly 1400. That is, opening 1228a is substantially aligned with diaphragm valve 242a. Opening 1228b is substantially aligned with diaphragm valve 242b. Opening 1228c is substantially aligned with diaphragm valve 242c. Opening 1228d is substantially aligned with diaphragm valve 242d.

Along opposite edges of opening 1222 are four openings 1230 for accessing and actuating the four diaphragm valves 244 of fluidic layer 200 of fluidic assembly 1400. That is, opening 1230a is substantially aligned with diaphragm valve 244a. Opening 1230b is substantially aligned with diaphragm valve 244b. Opening 1230c is substantially aligned with diaphragm valve 244c. Opening 1230d is substantially aligned with diaphragm valve 244d.

In addition, the base plate 1212 includes an opening 1232 for accessing and activating the four diaphragm valves 246 of the fluid layer 200 of the fluid assembly 1400. The base plate 1212 also includes an opening 1234 at the sequencing chamber 258. One corner of the base plate 1212 has a bevel 1236, which is used to orient the microfluidic cartridge assembly 1200 in the instrument level of the microfluidic system (not shown). Figures 32 and 33A also show four screws 1238 for fastening the base plate 1212 to the housing 1210. In addition, the rotatable valve assembly 1410 is shown relative to the reagent mixing and dispensing area 275 of the fluid layer 200 of the fluid assembly 1400. The rotatable valve assembly 1410 includes a knob with a handle portion 1240, and a user or device can rotate the flow controller portion 1242 (see Figure 35) by the handle portion 1240.

Beginning with the microfluidic cartridge assembly 1200 oriented with the base plate 1212 side facing upward, Figures 34 through 42 essentially illustrate a step-by-step deconstruction of the microfluidic cartridge assembly 1200, as a means of revealing the placement and installation of the internal components therein. First, Figure 34 shows the microfluidic cartridge assembly 1200 with the base plate 1212 removed to expose the fluidics assembly 1400. In doing so, the flexible PCB layer 260 side of the fluidics layer 200 is visible. Furthermore, one side of the flexible PCB heater 1412 is visible. Also revealed is the spacer 1244 between the fluidics layer 200 and the base plate 1212. In Figure 34, the diaphragm valves 242, 244, and 246 are visible.

35 , the handle portion 1240 of the rotatable valve assembly 1410 has been removed, making visible the flow controller portion 1242. The underside of the handle portion 1240 (not shown) is designed to engage the flow controller 1242 so that the flow controller portion 1242 can be rotated to direct the flow of liquid through one of the 13 reagent channels 226.

Referring now to FIG. 36 , the flow controller portion 1242 of the rotatable valve assembly 1410 is removed so that the fluid paths associated with the PCR output channel 224 , reagent channel 226 , and sequencing feed channel 228 of the fluidics layer 200 are visible.

Referring now to FIG. 37 , the fluidics layer 200 is shown having transparency such that the fluidic paths within the microfluidic cartridge assembly 1200 are visible.

Referring now to Figure 38, the fluidic layer 200 is removed and the flexible PCB heater 1412 is shown alone within the housing 1210. Referring now to Figure 39, the flexible PCB heater 1412 is removed and the fluidic layer 200 is shown alone within the housing 1210.

Referring now to Figure 40, the fluid layer 200 and the flexible PCB heater 1412 are removed from the housing 1210. In this view, the flow paths in the housing 1210 associated with the sample loading port 1214, the 13 reagent reservoirs 1216, and the waste reservoir 1218 are visible. For example, the housing 1210 includes an opening 1246 for the sample loading port 1214, an opening 1248 for the 13 reagent reservoirs 1216, and an opening 1250 for the waste reservoir 1218. Figure 40 also shows four threaded holes 1252 for receiving screws 1238. In addition, Figure 40 shows a portion of the CMOS image sensor 262 and a protective cover 1254 covering the CMOS image sensor 262. Referring now to Figure 41, the CMOS image sensor 262 is removed so that the protective cover 1254 is fully visible. Referring now to Figure 42, the protective cover 1254 is removed, showing a clearance area 1256 in the housing 1210 associated with the CMOS image sensor 262.

43 shows a transparent perspective view of the housing 1210 of the microfluidic cartridge assembly 1200 to illustrate the positions of the openings relative to the sample loading port 1214, the reagent reservoir 1216, and the waste reservoir 1218. That is, in this view, we can see the position of the opening 1246 relative to the sample loading port 1214, the position of the opening 1248 relative to the reagent reservoir 1216, and the position of the opening 1250 relative to the waste reservoir 1218.

Figure 44 shows a transparent perspective view of the housing 1210 of the microfluidic cartridge assembly 1200, with the various fluid channels superimposed thereon. That is, in this view, we can see the positions of the various fluid channels relative to the sample loading port 1214, the reagent reservoir 1216, and the waste reservoir 1218. Figure 45 shows a cross-sectional view of the microfluidic cartridge assembly 1200 of Figure 25, showing more details therein.

Figures 46A, 46B, 47A, 47B and 48 illustrate various views of the housing 1210 of the microfluidic cartridge assembly 1200 of Figure 25, which illustrate more details thereof. That is, Figures 46A and 46B illustrate a plan view and a side view of housing 1210, respectively. In one example, housing 1210 is from about 12mm to about 100mm in height, from about 100mm to about 200mm in length, and from about 100mm to about 200mm in width. Figure 47A illustrates a perspective view of housing 1210 without foil seal 1220. Figure 47B illustrates a perspective view of housing 1210 with foil seal 1220 installed. Although Figures 46A, 46B, 47A and 47B illustrate the exterior of housing 1210, Figure 48 illustrates an interior plan view of housing 1210.

Figures 49, 50, 51A, 51B, and 52 illustrate various views of the base plate 1212 of the microfluidic cartridge assembly 1200 of Figure 25, showing further details thereof. Specifically, Figures 49 and 50 illustrate perspective views of the exterior and interior of the base plate 1212, respectively. Figure 51A illustrates a plan view of the exterior of the base plate 1212, while Figure 51B illustrates a side view of the base plate 1212. Figures 49, 50, 51A, 51B, and 52 illustrate that the base plate 1212 further includes four holes 1258 for receiving screws 1238, a recessed area 1260 having a central opening 1262 for receiving the handle portion 1240 and the flow controller portion 1242 of the rotatable valve assembly 1410.

Figures 53A and 53B illustrate additional perspective views of fluidics assembly 1400 of microfluidic cartridge assembly 1200, showing greater detail therein. Specifically, Figures 53A and 53B each show a perspective view of fluidics assembly 1400. Figure 53A illustrates fluidics assembly 1400 without flexible PCB heater 1412, while Figure 53B illustrates fluidics assembly 1400 with flexible PCB heater 1412 installed. Furthermore, recess 1414 is provided on one edge of fluidics layer 200 and within PCR zone 270. Recess 1414 is designed to accommodate flexible PCB heater 1412.

Figures 54A, 54B, and 54C illustrate additional details of the flexible PCB heater 1412 of the fluidics assembly 1400 of the microfluidic cartridge assembly 1200. Specifically, Figures 54A and 54B each show a perspective view of each side of the flexible PCB heater 1412, while Figure 54C shows a side view of the flexible PCB heater 1412. The flexible PCB heater 1412 includes a U-shaped wraparound panel 1416 and a side extension panel 1418, both formed using flexible PCB technology. The U-shaped wraparound panel 1416 includes a panel 1420 and a panel 1422, each of which has heater traces 1500 patterned therein, namely, heater traces 1500a and 1500b. Examples of heater traces 1500 are shown in Figures 28A and 28B. The space between panels 1420 and 1422 is configured so that the flexible PCB heater 1412 can be press-fit onto the PCR region 270 of the fluidics layer 200 and into the recess 1414, as shown in Figure 53B. Figures 54B and 54C also show I/O pads 1226, which provide electrical connections to the two heater traces 1500 and to the CMOS image sensor 262.

Side extension panels 1418 extend from panel 1420 near the bend in the U-shaped wrap panel 1416. Side extension panels 1418 are designed to extend toward the CMOS image sensor 262. As shown in FIG53B , the end of side extension panel 1418 farthest from the U-shaped wrap panel 1416 is shaped to fit against the CMOS image sensor 262. The purpose of side extension panels 1418 is to provide electrical connection to the CMOS image sensor 262 assembled atop a rigid or flexible PCB.

Figures 55A and 55B show a perspective view and a plan view of the inlet/outlet layer 210 of the fluidic layer 200 shown in Figures 15 and 27, respectively. Again, the inlet/outlet layer 210 can be formed of, for example, polycarbonate or any other material suitable for use in the R2R process. The inlet/outlet layer 210 provides an interface between the fluidic layer 200 and the housing 1210 of the microfluidic cartridge assembly 1200. That is, the inlet/outlet layer 210 provides a fluid path from the sample loading port 1214, 13 reagent reservoirs 1216, and waste reservoir 1218 of the housing 1210 to the fluid channel layer 220 of the fluidic layer 200. For example, the inlet/outlet layer 210 includes a set of openings 212 that are substantially aligned with the opening 1246 of the sample loading port 1214 in the housing 1210. The inlet/outlet layer 210 includes a set of openings 214 that are substantially aligned with the openings 1248 of the reagent reservoir 1216 in the housing 1210. The inlet/outlet layer 210 also includes a set of openings 216 that are substantially aligned with the openings 1250 of the waste reservoir 1218 in the housing 1210.

Figures 56A and 56B respectively show a perspective view and a plan view of the fluidic channel layer 220 of the fluidic layer 200 shown in Figures 15 and 27 . Again, the fluidic channel layer 220 can be formed, for example, from polycarbonate or any other material suitable for use in the R2R process. The fluidic channel layer 220 is the layer within the fluidic layer 200 where the flow of all liquids is facilitated. In other words, all PCR and sequencing operations occur within the fluidic channel layer 220. PCR operations occur within the PCR channels 222 within the PCR region 270. The PCR output channels 224 feed the reagent mixing and dispensing region 275. Reagent dispensing occurs using the reagent channels 226 within the reagent mixing and dispensing region 275. Thirteen reagent channels 226 are patterned to feed the rotatable valve assembly 1410. Sequencing feed channels 228 feed the inlet of the sequencing chamber 258 of the sequencing chamber layer 250 shown in Figures 58A and 58B . The sequencing outlet channel 230 is then fluidly connected to the outlet of the sequencing chamber 258 .

Figures 57A and 57B illustrate perspective and plan views, respectively, of the flexible PCB layer 260 of the fluidic layer 200 shown in Figures 15 and 27 . Again, the flexible PCB layer 260 can be formed, for example, from polycarbonate or any other material suitable for use in an R2R process. The flexible PCB layer 260 includes a set of openings (or holes) 264 associated with the inlet/outlet of the diaphragm valve 242. The flexible PCB layer 260 also includes a set of openings (or holes) 266 associated with the inlet/outlet of the diaphragm valve 244. If the diaphragm valve 246 is present, the flexible PCB layer 260 includes a set of openings (or holes) 267 associated with the inlet/outlet of the diaphragm valve 246. Furthermore, the flexible PCB layer 260 includes a set of openings 268 that are substantially aligned with the rotatable valve assembly 1410 and provide a fluid path to the rotatable valve assembly 1410.

Figures 58A and 58B illustrate perspective and plan views, respectively, of the sequencing chamber bottom layer 280 of the fluidics layer 200 shown in Figures 15 and 27 . Again, the sequencing chamber bottom layer 280 can be formed, for example, from polycarbonate or any other material suitable for use in an R2R process. The sequencing chamber bottom layer 280 includes a set of openings 282 for the diaphragm valve 242 formed within the stack of layers of the fluidics layer 200. The sequencing chamber bottom layer 280 includes a set of openings 284 for the diaphragm valve 244 formed within the stack of layers of the fluidics layer 200. If the diaphragm layer 246 is present, the sequencing chamber bottom layer 280 includes a set of openings 286 for the diaphragm valve 246 formed within the stack of layers of the fluidics layer 200. Furthermore, the sequencing chamber bottom layer 280 includes a set of openings 288 that are substantially aligned with and provide a fluid path to the rotatable valve assembly 1410. Furthermore, the sequencing chamber bottom layer 280 includes a pair of openings 289 that are fluidly coupled to the sequencing chambers 258 of the sequencing chamber layer 250 .

Sequencing chamber bottom layer 280 is a layer of fluidic layer 200 in which CMOS technology is integrated. That is, CMOS image sensor 262 is mounted on sequencing chamber bottom layer 280. The position of CMOS image sensor 262 substantially corresponds to the position of sequencing chamber 258 of sequencing chamber layer 250.

Figures 59A and 59B show a perspective view and a plan view, respectively, of the sequencing chamber layer 250 of the fluidics layer 200 shown in Figures 15 and 27. Again, the sequencing chamber layer 250 can be formed, for example, from polycarbonate or any other material suitable for use in an R2R process. The sequencing chamber layer 250 is the layer of the fluidics layer 200 where sequencing operations occur; that is, using sequencing chamber 258.

Sequencing chamber layer 250 includes a set of openings 252 for diaphragm valve 242 formed within the stack of layers of fluidics layer 200. Sequencing chamber layer 250 also includes a set of openings 254 for diaphragm valve 244 formed within the stack of layers of fluidics layer 200. If diaphragm valve 246 is present, sequencing chamber layer 250 includes a set of openings 255 for diaphragm valve 246 formed within the stack of layers of fluidics layer 200. In addition, sequencing chamber layer 250 includes a set of openings 256 that are substantially aligned with and provide a fluid path to rotatable valve assembly 1410.

Figures 60A and 60B illustrate perspective and plan views, respectively, of the membrane layer 240 and sequencing chamber top layer 290 of the fluidics layer 200 shown in Figures 15 and 27 . The membrane layer 240 can be formed, for example, from a silicone elastomer, while the sequencing chamber top layer 290 can be formed, for example, from COC. The membrane layer 240 serves as an elastic membrane for opening and closing the membrane valves 242, 244, and 246 within the stack of fluidics layer 200, wherein the membrane valves 242, 244, and 246 are sequentially created by the combination of the flexible PCB layer 260, the sequencing chamber bottom layer 280, the sequencing chamber layer 250, and the membrane layer 240. Figures 60A and 60B also illustrate the sequencing chamber top layer 290 used to cover the sequencing chamber 258 of the sequencing chamber layer 250.

Figures 61A and 61B illustrate a flow chart of an example of a method 4800 for performing multiplex PCR and downstream mixing required for sequencing using a microfluidic cartridge assembly 1200. Because the microfluidic cartridge assembly 1200 is based on the microfluidic cartridge 1100 shown in Figure 24, the microfluidic cartridge assembly 1200 is configured to be used for 4X sample multiplexing. In addition, in method 4800, 13 reagent reservoirs 1216 are designated reagent reservoirs 1216a, 1216b, 1216c, 1216d, 1216e, 1216f, 1216g, 1216h, 1216i, 1216j, 1216k, 1216l, and 1216m. In addition, method 4800 utilizes an outlet pump 1114, which is fluidically connected to the microfluidic cartridge assembly 1200. The outlet pump 1114 is located downstream of the sequencing chamber 258. The outlet pump 1114 can provide positive and negative pressure (i.e., vacuum pressure). Method 4800 includes but is not limited to the following steps.

At step 4810, the microfluidic cartridge assembly 1200 is provided ready for use. That is, the microfluidic cartridge assembly 1200 is provided with one or more of its reservoirs filled with the desired liquids. For example, the reagent reservoirs 1216 can be filled with the same or different reagent liquids. In one example, all reagent reservoirs 1216a-m are filled with hydrogenation buffer (HT1). Method 4800 proceeds to step 4815.

In step 4815, all diaphragm valves are closed, and then sample/PCR MIX is loaded into. "PCR MIX" means the PCR Master Mix that is optimized for use in the conventional PCR for amplifying DNA template. In this step, diaphragm valve 242a and 244a are closed, diaphragm valve 242b and 244b are closed, diaphragm valve 242c and 244c are closed, and diaphragm valve 242d and 244d are closed. In this way, PCR passage 222a, 222b, 222c and 222d are all blocked completely. Then, the first sample liquid mixes with PCR MIX (hereinafter referred to as sample/PCR_MIX1) and is loaded in the sample loading port 1214a. The second sample liquid mixes with PCR MIX (hereinafter referred to as sample/PCR_MIX2) and is loaded in the sample loading port 1214b. The third sample liquid is mixed with the PCR MIX (hereinafter referred to as sample/PCR_MIX3) and loaded into sample loading port 1214c. The fourth sample liquid is mixed with the PCR MIX (hereinafter referred to as sample/PCR_MIX4) and loaded into sample loading port 1214d. Upon completion of this step, a volume of sample/PCR MIX is located in each sample loading port 1214 and is ready for processing. Method 4800 proceeds to step 4820.

At step 4820, the diaphragm valve for the first sample is opened. The first sample is then drawn into the PCR zone. The diaphragm valve for the first sample is then closed. For example, diaphragm valves 242a and 244a of PCR channel 222a are opened. Sample/PCR_MIX1 is then drawn into PCR channel 222a using outlet pump 1114. Diaphragm valves 242a and 244a of PCR channel 222a are then closed, with the volume of sample/PCR_MIX1 now sealed within PCR channel 222a. Method 4800 proceeds to step 4825.

At decision step 4825, a determination is made as to whether another sample is waiting to be loaded into the PCR area, i.e., PCR area 270. If so, method 4800 proceeds to step 4830. If not, method 4800 proceeds to step 4835.

In step 4830, the diaphragm valve for the next sample is opened. The next sample is then drawn into the PCR zone. The diaphragm valve for the next sample is then closed. In one example, diaphragm valves 242b and 244b of PCR channel 222b are opened. Sample/PCR_MIX2 is then drawn into PCR channel 222b using outlet pump 1114. Diaphragm valves 242b and 244b of PCR channel 222b are then closed, with the volume of sample/PCR_MIX2 now sealed within PCR channel 222b.

In another example, diaphragm valves 242c and 244c of PCR channel 222c are opened. Then, using outlet pump 1114, sample/PCR_MIX3 is drawn into PCR channel 222c. Then, diaphragm valves 242c and 244c of PCR channel 222c are closed, wherein the volume of sample/PCR_MIX3 is now sealed inside PCR channel 222c.

In yet another example, diaphragm valves 242d and 244d of PCR channel 222d are opened. Then, sample/PCR_MIX4 is drawn into PCR channel 222d using outlet pump 1114. Then, diaphragm valves 242d and 244d of PCR channel 222d are closed, wherein the volume of sample/PCR_MIX4 is now sealed inside PCR channel 222d.

Method 4800 returns to step 4825.

At step 4835, a PCR operation is performed using sample/PCR_MIX1 in PCR channel 222a, sample/PCR_MIX2 in PCR channel 222b, sample/PCR_MIX3 in PCR channel 222c, and sample/PCR_MIX4 in PCR channel 222d. When the PCR operation is complete, sample/PCR_MIX1 is now referred to as PCR_MIX1, sample/PCR_MIX2 is now referred to as PCR_MIX2, sample/PCR_MIX3 is now referred to as PCR_MIX3, and sample/PCR_MIX4 is now referred to as PCR_MIX4. Method 4800 proceeds to step 4840.

At step 4840 , the rotatable valve is rotated to the first PRC MIX position. For example, by rotating the handle portion 1240 of the rotatable valve assembly 1410 , the rotatable valve assembly 1410 is positioned to hold the PCR channel 222 a of PCR_MIX1. The method 4800 proceeds to step 4845 .

At step 4845, the diaphragm valve of the first PCR MIX is opened. The first PCR MIX is then pulled through the rotatable valve to the CMOS device. The diaphragm valve of the first PCR MIX is then closed. For example, diaphragm valves 242a and 244a of PCR channel 222a are opened. Then, using outlet pump 1114, PCR_MIX1 is pulled out of PCR channel 222a into PCR output channel 224 and through rotatable valve assembly 1410. Diaphragm valves 242a and 244a are then closed. Method 4800 proceeds to step 4850.

At step 4850, the rotatable valve is rotated to the hydrogenation buffer (HT1) position, indicating that reagent reservoir 1216 holds HT1. In method 4800, at least one reagent reservoir 1216 holds the volume of HT1. For example, reagent reservoir 1216k holds the volume of HT1. Therefore, by rotating handle portion 1240 of rotatable valve assembly 1410, the position of rotatable valve assembly 1410 is now set to reagent reservoir 1216k holding HT1. Method 4800 proceeds to step 4855.

At step 4855, the first PCR MIX is pushed into the HT1 reservoir. For example, using outlet pump 1114, PCR_MIX1 is pushed through rotatable valve assembly 1410 and into reagent reservoir 1216k where it mixes with the HT1 therein. Method 4800 proceeds to step 4860.

In decision step 4860, it is determined whether another PCR MIX is waiting to be mixed with HT1. If so, method 4800 proceeds to step 4865. If not, method 4800 proceeds to step 4885.

At step 4865, the rotatable valve is rotated to the next PRC MIX position. In one example, by rotating the handle portion 1240 of the rotatable valve assembly 1410, the position of the rotatable valve assembly 1410 is set to PCR channel 222b holding PCR_MIX2. In another example, by rotating the handle portion 1240 of the rotatable valve assembly 1410, the position of the rotatable valve assembly 1410 is set to PCR channel 222c holding PCR_MIX3. In yet another example, by rotating the handle portion 1240 of the rotatable valve assembly 1410, the position of the rotatable valve assembly 1410 is set to PCR channel 222d holding PCR_MIX4. Method 4800 proceeds to step 4870.

In step 4870, the diaphragm valve of the next PCR MIX is opened. Then, the next PCR MIX is pulled to the CMOS device through the rotatable valve. Then, the diaphragm valve of the next PCR MIX is closed. In one example, diaphragm valves 242b and 244b of PCR channel 222b are opened. Then, using outlet pump 1114, PCR_MIX2 is pulled out of PCR channel 222b into PCR output channel 224 and passes through rotatable valve assembly 1410. Then, diaphragm valves 242b and 244b are closed. In another example, diaphragm valves 242c and 244c of PCR channel 222c are opened. Then, using outlet pump 1114, PCR_MIX3 is pulled out of PCR channel 222c into PCR output channel 224 and passes through rotatable valve assembly 1410. Then, diaphragm valves 242c and 244c are closed. In another example, diaphragm valves 242d and 244d of PCR channel 222d are opened. Then, using outlet pump 1114, PCR_MIX4 is pulled out of PCR channel 222d into PCR output channel 224 and through rotatable valve assembly 1410. Diaphragm valves 242d and 244d are then closed. Method 4800 proceeds to step 4875.

At step 4875 , the rotatable valve is rotated to the HT1 position. For example, by rotating the handle portion 1240 of the rotatable valve assembly 1410 , the rotatable valve assembly 1410 is returned to the position of the reagent reservoir 1216 k holding HT1. The method 4800 proceeds to step 4880 .

In step 4880, the next PCR MIX is pushed into the HT1 reservoir. In one example, using the outlet pump 1114, PCR_MIX2 is pushed through the rotatable valve assembly 1410 and enters the reagent reservoir 1216k and mixes with the HT1 therein. In another example, using the outlet pump 1114, PCR_MIX3 is pushed through the rotatable valve assembly 1410 and enters the reagent reservoir 1216k and mixes with the HT1 therein. In yet another example, using the outlet pump 1114, PCR_MIX4 is pushed through the rotatable valve assembly 1410 and enters the reagent reservoir 1216k and mixes with the HT1 therein. Method 4800 returns to step 4860.

In step 4885, the mixture from the HT1 reservoir is pushed into the sequencing chamber, and the clustering/sequencing method is executed. For example, using reagent reservoir 1216k, which now holds a mixture of HT1, PCR_MIX1, PCR_MIX2, PCR_MIX3, and PCR_MIX4, this mixture is pulled out of reagent reservoir 1216k, then along sequencing feed channel 228 and into sequencing chamber 258. The clustering/sequencing method is then executed using CMOS image sensor 262. Method 4800 ends.

One or more embodiments may include a CMOS flow cell with accessible biosensor active areas. For example, the CMOS flow cell may be designed as a single-use consumable. Accordingly, it may be beneficial for the CMOS flow cell to be a small and inexpensive device. In a small CMOS flow cell, it is important to use as much biosensor active area as possible. However, current CMOS flow cell designs do not allow 100% utilization of the biosensor active area. Therefore, new methods are needed to provide increased utilization of the biosensor active area in a CMOS flow cell. The embodiments set forth herein may include a CMOS flow cell in which a majority or up to approximately 100% of the biosensor active area is accessible for reagent delivery and illumination, as shown and described herein below with reference to Figures 62 to 75.

FIG62 shows a side view of an example of a CMOS flow cell 4900, in which a majority, or up to approximately 100%, of the biosensor active area is accessible for reagent delivery and illumination. CMOS flow cell 4900 includes a PCB substrate 4910, which may be, for example, a flexible PCB substrate. Atop PCB substrate 4910 is a CMOS biosensor device 4920. CMOS biosensor device 4920 is a CMOS image sensor with a biolayer thereon. Also atop PCB substrate 4910 and surrounding CMOS biosensor device 4920 is a laminate film 4930. Laminate film 4930 may be formed, for example, from epoxy, polyimide or other plastic films, silicon, a bismaleimide triazine resin (BT) substrate, or the like. PCB substrate 4910 and laminate film 4930 may be formed using flexible PCB technology. Alternatively, a planarized surface may be created by machining cavities into the PCB substrate.

The purpose of the laminating film 4930 is to provide an extended surface around the perimeter of the CMOS biosensor device 4920 that is substantially coplanar with the top of the CMOS biosensor device 4920. In one example, if the wafer thickness of the CMOS biosensor device 4920 is approximately 100 μm, the thickness of the laminating film 4930 is approximately 100 μm + approximately 5 μm.

The elongated gap between the PCB substrate 4910 and the laminate film 4930 forms a trench or channel 4950 around the perimeter of the CMOS biosensor device 4920. The width of the trench or channel 4950 can be, for example, from about 100 μm to about 1000 μm. The trench or channel 4950 is filled with a filling material 4952 to form a substantially continuous, flat surface spanning the CMOS biosensor device 4920 and the laminate film 4930. The filling material 4952 is a material that does not interfere with the reactions occurring atop the CMOS biosensor device 4920. The filling material 4952 can be, for example, an ultraviolet (UV)-cured epoxy, a thermally cured epoxy, or the like.

Atop the CMOS biosensor device 4920 and the laminate film 4930 is a flow cell cover 4940 into which a flow channel 4942 is integrated. Furthermore, the flow cell cover 4940 includes a first port 4944 and a second port 4946 providing inlet/outlet access to the flow channel 4942. The flow cell cover 4940 is formed from a material that is optically transparent and has low or no autofluorescence in the portion of the spectrum used for analytical detection, such as, but not limited to, cyclic olefin copolymer (COC). The total thickness of the flow cell cover 4940 can be, for example, from about 300 μm to about 1000 μm. A bonding area is present on the exterior of the flow channel 4942 for bonding the flow cell cover 4940 to the laminate film 4930. Bonding can be via a low autofluorescence adhesive.

Because a substantially continuous flat surface exists across the CMOS biosensor device 4920 and the laminate film 4930, the flow channel 4942 within the flow cell cover 4940 can be sized to span the entire CMOS biosensor device 4920; that is, it can span approximately 100% of the biosensor active area. In one example, if the wafer size of the CMOS biosensor device 4920 is approximately 8 mm x 9 mm, the active area is approximately 7 mm x 8 mm. However, the wafer size of the CMOS biosensor device 4920 can range up to, for example, approximately 25 mm x 25 mm, with a proportionally larger active area.

FIG62 shows, for example, a reagent fluid 4954 filling a flow channel 4942. A chemical reaction occurs in the reagent fluid 4954 in the flow channel 4942, which is atop a CMOS biosensor device 4920. When illuminated through the flow cell cover 4940, the CMOS biosensor device 4920 senses the chemical reaction occurring in the flow channel 4942. Electrical connections (not shown) are provided through the PCB substrate 4910 for acquiring signals from the CMOS biosensor device 4920. In the CMOS flow cell 4900, approximately 100% of the biosensor active area of the CMOS biosensor device 4920 is accessible for reagent delivery and illumination.

FIG63 shows an exploded view of an illustrative example of the CMOS flow cell 4900 shown in FIG62 . FIG63 shows that the CMOS biosensor device 4920 includes an active area 4922. Any portion of the CMOS biosensor device 4920 outside the active area 4922 is an inactive area 4924. The CMOS biosensor device 4920 can be attached to the PCB substrate 4910 using, for example, flip-chip technology. Furthermore, the laminate film 4930 includes an opening or window 4932 sized to receive the CMOS biosensor device 4920 when laminated against the PCB substrate 4910. The opening or window 4932 is provided in the laminate film 4930 prior to lamination to the PCB substrate 4910. When the flow cell cover 4940 is bonded to the laminate film 4930, the flow channel 4942 is substantially aligned with the CMOS biosensor device 4920, and its area extends beyond the area of the CMOS biosensor device 4920. In Figure 63, the flow cell cover 4940 is shown as transparent. Figures 64 and 65 show perspective and side views, respectively, of the CMOS flow cell 4900 shown in Figure 63 when fully assembled.

FIG66 shows perspective views of an example of a flow cell cover 4940 of the CMOS flow cell 4900 shown in FIG63, 64, and 65. Specifically, FIG66 shows top and bottom perspective views of the flow cell cover 4940 of the CMOS flow cell 4900 shown in FIG63, 64, and 65. In this example, the diameters of the first port 4944 and the second port 4946 can be approximately 750 μm. Furthermore, the depth or height of the flow channel 4942 can be approximately 100 μm.

Figures 67, 68, 69 and 70 illustrate examples of processes for providing an extended planar surface in a CMOS flow cell onto which a flow cell cover can be mounted.

67 , a laminate film 4930 and a CMOS biosensor device 4920 are placed atop a PCB substrate 4910. A trench or channel 4950 exists around the perimeter of the CMOS biosensor device 4920. The trench or channel 4950 exists because the opening or window 4932 in the laminate film 4930 is slightly larger than the CMOS biosensor device 4920.

In the next step and referring now to FIG68 , the upper side of the groove or channel 4950 is sealed using, for example, an optically transparent elastomer 4960 that fits snugly against the groove or channel 4950. The elastomer 4960 is optically transparent so that UV light can pass through it. The purpose of the elastomer 4960 is to seal the top of the groove or channel 4950 in preparation for filling.

In the next step and now referring to Figure 69, using a pair of through-holes 4916 in the PCB substrate 4910, the groove or channel 4950 is filled with a filler material 4952, such as a UV cured epoxy, which is why the elastomer 4960 is optically transparent.

In the next step and now referring to Figure 70, once the filler material 4952 is cured, the elastomer 4960 is removed and a substantially continuous flat surface now exists in the flow cell for receiving a flow cell cover, such as flow cell cover 4940.

Figures 71A, 71B, 71C and 71D illustrate another example of a process for providing an extended planar surface in a CMOS flow cell onto which a flow cell cover can be mounted.

In a first step and referring now to FIG. 71A , a CMOS biosensor device 4920 is placed atop a PCB substrate 4910 .

71B , a mold 5510 (e.g., a clamshell type mold) is placed around the CMOS biosensor device 4920 and the PCB substrate 4910. The mold 5510 provides a space or void 5512 atop the PCB substrate 4910 and around the perimeter of the CMOS biosensor device 4920.

In the next step and referring now to FIG. 71C , the space or void 5512 in the mold 5510 is filled with a filler material 4952 , such as a UV-curable or heat-curable epoxy, using, for example, a low pressure injection molding process or a reaction injection molding process.

In the next step and now referring to Figure 71D, once the filling material 4952 is cured, the mold 5510 is removed and a substantially continuous flat surface now exists in the flow cell for receiving a flow cell cover, such as flow cell cover 4940.

72, 73, 74 and 75 illustrate yet another example of a process for providing an extended planar surface in a CMOS flow cell onto which a flow cell cover can be mounted.

In a first step, and referring now to FIG. 72 , a CMOS biosensor device 4920 is placed atop a PCB substrate 4910. Furthermore, a mechanical material sheet 5910 is placed atop the PCB substrate 4910 at one end of the CMOS biosensor device 4920. Similarly, a mechanical material sheet 5912 is placed atop the PCB substrate 4910 at the other end of the CMOS biosensor device 4920. Mechanical material sheets 5910 and 5912 can be, for example, blank silicon, glass, or plastic. A trench or channel 5914 is between the mechanical material sheet 5910 and the CMOS biosensor device 4920. Another trench or channel 5914 is between the mechanical material sheet 5912 and the CMOS biosensor device 4920.

73 , a set of barriers 5916 are placed at the ends of the trenches or channels 5914. For example, barriers 5916a and 5916b block the end of one trench or channel 5914, while barriers 5916c and 5916d block the end of another trench or channel 5914 in preparation for filling.

In the next step and referring now to Figure 74, the groove or channel 5914 is filled with a filler material 4952, such as a UV-cured or thermally cured epoxy. The filler material 4952 is held between the barriers 5916a and 5916b and between the barriers 5916c and 5916d.

In the next step and referring now to FIG. 75 , once the fill material 4952 is cured, a substantially continuous flat surface now exists in the flow cell for receiving a flow cell cover, such as flow cell cover 4940 .

It will be appreciated that various aspects of the present disclosure may be embodied as methods, systems, computer-readable media, and/or computer program products. Aspects of the present disclosure may take the form of hardware implementations, software implementations (including firmware, resident software, microcode, etc.), or combined software and hardware implementations, all of which may be referred to herein as "circuits," "modules," or "systems." Additionally, the methods of the present disclosure may take the form of a computer program product on a computer-usable storage medium having computer-usable program code embodied in the medium.

Any suitable computer-usable medium can be used for the software aspects of the present disclosure. A computer-usable or computer-readable medium can be, for example, but not limited to, an electronic, magnetic, optical, electromagnetic, infrared, or semiconductor system, device, apparatus, or propagation medium. A computer-readable medium can include temporary and/or non-temporary implementations. More specific examples of computer-readable media (a non-exhaustive list) would include some or all of the following: an electrical connection with one or more wires, a portable computer disk, a hard disk, a random access memory (RAM), a read-only memory (ROM), an erasable programmable read-only memory (EPROM or flash memory), an optical fiber, a portable compact disc read-only memory (CD-ROM), an optical storage device, a transmission medium such as one that supports the Internet or an intranet, or a magnetic storage device. Note that a computer-usable or computer-readable medium can even be paper or another suitable medium on which the program is printed, as the program can be electronically captured, for example, via an optical scan of paper or other medium, and then compiled, interpreted, or otherwise processed in an appropriate manner if necessary, and then stored in a computer memory. In the context of this document, a computer-usable or computer-readable medium may be any medium that can contain, store, communicate, propagate, or transport the program for use by or in connection with the instruction execution system, apparatus, or device.

The program code for the operation of the method and apparatus set forth herein can be written using an object-oriented programming language such as Java, Smalltalk, C++, etc. However, the program code for the operation of the method and apparatus set forth herein can also be written using a conventional process programming language such as "C" programming language or similar programming language. The program code can be executed by a processor, an application specific integrated circuit (ASIC) or other components that execute the program code. The program code can be simply referred to as a software application, which is stored in a memory (such as a computer-readable medium discussed above). The program code can cause a processor (or any processor-controlled device) to produce a graphical user interface ("GUI"). The graphical user interface can be produced visually on a display device, but the graphical user interface also can have audible features. However, the program code can be operated in any processor-controlled device such as a computer, server, personal digital assistant, telephone, television, or any processor-controlled device utilizing a processor and/or digital signal processor.

The program code can be executed locally and/or remotely. The program code can, for example, be stored in whole or in part in the local memory of the device controlled by the processor. However, the program code can also be stored, accessed and downloaded to the device controlled by the processor at least in part remotely. The user's computer can, for example, execute the program code in its entirety or only in part. The program code can be an independent software package executed at least in part on the user's computer and/or in part on a remote computer or entirely on a remote computer or server. In the latter case, the remote computer can be connected to the user's computer via a communication network.

The methods and devices described herein can be applied regardless of the networking environment. The communication network can be a cable network operating in the radio frequency domain and/or the Internet Protocol (IP) domain. The communication network can also include a distributed computing network, such as the Internet (sometimes optionally referred to as a "wide area network"), an intranet, a local area network (LAN) and/or a wide area network (WAN). The communication network can include coaxial cables, copper wires, fiber optic lines and/or hybrid-coaxial lines. The communication network can even include a wireless portion utilizing any part of the electromagnetic spectrum and any signaling standard (such as the IEEE 802 series of standards, GSM/CDMA/TDMA or any cellular standard and/or ISM band). The communication network can even include a power line portion, where signals are transmitted via electrical wiring. The methods and devices described herein can be applied to any wireless/wired communication network, regardless of physical components, physical configuration or communication standards.

Certain aspects of the present disclosure have been described with reference to various methods and method steps. It will be understood that each method step can be implemented by program code and/or by machine instructions. Program code and/or machine instructions can create a module for implementing the functions/actions specified in the method.

The program code may also be stored in a computer-readable memory that can direct a processor, a computer, or other programmable data processing device to function in a specific manner so that the program code stored in the computer-readable memory generates or converts an article of manufacture including an instruction module that implements various aspects of the method steps.

The program code may also be loaded onto a computer or another programmable data processing apparatus to cause a series of operational steps to be executed to produce a processor/computer implemented process, such that the program code provides steps for implementing the various functions/actions specified in the methods of the present disclosure.

In an embodiment, a system is provided that includes a removable cartridge having a cartridge housing. The removable cartridge further includes a fluid network disposed within the cartridge housing. The fluid network is configured to receive and fluidically direct a biological sample for at least one of sample analysis or sample preparation. The removable cartridge further includes a flow control valve that is operably coupled to the fluid network and movable relative to the fluid network to control the flow of the biological sample therethrough. The cartridge housing includes a housing side that defines an exterior of the removable cartridge and allows access to the flow control valve. The system further includes a base instrument having a control side configured to detachably engage the housing side of the removable cartridge. The housing side and the control side together define a system interface. The base instrument includes a valve actuator that engages the flow control valve via the system interface. The removable cartridge further includes a detection assembly held by at least one of the removable cartridge or the base instrument. The detection assembly includes an imaging detector and a reaction chamber in fluid communication with the fluid network. The imaging detector is configured to detect a specified reaction within the reaction chamber.

In one aspect, the control side of the base instrument as described herein and the housing side of the removable cartridge as described herein are generally planar and face each other. The system interface can be single-sided, wherein the base instrument and the removable cartridge are operatively coupled to each other only via the housing side and the control side. Alternatively, the base instrument and the removable cartridge can be operatively coupled such that the base instrument and the removable cartridge are secured to each other at the system interface using at least one of a fluidic coupling, an electrical coupling, or a thermal coupling established via the system interface.

In another aspect, the control side of the base instrument described herein can represent the top of the base instrument relative to gravity, such that the removable cartridge rests on and is supported by the base instrument.

In another aspect, the valve actuator of the base instrument described herein can include an elongated actuator body extending through the housing side and into the cartridge housing.

In another aspect, the flow control valve of the removable cassette described herein may include an elongated actuator body extending through a control side and into a base instrument.

In another aspect, the base instrument described herein may have an instrument side facing in an opposite direction relative to the control side. The base instrument may have an instrument dimension extending between the control side and the instrument side. The base instrument and the removable case may have a combined dimension that is greater than the instrument dimension.

In another aspect, each of the removable cartridge and the base instrument can include a contact array of electrical contacts. The contact arrays can be electrically coupled to each other at the system interface.

In another aspect, the housing side of the removable cartridge described herein can be a first housing side, and the cartridge housing can further include a second housing side. The first and second housing sides face in different directions. The system interface is a multi-sided interface, wherein the base instrument and the removable cartridge are operatively coupled to each other along the first and second housing sides.

Alternatively, the first and second housing sides of the removable cartridge described herein may be generally perpendicular to one another. The base instrument may have an instrument housing including first and second control sides, the first and second control sides oriented in perpendicular directions and forming an open-side recess of the base instrument. At least a portion of the removable cartridge may be disposed within the open-side recess such that the first and second housing sides engage the first and second control sides.

In one aspect, a valve actuator of the basic instrument described herein can include an elongated body extending through a system interface between a first housing side and a first control side. The second housing side and the second control side can include respective contact arrays of electrical contacts. The contact arrays can be electrically coupled to each other along the system interface.

In another aspect, the first and second housing sides of the removable cartridge described herein face generally opposite directions. The base instrument may have an instrument side and a cartridge receiving slot open to the instrument side. The removable cartridge may be disposed within the cartridge receiving slot.

In another aspect, the removable cartridge and the base instrument are fluidically coupled along a first housing side and electrically coupled along a second housing side. Optionally, the base instrument includes a locking mechanism that engages at least one of the first housing side or the second housing side to retain the removable cartridge within the base instrument.

In another aspect, each of the removable cartridge and the base instrument can include a flow port. The flow ports are fluidly coupled to each other at a system interface.

In another aspect, the systems described herein can include a locking mechanism attached to at least one of the removable cartridge or the base instrument. The locking mechanism is configured to removably secure the cartridge housing to the base instrument.

In another aspect, the imaging detector of the systems described herein can be held by a base instrument and the reaction chamber can be held by a removable cartridge.

In another aspect, a flow control valve of a removable cartridge as described herein may include a flexible membrane configured to control the flow of a biological sample through a fluidic network. The flexible membrane may be bent by a valve actuator between first and second conditions.

In another aspect, a housing side of a cartridge housing of a removable cartridge as described herein may include an access opening therethrough that receives a valve actuator.

In another aspect, the flow control valve of the basic apparatus described herein may include a rotatable valve configured to control the flow of a fluid through a fluid network.The rotatable valve may be rotated by a valve actuator.

In another aspect, the basic instrument described herein may include a thermal block, and the fluidic network of the cartridge housing may include a sample channel in which a designated reaction to a biological sample occurs. The housing side may include an access opening extending along the sample channel and configured to receive the thermal block for changing the temperature of the sample channel.

In another aspect, the fluidic network of the removable cartridge described herein may include a plurality of channels and a storage module. The storage module may include a plurality of reservoirs for storing reagents used for at least one of sample preparation or sample analysis.

In another aspect, a basic instrument described herein includes a system controller having a valve control module configured to control operation of a valve actuator to control the flow of a biological sample through a fluidic network.

In an embodiment, a method for sequencing nucleic acids is provided. The method includes providing a removable cartridge having a cartridge housing, a fluid network disposed within the cartridge housing, and a flow control valve operably coupled to the fluid network and movable relative to the fluid network. The cartridge housing includes a housing side defining the exterior of the removable cartridge. The method also includes contacting the removable cartridge with a base instrument. The housing side of the removable cartridge detachably engages a control side of the base instrument to jointly define a system interface. The base instrument includes a valve actuator that engages the flow control valve through the system interface. The method also includes fluidically directing a biological sample to flow through the fluid network of the cartridge to perform at least one of sample analysis or sample preparation in the cartridge. The biological sample is directed to flow into a reaction chamber, wherein the flow of the biological sample is controlled by the action of the valve actuator on the flow control valve. The method also includes detecting the biological sample using an imaging detector directed to the reaction chamber, wherein the detection assembly is held by at least one of the removable cartridge or the base instrument.

In one aspect, the methods described herein may further include removing the removable cartridge from the base instrument. The removable cartridge may be replaced by functionally mating a second removable cartridge to the base instrument. Several removable cartridges may be sequentially mated to the base instrument, used to prepare and/or analyze samples while mated to the base instrument, and then removed from the base instrument.

Accordingly, the method may include contacting a second removable cartridge with the base instrument, wherein a housing side of the second removable cartridge detachably engages a control side of the base instrument to collectively define a system interface.

In another aspect, the method described herein includes removing a removable cartridge from a base instrument. Optionally, the method includes contacting a second removable cartridge with the base instrument, wherein a housing side of the second removable cartridge detachably engages a control side of the base instrument to jointly define a system interface.

In another aspect of the methods described herein, the operations of fluidly directing and imaging the biological sample are repeated multiple times sequentially within a single removable cartridge.

In another aspect, the methods described herein include sealing a biological sample within a sample preparation zone of a fluidic network, and amplifying the biological sample while the biological sample is sealed within the sample preparation zone of the fluidic network.

In another aspect, a flow control valve used in the methods set forth herein includes a movable valve having at least one flow passage extending between two valve ports, a valve actuator configured to move the movable valve between different positions.

In another aspect, when a biological sample flows through a flow channel and is directed into a reaction chamber, the movable valve used in the method described herein is in a sample position, the method further comprising moving the movable valve to a component position and flowing a reagent through the flow channel into the reaction chamber, the reagent reacting with the biological sample in the reaction chamber.

In another aspect of the method described herein, the component location includes a plurality of component locations, and the method further includes moving the movable valve between the component locations according to a predetermined sequence to flow different reagents into the reaction chamber.

In another aspect, the biological sample used in the methods described herein comprises nucleic acids, and the predetermined sequence is according to a sequencing-by-synthesis (SBS) protocol.

In another aspect, a flow cell for use in the methods described herein comprises a reaction chamber. A biological sample is immobilized to one or more surfaces of the flow cell.

In an embodiment, a removable cartridge is provided that includes a cartridge housing having a sample port that is open to the exterior of the cartridge housing and configured to receive a biological sample. The cartridge housing has an array of electrical contacts and an externally exposed mechanical interface device. The cartridge housing is configured to be removably coupled to a basic instrument. The removable cartridge may also include a fluid network having a plurality of channels, a reaction chamber, and a storage module. The storage module includes a plurality of reservoirs for storing reagents. The fluid network is configured to guide the reagents from the reservoirs to the reaction chambers, wherein the mechanical interface device is movable relative to the fluid network to control the flow of fluids passing through the fluid network. The system also includes an imaging device disposed within the cartridge housing and positioned to detect a specified reaction within the reaction chamber. The imaging device is electrically coupled to the array of electrical contacts for communicating with the basic instrument. The mechanical interface device may be configured to be moved by the basic instrument when the removable cartridge is coupled to the basic instrument.

In one aspect, the mechanical interface device of the removable cartridge described herein may include a channel valve configured to control the flow of a fluid through one of the channels of the fluid network.

In another aspect, the cartridge housing of the removable cartridge described herein may include an access opening that allows access to the mechanical interface device. Optionally, the mechanical interface device includes a rotatable valve.

In another aspect, the cartridge housing of the removable cartridge described herein may include an externally exposed access opening, and the channel includes a sample channel in fluid communication with the sample port. The access opening may extend along the sample channel and may be configured to receive a thermal block for controlling the temperature of the sample channel.

In another aspect, the cartridge housing of the removable cartridge described herein may include a fluid coupling port exposed to the exterior and in fluid communication with the fluid network. The fluid coupling port is configured to engage an instrument port to receive fluid therethrough.

In another aspect, a cartridge housing of a removable cartridge as described herein may include first and second housing sides facing in opposite directions. The first housing side may include an array of electrical contacts. The second housing side may include a mechanical interface device.

In another aspect, the removable cartridge further comprises a locking mechanism attached to the cartridge housing. The locking mechanism may be configured to removably secure the cartridge housing to the base instrument.

In an embodiment, a removable cartridge is provided that includes a cartridge housing having a sample port, the sample port being open to the exterior of the cartridge housing and configured to receive a biological sample. The removable cartridge may further include a rotatable valve disposed within the cartridge housing. The rotatable valve has a fluid side and a plurality of valve ports open at the fluid side. The rotatable valve has at least one flow channel extending between the valve ports, wherein the rotatable valve is rotatable between different rotational positions. The removable cartridge may further include a microfluidic body having a body side slidably coupled to the fluid side of the rotatable valve. The microfluidic body may at least partially define a fluid network including a sample channel in fluid communication with the sample port. The sample channel has a network port open to the body side of the microfluidic body. The fluid network may further include a reservoir configured to hold a reagent. The reservoir is in fluid communication with the reservoir port open to the fluid side of the microfluidic body. The fluid network also includes a feed channel in fluid communication with a reaction chamber of the fluid network. The feed channel has a feed port open to the body side of the microfluidic body. The rotatable valve is configured to rotate between a first and a second rotational position. When the rotatable valve is in a first rotational position, the network port is fluidly coupled to the feed port through the rotatable valve. When the rotatable valve is in a second rotational position, the reservoir port is fluidly coupled to the feed port through the rotatable valve.

In one aspect, the cartridge housing of the removable cartridge described herein can have an exterior side configured to engage a base instrument.The rotatable valve can include a mechanical interface device accessible at the exterior side and configured to engage the base instrument.

In another aspect, the rotatable valve in a first rotational position can be configured to receive sample liquid in a removable cartridge as described herein when suction pulls the sample liquid toward the feed port. The rotatable valve in a second rotational position can be configured to allow sample liquid to move into the reservoir when a moving force pushes the sample fluid away from the feed port into the reservoir.

In another aspect, the rotatable valve of the removable cartridge described herein rotates about an axis. The feed port may be aligned with the axis.

In an embodiment, a removable cartridge is provided that includes a cartridge housing having a sample port that is open to the outside of the cartridge housing and configured to receive a biological sample. The cartridge housing may include a matching side configured to face and be removably coupled to a basic instrument. The removable cartridge also includes a fluid network disposed within the housing. The fluid network includes a sample channel in fluid communication with the sample port. The removable cartridge also includes a channel valve having a flexible member configured to move between a first and a second position. The flexible member blocks flow through the sample channel when in the first position and allows flow through the sample channel when in the second position. The matching side of the cartridge housing includes an access opening that exposes the channel valve to the outside of the cartridge housing. The access opening is configured to receive a valve actuator of a basic instrument for moving the flexible member between the first and second positions.

In another aspect, the flexible member of the removable cartridge described herein can include a flexible layer covering an interior cavity of a fluid network. The flexible layer can be configured to be pushed into the cavity to prevent flow therethrough.

In another aspect, the removable cartridge further comprises a rotatable valve disposed within the cartridge housing. The rotatable valve is configured to rotate between different positions to change a flow path of the fluid network. The rotatable valve may include a mechanical interface device accessible along a mating side.

In one aspect, the fluid network of the removable cartridge described herein can include a network port in fluid communication with a sample channel, a feed port in fluid communication with a reaction chamber, and a reservoir port in fluid communication with a reservoir configured to store a reagent. The removable cartridge can also include a rotatable valve disposed within the cartridge housing. The rotatable valve can fluidically couple the feed port and the network port when in a first rotational position and fluidically couple the feed port and the reservoir port when in a second rotational position.

In another aspect, the mating side of the removable cassette described herein can be a first mating side, and the removable cassette can include a second mating side. The first and second mating sides face in opposite directions. The second mating side is configured to mechanically, fluidically, or thermally engage the instrument.

In an embodiment, a base instrument is provided that includes a system housing having a mating side configured to engage a removable cartridge. The base instrument also includes a rotary motor configured to engage a rotatable valve of the removable cartridge. The base instrument also includes a valve actuator configured to engage a channel valve of the removable cartridge and an array of electrical contacts configured to be electrically coupled to the removable cartridge. The base instrument also includes a system controller configured to control the rotary motor and the valve actuator to execute an assay protocol within the removable cartridge. The system controller is configured to receive imaging data from the removable cartridge via the array of electrical contacts. Optionally, the base instrument includes a heat block for heating a portion of the removable cartridge.

In an embodiment, a removable cartridge is provided that includes a cartridge housing having a sample port, the sample port being open to the exterior of the cartridge housing and configured to receive a biological sample. The cartridge housing includes a mating side configured to face and removably couple to a primary instrument. The removable cartridge also includes a microfluidic body disposed within the cartridge housing. The microfluidic body has a body side and includes a fluid network. The fluid network has a plurality of discrete channels and corresponding ports open on the body side at a valve receiving area. The removable cartridge also includes a rotatable valve disposed within the cartridge housing. The rotatable valve has a fluid side and at least one flow channel extending between the plurality of valve ports. The valve ports are open to the fluid side. The fluid side is rotatably coupled to the valve receiving area on the body side of the microfluidic body, wherein the rotatable valve is movable between different rotational positions to fluidically couple the discrete channels. The rotatable valve has a mechanical interface device that is accessible along the mating side and configured to engage the primary instrument such that the rotatable valve is controlled by the primary instrument.

In an embodiment, a removable cartridge is provided that includes a cartridge housing having a sample port that is open to the exterior of the cartridge housing and configured to receive a biological sample. The cartridge housing includes a mating side configured to be removably coupled to a base instrument. The removable cartridge also includes a microfluidic structure disposed within the cartridge housing and comprising a plurality of stacked printed circuit board (PCB) layers. The PCB layers include a fluidic layer that defines channels and reaction chambers when the PCB layers are stacked. The PCB layers also include a wiring layer. The removable cartridge also includes a CMOS imager that is configured to be mounted to the microfluidic structure and electrically coupled to the conductive wiring layer. The CMOS imager is oriented to detect a specified reaction within the reaction chamber.

In one aspect, the removable cartridge includes input/output (I/O) contacts exposed to the exterior of the cartridge housing. The I/O contacts can be electrically coupled to the CMOS imager.

In one aspect, the microfluidic structure of the removable cartridge described herein includes a channel valve, wherein at least a portion of the channel valve is defined by the PCB layer. The channel valve is configured to be actuated to prevent and allow flow through one of the channels.

As used herein, elements or steps listed in the singular and preceded by the word "a" or "an" should be understood as not excluding the plural of said elements or steps, unless such exclusion is expressly stated. Furthermore, references to "one embodiment" are not intended to be interpreted as excluding the existence of additional embodiments that also incorporate the recited features. Moreover, unless expressly stated to the contrary, embodiments that "comprise" or "have" an element or multiple elements having a particular property may include additional elements, whether or not they have that property.

It should be noted that in various alternative embodiments, the specific arrangement (e.g., quantity, type, placement, etc.) of the components of the illustrated embodiments may be modified. In various embodiments, a different number of a given module or unit may be used, a different type of a given module or unit may be used, a given module or unit may be added, or a given module or unit may be omitted.

It should be understood that the above description is intended to be illustrative rather than restrictive. For example, the above embodiments (and/or aspects thereof) may be used in combination with each other. In addition, many modifications may be made to adapt specific situations or materials to the teachings of the various embodiments without departing from the scope thereof. The dimensions, material types, orientations, and the number and position of the various components of the various embodiments described herein are intended to define parameters of certain embodiments and are by no means restrictive but merely exemplary embodiments. Upon reviewing the above description, many other embodiments and modifications within the spirit and scope of the claims will be apparent to those skilled in the art. Therefore, the scope of the patentable invention should be determined with reference to the appended claims together with the full scope of equivalents to which such claims are entitled.

As used in the description, the phrase "in an exemplary embodiment" and the like mean that the embodiment described is merely an example. The phrase is not intended to limit the inventive subject matter to that embodiment. Other embodiments of the inventive subject matter may not include the recited features or structures. In the appended claims, the terms "including" and "inwhich" are used as the plain-English equivalents of the respective terms "comprising" and "wherein." Moreover, in the claims, the terms "first," "second," and "third," etc. are used merely as labels and are not intended to impose numerical requirements on their objects. Furthermore, the claim limitations are written in means-plus-function format and are not intended to be interpreted based on 35 U.S.C. §112(f), and until such a claim expressly uses the phrase "means for..." followed by a statement of function without another structure.

Claims (35)

1. A system for carrying out a biochemical reaction, the system comprising: A removable cartridge having a housing and including a fluid network disposed within the housing, the fluid network being configured to receive and fluidly guide a biological sample for at least one of sample analysis or sample preparation. The removable cartridge has a plurality of stacked layers to form sample channels having a first port in fluid communication with a valve chamber. The removable cartridge also includes a flow control valve operatively coupled to the fluid network and movable relative to the valve chamber to block the first port thereby controlling the flow rate of the biological sample flowing through it. The housing includes an accessible housing side defining the exterior of the removable cartridge and allowing operation of the flow control valve. A basic instrument having a control side configured to detachably engage the housing side of the removable case, wherein the housing side and the control side jointly define a system interface when the removable case is engaged with the basic instrument, the basic instrument including a valve actuator for engaging a flow control valve via the system interface, wherein the valve actuator includes an elongated actuator body extending through the housing side and into the case housing; and A detection assembly, held by at least one of the removable case or the basic instrument, the detection assembly including an imaging detector and a reaction chamber in fluid communication with the fluid network, the imaging detector being configured to detect a specified reaction within the reaction chamber. 2. The system of claim 1, wherein the control side and the housing side are generally planar and face each other, wherein the system interface is a one-sided interface, in which the basic instrument and the removable box are operatively coupled to each other only through the housing side and the control side. 3. The system of claim 2, wherein the basic instrument and the removable housing are operatively coupled such that the basic instrument and the removable housing are fixed to each other at the system interface using at least one of fluid coupling, electrical coupling or thermal coupling established through the system interface. 4. The system according to any one of claims 1-3, wherein the control side represents the top of the basic instrument relative to gravity, such that the removable box is located on and supported by the basic instrument. 5. The system of any one of claims 1-3, wherein the flow control valve includes an elongated actuator body extending through the control side and into the basic instrument. 6. The system of any one of claims 1-3, wherein the basic instrument has an instrument side facing in the opposite direction to the control side, the basic instrument has an instrument dimension extending between the control side and the instrument side, and the basic instrument and the removable housing have a combined dimension larger than the instrument dimension. 7. The system of any one of claims 1-3, wherein each of the removable housing and the basic instrument comprises an array of electrical contacts electrically coupled to each other at the system interface. 8. The system of any one of claims 1-3, wherein the housing side is a first housing side, and the housing further includes a second housing side, the first housing side and the second housing side facing different directions, wherein the system interface is a multi-sided interface, in which the basic instrument and the removable housing are operatively coupled to each other along each of the first housing side and the second housing side. 9. The system of claim 8, wherein the first housing side and the second housing side are substantially perpendicular to each other, the basic instrument has an instrument housing including a first control side and a second control side, the first control side and the second control side facing perpendicularly to each other and forming an open side recess of the basic instrument, the removable case being disposed within the open side recess such that the first housing side and the second housing side engage the first control side and the second control side. 10. The system of claim 9, wherein the valve actuator includes an elongated body extending through the system interface between the first housing side and the first control side, the second housing side and the second control side including respective contact arrays of electrical contacts electrically coupled to each other along the system interface. 11. The system of claim 8, wherein the first housing side and the second housing side face substantially opposite directions, the basic instrument has an instrument side and a box receiving slot open to the instrument side, and the removable box is disposed within the box receiving slot. 12. The system of claim 11, wherein the removable housing and the basic instrument are fluidly coupled along the first housing side and electrically coupled along the second housing side. 13. The system of claim 11, wherein the basic instrument includes a locking mechanism that engages at least one of the first housing side or the second housing side to retain the removable case within the basic instrument. 14. The system of any one of claims 1-3, wherein each of the removable housing and the basic instrument includes a flow port that is fluidly coupled to each other at the system interface. 15. The system of any one of claims 1-3, further comprising a locking mechanism attached to at least one of the removable housing or the basic instrument, the locking mechanism being configured to removably secure the housing to the basic instrument. 16. The system of any one of claims 1-3, wherein the imaging detector is held by the basic instrument and the reaction chamber is held by the removable housing. 17. The system of any one of claims 1-3, wherein the flow control valve includes a flexible membrane configured to control the flow rate of the biological sample through the fluid network, the flexible membrane being bent by the valve actuator between a first condition and a second condition. 18. The system of any one of claims 1-3, wherein the housing side of the housing includes an access opening therethrough for receiving the valve actuator. 19. The system of any one of claims 1-3, wherein the flow control valve comprises a rotatable valve configured to control the flow rate of fluid through the fluid network, the rotatable valve being rotated by the valve actuator. 20. The system of any one of claims 1-3, wherein the basic instrument includes a heat block, and the fluid network of the housing includes a sample channel in which a specified reaction to the biological sample occurs, and the housing side includes an access opening extending along the sample channel and configured to receive the heat block for changing the temperature of the sample channel. 21. The system of any one of claims 1-3, wherein the fluid network comprises a plurality of channels and a storage module, the storage module comprising a plurality of reservoirs for storing reagents for at least one of the sample preparation or sample analysis. 22. The system of any one of claims 1-3, wherein the basic instrument includes a system controller having a valve control module configured to control the operation of the valve actuator to control the flow rate of the biological sample through the fluid network. 23. The system of claim 22, wherein the valve control module is configured to control the operation of the valve actuator to perform the Synthesis Sequencing SBS protocol. 24. A method for sequencing nucleic acids, the method comprising: A removable cartridge is provided, the removable cartridge having a cartridge housing, a fluid network disposed within the cartridge housing, a plurality of stacked layers forming a sample channel of the fluid network, the sample channel having a first port in fluid communication with a valve chamber, and a flow control valve operatively coupled to the fluid network and movable relative to the valve chamber to block the first port, wherein the cartridge housing includes a housing side defining the exterior of the removable cartridge; The removable case is brought into contact with a basic instrument, wherein the housing side of the removable case is detachably engaged with the control side of the basic instrument to jointly define a system interface, wherein the basic instrument includes a valve actuator that operatively engages the flow control valve through the system interface, wherein the valve actuator includes an elongated actuator body that extends through the housing side and into the case housing. Fluidly guiding a biological sample through the fluid network of the removable cartridge for at least one of sample analysis or sample preparation within the removable cartridge, wherein the biological sample is guided to flow into a reaction chamber, and wherein the flow rate of the biological sample is controlled by the action of the flow control valve by the valve actuator; and The biological sample in the reaction chamber is imaged using a detection component held by at least one of the removable case or the basic instrument. 25. The method of claim 24, further comprising removing the removable case from the basic instrument. 26. The method of claim 25, further comprising contacting a second removable case with the base instrument, wherein the housing side of the second removable case is detachably engaged with the control side of the base instrument to jointly define the system interface. 27. The method of claim 26, wherein the fluidly guiding the biological sample and imaging the biological sample are repeated sequentially multiple times. 28. The method of any one of claims 24-27, further comprising sealing the biological sample within a sample preparation area of the fluid network, and amplifying the biological sample while it is sealed within the sample preparation area. 29. The method of any one of claims 24-27, wherein the flow control valve includes a movable valve having at least one flow passage extending between valve orifices, and the valve actuator is configured to move the movable valve between different positions. 30. The method of claim 29, wherein the movable valve is in the sample position when the biological sample flows through the at least one flow channel and is guided into the reaction chamber, the method further comprising moving the movable valve to the component position and allowing a reagent to flow through the at least one flow channel into the reaction chamber, the reagent reacting with the biological sample in the reaction chamber. 31. The method of claim 30, wherein the component locations include a plurality of component locations, and the method further includes moving the movable valve between the component locations in a predetermined order to allow different reagents to flow into the reaction chamber. 32. The method of claim 31, wherein the biological sample comprises nucleic acids, and the predetermined sequence is according to the Synthesis Sequencing-Breakthrough (SBS) protocol. 33. The method of any one of claims 24-27, wherein the flow cell includes the reaction chamber, and the biological sample is fixed to one or more surfaces of the flow cell. 34. A basic instrument for conducting biochemical reactions, said basic instrument comprising: A system housing having a control side configured to engage a detachable box; A rotary motor configured to engage a rotatable valve of the removable box; A valve actuator configured to engage a channel valve of the removable cartridge, wherein the valve actuator includes an elongated actuator body extending through the housing side of the removable cartridge and into the cartridge housing of the removable cartridge, the valve actuator engaging a flow control valve of the removable cartridge and being movable relative to the valve chamber to block a first opening of a sample channel formed by a plurality of stacked layers in the removable cartridge; An array of electrical contacts configured to be electrically coupled to the removable housing; and A system controller configured to control the rotary motor and the valve actuator to execute a measurement protocol within the removable housing, the system controller being configured to receive imaging data from the removable housing via an array of electrical contacts. 35. The basic instrument as claimed in claim 34, further comprising a heating block for heating a portion of the removable housing.