HK40012117A - 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 base instrument and a removable cartridge

This application is a divisional application filed on application No. 201580038608.6 entitled "system and method for biochemical analysis including a base instrument and a removable cartridge", filed on day 2015, 5, 27.

RELATED APPLICATIONS

The priority of U.S. application No. 62/003,264 filed on 27/5/2014, which is hereby incorporated by reference in its entirety.

Background

Embodiments of the present application relate generally 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 a reaction for at least one of sample preparation or biochemical analysis.

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

There is a general need for a system for automatically performing assays, such as those described above, where the system requires less work by or involvement of the user. Currently, most platforms require the user to separately prepare the biological sample before loading it into the system for analysis. It may be desirable for a user to load one or more biological samples into the system, select an assay for execution by the system, and have results from the analysis within a predetermined period of time, such as a day or less. At least some systems in use today are not capable of performing certain protocols, such as whole genome sequencing, which provides data with a sufficient level of quality and within a certain cost range.

Brief description of the drawings

In an embodiment, a system is provided that includes a removable cartridge having a cartridge housing. The removable cartridge also includes a fluidic network disposed within the cartridge housing. The fluidic network is configured to receive and fluidically conduct a biological sample for at least one of sample analysis or sample preparation. The removable cartridge also includes a flow control valve operably coupled to the fluidic network and movable relative to the fluidic network to control a 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 operation of the flow control valve. The system also 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 through a system interface. The removable cartridge also 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 substantially planar and face each other, wherein the system interface is a single-sided interface in which the base instrument and the removable cartridge are operatively 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 fluidic coupling, an electrical coupling, or a thermal coupling established through the system interface.

In some embodiments, the control side represents a top of the base instrument with respect to gravity, such that the removable cartridge is located on and 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 dimension extending between the control side and the instrument side, and the base instrument and the removable cartridge have a combined dimension greater than the instrument dimension.

In some embodiments, each of the removable cartridge and the base instrument includes a contact array of electrical contacts 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 and second housing sides facing different directions, wherein the system interface is a multi-sided interface in which the base instrument and the removable cartridge are operatively coupled to each other along each of the first and second housing sides.

In some embodiments, the first and second housing sides are substantially perpendicular to each other, the base instrument has an instrument housing including first and second control sides facing in a perpendicular direction and forming an open-sided recess of the base instrument, and the removable cartridge is disposed within the open-sided recess such that the first and second housing sides engage the first and second control sides.

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, the second housing side and the second control side including respective contact arrays of electrical contacts that 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 in 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, each of the removable cartridge and the base instrument includes a flow port that is 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 comprises a flexible membrane configured to control the flow of the biological sample through the fluidic network, the flexible membrane being flexed 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 fluidic network, the rotatable valve being rotated by the valve actuator.

In some embodiments, the base instrument comprises a thermal block and the fluidic network of the cartridge housing comprises a sample channel in which a specified reaction to the biological sample occurs, the housing side comprising an access opening extending along the sample channel and configured to receive the thermal block for changing a 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 conduct a sequencing-by-synthesis (SBS) protocol.

In embodiments, methods of sequencing nucleic acids are 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 an exterior of the removable cartridge. The method also includes contacting the removable cartridge to the base instrument. The housing side of the removable cartridge detachably engages the control side of the base instrument to collectively define a system interface. The base instrument includes a valve actuator that engages the flow control valve through a system interface. The method also includes fluidically directing the biological sample through a fluidic 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 the reaction chamber, wherein the flow of the biological sample is controlled by the action of a valve actuator on the flow control valve. The method also includes detecting the biological sample using an imaging detector oriented 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, the fluidically directing the biological sample and imaging the biological sample are repeated a plurality of times in sequence.

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 comprises a movable valve having at least one flow channel extending between valve ports, the valve actuator configured to move the movable valve between different positions.

In some embodiments, the movable valve is in a sample position when the biological sample flows through the flow channel and is directed into the reaction chamber, 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 within the reaction chamber.

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

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

In some embodiments, a flow cell comprises the reaction chamber, and the 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 an 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 base instrument. The removable cartridge may also include a fluidic network having a plurality of channels, reaction chambers, and a storage module. The storage module includes a plurality of reservoirs for storing reagents. The fluid network is configured to direct a reagent from the reservoir to the reaction chamber, wherein the mechanical interface device is movable relative to the fluid network to control a flow of fluid 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. An imaging device is electrically coupled to the array of electrical contacts for communicating with the base instrument. The mechanical interface device may be configured to be moved by the base instrument when the removable cartridge is coupled to the base instrument.

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

In some embodiments, the cartridge housing includes an access opening that allows 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 access opening exposed to the exterior, and the channel includes a sample channel in fluid communication with the sample port, the access opening extending along the sample channel and configured to receive a thermal block for controlling a 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 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, 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 memory module comprises reagents for performing sequencing-by-synthesis (SBS) protocols. In an embodiment, a removable cartridge is provided that includes a cartridge housing having a sample port that is open to an exterior of the cartridge housing and configured to receive a biological sample. The removable cartridge may also 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 valve ports, wherein the rotatable valve is rotatable between different rotational positions. The removable cartridge may also 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 fluidic 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 fluidic network may also include a reservoir configured to hold a reagent. The reservoir is in fluid communication with a reservoir port that is open to the fluid side of the microfluidic body. The fluidic network also includes a feed channel in fluid communication with the reaction chamber of the fluidic network. The feed channel has a feed opening that is open to the body side of the microfluidic body. The rotatable valve is configured to rotate between first and second rotational positions. The network port is fluidly coupled to the feed port through the rotatable valve when the rotatable valve is in the first rotational position. The reservoir port is fluidly coupled to the feed port through the rotatable valve when the rotatable valve is in the second rotational position.

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

In some embodiments, the memory module comprises reagents for performing sequencing-by-synthesis (SBS) protocols.

In some embodiments, the rotatable valve in the first rotational position is configured to receive a sample liquid as a 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 as 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 that includes a cartridge housing having a sample port that is open to an exterior of the cartridge housing and configured to receive a biological sample. The cartridge housing may include a mating side configured to face and removably couple to the base instrument. The removable cartridge also includes a fluidic network disposed within the housing. The fluidic 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 first and second positions. 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 mating side of the cartridge housing includes an access opening exposing the channel valve to the exterior of the cartridge housing. The access opening is configured to receive a valve actuator of a base instrument for moving the flexible member between the first and second positions.

In some embodiments, the flexible member comprises a flexible layer covering an inner lumen of the fluidic network, the flexible layer 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 fluidic network, the rotatable valve comprising a mechanical interface device accessible along the mating side.

In some embodiments, the fluidic network comprises a network port in fluid communication with the 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 further comprising a rotatable valve disposed within the cartridge housing that fluidly couples the feed port and the network port when in a first rotational position and fluidly couples 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 and second mating sides facing in opposite directions, the second mating side 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 the rotatable valve of the removable cartridge. The base instrument also includes a valve actuator configured to engage the channel valve of the removable cartridge and an array of electrical contacts configured to electrically couple to the removable cartridge. The base instrument also includes a system controller configured to control the rotary motor and the valve actuator to perform 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 thermal block for heating a portion of the removable cartridge.

In some embodiments, the base instrument further comprises a thermal 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 that is open to an 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 base instrument. The removable cartridge also includes a microfluidic body disposed within the cartridge housing. The microfluidic body has a body side and includes a fluidic network. The fluid network has a plurality of discrete channels and corresponding ports at the valve receiving area that open at the side of the body. 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 a plurality of valve ports. The valve port is open to the fluid side. The fluid side is rotatably coupled to a valve receiving region of the body side of the microfluidic body, wherein the rotatable valve is movable between different rotational positions to fluidly couple the discrete channels. The rotatable valve has a mechanical interface device accessible along the mating side and configured to engage the base instrument such that the rotatable valve is controlled by the base instrument.

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

In an embodiment, a removable cartridge is provided that includes a cartridge housing having a sample port that is open to an 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 including 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 layer also includes a routing layer. The removable cartridge also includes a CMOS imager configured to be mounted to the microfluidic structure and electrically coupled to the conductive routing layer. The CMOS imager is oriented to detect a specified reaction within the reaction chamber.

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

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

Brief description of the drawings

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

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

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

Fig. 3 is a side view of a system formed according to an embodiment including a base instrument and a removable cartridge.

Fig. 4 is a top view of a system formed according to an embodiment including a base instrument and a removable cartridge.

FIG. 5 is a cross-sectional view illustrating a portion of a system formed according to an embodiment having a flow control valve in a first position.

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

FIG. 7 is a cross-sectional view illustrating a portion of a system formed according to an embodiment having a flow control valve in a first position.

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

FIG. 9 is a cross-sectional view illustrating a portion of a system formed according to an embodiment having a flow control valve in a first position.

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

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

Fig. 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 can be fluidly interconnected using a rotatable valve.

Fig. 14 shows a flow chart of an example of a method of printing electronic devices using a flexible Printed Circuit Board (PCB) and roll-to-roll (R2R) for monolithically integrated and finger-like fluidic devices for CMOS technology.

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

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

Fig. 17A, 17B, 18, 19 and 20 show side views of structures and illustrate examples of processes for attaching CMOS devices to a flexible PCB using the method of fig. 14.

Fig. 21 shows a side view of an example of a structure formed using the method of fig. 14, wherein a fluidic layer and CMOS device are integrated together in a microfluidic cartridge.

Fig. 22A and 22B show perspective views of an example of a diaphragm valve, which can be integrated into a fluid layer.

Fig. 23A and 23B show cross-sectional views of a diaphragm valve in the open and closed states, respectively.

Fig. 24 shows a schematic of an example of a microfluidic cartridge comprising CMOS technology and finger fluidic devices integrated together.

Fig. 25 and 26 show perspective views of a microfluidic cartridge assembly that is a physical illustration of the integrated microfluidic cartridge shown in fig. 24.

Fig. 27A and 27B show perspective views of an example of a fluidic component mounted in the microfluidic cartridge assembly shown in fig. 25 and 26.

Fig. 28A and 28B show plan and cross-sectional views, respectively, of an example of a heater trace that may be mounted in the fluidic assembly shown in fig. 27A and 27B.

Fig. 29, 30, 31, 32, 33A, and 33B illustrate various other views of the microfluidic cartridge assembly of fig. 25 showing more detail therein.

Fig. 34 to 42 illustrate a process of deconstruction of the microfluidic cartridge assembly of fig. 25 as a means of revealing internal components therein.

Figure 43 shows a transparent perspective view of a portion of the microfluidic cartridge assembly of figure 25 and shows various reagent fluid reservoirs therein and sample loading ports thereof.

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

Figure 45 shows a cross-sectional view of the microfluidic cartridge assembly of figure 25 showing more detail therein.

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

Fig. 49, 50, 51A, 51B, and 52 show various views of the bottom plate of the microfluidic cartridge assembly of fig. 25, showing more detail therein.

Fig. 53A and 53B illustrate other perspective views of the fluidic components of the microfluidic cartridge assembly, showing more detail therein.

Fig. 54A, 54B, and 54C show additional views illustrating more details of the flexible PCB heater of the fluidic assembly of the microfluidic cartridge assembly.

Fig. 55A and 55B show perspective and plan views, respectively, of the inlet/outlet layers of the fluidic layers shown in fig. 15 and 27.

Fig. 56A and 56B show perspective and plan views, respectively, of the fluid channel layer of the fluidic layer shown in fig. 15 and 27.

Fig. 57A and 57B show perspective and plan views, respectively, of the flexible PCB layer of the fluidics layer shown in fig. 15 and 27.

FIGS. 58A and 58B show perspective and plan views, respectively, of the bottom layer of the sequencing chamber of the fluidic layer shown in FIGS. 15 and 27.

FIGS. 59A and 59B show perspective and plan views, respectively, of the sequencing chamber layer of the fluidic layer shown in FIGS. 15 and 27.

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

Fig. 61A and 61B show a flow diagram of an example of a method of performing multiplex PCR and downstream mixing required for sequencing using a microfluidic cartridge assembly.

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

Fig. 63 shows an exploded view of one illustrative example of the CMOS flow cell shown in fig. 49.

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

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

Fig. 67, 68, 69 and 70 show examples of processes that provide an extended flat surface in a CMOS flow cell on which a flow cell cover can be mounted.

Fig. 71A, 71B, 71C and 71D show another example of a process of providing an extended flat surface in a CMOS flow cell on which a flow cell cover can be mounted.

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

Detailed Description

The embodiments set forth herein may be used to perform a specified reaction for sample preparation and/or biochemical analysis. The term "biochemical analysis" may include at least one of a biological analysis or a chemical analysis. Fig. 1A is a schematic diagram of a system 100 configured to perform biochemical analysis and/or sample preparation. The system 100 includes a base instrument 102 and a removable cartridge 104 configured to detachably engage the base instrument 102. The base instrument 102 and the removable cartridge 104 may be configured to interact with each other to transport the biological sample to different locations within the system 100, perform a specified reaction involving the biological sample in order to prepare the biological sample for subsequent analysis, and optionally detect one or more events using the biological sample. An event may indicate a specified reaction with a biological sample. In some embodiments, the removable cartridge 104 is similar to the integrated microfluidic cartridge 1100 (shown in fig. 24) or the microfluidic cartridge assembly 1200 (shown in fig. 25 and 26).

While reference is made below to the base instrument 102 and the removable cartridge 104 as shown in FIG. 1A, it is to be understood that the base instrument 102 and the removable cartridge 104 illustrate only one exemplary embodiment of the system 100, as well as other embodiments that exist. For example, the base instrument 102 and the removable cartridge 104 include various components and features that collectively perform a plurality of operations for preparing and/or analyzing a biological sample. In the illustrated embodiment, each of the base instrument 102 and the removable cartridge 104 is capable of performing certain functions. However, it should be understood that the base instrument 102 and the 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 alternative embodiments, 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 fluid with the removable cartridge 104. In alternative embodiments, the base instrument 102 may provide reagents or other liquids to the removable cartridge 104, for example, that are subsequently consumed by the removable cartridge 104 (e.g., used in a given 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, the biological sample can 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, transudates, exudates, cystic fluid, bile, gastric fluid in urine, intestinal fluid, stool samples, fluids containing single or multiple cells, fluids containing organelles, fluidized tissues, fluidized organisms, fluids containing multicellular organisms, biological swabs, and biological washes.

In some embodiments, the biological sample may include added materials, such as water, deionized water, salt solutions, acid solutions, base solutions, detergent solutions, and/or pH buffers. The added materials may also include reagents used to perform biochemical reactions during a given assay protocol. For example, the added liquid may include material to perform multiple Polymerase Chain Reaction (PCR) cycles with the biological sample.

It should be understood, however, that the biological sample being analyzed may be in a different form or state than the biological sample loaded into the system 100. For example, a biological sample loaded into the system 100 may include whole blood or saliva that is subsequently processed (e.g., via a separation or amplification process) to provide the prepared nucleic acids. The prepared nucleic acids can then be analyzed by the system 100 (e.g., quantified by PCR or sequenced by SBS). Accordingly, when the term "biological sample" is used in describing a first operation, such as PCR, and is used again in describing a subsequent second operation, such as sequencing, it is understood that the biological sample in the second operation may be modified with respect to the biological sample prior to or during the first operation. For example, a sequencing step (e.g., SBS) can be performed on amplicon nucleic acids generated from template nucleic acids amplified in a previous amplification step (e.g., PCR). In this case, the amplicon is a copy of the template and is present in a higher amount than the amount of template.

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

As used herein, a "specified reaction" includes a change in at least one of a chemical, electrical, physical, or optical property (or mass) of an analyte of interest. In particular embodiments, the specified reaction is an associated binding event (e.g., the combination of a fluorescently labeled biomolecule with an analyte of interest). The specified reaction may be a separate binding event (e.g., the release of a fluorescently labeled biomolecule from the analyte of interest). The specified reaction may be a chemical transformation, a chemical change, or a chemical interaction. The specified reaction may also be a change in electrical properties. For example, the specified reaction may be a change in ion concentration within the solution. Exemplary reactions include, but are not limited to, chemical reactions such as reduction, oxidation, addition, elimination, rearrangement, esterification, amidation, etherification, cyclization, or displacement; a binding interaction in which the first chemical binds to the second chemical; dissociation reactions in which two or more chemicals are separated from each other; fluorescence is generated; 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. The designated reaction may also be the addition or elimination of a proton, which may be detected as a change in the pH of the surrounding solution or environment, for example. An additional specified reaction may be the detection of the flow of ions across a membrane (e.g. a natural or synthetic bilayer membrane), for example as ions flow through the membrane, the current is interrupted and the interruption may be detected. Field sensing of charged labels can also be used as thermal sensing and other analytical sensing techniques known in the art.

In particular embodiments, the specified reaction comprises the incorporation of a fluorescently labeled molecule into the analyte. The analyte may be an oligonucleotide and the fluorescently labeled molecule may be a nucleotide. A given reaction can be detected when excitation light is directed at the oligonucleotide with labeled nucleotides and the fluorophore emits a detectable fluorescent signal. In alternative embodiments, the detected fluorescence is the result of chemiluminescence or bioluminescence. The specified reaction may also increase fluorescence (or, for example, by bringing the donor fluorophore in close proximity to the acceptor fluorophore) Resonance Energy Transfer (FRET), lowering FRET by separating donor and acceptor fluorophores, increasing fluorescence by separating a quencher from a fluorophore or decreasing fluorescence by situating a quencher and a fluorophore.

As used herein, "reaction components" include any material that can be used to obtain a specified reaction. For example, reaction components include reagents, catalysts such as enzymes, reactants for the reaction, samples, products of the reaction, other biomolecules, salts, metal cofactors, chelators, and pH buffer solutions (e.g., hydrogenation buffers). The reaction components may be delivered to various locations in the fluid network, either individually in solution or in combination in one or more mixtures. For example, the reaction components may be delivered to a reaction chamber where the biological sample is immobilized. The reaction component may interact directly or indirectly with the biological sample. In some embodiments, the removable cartridge 104 is pre-loaded with one or more reaction components necessary to perform a specified assay protocol. The pre-load may occur at a location (e.g., a manufacturing facility) before the cartridge 104 is received by a user (e.g., at a customer's facility).

In some embodiments, the base instrument 102 may be configured to interact with the removable cartridge 104 per active period. After the active 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 active period. As used herein, the term "active period" includes performing at least one of a sample preparation and/or biochemical analysis protocol. Sample preparation may include separating, isolating, modifying, and/or amplifying one or more components of the biological sample such that the prepared biological sample is suitable for analysis. In some embodiments, the active period may comprise a continuous activity in which a plurality of 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 fault or malfunction is detected and/or (g) one or more resources used to react are exhausted. Optionally, the active period may include suspending system activity during a period of time (e.g., minutes, hours, days, weeks) and thereafter completing the active period until at least one of (a) - (g) occurs.

An assay protocol may include a sequence of operations for performing, detecting, and/or analyzing a specified reaction. Collectively, the removable cartridge 104 and the base instrument 102 may include the components necessary to perform different operations. The operation of the assay protocol may include fluidic operations, thermal control operations, sensing operations, and/or mechanical operations. Fluidic operations include controlling the flow of fluid (e.g., liquid or gas) through the system 100, which may be initiated by the base instrument 102 and/or by the removable cartridge 104. For example, the fluidic operation may include controlling a pump to introduce a flow of the biological sample or reaction components into the detection zone. The thermal control operation may include controlling the temperature of a designated portion of the system 100. As an example, the thermal control operation may include increasing or decreasing a temperature of a Polymerase Chain Reaction (PCR) zone in which a liquid including the biological sample is stored. The detecting operation may include controlling activation of the detector or monitoring activity of the detector to detect a predetermined characteristic, quantity, or characteristic of the biological sample. As one example, the detecting operation can include capturing an image of a specified region including the biological sample to detect fluorescent emissions from the specified region. The detecting operation may include controlling the light source to illuminate the biological sample or controlling the detector to observe the biological sample. The mechanical manipulation may include controlling the movement or position of a specified component. For example, the mechanical operation may include controlling a motor to move a valve control feature in the base instrument 102 that operably engages a rotatable valve in the removable cartridge 104. In some cases, combinations of different operations may occur simultaneously. For example, the detector may capture an image of the detection zone when the pump controls the flow of fluid through the detection zone. In some cases, different manipulations 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, a "liquid" is a relatively incompressible substance and has the ability to flow and conform to the shape of the container or channel holding the substance. The liquid may be aqueous based and include polar molecules that exhibit surface tension that holds the liquid together. The liquid may also comprise non-polar molecules, for example in oil-based or non-aqueous substances. It is to be understood that reference to a liquid in this application may include a liquid formed from a combination of two or more liquids. For example, separate reagent solutions may be later combined to perform a given reaction.

The removable cartridge 104 is configured to detachably engage or removably couple to the base instrument 102. As used herein, when the terms "detachably engaged" or "removably coupled" (or similar terms) are used to describe a relationship between a removable cartridge and a base instrument, the terms are intended to mean that the connection between the removable cartridge and the base instrument is easily separable without destroying the base instrument. Accordingly, the removable cartridge may be electrically detachably engaged to the base instrument such that electrical contacts of the base instrument are not damaged. The removable cartridge may be detachably engaged to the base instrument mechanically such that features of the base instrument that hold the removable cartridge are not disrupted. The removable cartridge may be detachably engaged to the base instrument in a fluid manner such that the port of the base instrument is not disrupted. For example, the basic instrument is not considered "damaged" if only a simple adjustment (or realignment) or a simple replacement of the component (e.g., replacement of the nozzle) is required. The components (e.g., the removable cartridge 104 and the base instrument 102) are readily separable when the components can be separated from one another without undue effort or a significant amount of time spent separating the components. In some embodiments, the removable cartridge 104 and the base instrument 102 are easily separable without damaging the removable cartridge 104 or the base instrument 102.

In some embodiments, the removable cartridge 104 may be permanently modified or partially damaged during periods of activity with the base instrument 102. For example, the container holding the liquid may include a foil lid that is pierced to allow the liquid to flow through the system 100. In such embodiments, the foil lid may be damaged, such that it may be necessary to replace the damaged container with another container. In certain embodiments, the removable cartridge 104 is a disposable cartridge, such that the removable cartridge 104 is replaced and optionally discarded after a single use.

In other embodiments, the removable cartridge 104 may be used during more than one active period when engaged with the base instrument 102, and/or may be removed from the base instrument 102, refilled with reagents, and re-engaged to the base instrument 102 for additional designated reactions. Accordingly, the removable cartridge 104 may be refurbished in some cases so that the same removable cartridge 104 may be used with different consumables (e.g., reaction components and biological samples). After removing the cartridge from the base instrument located at the customer's facility, refurbishment may be performed at the manufacturing facility.

As shown in fig. 1A, the removable cartridge 104 includes a fluidic network 106 that can hold and direct a fluid (e.g., a liquid or a gas) therethrough. The fluid network 106 includes a plurality of interconnected fluid elements capable of storing fluid and/or allowing fluid to flow therethrough. Non-limiting examples of fluidic elements include channels, ports of channels, cavities, storage modules, reservoirs of storage modules, reaction chambers, waste reservoirs, detection chambers, multi-purpose chambers for reactions and detections, and the like. The fluidic elements may be fluidically coupled to each other in a specified manner such that the system 100 is capable of performing sample preparation and/or analysis.

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

One or more embodiments can include retaining a biological sample (e.g., a template nucleic acid) at a specified location, wherein the biological sample is analyzed. As used herein, the term "retain," when used in reference to a biological sample, includes substantially attaching the biological sample to a surface or confining the biological sample within a specified space. As used herein, the term "immobilized" when used in reference to a biological sample includes surfaces that substantially attach the biological sample in or on a solid support. Immobilizing may include attaching the biological sample to the surface at a molecular level. For example, biological samples may be immobilized to the surface of a substrate using absorption techniques including non-covalent interactions (e.g., electrostatic forces, van der waals forces, and dehydration of hydrophobic interfaces) and covalent binding techniques in which functional groups or cross-linking agents facilitate attachment of the biological sample to the surface. The immobilization of the biological sample to the surface of the substrate may be 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 (e.g., chemically or physically modified) to facilitate immobilization of the biological sample to the substrate surface. The substrate surface may first be modified to have functional groups bound to the surface. The functional group can then bind to the biological sample to immobilize the biological sample thereon. In some cases, the biological sample may 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, the nucleic acid can be immobilized to a surface and amplified using bridge amplification. Useful bridge amplification methods are described, for example, in U.S. Pat. No. 5,641,658, WO 07/010251, U.S. Pat. No. 6,090,592, U.S. Pat. publication No. 2002/0055100A 1, U.S. Pat. No. 7,115,400, U.S. Pat. publication No. 2004/0096853A 1, U.S. Pat. publication No. 2004/0002090A1, U.S. Pat. publication No. 2007/0128624A 1, and U.S. Pat. publication No. 2008/0009420A 1, each of which is incorporated herein in its entirety. Another useful method for amplifying nucleic acids on a surface is Rolling Circle Amplification (RCA), for example, using the methods set forth in more detail below. In some embodiments, the nucleic acid can be attached to a surface and amplified using one or more primer pairs. For example, one of the primers may be in solution and the other primer may be immobilized on a surface (e.g., 5' -attached). As an example, a nucleic acid molecule may hybridize to one of the primers on the surface, followed by extension of the immobilized primer to produce a first copy of the nucleic acid. The primer in solution then hybridizes to a first copy of the nucleic acid, which can be extended using the first copy of the nucleic acid as a template. Alternatively, after the first copy of the nucleic acid is produced, the organic nucleic acid molecule may hybridize to the second immobilized primer on the surface and may be extended simultaneously or after the primers in solution are extended. In any embodiment, multiple copies of the nucleic acid are provided using repeated cycles of immobilization of the primer and extension (e.g., amplification) of the primer in solution. In some embodiments, a biological sample can be confined within a predetermined space along with reaction components configured for use during amplification (e.g., PCR) of the biological sample.

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

The fluidic network 106 is held by the cartridge housing 110 and includes a plurality of sample ports 116 that are open to the non-mating side 112. In alternative embodiments, the sample port 116 may be located along the non-mating side 111 or 113 or may be located along the mating 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 can be nucleic acids and other materials (e.g., reagents, buffers, etc.) used to perform PCR. Although three sample ports 116 are shown in fig. 1A, embodiments may include only one sample port, two sample ports, or more than three sample ports.

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

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

For example, in the illustrated embodiment, the fluidic 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 fig. 1A, but alternative embodiments may include multiple sample channels 131. The sample channel 131 may include a sample preparation zone 132. The valve assembly 120 includes a pair of passage valves 121, 122. The channel valves 121, 122 may be selectively activated by the base instrument 102 to block or prevent fluid flow through the sample channel 131. In certain embodiments, the channel valves 121, 122 can be activated to form a seal that retains a specified volume of liquid within the sample preparation region 132 of the sample channel 131. The designated volume within sample preparation area 132 may include a biological sample.

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

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 a rotatable valve 123. However, it should be understood that alternative embodiments may include a movable valve that does not rotate to a different position. In such embodiments, the movable valve may slide in one or more linear directions to align with the respective ports. The rotatable valves and linear motion valves set forth herein may be similar to the devices described in international application number PCT/US2013/032309 filed 3, 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. Optionally, the light source 158 may be incorporated with the removable cartridge 104. For example, the biological sample may include one or more fluorophores that provide light emission when excited by light having an appropriate wavelength. In the illustrated embodiment, the removable cartridge 104 has an optical pathway 154. The light path 154 is configured to allow illumination light 156 from a light source 158 of the base instrument 102 to be incident on the biological sample within the reaction chamber 126. Thus, 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 optics, etc., that actively direct the illumination light 156 to 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 assembly 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 the CMOS image sensor 262 (shown in fig. 40). In some embodiments, the imaging detector 109 can be positioned relative to the reaction chamber 126 to detect light signals (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 can detect the optical signal emitted from the chemiluminescence. In still other embodiments, the detection assembly 108 may not be limited to imaging applications. For example, the detection component 108 may be one or more electrodes that detect an electrical characteristic of the liquid.

As set forth 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 specified reactions and/or obtain data for a biological sample. To this end, the mating side 114 is configured to allow or permit the base instrument 102 to control 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 and 173 that allow the valves 121 and 123 to be controlled by the base instrument 102. The mating side 114 may also include an access opening 174 configured to receive a thermal block 206 of the base instrument 102. The inlet opening 174 extends along the sample channel 131. As shown, the access openings 171 and 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 cartridge 104. The mating side 114 of the removable cartridge 104 and the control side 202 of the base instrument 102 together define a system interface 204. The system interface 204 represents a common boundary between the removable cartridge 104 and the base instrument 102 through which the base instrument 102 and the removable cartridge 104 are operably engaged. More specifically, the base instrument 102 and the removable cartridge 104 are operably engaged along the system interface 204 such that the base instrument 102 can control various features of the removable cartridge 104 through the mating side 114. For example, the base instrument 102 may have one or more controllable components that control corresponding components of the removable cartridge 104.

In some embodiments, the base instrument 102 and the removable cartridge 104 are operably engaged 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 through the system interface 204. In the illustrated embodiment, the base instrument 102 and the removable cartridge 104 are configured to have an electrical coupling, a thermal coupling, a valve coupling, and an optical coupling. More specifically, the base instrument 102 and the removable cartridge 104 may communicate data and/or electrical power through an electrical coupling. The base instrument 102 and the removable cartridge 104 may transfer thermal energy to and/or from each other through a thermal coupling, and the base instrument 102 and the removable cartridge 104 may communicate an optical signal (e.g., illumination light) through an 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 cartridge 104 and the base instrument 102 are operatively coupled to each other only through the mating side 114 and the control side 202. In an alternative embodiment, the system interface may be a multi-sided interface. For example, at least 2, 3, 4, or 5 sides of the removable cartridge may be mating sides configured to couple with a base instrument. The multiple sides may be planar and may be arranged orthogonal or opposite to each other (e.g., around all or part of a rectangular volume).

To control the operation of the removable cartridge 104, the base instrument 102 may include a valve actuator 211 and 213 configured to operably engage the flow control valve 121 and 123, a thermal block 206 configured to provide and/or remove thermal energy from the sample preparation zone 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 latch 177 configured to engage a latch engagement element 178 of the removable cartridge 104. Optionally, the removable cartridge 104 may include a rotatable snap lock 177 and the base instrument 102 may include a snap lock engagement element 178. The snap lock 177 can rotate and engage the lock engagement element 176 when the removable cartridge 104 is mounted to the base instrument 102. The camming effect created by the locking mechanism 176 may push or drive the removable cartridge 104 to the base instrument 102 to secure the removable cartridge 104 thereto.

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

The base instrument 102 may also include a system controller 220 configured to control operation of at least one of the valve actuator 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 illustrated as a series of circuit blocks, but may be implemented using any combination of dedicated hardware boards, DSPs, processors, etc. 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 the remaining modular functions are performed using off-the-shelf PCs or the like.

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 actuator 211 and 213 and the system pump 119. The flow control module 221 may selectively activate the valve actuators 211 and 213 and the system pump 119 to induce and/or prevent the flow of fluid through one or more paths.

For example only, the valve actuator 213 may rotatably engage 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 may activate the valve actuator 213 to move the rotatable valve 123 to the first rotational position. With the rotatable valve 123 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 zone 132 and into the reaction chamber 126. The flow control module 221 may then activate the valve actuator 213 to move the rotatable valve 123 to the second rotational position. With the rotatable valve 123 in the second rotational position, the flow control module 221 can activate the system pump 219 to withdraw one or more of the reaction components from the respective reservoirs and into the reaction chamber 126. In some embodiments, the system pump 219 may be configured to provide positive pressure such that fluid is actively pumped in the opposite direction. Such operation may be used to add multiple liquids to a common reservoir, thereby mixing the liquids within the reservoir. Accordingly, fluid coupling port 118 may allow fluid (e.g., gas) to exit cartridge housing 110 or may receive fluid into cartridge housing 110.

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

The system controller 220 may also include a detection module 223 configured to control the detection component 108 to obtain data about the biological sample. The detection module 223 may control the operation of the detection assembly 108 through 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 are 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 or during a predetermined time. As an example, when the biological sample has a fluorophore attached thereto, the detection module 223 may control the detection assembly 108 to capture an image of the reaction chamber 126. Multiple images may be obtained.

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

System controller 220 and/or circuit modules 221 and 224 may comprise 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 executing the functions described herein. In the exemplary embodiment, system controller 220 and/or circuit module 221 and 224 execute a set of instructions stored therein to perform one or more metering protocols. The memory element may be in the form of an information source or a physical memory element within the base instrument 102 and/or the removable cartridge 104. The protocol performed by the assay system 100 may be to perform, for example, quantitative analysis of DNA or RNA, protein analysis, DNA sequencing (e.g., sequencing-by-synthesis (SBS)), sample preparation, and/or preparation of a library of fragments 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 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 exemplary only, and are thus not limiting as to the types of memory usable for storage of a computer program.

The software may be in various forms such as system software or application software. Further, the software may be in the form of a collection of separate programs or program modules or portions of program modules within a larger program. The software may also include modular programming in the form of object-oriented programming. After obtaining the detection data, the detection data may be automatically processed 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 over a communication link).

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

The system controller 220 may 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 may be configured and/or programmed to control data and/or power aspects of the various components. Although system controller 220 is represented in fig. 1A as a single structure, it should be understood that system controller 220 may include multiple separate components (e.g., processors) distributed throughout system 100 at different locations. In some embodiments, one or more components may be integrated with the base instrument, and one or more components may be remotely located with respect to the base instrument.

Fig. 1B is a flow diagram illustrating a method 180 of performing a specified reaction for at least one of sample preparation or sample analysis. In particular embodiments, method 180 may include sequencing a nucleic acid. The method 180 may use structures or aspects of various embodiments (e.g., systems and/or methods) discussed herein. In various embodiments, certain steps may be omitted or added, certain steps may be combined, certain steps may be performed simultaneously, certain steps may be performed in parallel, certain steps may be divided into multiple steps, certain steps may be performed in a different order, or certain steps or series of steps may be re-performed in an iterative manner.

For example, the method 180 may include providing 182 a removable cartridge having a cartridge housing. The removable cartridge may include a fluidic 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 movable cartridge.

The method 180 may also include mounting (e.g., contacting) the removable cartridge to the base instrument at 184. The housing side of the removable cartridge may detachably engage the control side of the base instrument to collectively define a system interface. The base instrument includes a valve actuator that engages the flow control valve through a system interface. For example, the valve actuator may include an elongated body that exits the control side and is inserted into the access 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 a biological sample to a sample port in fluid communication with a fluidic network. The reception at 186 may occur before or after the contact at 184. The method 180 may include, at 188, fluidically directing the biological sample through a fluidic network of a removable cartridge to perform at least one of sample analysis or sample preparation in the cartridge. For example, a biological sample may be directed to a sample preparation zone of a fluidic network, where the flow rate of the biological sample is controlled by the action of a valve actuator on a flow control valve. The biological sample may be subjected to an amplification process, such as PCR, while the biological sample is sealed within the sample preparation area. As another example, a flow of biological sample may be directed into the reaction chamber, where the flow of biological sample is controlled by the action of a valve actuator on a flow control valve.

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

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

Fig. 2 is a schematic diagram of a system 300 configured to perform at least one of biochemical analysis or sample preparation. System 300 may include the same or similar features as system 100 (fig. 1A). For example, the system 300 includes a base instrument 302 and a removable cartridge 304 configured to detachably engage the base instrument 302. The base instrument 302 and the removable cartridge 304 may have similar features as the base instrument 102 and the removable cartridge 104 (shown in fig. 1A), respectively. As shown in fig. 2, the base instrument 302 has an instrument housing 303 that includes an instrument side 306 and a cartridge receiving slot 308 that is open to the instrument side 306. In some embodiments, instrument side 306 may represent the top of base instrument 302 with respect to gravity and partially form the exterior of instrument housing 303. In the illustrated embodiment, the cartridge receiving slot 308 is defined by an interior docking side or control side 311 and 313 of the instrument housing 303. The control sides 311 and 313 are opposite each other, and the control side 312 extends between the control sides 311, 313. The control side 312 may face an opening 316 to the cartridge receiving slot 308.

The removable cartridge 304 is sized and shaped to be disposed within the cartridge receiving slot 308 and to operatively engage the base instrument 302. As shown, the removable cartridge 304 includes a cartridge housing 320 having a housing side 321 and 324. The housing side 321-323 is configured to operably engage the docking side or the control side 311-313 such that the base instrument 302 and the removable cartridge 304 establish at least one of an electrical coupling, a thermal coupling, an optical coupling, and/or a fluidic coupling. Thus, the shell side 321-323 is hereinafter referred to as the mating side 321-323. The housing side 324 does not operably engage the base instrument 302. Accordingly, the shell side 324 may be referred to as the 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 specified reactions. 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 may be open to one of the mating sides 321-323. In such embodiments, the biological sample may be disposed within 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 zone 334. The fluidic network 332 may include or fluidly interconnect various other components of the removable cartridge 304, such as a storage module 336, a movable 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 base instrument 302 may have corresponding components that operably engage the removable cartridge 304 to perform a specified reaction. For example, the base instrument 302 may include a thermal block 350, a valve actuator 352, a light source 356, a contact array 358, and a system pump 360. The removable cartridge 304 and the base instrument 302 may engage each other 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. More specifically, when the removable cartridge 304 is operably loaded into the base instrument 302, the thermal block 350 may be located proximate to the sample preparation zone 334, the valve actuator 352 may operably engage the movable valve 338, the light source 356 may be communicatively coupled to the light path 346, the contact array 358 may electrically engage the contact array 348, and the system pump 360 may communicatively engage the fluidic network 332. Accordingly, the removable cartridge 304 may be controlled by the base instrument 302 in a manner similar to the removable cartridge 104 being controlled by the base instrument 102.

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

The component holders 362, 364 may be automatically activated by the system controller 370. For example, the system controller 370 may determine that the removable cartridge 304 is loaded or has been loaded into the cartridge receiving slot 308. The system controller 370 may then activate the drive mechanism or mechanisms to drive the component holders 362, 364 toward the mating sides 321, 323. Alternatively, the component holders 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 holders 362, 364 toward the mating sides 321, 323, respectively. Accordingly, the base instrument 302 may be configured to allow the removable cartridge 304 to freely advance (e.g., without substantial hooking or kicking) into the cartridge receiving slot 308.

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

In some embodiments, at least one force 374, 376 facilitates providing intimate contact between the respective components. For example, force 374 may provide intimate contact between thermal block 350 and sample preparation zone 334 to enable thermal control of sample preparation zone 334. Likewise, force 374 may allow valve actuator 352 and movable valve 338 to properly engage each other such that valve actuator 352 may selectively control movable valve 338. The force 376 may achieve intimate contact between the respective electrical contacts of the contact arrays 348, 358.

Fig. 3 and 4 illustrate different systems including a removable cartridge with corresponding base instruments, and in particular, different multi-sided interfaces that may be utilized by one or more embodiments. For example, fig. 3 is an end view of a system 400 including a base instrument 402 and a removable cartridge 404. The base instrument 402 includes an open-sided recess 406 sized and shaped to receive the removable cartridge 404. As shown, the open-side recess 406 is formed by the first and second control sides 411, 412 facing in perpendicular directions relative to each other. More specifically, the first and second control sides 411, 412 form an L-shaped recess. The first and second control sides 411, 412 operatively engage first and second mating sides 413, 414, respectively, of the removable cartridge 404. A multi-sided interface 415 is formed collectively between the first control side 411 and the first mating side 413, and the second control side 412 and the 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 including a base instrument 422 and a removable cartridge 424. The base instrument 422 includes a cartridge receiving slot 426, which may be similar or identical to the cartridge receiving slot 308 (fig. 2). The cartridge receiving slot 426 is sized and shaped to receive the removable cartridge 424. As shown, the cartridge receiving slot 426 is formed by a control side 431 and 434. Control sides 431, 433 are opposite each other and control sides 432, 434 are opposite each other. The control sides 431-434 operably engage the mating sides 441-444 of the removable cartridge 424, respectively. A multi-sided interface 427 is cooperatively formed between the corresponding side of the removable cartridge 424 and the base instrument 422.

Fig. 5-12 illustrate various valving mechanisms by which the base instrument can control (e.g., regulate) flow through the fluidic network of the removable cartridge. Each of fig. 5-12 illustrate a cross-section of a system in which valve coupling is established between a base instrument and a removable cartridge through a system interface. Each of fig. 5-12 shows a channel valve, wherein the base instrument can activate the channel valve to open and close the corresponding channel. For example, fig. 5 and 6 illustrate a portion of a system 500 that may be similar to the systems described above, e.g., systems 100 (fig. 1A), 300 (fig. 2), 400 (fig. 3), 420 (fig. 4).

Fig. 5 and 6 show a cross-section of a portion of a system 500 having a base instrument 502 and a removable cartridge 504 operably engaged along a system interface 506. As shown, the removable cartridge 504 has a cartridge housing 508 and a microfluidic body 510 held by the cartridge housing 508. In the illustrated embodiment, the microfluidic body 510 includes a plurality of layers 521-523 stacked side-by-side. Layers 521- "523 may be Printed Circuit Board (PCB) layers such as those described below with respect to fig. 14-75. One or more of the layers 521- > 523 may be etched such that the microfluidic body 510 forms a sample channel 526 when the layers 5212- > 523 are stacked side-by-side. The sample channel 526 is part of a fluidic network, such as fluidic network 106 (fig. 1A), and includes a valve or lumen 528.

The removable cartridge 504 includes a channel valve 530 configured to regulate the flow of fluid through the sample channel 526. For example, channel valve 530 may allow for maximum clearance such that fluid may flow unimpeded. The channel valve 530 may also block fluid flow therethrough. As used herein, the term "impeding" may include slowing the flow of a fluid or completely stopping the flow of a fluid. As shown, the sample channel 530 includes first and second ports 532, 534 in fluid communication with the valve chamber 528. 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 channel valve 530 constitutes a flexible membrane that is capable of flexing between first and second conditions. The flexible membrane is in a first condition in fig. 5 and in a second condition in fig. 6. In a particular embodiment, the flexible film is a flexible layer, such as film layer 918 (shown in fig. 23A, 23B). The flexible layer is configured to be pushed into the valve cavity 528 to prevent fluid flow therethrough. In alternative embodiments, the channel valve 530 may be a physical element that is movable between different conditions or positions to regulate the flow of fluid.

Also shown, the base instrument 502 includes a valve actuator 540 configured to activate the channel valve 530. For example, the valve actuator 540 may bend the flexible membrane between the first and second conditions. The valve actuator 540 includes an elongated body 542, such as a post or rod, that extends through the system interface 506. More specifically, the elongated body 542 is spaced from the control side 544 of the base instrument 502. The removable cartridge 504 has an access opening 546 that receives the valve actuator 540. The access opening 546 is open to a mating side 548 of the removable cartridge 504. As shown, the elongated body 542 protrudes away from the control side 544 and into the access opening 546 of the mating side 548. The access opening 546 allows the valve actuator 540 to directly engage the channel valve 530, which in the illustrated embodiment is a flexible membrane. In fig. 5, the valve actuator 540 is in a first state or position. In fig. 6, the valve actuator 540 is in a second state or position. In the second position, the process actuator 540 has moved a distance toward the port valve 530 and is engaged with the port valve 530. The valve actuator 540 may deform the channel valve 530 such that the channel valve 530 covers the first port 532. Thus, fluid flow through the first port 532 is blocked by the channel valve 530.

In some embodiments, the system 500 can have first and second channel valves similar to or the same as the channel valve 530 shown in fig. 5 and 6, where the first channel valve is upstream with respect to a sample preparation zone (not shown) of the fluidic network and the second channel valve is downstream with respect to the sample preparation zone. Thus, the first and second channel valves can effectively seal the fluid that can contain the biological sample within the sample preparation zone. The fluid with the biological sample may then be heated to subject the fluid to an amplification protocol, such as a PCR protocol.

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

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

Fig. 11 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 (fig. 1) and other removable cartridges described herein. Rotatable valve 704 may be similar to movable valve 123 (fig. 1). Rotatable valve 704 is configured to be rotatably mounted to a body side or surface 706 of microfluidic body 702. Rotatable valve 704 has a fluid side 708 configured to slidably engage body side 706 when rotated about 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. Passages 763 and 766 are discrete passages. For example, passage 763 and 766 can be disconnected based on the rotational position of rotatable valve 704.

Passage 763 and 766 have corresponding ports open to body side 706. In the illustrated embodiment, four sample channels 763 are in fluid communication with a single sample channel 764. Thus, the sample channel 763 can be referred to as a channel portion, and the sample channel 764 can be referred to as a common sample channel. Each sample channel 763 is operatively coupled to a pair of channel valves 761, 762. The channel valves 761, 762 may be similar to the channel valves described herein, such as the channel valve 530. When in the respective closed position, the channel valves 761, 762 may seal the liquid containing the respective biological sample. In some embodiments, sample channel 763 extends adjacent to heat control region 770. A heating element (not shown) and thermal block (not shown) may be positioned adjacent to the thermal control region 770 when the biological sample is sealed in the respective sample channel 763. The heating element and thermal block can cooperate to increase and/or decrease the temperature experienced by the biological sample within the sample channel 763. In such embodiments, sample channel 763 can constitute a sample preparation zone.

The feed channel 766 is in fluid communication with the reaction chamber 716, and the reservoir channel 765 can be in fluid communication with a corresponding reservoir (not shown) of a storage module (not shown). The sample channel 764 has a network port 721, the feed channel 766 has a feed port 722, and the reservoir channel 765 has a corresponding reservoir port 723. Network port 721, feed port 722, and reservoir port 723 are open to body side 706. Reservoir ports 723 are in fluid communication with respective module ports 724 via respective reservoir channels 765. As shown, the module port 724 can be located at various locations along the body side 706 away from the feed port 722 or the shaft 710. The module port 724 is configured to fluidly couple to a reservoir (not shown). The module port 724 may have a location based on the size of the reservoir.

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

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

Fig. 12 shows a cross-section of a rotatable valve 704 operably engageable with a valve actuator 730. More specifically, 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 736 configured to engage the valve actuator 730. In the illustrated embodiment, the mechanical interface 736 comprises a planar body or fin that is coincident with the axis 710. The valve actuator 730 includes a slot 738 configured to receive a mechanical interface 736 such that the valve actuator 730 operably engages the rotatable valve 704. More specifically, the valve actuator 730 may engage the rotatable valve 704 such that the valve actuator 730 is able to rotate the rotatable valve 704 about the shaft 710.

Fluid side 708 includes a plurality of ports 740, 742 and flow channels 744 extending between ports 740, 742. Fluid side 708 is slidably engaged to body surface 706 at 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 ports and/or more than one flow channel.

As shown in FIG. 12, inlet 722 is fluidly aligned with and coupled to valve port 740, and valve port 742 is fluidly aligned with and coupled to network port 721. Valve port 742 may also be fluidly coupled to one of component ports 723 based on the rotational position of rotatable valve 704. As mentioned above, the rotatable valve 704 is configured to rotate about the shaft 710. In some embodiments, the inlet 722 and the valve port 740 are positioned such that the inlet 722 and the valve port 740 are aligned with the axis 710. More specifically, a shaft 710 extends through each of the inlet 722 and the valve port 740.

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

Accordingly, the rotatable valve 704 may 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. Rotatable valve 704 effectively changes the flow path of the fluid network as rotatable valve 704 is rotated between different rotational positions.

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

Fig. 13 is a top down view of the body side 706 showing the network port 721, feed port 722, and reservoir port 723. The flow channel 744 is shown in two different rotational positions. The memory ports 723 may include memory ports 723A-723D. Each reservoir port 723A-723D is fluidly coupled to a respective reservoir through a respective reservoir channel 765 (fig. 10). More specifically, a reservoir port 723A is fluidly coupled to the hydrogenation buffer, a reservoir port 723B is fluidly coupled to the nucleotide solution, a reservoir port 723C is fluidly coupled to the wash solution, and a reservoir port 723D is fluidly coupled to the lysis solution. As described above, the flow channel 744 can fluidly couple the feed port 722 to the sample channels 763, 764, or respective reservoirs based on the rotational position of the rotatable valve 704 (fig. 11).

Table 1 shows various states of the sequencing-by-synthesis (SBS) protocol, but it is understood that other assay protocols may be implemented. In stage 1, the flow channel 744 has a rotational position that fluidly couples the network port 721 and the feed port 722. In stage 1, channel valves (not shown) may be selectively activated to seal the second, third, and fourth biological samples within the respective sample preparation zones, but allow the first biological sample to flow through the network port 721. Accordingly, at stage 1, the system pump may apply suction that pulls the first biological sample into the flow channel 744. At stage 2, the rotatable valve 704 is rotated to a second rotational position while the first biological sample is stored within the flow channel 744, such that the flow channel 744 fluidly 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 so that the second biological sample can be pulled into flow channel 744. At stage 4, rotatable valve 704 is rotated back to the second rotational position while the first biological sample is stored within 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 respective sample preparation zones and added to the hydrogenation buffer. Accordingly, four biological samples may be stored in a single reservoir with a hydrogenation buffer. The reaction can occur with a biological sample and a hydrogenation buffer that prepares the biological sample for SBS sequencing.

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

TABLE 1

The above-mentioned embodiments may be used in conjunction with the subject matter of U.S. provisional patent application No. 61/951,462 (attorney docket No. IP-1210-PRV296PRV2) (hereinafter the' 462 application "), which is incorporated herein by reference in its entirety. At least a portion of the' 462 application is provided below.

The methods described herein can be used in conjunction with various nucleic acid sequencing techniques. Particularly applicable techniques are those in which the nucleic acids are attached at fixed positions in the array such that their relative positions do not change, and in which the array is repeatedly detected or imaged. The following embodiments are particularly applicable: images are obtained in different colour channels, for example coinciding with different labels for distinguishing one nucleotide base type from another. In some embodiments, the process of determining the nucleotide sequence of the target nucleic acid may be an automated process. Preferred embodiments include sequencing by synthesis ("SBS") techniques.

"sequencing-by-synthesis (" SBS ") techniques" typically involve enzymatic extension of a control template strand by a nascent nucleic acid strand of repeated additions of nucleotides. In the conventional method of SBS, a single nucleotide monomer may be provided to a target nucleotide in the presence of a polymerase in each delivery. However, in the methods described herein, more than one type of nucleotide monomer can be provided to the target nucleic acid in the presence of a polymerase in delivery.

SBS can utilize nucleotide monomers with a terminator moiety or nucleotide monomers lacking any terminator moiety. Methods of utilizing terminator-deficient nucleotide monomers include, for example, pyrosequencing and sequencing using gamma-phosphate labeled nucleotides, as set forth in more detail below. In methods using terminator-deficient nucleotide monomers, the number of nucleotides added in each cycle is generally variable and depends on the template sequence and the mode of nucleotide delivery. For SBS techniques that utilize nucleotide monomers with terminator moieties, the terminators may be practically irreversible under the sequencing conditions used, as is the case for traditional Sanger sequencing that utilizes dideoxynucleotides, or the terminators may be reversible, as is the case for sequencing methods developed by Solexa (now Illumina ltd.).

SBS techniques may utilize nucleotide monomers that have a label moiety or nucleotide monomers that lack a label moiety. Accordingly, incorporation events can be detected based on the characteristics of the label, e.g., fluorescence of the label, the characteristics of the nucleotide monomer, e.g., molecular weight or charge, the release of byproducts of nucleotide incorporation, e.g., protons or pyrocarbonates, and the like. In embodiments where two or more different nucleotides are present in the sequencing reagent, the different nucleotides may be distinguishable from each other, or alternatively, two or more different labels may be indistinguishable under the detection technique used. For example, different nucleotides present in a sequencing reagent may have different labels, and they may be distinguished using suitable optics as exemplified by the sequencing method developed by Solexa (now Illumina ltd).

In another exemplary type of SBS, cycle sequencing is accomplished by stepwise addition of reversible terminator nucleotides containing, 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 limited 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 efficient Cycle Reversible Termination (CRT) sequencing. The polymerases can also be co-designed to efficiently incorporate and extend from these modified nucleotides.

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

In particular embodiments, some or all of the nucleotide monomers may include a reversible terminator. In such embodiments, the reversible terminator/cleavable fluorophore can include a fluorophore linked to a nucleic acid moiety via a 3' ester linkage (Metzker, Genome Res.15: 1767-. Other methods separate terminator chemicals from the lysis of fluorescent labels (Ruparal et al, Proc Natl Acad Sci USA 102:5932-7(2005), which is incorporated herein by reference in its entirety). Ruparael et al describe the development of reversible terminators that use small 3' allyl groups to prevent extension but can be easily deblocked by brief handling using a palladium catalyst. The fluorophore is attached to the base via a photocleavable crosslinker that can be easily cleaved by 30 seconds exposure to long wavelength UV light. Thus, disulfide reduction or photocleavage can be used as a cleavable crosslinking agent. Another method of reversible termination is the use of natural terminators that are guaranteed after a large number of dyes are placed on the dntps. The presence of charged bulk dyes on dntps can act as an effective terminator by steric and/or electrostatic hindrance. The presence of one incorporation event prevents further incorporation unless the dye is removed. The cleavage of the dye removes the phosphor and effectively reverses the termination. Examples of modified nucleotides are described in U.S. patent 7,427,673 and U.S. patent 7,057,026, the disclosures of which are incorporated herein by reference in their entirety.

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 may utilize detection of four different nucleotides using less than four different labels. For example, SBS can be performed using the methods and systems described in the incorporated material of U.S. patent No. 2013/0079232. As a first example, a pair of nucleotide types may be detected at the same wavelength, but distinguished based on a difference in intensity of one member of the pair compared to the other member or based on a change (e.g., via chemical, photochemical, or physical modification) to one member of the pair that causes an apparent signal to appear or disappear compared to a signal detected to the other member of the pair. As a second example, three of the four different nucleotide types can be detected under particular conditions, while the fourth "dark state" nucleotide type lacks a label that is detectable or minimally detectable (e.g., due to minimal detection of background fluorescence, etc.) under those conditions. Incorporation of the first three nucleotide types into a nucleic acid can be determined based on the presence of their corresponding signals, and incorporation of the fourth nucleotide type into a nucleic acid can be determined based on the absence or minimal detection of any signal. As a third example, one nucleotide type may include labels that are detected in two different channels, while the other nucleotide type is detected in no more than one channel. The three exemplary configurations mentioned above are not considered mutually exclusive and may be used in various combinations. An exemplary embodiment combining all three examples is a fluorescence-based SBS method that uses a first nucleotide type detected in a first channel (e.g., dATP with a label detected in the first channel when excited by a first excitation wavelength), a second nucleotide type detected in a second channel (e.g., dCTP with a label detected in the second channel when excited by a second excitation wavelength), a third nucleotide type detected in the first and second channels (e.g., dTTP with at least one label detected in both channels when excited by the first and/or second excitation wavelengths), and a lack of a fourth nucleotide type (e.g., dGTP without a label) that is not or minimally detected in either channel.

In addition, a single channel can be used to obtain sequencing data, as described in the incorporated material of U.S. patent publication No. 2013/0079232. In such a so-called single dye sequencing method, a first nucleotide type is labeled, but the label is removed after the first image is generated, and a 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 may utilize sequencing through complex formation. Such techniques utilize DNA ligase to incorporate oligonucleotides and recognize the incorporation of such oligonucleotides. Oligonucleotides generally have different labels associated with the identity of a particular nucleotide in the sequence to which the oligonucleotide hybridizes. As with other SBS methods, images can be obtained using labeled sequencing reagents after processing of the array of nucleic acid features. Each image will show nucleic acid features with a specific type of incorporated label. Due to the different sequence content of each feature, different features are present or absent in different images, but the relative positions of the features will remain unchanged in the images. Images resulting from the complex formation-based sequencing methods can be stored, processed, and/or analyzed as set forth herein. Exemplary sequencing systems and methods that can be utilized with the methods and systems described herein are described in U.S. patent 6,969,488, U.S. patent 6,172,218, and U.S. patent 6,306,597.

Some embodiments may utilize Nanopore sequencing (Deamer, D.W. & Akeseon, M. "Nanopores and amplified nucleic acids", which "Trends Biotechnol.18,147-151 (2000); Deamer, D. and D.Branton," propagation of nucleic acids by nanopowder analysis ". Chem.35: 817. 825 (2002); Li, J, M.Gershow, D.Stem, E.G.Ind. and J.A.Goovchenko," DNA molecules and complexes in colloidal-colloidal nanoparticles "Nature.2. Mater.2: 2003 (sequence), which is disclosed by citation in U.S. Pat. No. 615, which is incorporated by reference herein, and which may be measured by other Nanopore sequencing-Nanopore sequencing methods, such as by applying a Nanopore sequencing, which is described by the aforementioned Nanopore.7. Ser. No. 32, which is incorporated by reference in the aforementioned U.S.7. Ser. No. 32, which is based on the Nanopore sequencing of the Nanopores, which is incorporated by the aforementioned Nanopore, and which may be detected by the aforementioned Nanopore sequencing by the aforementioned Nanopore sequencing, when the Nanopore sequencing of the Nanopore sequencing, the target nucleic acid is incorporated by the aforementioned Nanopore, the aforementioned, the sequence, the aforementioned Nanopore, the sequence, the aforementioned, the sequence of the DNA, the DNA sequence, the DNA is obtained by the aforementioned, the aforementioned Nanopore, and the aforementioned, may be measured by the method of the aforementioned Nanopore, by the aforementioned Nanopore, or by the other embodiments, by the application, the method of the application, the method of the aforementioned, the application, such that the DNA, the application of the aforementioned, the Nanopore, the method of the aforementioned, the application, the aforementioned, the method of which is distinguished by the application, the method of the Nanopore, the application, the Nanopore, the method of which is incorporated by the method of the application, the method of DNA, the method of which is described in U.s.s.s.7.7.7.7.7.7.7.7.7.7.7.7, the Nanopore, the application, the method, the application, the method, the.

Some embodiments may utilize methods involving real-time monitoring of DNA polymerase activity. Nucleotide incorporation can be detected by Fluorescence Resonance Energy Transfer (FRET) interaction between a fluorophore-bearing polymerase and a gamma phosphate-labeled nucleotide as described, for example, in U.S. patent 7,329,492 and U.S. patent 7,211,414, each of which is incorporated herein by reference, or can be detected using a zero mode waveguide as described, for example, in U.S. patent 7,315,019, which is incorporated herein by reference, and using a fluorescent nucleotide analog and an engineered polymerase as described, for example, in U.S. patent 7,405,281 and U.S. patent publication No. 2008/0108082, each of which is incorporated herein by reference. Illumination can be limited to a zeptometric volume around the surface bound polymerase so that incorporation of fluorescently labeled nucleotides can be observed with low background ("Zero-mode waveguides for single-molecule analysis at high concentrations" Science 299,682 686 (2003); Lundquist, p.m. et al, "Parallel focused detection of single molecules in time" operation. let. 33,1026-1028 (2008); Korlach, j. et al, "Selective amplification for targeted amplification of single DNA polymerase modules" nucleic acid.105, each incorporated by reference in the U.S. patent No. 105, USA 1186). The images resulting from such methods may be stored, processed, and analyzed as set forth herein.

Some SBS embodiments include detection of protons released when nucleotides are incorporated into the extension products. For example, sequencing based on detection of liberated protons may use electrical detectors and related technologies commercially available from Ion Torrent (Guilford, CT, a life technologies subsiadiory) or 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.

The SBS method described above can be advantageously performed in a number of formats such that a plurality of different target nucleic acids are manipulated simultaneously. In certain embodiments, different target nucleic acids may be processed in a common reaction vessel or on the surface of a particular substrate. This allows for convenient delivery of sequencing reagents, removal of unreacted reagents, and detection of incorporation events in a multiplexed manner. In embodiments using surface-bound target nucleic acids, the target nucleic acids may be in an array format. In an array format, target nucleic acids can be bound to a surface, typically in a spatially distinguishable manner. The target nucleic acid may be bound by direct covalent binding, attachment to beads or other particles, or binding to a polymerase or other molecule attached to a surface. The array may include a single copy of the target nucleic acid at each site (also referred to as a feature), or multiple copies having the same sequence may be present at each site or feature. Multiple copies may be generated by amplification methods such as bridge amplification or emulsion PCR as described further below.

The methods set forth herein may be used with a density of any of a variety of densities, including at least about 10 features/cm²100 features/cm²500 features/cm²1,000 features/cm²5,000 features/cm²10,000 features/cm²50,000 features/cm²100,000 features/cm²1,000,000 features/cm²5,000,000 features/cm²Or an array of features higher up. The methods and apparatus described herein may include having a density at least sufficient to resolve the sheet at one or more of these exemplified densitiesA detection component or device of the resolution of the unique features.

An advantage of the methods set forth herein is that they provide for the rapid and efficient detection of multiple target nucleic acids in parallel. Accordingly, the present disclosure provides integrated systems that enable the preparation and detection of nucleic acids using techniques known in the art, such as the techniques exemplified above. Thus, an integrated system of the present disclosure may include fluidic components capable of delivering amplification and/or sequencing reagents to one or more immobilized DNA fragments, the system including components such as pumps, valves, reservoirs, fluidic circuits, and the like. The flow cell may be configured and/or used in an integrated system for detection of target nucleic acids. Exemplary flow cells are described, for example, in U.S. patent publication No. 2010/0111768 a1 and U.S. patent application No. 13/273,666, each of which is incorporated herein by reference. As exemplified for the flow cell, one or more fluidic components of the integrated system may be used for amplification methods and for detection methods. Taking the nucleic acid sequencing embodiment as an example, one or more fluidic components of the integrated system may be used for the amplification methods set forth herein and for the delivery of sequencing reagents in sequencing methods, such as the sequencing methods exemplified above. Alternatively, the integrated system may comprise separate fluidic systems to perform the amplification method and to perform the detection method. Examples of integrated sequencing systems capable of creating amplified nucleic acids and also determining the sequence of the nucleic acids include, without limitation, MiSeq^TMOr NextSeq^TMPlatforms (Illumina, ltd, San Diego, CA) or devices described in U.S. patent application publication nos. 2012/0270305 a1 or 2013/0260372 a1, each of which is incorporated herein by reference.

By "activity detector" is meant any device or component capable of detecting an activity indicative of a particular reaction or process. The activity detector may be capable of detecting a predetermined event, characteristic, quantity, or characteristic within a predetermined volume or region. For example, the activity detector may be capable of capturing an image of a predetermined volume or region. The activity detector may be capable of detecting the concentration of ions within a predetermined volume of the solution or along a predetermined region. An exemplary activity detector includes: a Charge Coupled Device (CCD) (e.g., a CCD camera); a photomultiplier tube (PMT); molecular characterization devices or detectors, such as those used with nanopores; microcircuit arrangements such as those described in U.S. patent No. 7,595,883, which is incorporated herein by reference in its entirety; and CMOS fabricated sensors having Field Effect Transistors (FETs), including chemically sensitive field effect transistors (chemfets), Ion Sensitive Field Effect Transistors (ISFETs), and/or Metal Oxide Semiconductor Field Effect Transistors (MOSFETs). An exemplary activity detector is described, for example, in international patent publication No. WO 2012/058095.

The term "biosensor" includes any structure having a plurality of reaction sites. The biosensor may comprise a solid-state imaging device (e.g. a CCD or CMOS imager) and optionally a flow cell mounted thereto. The flow cell may comprise at least one flow channel in fluid communication with the reaction site. As one particular example, the biosensor is configured to be fluidically and electrically coupled to a biological assay system. The biological assay system may deliver the reactants to the reaction sites and perform a plurality of imaging events according to a predetermined protocol (e.g., sequencing-by-synthesis). For example, the biological assay system may direct the flow of solution along the reaction site. At least one solution may include four types of nucleotides having the same or different fluorescent labels. The nucleotide can bind to the corresponding oligonucleotide at the reaction site. The biological assay system may then use an excitation light source (e.g., 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 range of wavelengths. The excited fluorescent label provides an emission signal that can be detected by a photodetector.

In one aspect, a solid-state imager includes a CMOS image sensor including 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 may be substantially one-to-one. An exemplary biosensor is described, for example, in U.S. patent application No. 13/833,619.

By "detection surface" is meant any surface that includes a light detector. The detector may be based on any suitable technology, such as those including a Charge Coupled Device (CCD) or a Complementary Metal Oxide Semiconductor (CMOS). In particular embodiments, CMOS imagers with single-photon avalanche diodes (CMOS-SPADs) may 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. 2008/0037008A 1, "biomedical optics Express 1:1302-1308 (2010)" to Giraud et al, or "IEEE European Solid-State device Conference (ESCIRC), Athens, Greece, IEEE, pp.204-207 (2009)", to 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, the devices 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.

Further, it will be appreciated that other signal detection devices as are known in the art may be used to detect the signals generated in the methods set forth herein. For example, a detector for detecting pyrophosphate or protons is particularly useful. Pyrophosphate release can be detected using a detector such as that commercially available from 454Life Sciences (Branford, conn., a Roche Company) or 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 include those commercially available from IonTorrent (Guilford, conn., a ThermoFisher subsidiary) or described in U.S. patent publication Nos. 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 U.S. patent publication No. 2013/0116128a1, which is incorporated by reference herein in its entirety.

By "sequencing module" is meant a CMOS chip suitable for sequencing applications. The module may comprise a surface comprising a substrate for nucleic acid attachment and an amplified hydrophilic region surrounded by a hydrophobic region. For example, dynamic pads with hydrophilic patches, such as those described above, may be used. Alternatively or additionally, a stack of dynamic pads comprising some pads in a hydrophilic state surrounding pads in a hydrophobic state at the same time may form a hydrophilic region surrounded by a hydrophobic region. The surface for nucleic acid attachment will optionally comprise a plurality of isolation regions, such that each isolation region comprises a plurality of nucleic acid molecules, preferably derived 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, and the like. The hydrophobic regions are preferably located between the hydrophilic regions. The reagent is moved across the surface by any number of forces.

The subject matter described herein, in one or more embodiments, includes disposable microfluidic cartridges and methods of making and using the same. The method of manufacturing a disposable integrated microfluidic cartridge optionally utilizes a flexible Printed Circuit Board (PCB) and roll-to-roll (R2R) printed electronics for monolithic integration of CMOS technology and finger fluidic devices. That is, the disposable integrated microfluidic cartridge comprises a stack of fluidic layers, in which the CMOS sensors are integrated, all mounted in a housing. Accordingly, conventional injection molding fluidics may be integrated with flexible PCB technology. The fluid layer is formed using a material suitable for use in R2R printed electronics. In addition, the fluid layer includes a Polymerase Chain Reaction (PCR) region and a reagent mixing and distribution region. The fluidic layer also includes a set of membrane valves, and the PCR zone can be completely blocked by the membrane valves.

Methods of using disposable integrated microfluidic cartridges include multiplex PCR and downstream mixing required to perform sequencing.

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

Fig. 14 shows a flow chart of an example of a method 100 of printing electronic devices using a flexible Printed Circuit Board (PCB) and roll-to-roll (R2R) for monolithically integrated and finger-like fluidic devices for CMOS technology. That is, using the method 100, the multilayer laminated fluidic device may be integrated with flexible PCB technology (see fig. 15). Furthermore, conventional injection molding fluidics may be integrated with flexible PCB technology (see fig. 26-45) using the structures formed using the method 100. The method 100 may include, but is not limited to, the following steps.

At step 110, a fluid layer is formed and then laminated and bonded together. For example, fig. 15 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 layer 200 includes, in order, an inlet/outlet layer 210, a fluidic channel layer 220, a flexible PCB layer 260, a sequencing chamber bottom layer 280, a sequencing chamber layer 250, and a membrane layer 240 coplanar with a sequencing chamber top layer 290. Inlet/outlet layer 210, fluid channel layer 220, flexible PCB layer 260, sequencing chamber bottom layer 280, sequencing chamber layer 250, membrane layer 240, and sequencing chamber top layer 290 are suitable for formation using R2R printed electronics processes.

The inlet/outlet layer 210 may be formed of, for example, polycarbonate, polymethyl methacrylate (PMMA), Cyclic Olefin Copolymer (COC), and/or polyimide. The inlet/outlet layer 210 may 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 apertures) provide fluid paths that may be used, for example, as feed and/or discharge ports for various liquid supply reservoirs (not shown). Further details of the inlet/outlet layer 210 are shown and described herein below with reference to fig. 55A and 55B.

The fluid channel layer 220 may be formed of, for example, polycarbonate, PMMA, COC, and/or polyimide. The fluid channel layer 220 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 fluid channels is provided in the fluid channel layer 220. The fluid channels provide a fluid path along the fluid channel layer 220 from one destination to another. Because fluid channel layer 220 is sandwiched between inlet/outlet layer 210 and flexible PCB layer 260, fluid may be confined within the fluid channel by inlet/outlet layer 210 on the bottom and by flexible PCB layer 260 on the top. In one example, the fluidic 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 fig. 56A and 56B.

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

Sequencing chamber floor 280 can be formed of, for example, polycarbonate, PMMA, COC, and/or polyimide. Sequencing chamber floor 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 membrane valves within the stack of fluid layers 200. Sequencing chamber bottom layer 280 also includes a CMOS device, such as CMOS image sensor 262, positioned proximate to the sequencing chamber of sequencing chamber layer 250. Sequencing chamber bottom layer 280 is coplanar with the CMOS device and serves as a fluidic connection layer to the inlet/outlet of the sequencing chamber to sequencing chamber layer 250. Further details of sequencing chamber floor 280 are shown and described herein below with reference to FIGS. 58A and 58B.

The sequencing chamber layer 250 can be formed of, 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 membrane valves within the stack of fluid 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 fig. 59A and 59B.

The membrane layer 240 may be formed of, for example, a silicone elastomer. The membrane layer 240 may be from about 25 μm to about 1000 μm thick in one example, or about 250 μm thick in another example. Membrane layer 240 serves as an elastic membrane for opening and closing membrane valves within the stack of fluid layer 200, where the membrane valves are created in sequence from the combination of flexible PCB layer 260, sequencing chamber bottom layer 280, sequencing chamber layer 250, and membrane layer 240. Further details of the diaphragm valve are shown and described herein below with reference to fig. 22A, 22B, 23A and 23B. Further details of the membrane layer 240 are shown and described herein below with reference to fig. 60A and 60B.

The sequencing chamber top layer 290 may be formed of a low autofluorescent material with good optical properties, such as COC. The 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 the sequencing chamber top layer 290 are shown and described herein below with reference to FIGS. 60A and 60B.

Referring now again to fig. 14, at step 115, the CMOS device is attached to the flexible PCB. For example, CMOS image sensor 262 (see fig. 15) is attached to sequencing chamber floor 280 of fluidic layer 200. Fig. 16 shows a perspective view of an example of the CMOS image sensor 262. In one example, the CMOS image sensor 262 is about 9200 μm long, about 8000 μm wide and about 800-1000 μm thick; and may have approximately 50I/O pads. The CMOS image sensor 262 may include a pixel array. In one example, the pixel array is 4384x 3292 pixels, with an overall size of about 7272 μm x 5761 μm. It will be appreciated that CMOS wafers may have a wide range of sizes and I/O pad counts. For example, a rectangular wafer (e.g., a non-square size that appears slim) may be used with finger fluidic devices to utilize only a portion of the wafer in any given analysis protocol.

Continuing with step 115, fig. 17A, 17B, 18, 19, and 20 show side views of structure 400 showing an example of a process of attaching a CMOS device to a flexible PCB. Structure 400 is a multi-layer structure. Referring now to fig. 17A, the initial formation of the structure 400 begins with a flexible PCB. For example, the flexible PCB includes a polyimide layer 410, a PCB heater layer 412, a polyimide layer 414, a PCB wiring layer 416, and a polyimide layer 418 in this order. That is, FIG. 17 shows a flexible PCB having a PCB heater layer and a PCB wiring layer, also called a roll of foil.

Next and referring now to fig. 17B, a low temperature isotropic conductive adhesive (low temperature ICA)420 is dispensed on top of the polyimide layer 418.

Next and referring now to fig. 18, a CMOS device such as a CMOS image sensor 262 is placed on the roll of foil; that is, on top of the low-temperature ICA 420. In one example, the CMOS image sensor 262 is placed on top of the low temperature ICA 420 using a well known pick and place process. Fig. 18 shows that the I/O pad 422 of the CMOS image sensor 262 is in contact with the low-temperature ICA 420 and thus electrically connected to the PCB wiring layer 416. Other attachment options also exist, including but not limited to controlled collapse/flip chip bonding, wire bonding, and the like. Fig. 18 also shows that the CMOS image sensor 262 includes a bio-layer 424 facing away from the polyimide layer 418. Protective film 426 may be placed on top of bio-layer 424 until ready for use.

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

Next and referring now to fig. 20, flip chip bonding of the CMOS image sensor 262 on the roll foil is accomplished 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 fluidic 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 fluidic portion 810 and a CMOS portion 812, which are based on the structure 400 shown in fig. 20. 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) at the CMOS portion 812, laminating a low autofluorescent Cyclic Olefin Copolymer (COC) layer 816 to the CMOS portion 812 of the microfluidic cartridge 800, and laminating a flexible PCB heater 818 on both sides of the fluidic portion 810. In forming the microfluidic cartridge 800, it is important to use a self-aligned process flow so that the surfaces of the CMOS device and the fluidic layer are flush with each other.

A fluid path is formed through the microfluidic cartridge 800. That is, sample inlet 820 is disposed at the input of fluidic portion 810 and outlet 822 is disposed downstream of CMOS portion 812. Sample inlet 820 feeds PCR chamber 824. The PCR chamber 824 then feeds reagent dispensing region 826. Reagent dispensing region 826 then feeds sequencing chamber 828. The bio-layer 424 of the CMOS image sensor 262 is oriented toward the sequencing chamber 828. Sequencing chamber 828 then feeds outlet 822. In addition, microfluidic cartridge 800 includes certain membrane valves 830 that control the flow of liquid into and out of PCR chamber 824.

Fig. 22A and 22B show perspective views of an example of a diaphragm valve 830, which can be integrated into, for example, the fluidic layer 200. Reference is now made to fig. 22A, which is a perspective view of the diaphragm valve 830. In this example, the membrane valve 830 includes, 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 may be formed of, for example, polycarbonate, PMMA, COC, and/or polyimide. The reservoir layer 914 has recessed areas of small reservoirs 916 created in the reservoir layer 914. The membrane layer 918 is stretched across the reservoir 916. The reservoir 916 has an inlet 920 and an outlet 922 that provide a flow path to a respective fluid channel 924. To better illustrate the features of the reservoir 916 and the inlet 920 and outlet 922, fig. 22B shows the membrane valve 830 without the membrane layer 918 covering the reservoir 916. The membrane layer 918 is formed of a flexible and stretchable elastomeric film material (e.g., a silicone elastomer).

Fig. 23A and 23B each show a cross-sectional view of the diaphragm valve 830 taken along line a-a of fig. 22A. An actuator such as actuator 1010 may be used to open and close the diaphragm valve 830. For example, fig. 23A shows the diaphragm valve 830 in an open state, where the actuator 1010 is not engaged with the diaphragm layer 918. In contrast, fig. 23B shows the membrane valve 830 in a closed state, where the actuator 1010 is engaged with the membrane layer 918. That is, the tip of the actuator 1010 acts to push the central portion of the membrane layer 918 against the outlet 922 and thereby prevent the flow of liquid therethrough. Diaphragm valve 830 (i.e., diaphragm valves 242, 244, and 246) may be activated using, for example, mechanical or pneumatic actuation such as a solenoid or pneumatic pump.

Fig. 24 shows a schematic diagram of an example of a microfluidic cartridge 1100 comprising integrated CMOS technology and finger fluidic devices. That is, microfluidic cartridge 1100 includes fluidics layer 200 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 fluidic layer 200 includes a PCR region 270 and a reagent mixing and dispensing region 275. PCR region 270 includes, for example, four PCR channels 222 (e.g., PCR channels 222a, 222b, 222c, 222 d). The inlets of PCR channels 222a, 222b, 222c, and 222d are fed by sample supplies 1110a, 1110b, 1110c, and 1110d, respectively. Because microfluidic cartridge 1100 includes four PCR channels 222 fed by four sample supplies 1110, microfluidic cartridge 1100 is configured for 4X sample multiplexing.

Four membrane valves 242 are used to control the input to four PCR channels 222. That is, membrane valves 242a, 242b, 242c, and 242d are used to control the inputs to PCR channels 222a, 222b, 222c, and 222d, respectively. Similarly, four membrane valves 244 are used to control the output of four PCR channels 222. That is, the outputs of PCR channels 222a, 222b, 222c, and 222d are controlled using membrane valves 244a, 244b, 244c, and 244d, respectively. The outputs of the four PCR channels 222 feed a common PCR output channel 224, which common PCR output channel 224 in turn feeds a reagent mixing and dispensing zone 275. The presence of membrane valve 242 and membrane valve 244 in fluid layer 200 allows PCR region 270 to be completely blocked.

The reagent mixing and distribution zone 275 includes an arrangement of 13 reagent channels 226 (e.g., reagent channels 226a-226 m). 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 fluidly connect a certain PCR channel 222 to a certain reagent supply 1112. In doing so, a certain PCR mix may be created. A rotatable valve assembly (not shown) is also used to fluidly connect a certain PCR mix to the 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 disposed at the outlet of the sequencing chamber 258. Outlet pump 1114 is fluidly and operatively connected to sequencing outlet channel 230. Outlet pump 1114 is used to provide positive or negative pressure to move liquid in any direction along the flow path of fluid layer 200. In addition, a series of three membrane valves 246 are provided along the length of the sequencing outlet channel 230. Diaphragm valves 242, 244, and 246 may be implemented in accordance with diaphragm valve 830 shown and described in fig. 22A, 22B, 23A, and 23B.

Three membrane valves 246 at the sequencing outlet channel 230 can be used as pumps in place of or in conjunction with the outlet pump 1114. Thus, in one embodiment, microfluidic cartridge 1100 includes only outlet pump 1114 and three membrane valves 246 are omitted. In another embodiment, microfluidic cartridge 1100 includes only three membrane valves 246 and outlet pump 1114 is omitted. In yet another embodiment, microfluidic cartridge 1100 includes outlet pump 1114 and three membrane valves 246. In yet another embodiment, microfluidic cartridge 1100 includes any other type of pumping mechanism in place of outlet pump 1114 and/or three membrane valves 246. Further details of examples of implementing the microfluidic cartridge 1100 are shown and described herein below with reference to fig. 25 through 60B.

Fig. 25 and 26 show perspective views of a microfluidic cartridge assembly 1200, which is one example of a physical illustration of the integrated microfluidic cartridge 1100 shown in fig. 24. The microfluidic cartridge assembly 1200 is an example of a conventional injection molded fluidic device integrated with flexible PCB technology. In this example, the microfluidic cartridge assembly 1200 is a multi-compartment microfluidic cartridge that includes a housing 1210 secured atop a base plate 1212. The housing 1210 and the base plate 1212 may be formed of, for example, a molding compound and fastened together via screws (see fig. 32). The total height of the microfluidic cartridge assembly 1200 may be, for example, from about 12mm to about 100 mm. The overall length of the microfluidic cartridge assembly 1200 may be, for example, from about 100mm to about 200 mm. The overall width of the microfluidic cartridge assembly 1200 may be, for example, from about 100mm to about 200 mm.

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

In addition, fluidic assembly 1400 includes flexible PCB heaters 1412 wrapped around both sides of PCR region 270 of fluidic layer 200. Two separately controlled heater traces are provided in flexible PCB heater 1412 such that there is one heater trace on one side of PCB section 270 and another heater trace on the other side of PCB section 270. The flexible PCB heater 1412 is an example of the flexible PCB heater 818 of the microfluidic cartridge 800 shown in fig. 21. Further details of examples of heater traces are shown and described herein below with reference to fig. 28A through 28B. Further details of examples of flexible PCB heaters 1412 are shown and described herein below with reference to fig. 54A, 54B, and 54C.

Referring now again to fig. 25 and 26, the housing 1210 of the microfluidic cartridge assembly 1200 also 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 also 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 the same size or different. For example, reagent reservoir 1216 can hold a volume of liquid ranging from about 0.001ml to about 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 may hold a volume of liquid ranging from about 25ml to about 100ml, for example. Figure 26 shows that the reagent reservoir 1216 and waste reservoir 1218 can be covered and sealed using, for example, a foil seal 1220.

Fig. 28A and 28B show a plan view and a cross-sectional view, respectively, of an example of a heater trace 1500 that may be mounted in the fluidic assembly 1400 shown in fig. 27A and 27B. That is, fig. 28A shows a plan view of an example of a heater trace 1500 having a serpentine type layout. Fig. 28B shows a cross-sectional view of one side of the flexible PCB heater 1412 of the fluidic assembly 1400 including the heater trace 1500. The flexible PCB heater 1412 is comprised of, in order, for example, a single-sided flexible copper layer 1510, an adhesive layer 1512, a dielectric layer 1514, a copper heater layer 1516 (with heater trace 1500 patterned), andthe multilayer structure of layer 1518. Copper heater layer 1516 shows a cross-section of heater trace 1500 taken along line a-a of fig. 28A.

Fig. 29, 30, 31, 32, 33A, and 33B illustrate various other views of the microfluidic cartridge assembly 1200 of fig. 25 showing more detail therein. That is, fig. 29 shows a perspective view of the housing 1210 side of the microfluidic cartridge assembly 1200, while fig. 30 shows a plan view, both of which show more detail of the configuration of the 13 reagent reservoirs 1216 and the waste reservoirs 1218. Fig. 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 so that the four sample loading ports 1214 remain exposed and accessible.

Fig. 32 shows a perspective view of the housing 1212 side of the microfluidic cartridge assembly 1200. Fig. 33A shows a plan view of the bottom plate 1212 side of the microfluidic cartridge assembly 1200. Fig. 33B shows a side view of the microfluidic cartridge assembly 1200. Fig. 32, 33A, and 33B show more detail of the bottom plate 1212. That is, the bottom plate 1212 includes openings 1222 and 1224 to reveal portions of the PCR region 270 of the fluidics layer 200 of the fluidics assembly 1400. Shown through opening 1224 is a set of I/O pads 1226 for contacting a flexible PCB heater 1412 of fluidic assembly 1400.

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

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

In addition, bottom plate 1212 includes openings 1232 for accessing and actuating four membrane valves 246 of fluidic layer 200 of fluidic assembly 1400. The bottom plate 1212 also includes an opening 1234 at the sequencing chamber 258. One corner of the bottom plate 1212 has a bevel 1236 that is used to orient the microfluidic cartridge assembly 1200 in the instrument plane of the microfluidic system (not shown). Fig. 32 and 33A also show four screws 1238 for fastening the base plate 1212 to the housing 1210. Further, rotatable valve assembly 1410 is shown relative to reagent mixing and dispensing region 275 of fluidic layer 200 of fluidic assembly 1400. Rotatable valve assembly 1410 includes a knob having a handle portion 1240 through which a user or device can rotate a flow controller portion 1242 (see fig. 35).

Starting with the microfluidic cartridge assembly 1200 oriented with the bottom plate 1212 side facing upward, fig. 34-42 essentially illustrate a step-by-step deconstruction of the microfluidic cartridge assembly 1200 as a means of revealing the placement and installation of internal components therein. First, fig. 34 shows a microfluidic cartridge assembly 1200 with a bottom plate 1212 removed to reveal a fluidic assembly 1400. In doing so, the flexible PCB layer 260 side of the fluidic layer 200 is visible. In addition, one side of the flexible PCB heater 1412 is visible. Also disclosed is a spacer 1244 between the fluid layer 200 and the bottom plate 1212. In fig. 34, membrane valves 242, 244 and 246 are visible.

Referring now to fig. 35, the handle portion 1240 of the rotatable valve assembly 1410 is removed so that the flow controller portion 1242 is now visible. The underside (not shown) of the grip portion 1240 is designed to engage with the flow controller 1242 such 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, flow controller portion 1242 of rotatable valve assembly 1410 is removed such that the fluid paths associated with PCR output channel 224, reagent channel 226, and sequencing feed channel 228 of fluidic layer 200 are visible.

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

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

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

Fig. 43 shows a transparent perspective view of the housing 1210 of the microfluidic cartridge assembly 1200 to illustrate the location 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 location of the opening 1246 relative to the sample loading port 1214, the location of the opening 1248 relative to the reagent reservoir 1216, and the location of the opening 1250 relative to the waste reservoir 1218.

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

Fig. 46A, 46B, 47A, 47B, and 48 illustrate various views of the housing 1210 of the microfluidic cartridge assembly 1200 of fig. 25 showing more detail therein. That is, fig. 46A and 46B show a plan view and a side view of the case body 1210, respectively. In one example, the 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. Fig. 47A shows a perspective view of the housing 1210 without the foil seal 1220 installed. Fig. 47B shows a perspective view of the housing 1210 with the foil seal 1220 installed. Although fig. 46A, 46B, 47A, and 47B illustrate the exterior of the housing 1210, fig. 48 illustrates a plan view of the interior of the housing 1210.

Fig. 49, 50, 51A, 51B, and 52 illustrate various views of the bottom plate 1212 of the microfluidic cartridge assembly 1200 of fig. 25 showing further details therein. That is, fig. 49 and 50 show perspective views of the exterior and interior of the bottom plate 1212, respectively. Fig. 51A shows a plan view of the exterior of the base plate 1212, while fig. 51B shows a side view of the base plate 1212. Fig. 49, 50, 51A, 51B and 52 show the base plate 1212 further including four holes 1258 for receiving screws 1238, a recessed area 1260 with a centrally located opening 1262 for receiving the handle portion 1240 and the flow controller portion 1242 of the rotatable valve assembly 1410.

Fig. 53A and 53B illustrate other perspective views of the fluidic assembly 1400 of the microfluidic cartridge assembly 1200 showing more detail therein. That is, fig. 53A and 53B each show a perspective view of the fluidic assembly 1400. Fig. 53A shows the fluidic assembly 1400 without the flexible PCB heater 1412, while fig. 53B shows the fluidic assembly 1400 with the flexible PCB heater 1412 installed. In addition, there is a notch 1414 on one edge of the fluid layer 200 and within the PCR zone 270. Notch 1414 is designed to receive flexible PCB heater 1412.

Fig. 54A, 54B, and 54C show additional views illustrating more details of the flexible PCB heater 1412 of the fluidic assembly 1400 of the microfluidic cartridge assembly 1200. That is, fig. 54A and 54B show a perspective view of each side of the flexible PCB heater 1412, respectively, and fig. 54C shows a side view of the flexible PCB heater 1412. The flexible PCB heater 1412 includes a U-shaped wrap panel 1416 and a side extension panel 1418 both formed using flexible PCB technology. The U-wrap panel 1416 includes a panel 1420 and a panel 1422, each having heater traces 1500, namely heater traces 1500a and 1500b, patterned therein. An example of a heater trace 1500 is shown in fig. 28A and 28B. The space between panel 1420 and panel 1422 is configured such that flexible PCB heater 1412 can be press-fit onto PCR region 270 of fluidic layer 200 and into notch 1414, as shown in fig. 53B. Fig. 54B and 54C also show I/O pads 1226 that provide electrical connections to both heater traces 1500 and to CMOS image sensor 262.

A side extension panel 1418 extends from the panel 1420 near the bend in the U-shaped wrap panel 1416. The side extension panel 1418 is designed to extend toward the CMOS image sensor 262. As shown in fig. 53B, the end of the side extension panel 1418 furthest from the U-wrap panel 1416 is shaped to fit against the CMOS image sensor 262. The purpose of the side extension panel 1418 is to provide electrical connections to the CMOS image sensor 262 assembled atop a rigid or flexible PCB.

Fig. 55A and 55B show perspective and plan views, respectively, of the inlet/outlet layer 210 of the fluidic layer 200 shown in fig. 15 and 27. Again, the inlet/outlet layer 210 may 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, the 13 reagent reservoirs 1216, and the waste reservoirs 1218 of the housing 1210 to the fluid passage layer 220 of the fluidics layer 200. For example, the inlet/outlet layer 210 includes a set of openings 212 that are substantially aligned with the openings 1246 of the sample loading port 1214 in the housing 1210. The inlet/outlet layer 210 includes a set of openings 214 that substantially align with openings 1248 of the reagent reservoirs 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.

Fig. 56A and 56B show perspective and plan views, respectively, of the fluid channel layer 220 of the fluidic layer 200 shown in fig. 15 and 27. Again, the fluid passage layer 220 may be formed of, for example, polycarbonate or any other material suitable for use in the R2R process. Fluid channel layer 220 is the layer in fluid layer 200 where the flow of all liquids is promoted. That is, all PCR and sequencing operations occur at the fluidic channel layer 220. The PCR operation occurs in PCR channel 222 at PCR region 270. The PCR output channel 224 feeds the reagent mixing and dispensing region 275. Reagent dispensing occurs using the reagent channels 226 at the reagent mixing and dispensing zone 275. 13 reagent channels 226 are patterned to feed rotatable valve assembly 1410. The sequencing feed channel 228 feeds the inlet of the sequencing chamber 258 of the sequencing chamber layer 250 shown in FIGS. 58A and 58B. The sequencing outlet channel 230 is then fluidly connected to an outlet of the sequencing chamber 258.

Fig. 57A and 57B show perspective and plan views, respectively, of the flexible PCB layer 260 of the fluidic layer 200 shown in fig. 15 and 27. Again, the flexible PCB layer 260 may be formed of, for example, polycarbonate or any other material suitable for use on the R2R process. Flexible PCB layer 260 includes a set of openings (or holes) 264 associated with the inlet/outlet of membrane 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 membrane valve 246 is present, flexible PCB layer 260 includes a set of openings (or holes) 267 associated with the inlet/outlet of membrane valve 246. In addition, flexible PCB layer 260 includes a set of openings 268 that are substantially aligned with rotatable valve assembly 1410 and provide a fluid path to rotatable valve assembly 1410.

Fig. 58A and 58B show perspective and plan views, respectively, of sequencing chamber floor 280 of fluidics layer 200 shown in fig. 15 and 27. Again, sequencing chamber bottom layer 280 may be formed of, for example, polycarbonate or any other material suitable for use on the R2R process. Sequencing chamber bottom layer 280 includes a set of openings 282 for membrane valves 242 formed in the stack of fluid layer 200. Sequencing chamber bottom layer 280 includes a set of openings 284 for membrane valves 244 formed within the stack of fluid layer 200. If membrane layer 246 is present, sequencing chamber bottom layer 280 includes a set of openings 286 for membrane valves 246 formed within the stack of fluid layer 200. In addition, sequencing chamber bottom layer 280 includes a set of openings 288 that are substantially aligned with rotatable valve assembly 1410 and provide a fluid path to rotatable valve assembly 1410. In addition, the sequencing chamber bottom layer 280 includes a pair of openings 289 fluidly coupled to the sequencing chamber 258 of the sequencing chamber layer 250.

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

Fig. 59A and 59B show perspective and plan views, respectively, of the sequencing chamber layer 250 of the fluidics layer 200 shown in fig. 15 and 27. Again, the sequencing chamber layer 250 may be formed of, for example, polycarbonate or any other material suitable for use on the R2R process. Sequencing chamber layer 250 is the layer in which the sequencing operation of fluidic layer 200 occurs; i.e., using sequencing chamber 258.

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

Fig. 60A and 60B show perspective and plan views, respectively, of the membrane layer 240 and the sequencing chamber top layer 290 of the fluidic layer 200 shown in fig. 15 and 27. The membrane layer 240 can be formed of, for example, a silicone elastomer, while the sequencing chamber top layer 290 can be formed of, for example, COC. Membrane layer 240 serves as an elastic membrane for opening and closing membrane valves 242, 244, and 246 within the stack of fluid layer 200, where membrane valves 242, 244, and 246 are created in sequence from the combination of flexible PCB layer 260, sequencing chamber bottom layer 280, sequencing chamber layer 250, and membrane layer 240. Fig. 60A and 60B also show sequencing chamber top layer 290 for sequencing chamber 258 covering sequencing chamber layer 250.

Fig. 61A and 61B show a flow diagram of an example of a method 4800 of 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 fig. 24, the microfluidic cartridge assembly 1200 is configured for 4X sample multiplexing. Further, in method 4800, 13 reagent reservoirs 1216 are designated reagent reservoirs 1216a, 1216b, 1216c, 1216d, 1216e, 1216f, 1216g, 1216h, 1216i, 1216j, 1216k, 12161, and 1216 m. In addition, method 4800 utilizes an outlet pump 1114 that is fluidly connected to the microfluidic cartridge assembly 1200. An outlet pump 1114 is located downstream of the sequencing chamber 258. Outlet pump 1114 is capable of providing both positive and negative pressures (i.e., vacuum pressure). Method 4800 includes, but is not limited to, the following steps.

At step 4810, a microfluidic cartridge assembly 1200 is provided that is ready for use. That is, the microfluidic cartridge assembly 1200 is provided with its reservoir or reservoirs filled with the desired liquid. For example, reagent reservoirs 1216 can be filled with the same or different reagent liquids. In one example, all of the reagent reservoirs 1216a-m are filled with a hydrogenation buffer (HT 1). Method 4800 proceeds to step 4815.

At step 4815, all membrane valves are closed and then sample/PCR MIX is loaded. "PCR MIX" means a PCR Master MIX optimized for use in conventional PCR for amplifying a DNA template. In this step, membrane valves 242a and 244a are closed, membrane valves 242b and 244b are closed, membrane valves 242c and 244c are closed, and membrane valves 242d and 244d are closed. In this manner, PCR channels 222a, 222b, 222c, and 222d are all completely blocked. Then, the first sample liquid is mixed with PCR MIX (hereinafter referred to as sample/PCR MIX1) and loaded into the sample loading port 1214 a. The second sample liquid is mixed with PCR MIX (hereinafter referred to as sample/PCR MIX2) and loaded into the sample loading port 1214 b. The third sample liquid is mixed with PCR MIX (hereinafter referred to as sample/PCR MIX3) and loaded into the sample loading port 1214 c. The fourth sample liquid is mixed with PCR MIX (hereinafter referred to as sample/PCR MIX4) and loaded into the sample loading port 1214 d. At the 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 membrane valve of the first sample is opened. The first sample is then pulled into the PCR region. The membrane valve for the first sample is then closed. For example, membrane valves 242a and 244a of PCR channel 222a are opened. sample/PCR MIX1 is then pulled into PCR channel 222a using outlet pump 1114. Membrane valves 242a and 244a of PCR channel 222a are then closed, with the volume of sample/PCR MIX1 now sealed inside PCR channel 222 a. Method 4800 proceeds to step 4825.

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

At step 4830, the membrane valve for the next sample is opened. The next sample is then pulled into the PCR region. Then, the membrane valve for the next sample is closed. In one example, membrane valves 242b and 244b of PCR channel 222b are opened. sample/PCR MIX2 is then pulled into PCR channel 222b using outlet pump 1114. Membrane valves 242b and 244b of PCR channel 222b are then closed, with the volume of sample/PCR MIX2 now sealed inside PCR channel 222 b.

In another example, membrane valves 242c and 244c of PCR channel 222c are opened. sample/PCR MIX3 is then pulled into PCR channel 222c using outlet pump 1114. Membrane valves 242c and 244c of PCR channel 222c are then closed, with the volume of sample/PCR MIX3 now sealed inside PCR channel 222 c.

In yet another example, membrane valves 242d and 244d of PCR channel 222d are opened. sample/PCR MIX4 is then pulled into PCR channel 222d using outlet pump 1114. Membrane valves 242d and 244d of PCR channel 222d are then closed, with the volume of sample/PCR MIX4 now sealed inside PCR channel 222 d.

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 222 d. 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 MIX 4. Method 4800 proceeds to step 4840.

At step 4840, the rotatable valve is rotated to the first PRC MIX position. For example, by rotating handle portion 1240 of rotatable valve assembly 1410, the position of rotatable valve assembly 1410 is set to PCR channel 222a, which holds PCR MIX 1. Method 4800 proceeds to step 4845.

At step 4845, the membrane valve of the first PCR MIX is opened. The first PCR MIX is then pulled through the rotatable valve to the CMOS device. Then, the membrane valve of the first PCR MIX was closed. For example, membrane 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. Then, membrane valves 242a and 244a are closed. Method 4800 proceeds to step 4850.

At step 4850, the rotatable valve is rotated to the hydrogenation buffer (HT1) position, meaning to reagent reservoir 1216 holding HT 1. In method 4800, at least one reagent reservoir 1216 is holding a volume of HT 1. By way of example, reagent reservoir 1216k holds a volume of HT 1. Thus, by rotating handle portion 1240 of rotatable valve assembly 1410, the position of rotatable valve assembly 1410 is now set to reagent reservoir 1216k, which holds HT 1. 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 and mixed with HT1 therein. Method 4800 proceeds to step 4860.

At decision step 4860, it is determined whether another PCR MIX is waiting to be mixed with HT 1. 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 handle portion 1240 of rotatable valve assembly 1410, the position of rotatable valve assembly 1410 is set to PCR channel 222b, which holds PCR MIX 2. In another example, by rotating handle portion 1240 of rotatable valve assembly 1410, the position of rotatable valve assembly 1410 is set to PCR channel 222c, which holds PCR MIX 3. In yet another example, by rotating handle portion 1240 of rotatable valve assembly 1410, the position of rotatable valve assembly 1410 is set to PCR channel 222d, which holds PCR MIX 4. Method 4800 proceeds to step 4870.

At step 4870, the membrane valve for the next PCR MIX is opened. The next PCR MIX is then pulled through the rotatable valve to the CMOS device. Then, the membrane valve of the next PCR MIX is closed. In one example, membrane 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 through rotatable valve assembly 1410. Then, membrane valves 242b and 244b are closed. In another example, membrane 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 through rotatable valve assembly 1410. Then, membrane valves 242c and 244c are closed. In yet another example, membrane 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. Then, membrane valves 242d and 244d are closed. Method 4800 proceeds to step 4875.

At step 4875, the rotatable valve is rotated to the HT1 position. For example, by rotating handle portion 1240 of rotatable valve assembly 1410, the position of rotatable valve assembly 1410 is returned to reagent reservoir 1216k, which holds HT 1. Method 4800 proceeds to step 4880.

At step 4880, the next PCR MIX is pushed into the HT1 reservoir. In one example, using outlet pump 1114, PCR MIX2 is pushed through rotatable valve assembly 1410 and into reagent reservoir 1216k and mixed with HT1 therein. In another example, using outlet pump 1114, PCR MIX3 is pushed through rotatable valve assembly 1410 and into reagent reservoir 1216k and mixed with HT1 therein. In yet another example, using outlet pump 1114, PCR MIX4 is pushed through rotatable valve assembly 1410 and into reagent reservoir 1216k and mixed with HT1 therein. Method 4800 returns to step 4860.

At step 4885, the mix from HT1 reservoir was pushed into the sequencing chamber and the clustering/sequencing method was performed. For example, using reagent reservoir 1216k, which now holds a mixture of HT1, PCR MIX1, PCR MIX2, PCR MIX3, and PCR MLX4, this mixture is pulled out of reagent reservoir 1216k and then along sequencing feed channel 228 and into sequencing chamber 258. Then, using the CMOS image sensor 262, a clustering/sequencing method is performed. Method 4800 ends.

One or more embodiments may include a CMOS flow cell with an accessible biosensor active area. For example, CMOS flow cells can be designed as single use consumables. Accordingly, it may be beneficial for a CMOS flow cell to be a small and inexpensive device. In small CMOS flow cells it is important to use as much of the biosensor active area as possible. However, current CMOS flow cell designs do not allow 100% utilization of the biosensor active area. Therefore, new approaches are needed to provide increased utilization of the biosensor active area in CMOS flow cells. Embodiments set forth herein may include CMOS flow cells in which most or up to about 100% of the biosensor active area is accessible for reagent delivery and illumination, as shown and described herein below with reference to fig. 62 through 75.

Fig. 62 shows a side view of an example of a CMOS flow cell 4900 in which most or up to about 100% of the biosensor active area is accessible for reagent delivery and illumination. CMOS flow cell 4900 includes a PCB substrate 4910, which is, for example, a flexible PCB substrate. Atop PCB substrate 4910 is CMOS biosensor device 4920. CMOS biosensor device 4920 is a CMOS image sensor having a bio-layer thereon. Also atop PCB substrate 4910 and surrounding CMOS biosensor device 4920 is a laminate film 4930. Laminate film 4930 may be made of, for example, epoxy, polyimide or other plastic film, silicon, glass, or glass,A bismaleimide triazine resin (BT) substrate, and the like. PCB substrate 4910 and laminate film 4930 may be formed using flexible PCB technology. The planarized surface can also be created by machining a cavity in the PCB substrate.

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

The elongated gap between PCB substrate 4910 and laminate film 4930 forms a trench or channel 4950 around the perimeter of CMOS biosensor device 4920. The width of the trench or channel 4950 may be, for example, from about 100 μm to about 1000 μm. Trench or channel 4950 is filled with a filler material 4952 so as to form a substantially continuous planar surface across CMOS biosensor device 4920 and laminate film 4930. The fill material 4952 is a material that does not interfere with reactions occurring atop the CMOS biosensor device 4920. The filler material 4952 may be, for example, an Ultraviolet (UV) cured epoxy, a thermal cured epoxy, or the like.

Atop CMOS biosensor device 4920 and laminate film 4930 is flow cell lid 4940, with flow channel 4942 integrated in flow cell lid 4940. Additionally, flow cell lid 4940 includes a first port 4944 and a second port 4946 that provide an inlet/outlet to flow channel 4942. Flow cell lid 4940 is formed of 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 flow cell lid 4940 may be, for example, from about 300 μm to about 1000 μm. A bonding area exists outside of flow channel 4942 for bonding flow cell lid 4940 to laminate film 4930. The bonding may be via a low autofluorescence adhesive.

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

Fig. 62 shows reagent fluid 4954 filling flow channel 4942, for example. The chemical reaction takes place in reagent fluid 4954 in flow channel 4942, flow channel 4942 being atop CMOS biosensor device 4920. CMOS biosensor device 4920 is used to sense chemical reactions occurring in flow channel 4942 when illuminated through flow cell lid 4940. Electrical connections (not shown) are provided through PCB substrate 4910 for acquiring signals from CMOS biosensor device 4920. In CMOS flow cell 4900, approximately 100% of the biosensor active area of CMOS biosensor device 4920 is accessible for reagent delivery and illumination.

Fig. 63 shows an exploded view of one illustrative example of CMOS flow cell 4900 shown in fig. 62. Fig. 63 shows that CMOS biosensor device 4920 includes an active area 4922. Any portion of CMOS biosensor device 4920 outside of active area 4922 is inactive area 4924. CMOS biosensor device 4920 may be attached to PCB substrate 4910 using, for example, flip chip technology. Further, laminate film 4930 includes an opening or window 4932 that is sized to receive CMOS biosensor device 4920 when laminated against PCB substrate 4910. Opening or window 4932 is provided in laminate film 4930 prior to laminating laminate film 4930 to PCB substrate 4910. When flow cell lid 4940 is bonded to laminate film 4930, flow channel 4942 is substantially aligned with CMOS biosensor device 4920 and its area extends beyond the area of CMOS biosensor device 4920. In fig. 63, flow cell lid 4940 is shown as transparent. Fig. 64 and 65 show a perspective view and a side view, respectively, of CMOS flow cell 4900 shown in fig. 63 when fully assembled.

Fig. 66 shows a perspective view of an example of flow cell lid 4940 of CMOS flow cell 4900 shown in fig. 63, 64, and 65. That is, fig. 66 shows a top and bottom perspective view of flow cell lid 4940 of CMOS flow cell 4900 shown in fig. 63, 64, and 65. In this example, the diameter of the first port 4944 and the second port 4946 may be about 750 μm. Further, the depth or height of the flow channel 4942 may be about 100 μm.

Fig. 67, 68, 69 and 70 show examples of processes that provide an extended flat surface in a CMOS flow cell on which a flow cell cover can be mounted.

In a first step and referring now to fig. 67, laminate film 4930 and CMOS biosensor device 4920 are disposed atop PCB substrate 4910. A trench or channel 4950 exists around the perimeter of CMOS biosensor device 4920. Trench or channel 4950 exists because opening or window 4932 in laminate film 4930 is slightly larger than CMOS biosensor device 4920.

In the next step and referring now to fig. 68, the upper side of the trench or channel 4950 is sealed using, for example, an optically transparent elastomer 4960 with a surface for mating against the trench or channel 4950. Elastomer 4960 is optically transparent so that UV light can pass through it. The purpose of the elastomer 4960 is to seal off the top of the trench or channel 4950 in preparation for filling.

In the next step and referring now to fig. 69, using a pair of perforations 4916 in PCB substrate 4910, the trenches or channels 4950 are filled with a filler material 4952, such as a UV cured epoxy, which is why elastomer 4960 is optically transparent.

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

Fig. 71A, 71B, 71C and 71D show another example of a process of providing an extended flat surface in a CMOS flow cell on which a flow cell cover can be mounted.

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

In the next step and referring now to fig. 71B, a mold 5510 (e.g., a clamshell mold) is disposed around CMOS biosensor device 4920 and PCB substrate 4910. Mold 5510 provides a space or void 5512 atop PCB substrate 4910 and around the perimeter of 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 cured or thermally cured epoxy, using, for example, a low pressure injection molding process or a reaction injection molding process.

In the next step and referring now to fig. 71D, once filler material 4952 is cured, mold 5510 is removed and a substantially continuous planar surface is now present in the flow cell for receiving a flow cell lid, such as flow cell lid 4940.

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

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

In the next step and referring now to fig. 73, a set of stops 5916 are provided at the ends of a groove or channel 5914. For example, barrier 5916a and 5916b block the ends of one groove or channel 5914, while barrier 5916c and 5916d block the ends of another groove or channel 5914 in preparation for filling.

In a next step and referring now to fig. 74, the trench or channel 5914 is filled with a filler material 4952, such as a UV cured or thermally cured epoxy. Filler material 4952 is held between barrier 5916a and 5916b and between barrier 5916c and 5916 d.

In the next step and referring now to fig. 75, once the filler material 4952 is cured, a substantially continuous planar surface is now present in the flow cell for receiving a flow cell lid, such as flow cell lid 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 a hardware embodiment, a software embodiment (including firmware, resident software, micro-code, etc.) or an embodiment combining software and hardware aspects that may all be referred to herein as a "circuit," module "or" system. Furthermore, 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 may be used for the software aspects of the present disclosure. The computer-usable or computer-readable medium may be, for example but not limited to, an electronic, magnetic, optical, electromagnetic, infrared, or semiconductor system, apparatus, device, or propagation medium. The computer-readable medium may include transitory and/or non-transitory embodiments. More specific examples (a non-exhaustive list) of the computer-readable medium would include some or all of the following: an electrical connection having one or more wires, a portable computer diskette, 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 media such as those supporting the Internet or an intranet, or a magnetic storage device. Note that the computer-usable or computer-readable medium could even be paper or another suitable medium upon which the program is printed, as the program can be electronically captured, via, for instance, optical scanning of the paper or other medium, then compiled, interpreted, or otherwise processed in a suitable 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.

Program code for carrying out operations of the methods and apparatus set forth herein may be written in an object oriented programming language such as Java, Smalltalk, C + +, or the like. However, program code for carrying out operations of the methods and apparatus set forth herein may also be written in conventional procedural programming languages, such as the "C" programming language or similar programming languages. The program code may be executed by a processor, an Application Specific Integrated Circuit (ASIC), or other component executing the program code. The program code may be referred to simply as a software application, which is stored in a memory (e.g., the computer-readable medium discussed above). The program code may cause the processor (or any processor-controlled device) to generate a graphical user interface ("GUI"). The graphical user interface may be visually generated on the display device, however the graphical user interface may also have audible features. However, the program code may operate in any processor controlled device such as a computer, server, personal digital assistant, telephone, television, or any processor controlled device that utilizes a processor and/or digital signal processor.

The program code may be executed locally and/or remotely. The program code may be stored in whole or in part in a local memory of a processor-controlled device, for example. However, the program code may also be stored, accessed and downloaded to the processor-controlled device at least partially remotely. The user's computer may execute the program code in its entirety or only partially, for example. The program code may be a stand-alone software package that executes at least in part on the user's computer and/or in part on a remote computer or entirely on the remote computer or server. In the latter scenario, the remote computer may be connected to the user's computer through a communications network.

The methods and apparatus set forth herein may be applied regardless of the networked environment. The communication network may be a cable network operating in the radio frequency domain and/or the Internet Protocol (IP) domain. The communication network may, however, also include a distributed computing network, such as the internet (sometimes alternatively referred to as a "wide area network"), an intranet, a Local Area Network (LAN), and/or a Wide Area Network (WAN). The communication network may include coaxial cables, copper wire, fiber optic lines, and/or hybrid-coaxial lines. The communication network may even include a wireless portion that utilizes any portion of the electromagnetic spectrum and any signaling standard (e.g., the IEEE 802 family of standards, GSM/CDMA/TDMA or any cellular standard and/or ISM band). The communication network may even comprise a power line section, wherein the signals are transferred via electrical wiring. The methods and apparatus set forth herein may be applied to any wireless/wired communication network regardless of physical components, physical configuration, or communication standards.

Certain aspects of the present disclosure are 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. The program code and/or machine instructions may create modules for implementing the functions/acts specified in the methods.

The program code may also be stored in a computer-readable memory that can direct a processor, computer, or other programmable data processing apparatus to function in a particular manner, such that the program code stored in the computer-readable memory produces or transforms an article of manufacture including instruction modules that implement 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 performed to produce a processor/computer implemented process such that the program code provides steps for implementing the various functions/acts specified in the method of the disclosure.

In an embodiment, a system is provided that includes a removable cartridge having a cartridge housing. The removable cartridge also includes a fluidic network disposed within the cartridge housing. The fluidic network is configured to receive and fluidically conduct a biological sample for at least one of sample analysis or sample preparation. The removable cartridge also includes a flow control valve operably coupled to the fluidic network and movable relative to the fluidic network to control a 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 operation of the flow control valve. The system also 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 through a system interface. The removable cartridge also 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 set forth herein and the housing side of the removable cartridge set forth herein are generally planar and face each other. The system interface may be single-sided, wherein the base instrument and the removable cartridge are operatively coupled to each other only through the housing side and the control side. Optionally, the base instrument and the removable cartridge may be 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 fluidic coupling, an electrical coupling, or a thermal coupling established through the system interface.

In another aspect, the control side of the base instrument set forth herein may represent the top of the base instrument with respect to gravity, such that the removable cartridge is located on and supported by the base instrument.

In another aspect, the valve actuator of the basic instrument set forth herein can include an elongated actuator body extending through a side of the housing and into the cartridge housing.

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

In another aspect, the basic instruments set forth herein may have an instrument side facing in an opposite direction relative to a 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 cartridge may have a combined size that is larger than the instrument size.

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

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

Alternatively, the first and second housing sides of the removable cartridge set forth herein may be generally perpendicular to each other. The base instrument may have an instrument housing including first and second control sides facing in perpendicular directions and forming an open-sided recess of the base instrument. At least a portion of the removable cartridge may be disposed within the open-sided recess such that the first and second housing sides engage the first and second control sides.

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

In another aspect, the first and second housing sides of the removable cartridge set forth herein face in 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 fluidly 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 may include a flow port. The flow ports are fluidly coupled to each other at a system interface.

In another aspect, the systems set forth 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 the base instrument and the reaction chamber can be held by the removable cartridge.

In another aspect, the flow control valve of the removable cartridge set forth herein may comprise a flexible membrane configured to control the flow of the biological sample through the fluidic network. The flexible membrane is bendable between first and second conditions by the valve actuator.

In another aspect, the housing side of the cartridge housing of the removable cartridge set forth herein may include an access opening therethrough that receives the valve actuator.

In another aspect, the flow control valve of the basic instrument set forth herein may comprise a rotatable valve configured to control the flow of fluid through the fluid network. The rotatable valve may be rotated by a valve actuator.

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

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

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

In embodiments, methods of sequencing nucleic acids are 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 an exterior of the removable cartridge. The method also includes contacting the removable cartridge to the base instrument. The housing side of the removable cartridge detachably engages the control side of the base instrument to collectively define a system interface. The base instrument includes a valve actuator that engages the flow control valve through a system interface. The method also includes fluidically directing the biological sample through a fluidic 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 the reaction chamber, wherein the flow of the biological sample is controlled by the action of a valve actuator on the flow control valve. The method also includes detecting the biological sample using an imaging detector oriented 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 set forth herein can further include removing the removable cartridge from the base instrument. The removable cartridge may be replaced by functionally mating the second removable cartridge with the base instrument. Several removable cartridges may be sequentially mated with the base instrument for preparing and/or analyzing samples while mated with 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 methods set forth herein include removing a removable cartridge from a base instrument. Optionally, the method includes contacting the 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 another aspect of the methods set forth herein, the operations of fluidically conducting and imaging a biological sample are repeated a plurality of times in sequence in a single removable cartridge.

In another aspect, the methods set forth herein comprise sealing a biological sample within a sample preparation region of a fluidic network, and amplifying the biological sample while the biological sample is sealed within the sample preparation region of the fluidic network.

In another aspect, a flow control valve for use 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, the moveable valve used in the methods set forth herein is in the sample position when the biological sample flows through the flow channel and is directed into the reaction chamber, the method further comprising moving the moveable valve to the component position and flowing a reagent through the flow channel into the reaction chamber, the reagent reacting with the biological sample within the reaction chamber.

In another aspect of the methods set forth herein, the component position comprises a plurality of component positions, the method further comprising moving the movable valve between the component positions according to a predetermined sequence to cause different reagents to flow into the reaction chamber.

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

In another aspect, a flow cell for use in the methods set forth herein comprises a reaction chamber. The 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 an 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 base instrument. The removable cartridge may also include a fluidic network having a plurality of channels, reaction chambers, and a storage module. The storage module includes a plurality of reservoirs for storing reagents. The fluid network is configured to direct a reagent from the reservoir to the reaction chamber, wherein the mechanical interface device is movable relative to the fluid network to control a flow of fluid 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. An imaging device is electrically coupled to the array of electrical contacts for communicating with the base instrument. The mechanical interface device may be configured to be moved by the base instrument when the removable cartridge is coupled to the base instrument.

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

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

In another aspect, the cartridge housing of the removable cartridge set forth herein may include an access opening exposed to the exterior, 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 a temperature of the sample channel.

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

In another aspect, the cartridge housing of the removable cartridge set forth 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 comprise mechanical interface means.

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 that is open to an exterior of the cartridge housing and configured to receive a biological sample. The removable cartridge may also 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 valve ports, wherein the rotatable valve is rotatable between different rotational positions. The removable cartridge may also include a microfluidic body having a body side slidably coupled to the fluidic side of the rotatable valve. The microfluidic body may at least partially define a fluidic 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 fluidic network may also include a reservoir configured to hold a reagent. The reservoir is in fluid communication with a reservoir port that is open to the fluid side of the microfluidic body. The fluidic network also includes a feed channel in fluid communication with the reaction chamber of the fluidic network. The feed channel has a feed opening that is open to the body side of the microfluidic body. The rotatable valve is configured to rotate between first and second rotational positions. The network port is fluidly coupled to the feed port through the rotatable valve when the rotatable valve is in the first rotational position. The reservoir port is fluidly coupled to the feed port through the rotatable valve when the rotatable valve is in the second rotational position.

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

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

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

In an embodiment, a removable cartridge is provided that includes a cartridge housing having a sample port that is open to an exterior of the cartridge housing and configured to receive a biological sample. The cartridge housing may include a mating side configured to face and removably couple to the base instrument. The removable cartridge also includes a fluidic network disposed within the housing. The fluidic 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 first and second positions. 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 mating side of the cartridge housing includes an access opening exposing the channel valve to the exterior of the cartridge housing. The access opening is configured to receive a valve actuator of a primary instrument for moving the flexible member between the first and second positions.

In another aspect, the flexible member of the removable cartridge set forth herein may include a flexible layer covering the inner lumen of the fluidic network. The flexible layer may 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 comprise a mechanical interface device accessible along the mating side.

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

In another aspect, the mating side of the removable cartridge set forth herein can be a first mating side, and the removable cartridge can include a second mating side. The first and second mating sides face in opposite directions. The second mating side is 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 the rotatable valve of the removable cartridge. The base instrument also includes a valve actuator configured to engage the channel valve of the removable cartridge and an array of electrical contacts configured to electrically couple to the removable cartridge. The base instrument also includes a system controller configured to control the rotary motor and the valve actuator to perform 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 thermal 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 that is open to an 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 base instrument. The removable cartridge also includes a microfluidic body disposed within the cartridge housing. The microfluidic body has a body side and includes a fluidic network. The fluid network has a plurality of discrete channels and corresponding ports that open at the side of the body at the 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 a plurality of valve ports. The valve port is open to the fluid side. The fluid side is rotatably coupled to a valve receiving region of the body side of the microfluidic body, wherein the rotatable valve is movable between different rotational positions to fluidly couple the discrete channels. The rotatable valve has a mechanical interface device accessible along the mating side and configured to engage the base instrument such that the rotatable valve is controlled by the base instrument.

In an embodiment, a removable cartridge is provided that includes a cartridge housing having a sample port that is open to an 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 including 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 layer also includes a routing layer. The removable cartridge also includes a CMOS imager configured to be mounted to the microfluidic structure and electrically coupled to the conductive routing 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 an exterior of the cartridge housing. The I/O contacts may be electrically coupled to the CMOS imager.

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

As used herein, an element or step recited in the singular and proceeded with the word "a" or "an" should be understood as not excluding plural said elements or steps, unless such exclusion is explicitly recited. 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 explicitly stated to the contrary, embodiments "comprising" or "having" an element or a plurality of 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 particular arrangement (e.g., number, type, placement, etc.) of the components of the illustrated embodiments may be modified. In various embodiments, different numbers of given modules or units may be used, different types of given modules or units may be used, given modules or units may be added, or given modules or units may be omitted.

It is to be understood that the above description is intended to be illustrative, and not restrictive. For example, the above-described embodiments (and/or aspects thereof) may be used in conjunction with one another. In addition, many modifications may be made to adapt a particular situation or material to the teachings of the various embodiments without departing from their scope. The dimensions, types of materials, orientations of the various embodiments, and the numbers and locations of the various components described herein are intended to define the parameters of the certain embodiments, and are by no means limiting and are merely exemplary embodiments. Many other embodiments and modifications within the spirit and scope of the claims will be apparent to those skilled in the art upon review of the foregoing description. The patentable scope should, therefore, be determined with reference to the appended claims, along with the full scope of equivalents to which such claims are entitled.

As used in the description, the phrases "in an exemplary embodiment," and the like, mean that the embodiment described is merely one 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 enumerated features or structures. In the appended claims, the terms "including" and "in which" are used as the plain-English equivalents of the respective terms "comprising" and "where". 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 limitations of the claims are written in a device-plus-function format and are not intended to be interpreted based on 35u.s.c. § 112(f), and until such claims expressly use the phrase "means for …" followed by a statement that there is no further structure for the function.

Claims (10)

1. A method of sequencing a nucleic acid, the method comprising:

providing a removable cartridge having a cartridge housing, a fluidic network disposed within the cartridge housing, and a flow control valve operably coupled to the fluidic network and movable relative to the fluidic network, wherein the cartridge housing comprises a housing side defining an exterior of the removable cartridge;

contacting the removable cartridge to a base instrument, wherein the housing side of the removable cartridge is separably engageable with a control side of the base instrument to collectively define a system interface, wherein the base instrument includes a valve actuator operatively engaging the flow control valve through the system interface;

fluidically directing a biological sample through the fluidic network of the removable cartridge for at least one of sample analysis or sample preparation in the removable cartridge, wherein the biological sample is directed to flow into a reaction chamber, wherein a flow rate of the biological sample is controlled by an action of the valve actuator on the flow control valve; and

imaging the biological sample in the reaction chamber using a detection assembly held by at least one of the removable cartridge or the base instrument.

2. The method of claim 1, further comprising removing the removable cartridge from the base instrument.

3. The method of claim 2, further comprising 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.

4. The method of claim 3, wherein the fluidically directing the biological sample and imaging the biological sample are repeated a plurality of times in sequence.

5. The method of any one of claims 1-4, further comprising 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.

6. The method of any of claims 1-4, wherein the flow control valve comprises a movable valve having at least one flow channel extending between valve ports, the valve actuator configured to move the movable valve between different positions.

7. The method of claim 6, wherein the movable valve is in a sample position when the biological sample flows through the at least one flow channel and is directed into the reaction chamber, the method further comprising moving the movable valve to a component position and flowing a reagent through the at least one flow channel into the reaction chamber, the reagent reacting with the biological sample within the reaction chamber.

8. The method of claim 7, wherein the component position comprises a plurality of component positions, the method further comprising moving the movable valve between the component positions according to a predetermined sequence to cause different reagents to flow into the reaction chamber.

9. The method of claim 8, wherein the biological sample comprises nucleic acids and the predetermined order is according to sequencing-by-synthesis SBS protocol.

10. The method of any one of claims 1-4, wherein a flow cell comprises the reaction chamber, the biological sample being immobilized to one or more surfaces of the flow cell.