RU2785864C2 - Replaceable cartridge for biochemical reactions - Google Patents

Abstract

FIELD: biochemistry.

SUBSTANCE: invention relates to a cartridge for biochemical reactions. The cartridge contains a case including a set of reservoirs for reagents and a hole for loading a sample; a reaction chamber; a jet equipment node including a hydraulic network and a rotary valve; and a seal closing and sealing the set of reservoirs for reagents in the case. The hydraulic network contains a channel for a sample, a set of reservoir channels, and a supplying channel, wherein the channel for a sample is in flow communication with the hole for loading a sample and a network hole, each of the set of reservoir channels is in flow communication with the corresponding reservoir for a reagent of the set of reservoirs for reagents and the corresponding reservoir hole of the set of reservoir holes, the supplying channel is in flow communication with a supplying hole and the reaction chamber. The rotary valve contains a valve case with a flow channel, the first hole, and the second hole. The second hole is in flow communication with the supplying hole, the valve case is capable of rotating around an axis from the first position, in which the first hole is in flow communication with the network hole, to the second position, in which the first hole is in flow communication with the corresponding reservoir hole of the set of reservoir holes.

EFFECT: provision of automatic implementation of biochemical analyses, including preparation of a sample, without participation of a user, as well as expansion of device capabilities of performing certain protocols.

10 cl, 75 dwg

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority of U.S. Provisional Application No. 62/003264, filed May 27, 2014, which is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

[0002] Embodiments of the present patent application generally relate to systems and methods for conducting biochemical reactions, and more specifically, to systems and methods in which a main instrument interacts with a removable cartridge to conduct reactions for at least one of sample preparation or biochemical analysis.

[0003] Various biochemical protocols involve performing a large number of controlled reactions on substrate surfaces or within dedicated reaction chambers. Controlled reactions may be performed to analyze a biological sample or to prepare a biological sample for subsequent analysis. The analysis can identify or reveal the properties of the chemicals involved in these reactions. For example, in template cyclic analysis of a nucleotide sequence (eg, sequencing by synthesis (from English, sequencing-by-synthesis - SBS)) a dense array of DNA fragments (eg, template nucleic acids) is sequenced through iterative cycles of enzymatic processing. After each cycle, an image can be captured, which can then be analyzed in conjunction with other images in order to determine the sequence of the DNA fragments. In another biochemical assay, an unknown analyte with an identifiable label (eg, a fluorescent label) may be exposed to an array of known probes having predetermined addresses in the array. Observing the chemical reactions that occur between these probes and an unknown analyte can help identify or reveal the properties of the analyte.

[0004] There is a general need for systems that automatically perform assays, such as those described above, that would require less work or less user intervention. Currently, most platforms require the user to separately prepare a biological sample before loading that biological sample into the system for analysis. It may be desirable for a user to load one or more biological samples into the system, select the type of analysis to be performed by the system, and receive the results of the analysis after a predetermined amount of time, such as a day or less. At least some of the systems in use today are not capable of performing certain protocols, such as whole genome sequencing, that would provide data at a sufficient level of quality and within a particular price range.

SHORT DESCRIPTION

[0005] In one embodiment, a system is provided that includes a removable cartridge having a cartridge body. The removable cartridge also includes a hydraulic network that is located inside the cartridge housing. This hydraulic network is configured to receive and hydraulically guide a biological sample to perform at least one of sample analysis or sample preparation. The removable cartridge also includes a flow control valve that is operatively connected to the hydraulic network and is movable relative to the hydraulic network to control the flow of the biological sample through it. The cartridge body includes a body side that defines the appearance of the removable cartridge and allows functional access to the flow control valve. The system also includes a host device having a control side that is configured to removably interact with said side of the removable cartridge body. The housing side and the control side together form the system interface. The main instrument includes a valve actuator that communicates with the flow control valve via a system interface. The removable cartridge also includes a sensing assembly that is held by at least one of the removable cartridge or host. The detection unit includes an image detector and a reaction chamber that is in fluid communication with the hydraulic network. The image detector is configured to detect intended reactions within the reaction chamber.

[0006] In one embodiment, a method for sequencing nucleic acids is provided. This method includes providing a removable cartridge having a cartridge body, a hydraulic network located within the cartridge body, and a flow control valve that is operatively connected to the hydraulic network and movable with respect to the hydraulic network. The cartridge body includes a body side that defines the appearance of the removable cartridge. This method also includes bringing the removable cartridge into contact with the main apparatus. The body side of the removable cartridge is releasably connected to the control side of the main unit, together forming a system interface. The main instrument includes a valve actuator that communicates with the flow control valve via a system interface. The method also includes hydraulically guiding the biological sample through the hydraulic 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, the flow of the biological sample being controlled by a valve actuator on the flow control valve. The method also includes detecting a biological sample using an image detector directed at the reaction chamber, wherein the detection assembly is held by at least one of the removable cartridge or main instrument.

[0007] In one embodiment, a removable cartridge is provided that includes a cartridge body having a sample opening that opens to the outside of the cartridge body and is configured to receive a biological sample. The cartridge body has a set of electrical contacts and a mechanical interface that are accessible from the outside. The cartridge body is designed to be detachably attached to the main device. The removable cartridge may also include a hydraulic network having a plurality of channels, a reaction chamber, and a storage module. The storage module includes a plurality of reagent storage tanks. The hydraulic network is configured to direct the reactants from the reservoirs to the reaction chamber, wherein the mechanical interface is movable relative to the hydraulic network to control fluid flow through the hydraulic network. The system also includes an imaging device located within the cartridge body and positioned to detect intended reactions within the reaction chamber. The visualization device is electrically connected to a set of electrical contacts for information exchange with the main device. The mechanical interface may be movable by the host when the removable cartridge is attached to the host.

[0008] In one embodiment, a removable cartridge is provided that includes a cartridge body having a sample opening that opens to the outside of the cartridge body and is configured to receive a biological sample. The removable cartridge may also include a rotary valve located within the cartridge housing. This rotary valve has a hydraulic side and a plurality of valve openings that open onto the hydraulic side. This rotary valve has at least one flow channel extending between valve openings, wherein the rotary valve is capable of being rotated between different angular positions. The removable cartridge may also include a microfluidic body having a body side that is slidably mated with the hydraulic side of the rotary valve. This microfluidic body may at least partially form a hydraulic network that includes a sample conduit in fluid communication with the sample orifice. The sample channel has a network opening that opens onto said side of the microfluidic body. The hydraulic network may also include a reservoir configured to hold the reagent. This reservoir is in fluid communication with a reservoir opening that opens onto the hydraulic side of the microfluidic body. The hydraulic network also includes a supply channel in fluid communication with the reaction chamber of the hydraulic network. The feed channel has a feed opening that extends to said side of the microfluidic body. The rotary valve is rotatable between the first and second angular positions. The network opening is hydraulically connected to the supply opening through a rotary valve when this rotary valve is in the first angular position. The reservoir opening is in fluid communication with the supply opening via the rotary valve when the rotary valve is in the second angular position.

[0009] In one embodiment, a removable cartridge is provided that includes a cartridge body having a sample opening that opens to the outside of the cartridge body and is configured to receive a biological sample. The cartridge body may include a docking side that is designed to face the main device and is configured to be removably connected to it. The removable cartridge also includes a hydraulic network that is located inside the housing. This hydraulic network includes a sample conduit that is in fluid communication with the sample port. The removable cartridge also includes a channel valve having a flexible member that is movable between first and second positions. The flexible member blocks flow through the sample port when it is in the first position and allows flow through the sample port when it is in the second position. The mating side of the cartridge body includes an access port that allows access to the channel valve from the outside of the cartridge body. The access hole is configured to receive a valve actuator of the main device to move the flexible part between the first and second positions.

[0010] In one embodiment, a host device is provided that includes a system housing having a mating side that is configured to interact with a removable cartridge. The main instrument also includes a rotary motor that is configured to interact with the rotary valve of the removable cartridge. The host also includes a valve actuator that is configured to interact with the removable cartridge channel valve and a set of electrical contacts configured to be electrically connected to the removable cartridge. The main instrument also includes a system controller that is configured to control a rotating motor and drive to execute the assay protocol within the removable cartridge. The system controller is configured to receive imaging data from a removable cartridge through a set of electrical contacts. Optionally, the main apparatus includes a thermal block for heating a portion of the removable cartridge.

[0011] In one embodiment, a removable cartridge is provided that includes a cartridge body having a sample opening that opens to the outside of the cartridge body and is configured to receive a biological sample. The cartridge body includes a docking side that is designed to face the main instrument and is configured to be removably connected to it. The removable cartridge also includes a microfluidic body located within the cartridge body. The microfluidic body has a body side and includes a hydraulic network. The hydraulic network has a plurality of discrete channels and corresponding openings that extend to this side of the body in the valve-receiving region. The removable cartridge also includes a rotary valve located within the cartridge body. The rotary valve has a hydraulic side and at least one flow channel that extends between a plurality of valve openings. The valve ports face the hydraulic side. The hydraulic side is rotatably connected to the valve-receiving area of said side of the microfluidic body, the rotary valve being able to move between different angular positions in order to hydraulically couple the discrete channels. The rotary valve has a mechanical interface that is accessible along the docking side and is configured to interact with the main instrument such that the rotary valve is controlled by the main instrument.

[0012] In one embodiment, a removable cartridge is provided that includes a cartridge body having a sample opening that opens to the outside of the cartridge body and is configured to receive a biological sample. The cartridge body has a docking side, which is configured to be detachably attached to the main device. The removable cartridge also includes a microfluidic structure that resides within the cartridge body and includes a plurality of stacked printed circuit board (PCB) layers. The PP layers include hydraulic layers that form channels and a reaction chamber when the PP layers are stacked on top of each other. The PP layers also include a wiring layer. The removable cartridge also includes a CMOS imager that is configured to be mounted on the microfluidic structure and electrically connected to the wiring layer. The CMOS imager is oriented to detect intended reactions within the reaction chamber.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013] FIG. 1A is a schematic representation of a system in accordance with one embodiment that is configured to perform at least one of biochemical analysis or sample preparation.

[0014] FIG. 1B is a flow diagram illustrating a method for carrying out the envisaged reactions for at least one of sample preparation or sample analysis.

[0015] FIG. 2 is a schematic representation of a system in accordance with one embodiment that is configured to perform at least one of biochemical analysis or sample preparation.

[0016] FIG. 3 is a side view of a system in accordance with one embodiment that includes a main instrument and a removable cartridge.

[0017] FIG. 4 is a plan view of a system in accordance with one embodiment that includes a main instrument and a removable cartridge.

[0018] FIG. 5 is a sectional view of a portion of a system in accordance with one embodiment illustrating the flow control valve in the first position.

[0019] FIG. 6 is a sectional view of part of the system of FIG. 5 illustrating the flow control valve in its second position.

[0020] FIG. 7 is a sectional view of a portion of a system in accordance with one embodiment illustrating the flow control valve in the first position.

[0021] FIG. 8 is a sectional view of part of the system of FIG. 5 illustrating the flow control valve in its second position.

[0022] FIG. 9 is a sectional view of a portion of a system in accordance with one embodiment illustrating the flow control valve in the first position.

[0023] FIG. 10 is a sectional view of part of the system of FIG. 5 illustrating the flow control valve in its second position.

[0024] FIG. 11 is a perspective view of an exposed portion of a removable cartridge according to one embodiment.

[0025] FIG. 12 is a sectional view of a rotary valve that can be used in conjunction with the removable cartridge of FIG. eleven.

[0026] FIG. 13 illustrates one arrangement of ports that can be hydraulically interconnected using a rotary valve.

[0027] FIG. 14 illustrates a flowchart of one example of a method for using a flexible printed circuit board (FCB) and roll-to-roll (R2R) printed electronics to seamlessly integrate CMOS technology and digital inkjet technology.

[0028] FIG. 15 illustrates an exploded view of one example of an inkjet stack with defined layers that can be laminated and bonded together using the method of FIG. 16.

[0029] FIG. 16 illustrates a perspective view of one example of a CMOS device that can be integrated into the inkjet layers of a microfluidic cartridge using the method of FIG. fourteen.

[0030] FIG. 17A, 17B, 18, 19 and 20 illustrate side views of the structure and show one example of a process for attaching a CMOS device to a flexible printed circuit board using the method of FIG. fourteen.

[0031] FIG. 21 illustrates a side view of one example of a structure formed using the method of FIG. 14 in which the inkjet technology layers and the CMOS device are integrated together in a microfluidic cartridge.

[0032] FIG. 22A and 22B illustrate perspective views of one example of a diaphragm valve, wherein the diaphragm valves may be integrated into fluid technology layers.

[0033] FIG. 23A and 23B illustrate cross-sectional views of the diaphragm valve in the open and closed states, respectively.

[0034] FIG. 24 illustrates a schematic diagram of one example of a microfluidic cartridge that includes CMOS technology and digital inkjet technology integrated together.

[0035] FIG. 25 and 26 illustrate perspective views of the microfluidic cartridge assembly, which is one physical embodiment of the integrated microfluidic cartridge shown in FIG. 24.

[0036] FIG. 27A and 27B illustrate perspective views of one example of an inkjet assembly that fits into the microfluidic cartridge assembly shown in FIG. 25 and 26.

[0037] FIG. 28A and 28B illustrate, respectively, a plan view and a sectional view of one example of a heater track that may be installed in the jetting assembly shown in FIG. 27A and 27B.

[0038] FIG. 29, 30, 31, 32, 33A and 33B illustrate various other views of the microfluidic cartridge assembly of FIG. 25 showing it in more detail.

[0039] FIG. 34-42 illustrate the process of disassembling the microfluidic cartridge assembly of FIG. 25 as a means of showing its internal components.

[0040] FIG. 43 shows a transparent perspective view of a portion of the microfluidic cartridge assembly of FIG. 25 showing the various hydraulic reagent reservoirs as well as its sample loading ports.

[0041] FIG. 44 shows another transparent perspective view of a portion of the microfluidic cartridge assembly of FIG. 25, further showing his inkjet channels.

[0042] FIG. 45 shows a sectional view of the microfluidic cartridge assembly of FIG. 25, which shows it in more detail.

[0043] FIG. 46A, 46B, 47A, 47B, and 48 show various views of the microfluidic cartridge body assembly of FIG. 25, which show it in more detail.

[0044] FIG. 49, 50, 41A, 41B, and 52 show various views of the microfluidic cartridge baseplate assembly of FIG. 25, which show it in more detail.

[0045] FIG. 53A and 53B illustrate other perspective views of the microfluidic cartridge inkjet assembly, showing it in more detail.

[0046] FIG. 54A, 54B, and 54C are other views showing more details of the flexible PP heater of the microfluidic cartridge inkjet assembly.

[0047] FIG. 55A and 55B show respectively a perspective view and a top view of the inlet/outlet layer of the inkjet layers shown in FIG. 15 and FIG. 27.

[0048] FIG. 56A and 56B show respectively a perspective view and a plan view of the hydraulic channel layer of the inkjet layers shown in FIG. 15 and FIG. 27.

[0049] FIG. 57A and 57B show respectively a perspective view and a plan view of the flexible PP layer of the inkjet layers shown in FIG. 15 and FIG. 27.

[0050] FIG. 58A and 58B show respectively a perspective view and a plan view of the bottom layer of the inkjet layer sequencing chamber shown in FIG. 15 and FIG. 27.

[0051] FIG. 59A and 59B show respectively a perspective view and a plan view of a layer of the inkjet layer sequencing chamber shown in FIG. 15 and FIG. 27.

[0052] FIG. 60A and 60B show respectively a perspective view and a plan view of the membrane layer and the top layer of the inkjet layer sequencing chamber shown in FIG. 15 and FIG. 27.

[0053] FIG. 61A and 61B illustrate a flowchart of one example of a method for using a microfluidic cartridge assembly to perform a multilocus polymerase chain reaction (PCR) and subsequent mixing required for sequencing.

[0054] FIG. 62 illustrates a side view of one example of a CMOS flow cell in which up to about 100% of the biosensor active area is available for reagent delivery and illumination.

[0055] FIG. 63 illustrates an exploded view of an example of one embodiment of the CMOS flow cell shown in FIG. 49.

[0056] FIG. 64 and 65 illustrate respectively a perspective view and a side view of the CMOS flow cell shown in FIG. 63, in its fully assembled state.

[0057] FIG. 66 illustrates perspective views of one example of the lid of the CMOS flow cell shown in FIG. 63, 64 and 65.

[0058] FIG. 67, 68, 69, and 70 illustrate one example of a process for providing an extended flat surface in a CMOS flow cell onto which the flow cell cover can be placed.

[0059] FIG. 71A, 71B, 71C, and 71D illustrate another example of a process for providing an extended flat surface in a CMOS flow cell onto which the flow cell cover can be placed.

[0060] FIG. 72, 73, 74, and 75 illustrate yet another example of a process for providing an extended flat surface in a CMOS flow cell onto which the flow cell cover can be placed.

DETAILED DESCRIPTION OF THE INVENTION

[0061] The embodiments set forth herein may be used to perform the intended reactions for sample preparation and/or biochemical analysis. The term "biochemical analysis" may encompass at least one of biological analysis or chemical analysis. Fig. 1A is a schematic representation of a system 100 configured for biochemical analysis and/or sample preparation. The system 100 includes a main device 102 and a removable cartridge 104 that is configured to releasably interact with the main device 102. The main device 102 and the removable cartridge 104 can be configured to interact with each other to transfer a biological sample to various locations within the system 100 , performing the intended reactions involving the biological sample in order to prepare the biological sample for subsequent analysis, and optionally, to detect one or more events with the biological sample. These events may indicate an intended reaction with a biological sample. In some embodiments, the removable cartridge 104 is similar to an integrated microfluidic cartridge 1100 (shown in FIG. 24) or an assembled microfluidic cartridge 1200 (shown in FIGS. 25 and 26).

[0062] Although the following description is made with reference to the main apparatus 102 and the removable cartridge 104 shown in FIG. 1A, it should be understood that the main device 102 and the removable cartridge 104 illustrate only one exemplary embodiment of the system 100 and that other embodiments also exist. For example, the main instrument 102 and the removable cartridge 104 include various components and features that together perform a series of operations for preparing a biological sample and/or analyzing a biological sample. In the illustrated embodiment, the main device 102 and the removable cartridge 104 are each capable of performing certain functions. It should be understood, however, that the main device 102 and the removable cartridge 104 may perform different functions and/or may jointly perform such functions. For example, in the illustrated embodiment, the removable cartridge 104 is configured to detect intended reactions using an imaging device. In alternative embodiments, main instrument 102 may include an imaging device. As another example, in the illustrated embodiment, main apparatus 102 is a "dry" apparatus that does not supply liquids, receive liquids, or exchange liquids with removable cartridge 104. In alternative embodiments, main apparatus 102 may supply, for example, reagents or other liquids to the removable cartridge 104, which are subsequently consumed (eg, used in the intended reactions) by the removable cartridge 104.

[0063] As used herein, the term "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(s) compound(s), such as analogs or mimetics of the aforementioned substances. In some cases, the biological sample may include whole blood, lymphatic fluid, blood serum, blood plasma, sweat, tears, saliva, sputum, cerebrospinal fluid, amniotic fluid, seminal fluid, vaginal secretions, serous fluid, synovial fluid, pericardial fluid, peritoneal fluid, pleural fluid, transudates, exudates, cystic fluid, bile, urine, gastric fluid, intestinal fluid, fecal specimens, fluids containing single or multiple cells, fluids containing organelles, fluidized (i.e. brought into a fluid state) tissues, fluidized organisms, fluids containing multicellular organisms, biological swabs and biological swabs.

[0064] In some embodiments, the biological sample may include added material such as water, demineralized water, saline solutions, acidic solutions, basic solutions, detergent solutions, and/or pH buffers. The added material may also include reagents that will be used during the intended assay protocol to carry out biochemical reactions. For example, the added fluids may include material for conducting multiple cycles of polymerase chain reaction (PCR) with a biological sample.

[0065] 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 system 100. For example, the biological sample loaded into system 100 may include whole blood or saliva, which subsequently processed (eg, through separation or amplification procedures) to provide prepared nucleic acids. The prepared nucleic acids can then be analyzed (for example, quantitatively by PCR, or sequenced by SBS) by the system 100. Accordingly, when the term "biological sample" is used when describing a first operation, such as PCR, and then used again when describing a subsequent second operation, such as sequencing, it should be understood that the biological sample in the second operation may be modified relative to the biological sample before or during the first operation. For example, a sequencing step (eg SBS) can be performed on amplicon nucleic acids that have been derived from template nucleic acids that have been amplified in a previous amplification step (eg PCR). In this case, the amplicons are copies of the templates, and the amplicons are present in greater numbers than the templates.

[0066] In some embodiments, the system 100 can automatically prepare a sample for biochemical analysis based on a user-provided substance (eg, whole blood or saliva). However, in other embodiments, the system 100 may analyze biological samples that are partially or pre-prepared for analysis by a user. For example, the user may provide a solution including nucleic acids that have already been isolated and/or amplified from whole blood.

[0067] As used herein, the term "intended reaction" includes a change in at least one (one) of the chemical, electrical, physical, or optical properties (or quantities) of the analyte of interest. In particular embodiments, the reaction provided is an association event (eg, incorporation of a fluorescently labeled biomolecule into an analyte of interest). The envisaged reaction may be a dissociative binding event (eg, release of a fluorescently labeled biomolecule from the analyte of interest). The envisaged reaction may be a chemical transformation, a chemical change, or a chemical reaction. The envisaged reaction may also be a change in electrical properties. For example, the intended reaction may be a change in the concentration of ions in the solution. Exemplary reactions include, but are not limited to, chemical reactions such as reduction, oxidation, addition, elimination, rearrangement, esterification, amidation, esterification, cyclization, or substitution; bonding interactions in which a first chemical binds to a second chemical; dissociation reactions, in which two or more chemicals are separated from each other; fluorescence; luminescence; bioluminescence; chemiluminescence; and biological reactions such as nucleic acid replication, nucleic acid amplification, nucleic acid hybridization, nucleic acid ligation, phosphorylation, enzymatic catalysis, receptor binding, or ligand binding. The envisaged reaction may also be the addition or removal of a proton, detected, for example, as a change in the pH of the surrounding solution or environment. An additional response provided may be to detect the flow of ions across the membrane (eg natural or synthetic bilayer membrane), eg as ions pass through the membrane the current is interrupted and the interruption can be detected. Reading fields of charged marks can also be used, as well as thermal reading and other analytical reading or measurement methods known in the art.

[0068] In specific embodiments, the reaction provided includes incorporating the fluorescently labeled molecule into the analyte. The analyte may be an oligonucleotide and the fluorescently labeled molecule may be a nucleotide. The intended reaction can be detected when excitation light is directed to the labeled nucleotide oligonucleotide and the fluorophore emits a detectable (detectable) fluorescent signal. In alternative embodiments, the detectable fluorescence is the result of chemiluminescence or bioluminescence. The envisaged reaction can also increase the (Förster) fluorescence resonance energy transfer (FRET), for example, by bringing the donor fluorophore into close proximity to the acceptor fluorophore, decrease FRET by separating donor and acceptor fluorophores, increase fluorescence by separating the quencher from the quencher. fluorophore or reduce fluorescence by bringing the quencher and fluorophore closer together.

[0069] As used herein, the term "reaction component" includes any substance that can be used to produce the intended reaction. For example, reaction components include reactants, catalysts such as enzymes, reaction reagents, samples, reaction products, other biomolecules, salts, metal cofactors, chelating agents, and pH buffer solutions (eg, hydrogenation buffer). The reaction components may be delivered, individually in solutions or combined in one or more mixtures, to various locations in the hydraulic network. For example, the reaction component can be delivered to the 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 preloaded with one or more reaction components that are necessary to carry out the intended assay protocol. Preloading may occur at one location (eg, at a manufacturing facility) before the cartridge 104 is received by the user (eg, at a customer's facility).

[0070] In some embodiments, the primary device 102 may be configured to interact with one removable cartridge 104 per session. After a session, the removable cartridge 104 may be replaced with another removable cartridge 104. In other embodiments, the main device 102 may be configured to interact with more than one removable cartridge 104 per session. As used herein, the term "session" includes performing at least one of sample preparation and/or biochemical analysis protocol. Sample preparation may include separating, isolating, modifying and/or amplifying one or more components of a biological sample such that the prepared biological sample is suitable for analysis. In some embodiments, the session may include continuous activity during which a number of controlled reactions are performed until (a) the prescribed number of reactions has been carried out, (b) the prescribed number of events has been detected, (c) the the specified period of system time, (d) the signal-to-noise ratio does not fall below the specified threshold; (e) the target component will not be identified; (f) no system failure or failure will be detected; and/or (g) one or more of the resources to carry out the reactions will not be exhausted. Alternatively, the session may include suspending system activity for a period of time (eg, minutes, hours, days, weeks) and later terminating the session until at least one of (a)-(g) occurs.

[0071] The assay protocol may include a sequence of steps for performing predicted reactions, detecting predicted reactions, and/or analyzing predicted reactions. Collectively, the removable cartridge 104 and the main device 102 may include components that are necessary to perform these various operations. The analysis protocol operations may include fluid operations, thermal control operations, detection operations, and/or mechanical operations. Fluid operation includes controlling the flow of a fluid (e.g., liquid or gas) through system 100, which may be performed by host device 102 and/or removable cartridge 104. For example, fluid operation may include controlling a pump to cause flow of a biological sample or component reactions to the detection zone. The temperature control operation may include controlling the temperature of a provided portion of the system 100. As an example, the temperature control operation may include raising or lowering the temperature of a polymerase chain reaction (PCR) zone where a fluid is stored that includes a biological sample. The detection operation may include controlling the activation of the detector or controlling the activity of the detector to detect predetermined properties, quantities, or characteristics of the biological sample. As one example, the detection operation may include capturing an image of a provided area that includes a biological sample to detect fluorescent radiation from the provided area. The detection operation may include driving a light source to illuminate the biological sample or driving a detector to observe the biological sample. The mechanical operation may include controlling the movement or position of the provided component. For example, the mechanical operation may include driving a motor to move a valve control component in the main apparatus 102 that is operable with a rotary valve in the removable cartridge 104. In some cases, some combination of different operations may occur simultaneously. For example, the detector may capture images of the detection zone while a pump controls fluid flow through the detection zone. In some cases, different operations may occur simultaneously, aimed at different biological samples. For example, the first biological sample may be subject to amplification (eg, PCR), while the second biological sample may be subject to detection.

[0072] As used herein, the term "liquid" means a substance that is relatively incompressible and has the ability to flow and take the form of a container or channel that holds this substance. The liquid may be a water-based liquid and may include polar molecules that create a surface tension that holds the liquid. The liquid may also include non-polar molecules, such as in an oily or non-aqueous material. It is understood that references to a liquid in this application may include a liquid that has been formed from a combination of two or more liquids. For example, separate reagent solutions can be combined later to carry out the intended reactions.

[0073] The removable cartridge 104 is configured to be removably interoperable or releasably connected to the host device 102. In this context, when the terms "removably cooperating" or "removably connected" (etc.) are used to describe the relationship between the removable cartridge and the main device, these terms are meant to mean that the connection between the removable cartridge and the main device is easily detachable without destroying the main device. Accordingly, the removable cartridge can be releasably connected to the main apparatus electrically so that the electrical contacts of the main apparatus are not destroyed. The removable cartridge can be releasably attached to the main device mechanically in such a way that the parts of the main device holding the removable cartridge are not destroyed. The removable cartridge can be releasably attached to the main device hydraulically in such a way that the openings of the main device are not destroyed. The main fixture is not considered "destroyed" if, for example, only a simple adjustment of a component (eg, repositioning) or a simple replacement (eg, replacement of a nozzle) is required. Components (eg, removable cartridge 104 and main device 102) can be easily detachable when these components can be separated from each other without undue effort or a significant amount of time spent separating the components. In some embodiments, removable cartridge 104 and main device 102 can be easily detachable without destroying the removable cartridge 104 or main device 102.

[0074] In some embodiments, the removable cartridge 104 may be permanently modified or partially damaged during a session with the main device 102. For example, containers containing liquids may include foil covers that are pierced to allow liquid to flow. through the system 100. In such embodiments, the foil covers may be damaged such that it may be necessary to replace the damaged container with another container. In particular embodiments, the removable cartridge 104 is a disposable cartridge such that the removable cartridge 104 can be replaced and optionally discarded after a single use.

[0075] In other embodiments, the removable cartridge 104 may be used for more than one session while connected to the main instrument 102 and/or may be removed from the main instrument 102, reloaded with reagents, and reattached to the main instrument 102 in order to carry out the additional reactions envisaged. Accordingly, the removable cartridge 104 can be remanufactured in some cases so that the same removable cartridge 104 can be used with different consumables (eg, reaction components and biological samples). Remanufacturing can be performed at the manufacturing facility after the cartridge has been removed from the main instrument located at the customer's facility.

[0076] As shown in FIG. 1A, removable cartridge 104 includes a hydraulic network 106 that can contain and direct fluids (eg, liquids or gases) therethrough. Hydraulic network 106 includes a plurality of interconnected hydraulic elements that are capable of storing fluid and/or allowing fluid to flow through them. Non-limiting examples of hydraulic elements include channels, channel openings, cavities, storage modules, storage module reservoirs, reaction chambers, waste reservoirs, detection chambers, multipurpose reaction and detection chambers, and the like. The hydraulic elements may be fluidly coupled to each other in a provided manner such that the system 100 is capable of performing sample preparation and/or analysis.

[0077] As used herein, the term "fluidically connected" (or a similar term) refers to two spatial regions connected together in such a way that a liquid or gas can be directed between these two spatial regions. In some instances, fluid communication allows fluid to be directed back and forth between these two spatial regions. In other cases, the hydraulic connection is unidirectional, so that there is only one direction of flow between the two spatial regions. For example, the analysis reservoir may be in fluid communication with the channel such that liquid can be transferred into the channel from the analysis reservoir. However, in some embodiments, it may not be possible to direct the fluid through the channel back to the analysis reservoir. In specific embodiments, hydraulic network 106 is configured to receive a biological sample and direct that biological sample for sample preparation and/or sample analysis. The hydraulic network 106 may direct the biological sample and other reaction components to the waste reservoir.

[0078] One or more embodiments may include holding a biological sample (eg, a template nucleic acid) at a designated location where the biological sample is analyzed. As used herein, the term "retained" when used in relation to a biological sample includes essentially attaching the biological sample to a surface or enclosing the biological sample within a provided space. As used herein, the term "immobilized" when used in relation to a biological sample includes essentially attaching the biological sample to a surface in or on a solid support. Immobilization may involve attaching a biological sample to a surface at the molecular level. For example, a biological sample can be immobilized to a substrate surface using adsorption techniques, including non-covalent interactions (e.g., electrostatic forces, van der Waals forces, and dehydration of hydrophobic interfaces), as well as covalent bonding techniques, where functional groups or linkers facilitate attachment of the biological sample to the surface. The immobilization of a biological sample on the substrate surface can be based on the properties of the surface of the substrate, the liquid medium that is the carrier of the biological sample, and also on the properties of the biological sample itself. In some cases, the surface of the substrate may be functionalized (eg, chemically or physically modified) in order to facilitate the immobilization of the biological sample on the surface of the substrate. The surface of the substrate may first be modified in order for the functional groups to bind to the surface. These functional groups can then bind to the biological sample to immobilize the biological sample on itself. In some cases, the biological sample can be immobilized on the surface by means of a gel, for example, as described in US patent publications No. 2011/0059865 A1 and No. 2014/0079923 A1, each of which is incorporated here by reference in its entirety.

[0079] In some embodiments, the implementation of nucleic acids can be immobilized on the surface and amplified using bridged amplification. Useful bridge amplification techniques are described, for example, in US Pat. No. 5,641,658; WO 07/010251, US patent No. 6090592; US Patent Publication No. 2002/0055100 A1; US patent No. 7115400; US Patent Publication No. 2004/0096853 A1; US Patent Publication No. 2004/0002090 A1; US Patent Publication No. 2007/0128624 A1; and US Patent Publication No. 2008/0009420 A1, all of which are incorporated herein by reference in their entirety. Another useful method for amplifying nucleic acids at the surface is rolling ring amplification (RCA), for example, using the methods described in more detail below. In some embodiments, nucleic acids 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 the surface (eg, attached at the 5' end). As an example, a nucleic acid molecule can hybridize to one of the primers on the surface, followed by extension of the immobilized primer to obtain the first copy of the nucleic acid. The primer in solution then hybridizes to the first copy of the nucleic acid, which can be extended using the first copy of the nucleic acid as a template. Optionally, after the creation of the first copy of the nucleic acid, the original nucleic acid molecule may hybridize to the second immobilized primer on the surface and may be extended at the same time or after the primer in solution is extended. In any embodiment, repeated rounds of extension (eg, amplification) using the immobilized primer and primer in solution provide multiple copies of the nucleic acid. In some embodiments, the biological sample may be enclosed within a predetermined space along with reaction components that are configured to be used during amplification of the biological sample (eg, PCR).

[0080] In the illustrated embodiment, the removable cartridge 104 includes a cartridge body 110 having a plurality of body sides 111-114. Housing sides 111-114 include non-docking sides 111-113 and a docking side 114. Docking side 114 is configured to interact with host device 102. In the illustrated embodiment, cartridge body 110 forms a substantially integral structure. In alternative embodiments, the cartridge body 110 may be composed of one or more sub-components that are combined by the user of the system 100. These sub-components may be combined before the removable cartridge 104 is releasably attached to the main device 102, or after one of the sub-components is releasably attached. to the main instrument 102. For example, the storage module 150 may be held by a first sub-body (not shown) and the remainder of the removable cartridge 104 (eg, hydraulic network and imaging device) may be included in a second sub-body (not shown). The first and second sub-hulls may be combined to form the cartridge body 110.

[0081] The hydraulic network 106 is held by the cartridge body 110 and includes a plurality of sample holes 116 extending to the non-mating side 112. In alternative embodiments, the sample holes 116 may be located along the non-mating sides 111 or 113, or may be located along the mating side 114. Each of the sample holes 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 implementation of the biological sample may be nucleic acids and other materials (eg, reagents, buffers, etc.) for PCR. Although in Fig. 1A shows three sample holes 116, embodiments may include only one sample hole, two sample holes, or more than three sample holes.

[0082] The fluid network 106 also includes a fluid communication opening 118 that extends to the mating side 114 and is open to the outside of the cartridge body 110. The fluid communication port 118 is provided for fluid communication with the system pump 119 of the host 102. The fluid communication port 118 is in fluid communication with the pump channel 133 which is part of the hydraulic network 106. During operation of the system 100, the system pump 119 is configured to provide negative pressure to cause fluid to flow through pump channel 133 and through the remainder of hydraulic network 106. For example, system pump 119 may cause a biological sample to flow from sample port 116 to sample preparation area 132, in which the biological sample can be prepared for subsequent analysis. . The system pump 119 may cause the biological sample to flow from the sample preparation area 132 to the reaction chamber 126 in which detection operations are performed to obtain data (eg, image data) about the biological sample. System pump 119 may also cause fluid to flow from reservoirs 151, 152 of storage module 150 to reaction chamber 126. After detection operations have been performed, system pump 119 may cause fluid to flow to waste reservoir 128.

[0083] In addition to the hydraulic network 106, the removable cartridge 104 may include one or more mechanical interfaces 117 that can be controlled by the host 102. For example, the removable cartridge 104 may include a valve assembly 120 having a plurality of valves 121-123 flow control valves that are operatively connected to the hydraulic network 106. Each of the flow control valves 121-123 may provide a mechanical interface 117 that is controlled by the main instrument 102. For example, the flow control valves 121-123 may be selectively actuated or controlled by the main instrument 102, in combination with selective actuation of the system pump 119 in order to control the flow of fluid within the hydraulic network 106.

[0084] For example, in the illustrated embodiment, fluid network 106 includes a sample conduit 131 that is located immediately downstream of and in fluid communication with sample holes 116. On FIG. 1A shows only one sample channel 131, but alternative embodiments may include multiple sample channels 131. The sample channel 131 may include a sample preparation area 132. The valve assembly 120 includes a pair of channel valves 121, 122. Channel valves 121, 122 can be selectively actuated by main instrument 102 to prevent or block fluid flow through sample channel 131. In particular embodiments, the valves 121, 122 of the channel may be actuated to form a seal that retains a prescribed volume of liquid within the sample preparation area 132 of the sample channel 131. The provided volume within the sample preparation area 132 may include a biological sample.

[0085] The valve assembly 120 may also include a movable valve 123. The movable valve 123 may be similar to the rotary valve assembly 1410 (shown in FIGS. 27A and 27B). The movable valve 123 has a valve body 138 that may include at least one flow passage 140 extending between respective openings. The valve body 138 is movable between various positions in order to align the openings with the corresponding openings of the hydraulic network 106. For example, the position of the movable valve 123 may determine the type of fluid that flows into the reaction chamber 126. In the first position, the movable valve 123 may be aligned with the corresponding opening of the sample channel 131 so as to introduce a biological sample into the reaction chamber 126. In the second position, the movable valve 123 can be aligned with one or more corresponding openings of the channels 161, 162 of the reservoir, which are in fluid communication with the reservoirs 151, 152 of the module 150, respectively. storage. Each reservoir 151, 152 is configured to store a reaction component that can be used to carry out the intended reactions. Channels 161, 162 of the tank are located respectively downstream of the tanks 151, 152 and are in fluid communication with them. In some embodiments, the implementation of the mobile valve 123 can move, separately, in various positions in order to be aligned with the corresponding openings of the reservoir channels.

[0086] In the illustrated embodiment, the movable valve 123 is a rotary valve that is rotatable about an axis 142. Accordingly, the movable valve 123 is hereinafter referred to as the rotary valve 123. However, it should be understood that alternative embodiments may include movable valves that do not rotate to different positions. In such embodiments, the movable valve may slide in one or more linear directions in order to align the corresponding holes. The rotary and linear valves referred to herein may be similar to those described in International Application No. PCT/US2013/032309, filed March 15, 2013, which is incorporated here by reference in its entirety.

[0087] In some embodiments, the biological sample is illuminated by the light source 158 of the main instrument 102. Alternatively, the light source 158 may be built into the removable cartridge 104. For example, the biological sample may include one or more fluorophores that provide light emission when excited. light with the right wavelength. In the illustrated embodiment, the removable cartridge 104 has an optical path 154. The optical path 154 is designed to allow illumination light 156 from the light source 158 of the main instrument 102 to fall on the biological sample within the reaction chamber 126. Thus, the reaction chamber may have one or more optically transparent sides. or windows. Optical path 154 may include one or more optical elements, such as lenses, reflectors, fiber optic lines, and the like, that actively direct illumination light 156 to reaction chamber 126. In one exemplary embodiment, light source 158 may be a light emitting diode. (LED). However, in alternative embodiments, light source 158 may include other types of light generating devices such as lasers or lamps.

[0088] In some embodiments, detection assembly 108 includes an image detector 109 and a reaction chamber 126. Image detector 109 is configured to detect intended reactions within reaction chamber 126. Image detector 109 may be similar to CMOS image sensor 262 (shown in FIG. 40). In some embodiments, the image detector 109 may be positioned relative to the reaction chamber 126 so as to detect light signals (eg, absorption, reflection/refraction, or light emission) from the reaction chamber 126. The image 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 image detector 109 can detect light signals that are emitted by chemiluminescence. In other embodiments, the discovery node 108 may not be limited to imaging applications. For example, detection assembly 108 may be one or more electrodes that detect an electrical property of a fluid.

[0089] As indicated herein, the host device 102 is configured to operably interact with the removable cartridge 104 and control various operations within the removable cartridge 104 in order to perform the intended reactions and/or obtain biological sample data. To this end, docking side 114 is configured to permit or allow host device 102 to control the operation of one or more components of removable cartridge 104. For example, docking side 114 may include a plurality of access holes 171-173 that allow host device 102 to control valves 121- 123. The docking side 114 may also include an access port 174 configured to receive a fuser 206 of the main instrument 102. The access port 174 extends along the sample path 131. As shown, the access holes 171-174 open onto the docking side 114.

[0090] The host device 102 has a control side 202 configured to be releasably coupled to the docking side 114 of the removable cartridge 104. The docking side 114 of the removable cartridge 104 and the control side 202 of the host device 102 may collectively form a system interface 204. The system interface 204 represents a common boundary between the removable cartridge 104 and the main apparatus 102, through which the main apparatus 102 and the removable cartridge 104 are operatively cooperating. More specifically, main device 102 and removable cartridge 104 are operatively docked along system interface 204 such that main device 102 can control various elements of removable cartridge 104 through docking side 114. For example, main device 102 may have one or more controllable components that control corresponding components of the removable cartridge 104.

[0091] In some embodiments, the main device 102 and the removable cartridge 104 are operatively coupled such that the main device 102 and the removable cartridge 104 are attached to each other at the system interface 204 in at least one of electrical coupling, thermal coupling, optical coupling. , valve communication, or hydraulic communication through the system interface 204. In the illustrated embodiment, the host device 102 and the removable cartridge 104 are configured to have electrical communication, thermal communication, valve communication, and optical communication. More specifically, through electrical communication, the main device 102 and the removable cartridge 104 can exchange data and/or power. Through thermal communication, the host 102 and removable cartridge 104 can transfer thermal energy to and/or from each other, and through optical communication, the host 102 and removable cartridge 104 can exchange light signals (eg, illumination light).

[0092] In the illustrated embodiment, system interface 204 is a one-way interface 204. For example, control side 202 and docking side 114 are generally flat and facing in opposite directions. The system interface 204 is one-way such that the removable cartridge 104 and the host device 102 are operatively connected to each other only through the docking side 114 and the control side 202. In alternative embodiments, the system interface may be a multi-way interface. For example, at least 2, 3, 4, or 5 sides of the removable cartridge may be mating sides that are configured to connect to the host device. These multiple sides may be planar and may be orthogonal or opposite to each other (eg, surrounding all or part of the rectangular volume).

[0093] In order to control the operations of the removable cartridge 104, the main apparatus 102 may include valve actuators 211-213 that are configured to operatively interact with the flow control valves 121-123, a thermal block 206 that is configured to supply and/or or removal of thermal energy to/from the sample preparation area 132, as well as a contact set 208 of electrical contacts 209. The main device 102 may also include a light source 158 located along the control side 202. The main device 102 may also include a system pump 119 with a control hole 210 located along the control side 202.

[0094] The system 100 may also include a locking mechanism 176. In the illustrated embodiment, the locking mechanism 176 includes a rotary latch 177 configured to engage the latch engaging member 178 of the removable cartridge 104. Alternatively, the removable cartridge 104 may include rotary valve 177, and the main appliance 102 may include a latch engaging member 178. When the removable cartridge 104 is installed in the main appliance 102, the latch 177 can be rotated and pushed behind the latch engaging member 176. The camming effect generated by the locking mechanism 176 may push or bring the removable cartridge 104 to the main device 102 to secure the removable cartridge 104 thereon.

[0095] The main instrument 102 may include a user interface 125 that is configured to receive user input to perform a provided analysis protocol and/or is configured to inform the user of information about the analysis. The user interface 125 may be embedded in the main device 102. For example, the user interface 125 may include a touch screen attached to the body of the main device 102 and configured to identify a user's touch and localize that touch with respect to information displayed on the touch screen. Alternatively, the user interface 125 may be located remotely from the main instrument 102.

[0096] The host 102 may also include a system controller 220 that is configured to control the operation of at least one of the valve actuators 211-213, the fuser 206, the contact set 208, the light source 158, or the system pump 119. The system controller 220 illustrated conceptually as a collection of electronic modules, but may be implemented using any combination of dedicated hardware boards, processors, digital signal processors (DSPs), etc. Alternatively, system controller 220 may be implemented using a commercially available personal computer (PC) with a single processor or multiple processors with functional operations distributed among these processors. As a further option, the electronic modules described below can be implemented using a hybrid configuration in which some of the modular functions are performed using dedicated hardware while the rest of the modular functions are performed using a commercial PC, and the like.

[0097] The system controller 220 may include a plurality of electronic modules 221-224 configured to control the operation of certain components of the main device 102 and/or removable cartridge 104. For example, the electronic module 221 may be a flow control module 221 configured to control flow of fluids through hydraulic network 106. Flow control module 221 may be operatively coupled to valve actuators 211-213 and to system pump 119. Flow control module 221 may selectively actuate valve actuators 211-213 and system pump 119 to create fluid flow through one or more paths and/or to block the flow of fluid through one or more paths.

[0098] By way of example only, the valve actuator 213 may rotate with the rotary valve 123. The valve actuator 213 may include a rotary motor 214 configured to drive (eg, rotate) the valve actuator 213. The flow control module 221 may actuate the valve actuator 213 to move the rotary valve 123 to the first angular position. With the rotary valve 123 in the first angular position, the flow control module 221 may actuate the system pump 219, thereby drawing the biological sample from the sample preparation area 132 into the reaction chamber 126. The flow control module 221 may actuate the valve actuator 213 in order to move the rotary valve 123 to the second angle position. With the rotary valve 123 in the second angle position, the flow control module 221 may actuate the system pump 219, thereby drawing one or more reaction components from the respective reservoir(s) into the reaction chamber 126. In some embodiments, the system pump 219 may be configured to provide positive pressure so that the fluid is actively pumped in the opposite direction. Such operations can be used to add multiple liquids to a common reservoir, thereby mixing the liquids within this reservoir. Accordingly, fluid communication opening 118 may allow fluid (eg, gas) to exit cartridge body 110 or may receive fluid into cartridge body 110.

[0099] The system controller 220 may also include a temperature control module 222. Thermal control module 222 may control thermal block 206 to supply and/or remove thermal energy to/from sample preparation area 132. In one specific example, the fuser 206 may increase and/or decrease the temperature that the biological sample is exposed to within the sample channel 131 in accordance with the PCR protocol. Although not shown, system 100 may include additional thermal devices that are adjacent to sample preparation area 132. For example, the removable cartridge 104 may include a thermal device similar to the flexible PP heater 1412 (shown in FIGS. 27A and 27B).

[00100] The system controller 220 may also include a detection module 223 configured to control the detection node 108 to obtain data about a biological sample. The detection module 223 can control the operation of the detection node 108 through the contact set 208. For example, the detection node 108 can be connected with the possibility of communication with the contact set 194 of electrical contacts 196 along the docking side 114. In some embodiment, the electrical contacts 196 may be flexible contacts (for example , pogo pins, or contact bars) that are capable of repositioning to and from docking side 114. Electrical contacts 196 are accessible from the outside of the cartridge body and are electrically connected to detection assembly 108. The electrical contacts 196 may be referred to as input/output (I/O) contacts. When the main device 102 and the removable cartridge 104 are operatively docked, the detection module 223 can control the detection node 108 to acquire data at predetermined times or within predetermined time intervals. As an example, detection module 223 may control detection assembly 108 to capture an image of reaction chamber 126 when a biological sample has a fluorophore attached to it. A number of images may be taken.

[00101] Optionally, system controller 220 includes an analysis module 224 configured to analyze data to provide at least partial results to a user of system 100. For example, analysis module 224 may analyze imaging data provided by image detector 109. This analysis may include identification of the nucleic acid sequence of the biological sample.

[00102] The system controller 220 and/or the electronic modules 221-224 may include one or more logical devices, including one or more microcontrollers, processors, reduced instruction set computers (RISC), user programmable application specific integrated circuits (ASICs). gate arrays (FPGAs), logic circuits, and any other circuit capable of performing the functions described here. In one exemplary embodiment, system controller 220 and/or electronic modules 221-224 execute a set of instructions stored therein in order to perform one or more analysis protocols. The memory elements may be in the form of information sources or physical memory elements within the main instrument 102 and/or the removable cartridge 104. The protocols performed by the assay system 100 may be for performing, for example, DNA or RNA quantitation, protein analysis, DNA sequencing (e.g. , sequencing by synthesis (SBS)), sample preparation, and/or preparation of fragment libraries for sequencing.

[00103] The instruction set may include various instructions that instruct the system 100 to perform specific operations, such as the methods and processes of the various embodiments described herein. The instruction set can be in the form of a software system program. As used herein, the terms "software" and "firmware" are used interchangeably and include any computer program stored in memory for execution by a computer, including random access memory (RAM), read-only memory (ROM), erasable programmable read-only memory (EPROM), electronically erasable ROM and non-volatile RAM (non-volatile random access memory, NVRAM). The above types of memory are just examples, and therefore do not limit the types of memory suitable for storing a computer program.

[00104] The software may be in various forms such as system software or application software. In addition, software may take the form of a collection of individual programs, or a program module within a larger program, or part of a program module. The software may also include modular programming in the form of object-oriented programming. Once the discovery data is received, the discovery data may be processed by the system 100 automatically, may be processed in response to user input, or may be processed in response to a request made by another information processing engine (eg, a remote request over a communication channel).

[00105] System controller 220 may be connected to other components or subsystems of system 100 via communication channels, which may be wired or wireless. The system controller 220 may also be communicatively connected to external systems or servers. The system controller 220 may receive user input or commands from a user interface (not shown). The user interface may include a keyboard, mouse, touch screen panel and/or speech recognition system, and the like.

[00106] System controller 220 may serve to provide processing capabilities such as storing, interpreting, and/or executing software instructions, and controlling the overall operation of system 100. System controller 220 may be configured and programmed to control data and/or features power supply of various components. Although the system controller 220 is shown in FIG. 1A as a single construct, it should be understood that system controller 220 may include multiple individual components (eg, processors) that are distributed throughout system 100 at various locations. In some embodiments, one or more components may be integrated with the main device, and one or more components may be located remotely from the main device.

[00107] FIG. 1B is a flow diagram illustrating a method 180 for performing the provided reactions for at least one of sample preparation or sample analysis. In specific embodiments, method 180 may include nucleic acid sequencing. Method 180 may use structures or aspects of various embodiments (eg, systems and/or methods) discussed here. In various embodiments, some steps may be omitted or added, some steps may be combined, some steps may be performed simultaneously, some steps may be performed in parallel, some steps may be split into multiple steps, some steps may be performed in a different order, or some steps or sequences of steps may be repeated in an iterative manner.

[00108] For example, method 180 may include providing, at 182, a removable cartridge with a cartridge body. The removable cartridge may include a hydraulic network located within the cartridge body. The removable cartridge may also include a flow control valve that is operatively connected to and movable with respect to the hydraulic network. The flow control valve may be, for example, a channel valve or a movable valve such as a rotary valve. The cartridge body includes a body side that defines the appearance of the removable cartridge.

[00109] Method 180 may also include installing, at step 184, a removable cartridge on the main device (eg, bringing them into contact with each other). The body side of the removable cartridge can be releasably connected to the control side of the main apparatus to form a system interface together. The main instrument includes a valve actuator that communicates with the flow control valve via a system interface. For example, the valve actuator may include an elongated body that extends through the control side and is inserted into an access hole along the side of the removable cartridge body. Optionally, the valve actuator interacts directly with the flow control valve part.

[00110] At 186, one or more biological samples may be received by the removable cartridge. For example, the user may use a pipette to introduce the biological sample(s) into sample holes that are in fluid communication with a hydraulic network. Reception at step 186 may occur before or after contact at step 184. Method 180 may include hydraulically directing, at step 188, a biological sample with flow through the hydraulic network of a removable cartridge to conduct at least one of sample analysis or sample preparation in the cartridge . For example, the biological sample may be directed to the sample preparation area of the hydraulic network, with the flow of the biological sample being controlled by a valve actuator on the flow control valve. The biological sample may undergo an amplification process, such as PCR, as long as the biological sample is sealed within the sample preparation area. As another example, the biological sample may be directed to flow into the reaction chamber, the flow of the biological sample being controlled by a valve actuator on the flow control valve.

[00111] Optionally, at step 190, method 180 includes detecting a biological sample using an image detector directed at the reaction chamber. The detection node may be held by at least one of the removable cartridge or the main device. For example, the detection unit may be embedded within the removable cartridge. The main instrument may be electrically connected to the detection node to control the operation of the detection node. Optionally, the hydraulic guidance of the biological sample at step 186 and/or the imaging (imaging) of the biological sample at step 190 may be repeated multiple times in accordance with a predetermined schedule or sequence.

[00112] In some embodiments, method 180 includes removing, at step 192, a removable cartridge from the host device. After the analysis protocol is completed, the removable cartridge can be removed from the main instrument. In some cases, the removable cartridge can be refilled or remanufactured. For example, the removable cartridge may be decontaminated and/or sterilized and the used storage module may be replaced with a new storage module. Method 180 may then return to step 182 where another removable cartridge is taken and installed at step 184 on the same main appliance. Similar to the first removable cartridge, the body side of the second removable cartridge can be releasably mated with the control side of the host device to form a system interface together.

[00113] FIG. 2 is a schematic representation of a system 300 configured to perform at least one of biochemical analysis or sample preparation. System 300 may include features identical or similar to those of system 100 (FIG. 1A). For example, system 300 includes a main device 302 and a removable cartridge 304 configured to releasably dock with the main device 302. The main device 302 and the removable cartridge 304 may have similar features to the main device 102 and the removable cartridge 104, respectively (shown in Fig. 1A). As shown in FIG. 2, the main instrument 302 has an instrument body 303 that includes an instrument side 306 and a cartridge receiving socket 308 extending to that instrument side 306 . In some embodiments, the side 306 of the instrument may represent the top of the main instrument 302 due to gravity and partially form the exterior of the instrument body 303. In the illustrated embodiment, the cartridge receiving seat 308 is formed by the internal mounting or control sides 311-313 of the instrument body 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 the opening 316 of the cartridge receiving slot 308.

[00114] The removable cartridge 304 is sized and shaped to reside within the cartridge receiving socket 308 and operatively interact with the host device 302. As shown, the removable cartridge 304 includes a cartridge body 320 that has body sides 321-324. Sides 321-323 of the body are configured to operably interact (mate) with mounting or control sides 311-313 such that the main instrument 302 and the removable cartridge 304 establish at least one of electrical coupling, thermal coupling, optical coupling, and/or hydraulic connections. As such, these body sides 321-323 are hereinafter referred to as docking sides 321-323. The body side 324 does not operably interact with the host 302. Accordingly, the body side 324 may be referred to as the non-mating side 324.

[00115] Similar to the removable cartridge 104 (FIG. 1A), the removable cartridge 304 includes a plurality of elements and components for controlling operations within the removable cartridge 304 so as to carry out the intended reactions. For example, the removable cartridge 304 has sample openings 330 that extend to the non-mating side 324 and are configured to receive one or more biological samples. Alternatively, the sample holes 330 may extend to one of the mating sides 321-323. In such embodiments, the biological sample(s) may be applied(s) to the inside of the sample holes 330 before the removable cartridge 304 is loaded into the cartridge receiving seat 308.

[00116] The removable cartridge 304 may also include a hydraulic network 332 having a sample preparation area 334. The hydraulic network 332 may include or fluidly interconnect a number of other components of the removable cartridge 304, such as a storage module 336, a movable valve 338, a detection assembly 340 with an image detector 342, and a waste reservoir 344. Optionally, removable cartridge 304 may also include an optical path 346 and contact set 348. Components of removable cartridge 304 may be similar to those described above with respect to removable cartridge 304.

[00117] The main apparatus 302 may have appropriate components that are operable with the removable cartridge 304 to carry out the intended reactions. For example, the main appliance 302 includes a fuser 350, a valve actuator 352, a light source 356, a contact set 358, and a system pump 360. cartridge, the various components of the removable cartridge 304 and the main apparatus 302 can interact with each other. More specifically, when the removable cartridge 304 is operatively loaded into the main instrument 302, the fuser 350 may be positioned adjacent to the sample preparation area 334, the valve actuator 352 may be operatively coupled to the movable valve 338, the light source 356 may be communicatively coupled to the optical path 346, contact set 358 may be electrically connected to contact set 348, and system pump 360 may be communicatively coupled to hydraulic network 332. Accordingly, host instrument 302 may control removable cartridge 304 in a manner similar to host instrument 102 controlling removable cartridge 104.

[00118] The main device 302 may be configured to allow the removable cartridge 304 to be freely inserted into the cartridge receiving socket 308 without damaging the components located on the control sides 311-313 or docking sides 321-323. For example, one or more components of main appliance 302 are biased towards or moved towards removable cartridge 304. In some embodiments, fuser 350 and valve actuator 352 are attached to component support 362. The component support 362 can be offset toward the docking side 321 or moved towards the docking side 321 after the removable cartridge 304 is positioned inside the cartridge receiving slot 308 . Similarly, system pump 360 may be attached to component support 364. The component support 364 can be offset towards the docking side 323 or moved towards the docking side 323 after the removable cartridge 304 is positioned inside the cartridge receiving slot 308 .

[00119] The component supports 362, 364 may be automatically actuated by the system controller 370. For example, the system controller 370 may determine that the removable cartridge 304 is being loaded or has already been loaded into the cartridge receiving slot 308. The system controller 370 may then activate a drive mechanism or a plurality of mechanisms to move the component supports 362, 364 to the docking sides 321, 323. when activated by the user, the system 300 can shift the component supports 362, 364 to the mating sides 321, 323, respectively. Accordingly, the host device 302 may be configured to slide the removable cartridge 304 freely (eg, without significant nicks or jams) into the cartridge receiving seat 308.

[00120] The embodiments set forth herein include systems in which a removable cartridge and a mainstream device can form a system interface that is versatile. For example, each of the docking sides 321-323 is operable with a respective control side that forms the cartridge receiving socket 308. Collectively, docking sides 321-323 and respective control sides 311-313 form a system interface, which may be referred to as a multi-party interface. Such embodiments may be desirable in order to balance the forces applied to the removable cartridge 304. For example, the fuser 350 and valve actuator 352 may apply force 374 in a first direction (as indicated by the arrow). The system pump 360 may apply force 376 in the opposite second direction (as indicated by the arrow). The interaction between the contact sets 348, 358 may also provide part of the force 376.

[00121] In some embodiments, at least one of the forces 374, 376 contributes to ensuring tight contact between the respective components. For example, force 374 may provide intimate contact between fuser 350 and sample preparation area 334 to allow temperature control of sample preparation area 334. Likewise, force 374 may allow valve actuator 352 and mobile valve 338 to appropriately interact with each other so that valve actuator 352 can selectively operate mobile valve 338. Force 376 can provide intimate contact between respective electrical contacts of contact sets 348, 358.

[00122] FIG. 3 and 4 illustrate various systems with associated main appliances and removable cartridges, and in particular illustrate various multi-party interfaces that may be used by one or more embodiments. For example, Fig. 3 is an end view of a system 400 that includes a main tool 402 and a removable cartridge 404. The main tool 402 includes an open recess 406 that is sized and shaped to receive a removable cartridge 404. As shown, the open recess 406 formed by the first and second control sides 411, 412, which "look" 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 are operable respectively with the first and second docking sides 413, 414 of the removable cartridge 404. Together, a multi-way interface 415 is formed between the first control side 411 and the first docking side 413 and between the second control side 412 and the second docking side 414. More specifically, along each of the first and second docking sides 413, 414, at least one of a valve connection, hydraulic connection, electrical connection, optical connection, or thermal connection can be installed.

[00123] FIG. 4 is a plan view of a system 420 that includes a host 422 and a removable cartridge 424. The host 422 includes a cartridge receiving socket 426, which may be similar or identical to the cartridge receiving socket 308 (FIG. 2). The cartridge receiving socket 426 is sized and shaped to receive the removable cartridge 424. As shown, the cartridge receiving socket 426 is formed by the operating sides 431-434. The control sides 431, 433 are opposite to each other, and the control sides 432, 434 are also opposite to each other. The control sides 431-434 operably interact with the mating sides 441-444 of the detachable cartridge 424, respectively.

[00124] FIG. 5-12 illustrate the various valve mechanisms by which the main instrument can control (eg, regulate) the flow through the hydraulic network of the removable cartridge. Each of Fig. 5-12 illustrates a sectional view of a system in which valve communication is established between the main instrument and the removable cartridge via the system interface. Each of Fig. 5-12 illustrates a channel valve in which the main instrument may actuate the channel valve to open and close the corresponding channel. For example, Fig. 5 and FIG. 6 illustrate a portion of system 500 that may be similar to systems described above, such as systems 100 (FIG. 1A), 300 (FIG. 2), 400 (FIG. 3) and 420 (FIG. 4).

[00125] FIG. 5 and 6 illustrate a cross section of a portion of system 500 with a main instrument 502 and a removable cartridge 504 that are operatively cooperating along a system interface 506. As shown, the removable cartridge 504 has a cartridge body 508 and a microfluidic body 510 that is held by the cartridge body 508. In the illustrated embodiment, the microfluidic body 510 includes a plurality of layers 521-523 that are stacked on top of each other. Layers 521-523 may be printed circuit board (PCB) layers, such as the layers described below with reference to FIG. 14-75. One or more layers 521-523 may be etched such that when layers 5212-523 are stacked, the microfluidic body 510 forms a sample channel 526. Sample conduit 526 is part of a hydraulic network, such as hydraulic network 106 (FIG. 1A), and includes a valve or internal cavity 528.

[00126] The removable cartridge 504 includes a channel valve 530 configured to control the flow of fluid through the sample channel 526. For example, the channel valve 530 may allow a maximum opening so that the fluid can flow unhindered. Channel valve 530 may also prevent fluid from flowing through it. As used herein, the term "obstruct" may encompass slowing down the flow of fluid or completely blocking the flow of the fluid. As shown, sample passage 530 includes first and second ports 532, 534 that are in fluid communication with valve cavity 528. Fluid is intended to flow into valve cavity 528 through first opening 532 and out of valve cavity 528 through second opening 534. In the illustrated embodiment, channel valve 530 is a flexible membrane that is capable of flexing between the first and second states. This flexible membrane is in the first state in FIG. 5 and in the second state in FIG. 6. In particular embodiments, the flexible membrane is a flexible layer, such as membrane layer 918 (shown in FIGS. 23A, 23B). This flexible layer is configured to be pressed into the valve cavity 528 to block the flow of fluid through it. In alternative embodiments, channel valve 530 may be another physical element capable of moving between different states or positions to control fluid flow.

[00127] The main instrument 502 is also shown to include a valve actuator 540 that is configured to actuate the channel valve 530. For example, valve actuator 540 may bend the flexible membrane between the first and second states. The valve actuator 540 includes an elongated body 542, such as a pin or rod, that extends through the system interface 506. More specifically, the elongated body 542 extends through the control side 544 of the main instrument 502. The removable cartridge 504 has an access port 546 that receives the actuator 540 valves. This access hole 546 extends onto the docking side 548 of the removable cartridge 504. As shown, the elongated body 542 protrudes from the control side 544 and enters the access hole 546 of the docking side 548. This access hole 546 allows the valve actuator 540 to interact directly with the valve 530 channel, which is a flexible membrane in the illustrated embodiment. On FIG. 5, the valve actuator 540 is in a first state or position. On FIG. 6, the valve actuator 540 is in a second state or position. In the second position, the valve actuator 540 is moved some distance towards the channel valve 530 and contacts the channel valve 530. The valve actuator 540 may deform the channel valve 530 such that the channel valve 530 closes the first port 532. As such, the flow of fluid through the first port 532 is blocked by the port valve 530.

[00128] In some embodiments, system 500 may have first and second channel valves that are similar or identical to channel valve 530 shown in FIG. 5 and 6, wherein the first port valve is upstream of the sample preparation area (not shown) of the hydraulic network and the second port valve is downstream of the sample preparation area. As such, the first and second conduit valves can effectively seal fluid, which may contain a biological sample, within the sample preparation area. The biological sample fluid may then be heated in order to subject the fluid to an amplification protocol, such as a PCR protocol.

[00129] FIG. 7 and 8 illustrate a cross-section of a portion of system 550 with a main device 552 and a removable cartridge 554 that are operable along a system interface 556. The main device 552 and removable cartridge 554 may be similar to the main device 502 and removable cartridge 504 shown in FIG. 5 and 6. The host 552 has a valve actuator 590 having an elongated body 592, such as a nozzle, that extends through the control side 594 of the host 552 and is inserted into the access port 596 of the mating side 598 of the removable cartridge 554. The valve actuator 590 extends through the system interface 556. Optionally, the main apparatus 552 may include a sealing member 595, such as an O-ring, that surrounds the elongated body 592 and plugs the access port 596 so as to provide a closed chamber. In one exemplary embodiment, the removable cartridge 554 includes a port valve 580, which may be a flexible membrane that is pneumatically actuated by a valve actuator 590. More specifically, the valve actuator 590 is configured to supply a fluid (eg, air) to increase pressure within the closed chamber, thereby causing the channel valve 580 to deform. When the channel valve 580 deforms, it may close the first opening 582 of the sample channel 576, thereby blocking the flow through the sample channel 576.

[00130] FIG. 9-10 illustrate a system 600 that is similar to systems 500 and 550. More specifically, FIG. 9 and 10 illustrate a system 600 with a host 602 and a removable cartridge 604 that are operable along a system interface 606. The removable cartridge 604 includes a movable valve 630 that rotates with a valve actuator 640 of the host 602. The movable valve 630 is a flat body. , which is shaped to allow flow through the sample port 626 when it is in the first angular position (shown in FIG. 9) and block flow through the sample port 626 when it is in the second angular position (shown in FIG. . 10). More specifically, the movable valve 630 may close the opening 632 while in the second angular position.

[00131] FIG. 11 is a perspective view of the exposed portion of a removable cartridge 700 having a microfluidic body 702 and a rotary valve 704. The removable cartridge 700 may be similar to the removable cartridge 104 (FIG. 1) as well as other removable cartridges described herein. The rotary valve 704 may be similar to the mobile valve 123 (FIG. 1). The rotary valve 704 is rotatable on the body side or surface 706 of the microfluidic body 702. The rotary valve 704 has a hydraulic side 708 that is configured to slide with the body side 706 when rotated about the axis 710. The microfluidic body 702 may include a hydraulic a network 760 having a plurality of sample channels 763, 764, a plurality of reservoir channels 765, and a feed channel 766. Channels 763-766 are discrete channels. For example, channels 763-766 are able to separate depending on the angular position of rotary valve 704.

[00132] Channels 763-766 have corresponding openings that open onto side 706 of the body. In the illustrated embodiment, four sample channels 763 are in fluid communication with a single sample channel 764. As such, sample channels 763 may be referred to as channel parts, and sample channel 764 may be referred to as a common sample channel. Each of the sample channels 763 is operatively connected to a pair of channel valves 761, 762. Channel valves 761, 762 may be similar to channel valves described herein, such as channel valve 530. When in their respective closed positions, the channel valves 761, 762 can seal the fluid containing the respective biological sample. In some embodiments, sample channels 763 extend adjacent to thermoregulation region 770. When the biological samples are sealed within the respective sample channels 763, a heating element (not shown) and a thermal block (not shown) can be located adjacent to the thermoregulation area 770. The heating element and the fuser can work together to increase and/or decrease the temperature experienced by the biological samples within the sample channels 763. In such embodiments, sample channels 763 may constitute sample preparation areas.

[00133] Feed conduit 766 is in fluid communication with reaction chamber 716, and reservoir conduits 765 may be in fluid communication with respective reservoirs (not shown) of a storage module (not shown). The sample conduit 764 has a network opening 721, the feed conduit 766 has a feed opening 722, and the reservoir conduits 765 have corresponding reservoir openings 723. The network opening 721, the supply opening 722, and the reservoir openings 723 face the body side 706. The reservoir openings 723 are in fluid communication with the corresponding module openings 724 through a corresponding reservoir channel 765 . As shown, module openings 724 may be located at various locations along body side 706 away from supply opening 722 or axis 710. Module openings 724 are in fluid communication with reservoirs (not shown). Module openings 724 may have locations depending on the size of the tanks.

[00134] In the illustrated embodiment, the microfluidic body 702 has a total of fifteen channels that are directly interconnected with the rotary valve 704. More specifically, only one sample channel 764 and only one supply channel 766, but thirteen reservoir channels 765 can communicate directly ( hydraulically) with a rotary valve 704. In other embodiments, the microfluidic body 702 may include a plurality of sample channels 764 and/or a plurality of supply channels 766 that are directly connected to the rotary valve 704. Each of the sample channels 763 may be fluidly coupled to a corresponding sample port (not shown) configured to receive a biological sample from a user.

[00135] Hydraulic side 708 is configured to slidably mate with body side 706 in valve receiving region 728. Rotary valve 704 is sized and shaped such that hydraulic side 708 covers valve receiving region 728 and one or more openings 721-723 along side 706 body. Rotary valve 704 includes a flow passage 744 (shown in FIG. 12) that is configured to fluidly connect supply port 722 to one or more of ports 721, 723. Rotary valve 704 can block flow through one or more ports and permit flow. through one or more other ports depending on the position and configuration of the rotary valve 704.

[00136] FIG. 12 illustrates a sectional view of a rotary valve 704 that operably interacts with a valve actuator 730. More specifically, the rotary valve 704 includes a valve body 732 having a hydraulic side 708 and an operating side 734. The operating side 734 may include a mechanical interface 736 that is configured to interact with the valve actuator 730. In the illustrated embodiment, the mechanical interface 736 includes a flat body or rib that coincides with the axis 710. The valve actuator 730 includes a slot 738 configured to receive the mechanical interface 736 such that the valve actuator 730 is operably engaged with the rotary valve 704 More specifically, valve actuator 730 may cooperate with rotary valve 704 such that valve actuator 730 may rotate rotary valve 704 about axis 710.

[00137] The hydraulic side 708 includes a plurality of valve openings 740, 742 and a flow passage 744 extending between the valve openings 740, 742. Hydraulic side 708 slidably mates with body surface 706 in valve receiving region 728. In one exemplary embodiment, rotary valve 704 includes only two valve openings 740, 742 and only one flow path 744. In other embodiments, rotary valve 704 valve 704 may include more than two valve openings and/or more than one flow channel.

[00138] As shown in FIG. 12, the supply port 722 is fluidly aligned and connected to the valve port 740, and the valve port 742 is fluidly aligned and connected to the network port 721. Depending on the angular position of the rotary valve 704, the valve opening 742 may also be hydraulically connected to one of the component openings 723. As noted above, rotary valve 704 is rotatable about axis 710. In some embodiments, supply port 722 and valve port 740 are positioned such that supply port 722 and valve port 740 are aligned with axis 710. More specifically, axis 710 extends through each of the supply port 722 and the valve port 740.

[00139] When the valve actuator 730 is operatively engaged with the rotary valve 704, the valve actuator 730 may apply an actuator force 748 in a direction against the side 706 of the body. In such embodiments, the actuator force 748 may be sufficient to seal the flow path 744 between valve openings 740, 742 and to seal reservoir openings 723 and/or network opening 721.

[00140] Accordingly, the rotary valve 704 can fluidly communicate the supply port 722 and the network port 721 at the first corner position, and hydraulically link the supply port 722 and the corresponding reservoir port 723 at the second corner position. As the rotary valve 704 rotates between different angle positions, the rotary valve 704 actually changes the flow path in the hydraulic network.

[00141] Fluid may flow in any direction through flow channel 744. For example, a system pump (not shown), such as system pump 119 (FIG. 1), may be in fluid communication with supply port 722. The system pump may generate force suction that draws fluid through the network opening 721 (or the corresponding tank opening 723), then into the flow channel 744, and then through the supply hole 722. Alternatively, the system pump can provide positive pressure that displaces the fluid inside the flow channel 744 in this way that the fluid flows through the supply opening 722, then into the flow channel 744, and then through the opening 721 of the network (or the corresponding opening 723 of the tank).

[00142] FIG. 13 is a plan view of the body side 706 illustrating the net opening 721 feeding the opening 722 and the reservoir openings 723. The flow channel 744 is presented in two different angular positions. Tank openings 723 may include tank openings 723A-723D. Each of the reservoir openings 723A-723D is in fluid communication with a respective reservoir through a respective reservoir channel 765 (FIG. 10). More specifically, reservoir port 723A is in fluid communication with the hydrogenation buffer, reservoir port 723B is in fluid communication with the nucleotide solution, reservoir port 723C is in fluid communication with the wash solution, and reservoir port 723D is in fluid communication with the digest solution. As described above, depending on the angular position of the rotary valve 704 (FIG. 11), the flow passage 744 may fluidly couple the supply port 722 to the sample passages 763, 764 or to an appropriate reservoir.

[00143] Table 1 illustrates the various steps of the sequencing by synthesis (SBS) protocol, however, it is understood that other analysis protocols may be implemented. In step 1, flow channel 744 is at an angle that hydraulically couples network opening 721 and supply port 722. In step 1, channel valves (not shown) can be selectively actuated to seal second, third, and fourth biological samples within corresponding area of sample preparation, but allow the first biological sample to flow through the opening 721 of the network. Accordingly, in step 1, the system pump may apply a suction force that draws the first biological sample into the flow channel 744. In step 2, the rotary valve 704 is rotated to this second angular position while the first biological sample is stored within the flow channel 744 so that the flow conduit 744 fluidly coupled reservoir port 723A and supply port 722. In the second angular position, the system pump can provide a positive displacement force that pushes the first biological sample through reservoir port 723A and into the hydrogenation buffer reservoir.

[00144] In step 3, the rotary valve 704 is rotated back to the first angle position and the channel valves are selectively actuated so that the second biological sample can be drawn into the flow channel 744. In step 4, the rotary valve 704 is rotated back to the second angular position, at while the first biological sample is stored within the flow channel 744 and the second biological sample is added to the hydrogenation buffer with the first biological sample. During steps 5-8, the third and fourth biological samples are removed from the respective sample preparation areas and added to the hydrogenation buffer. Accordingly, four biological samples can be stored within one hydrogenation buffer tank. Biological samples and hydrogenation buffer can undergo reactions that prepare biological samples for SBS sequencing.

[00145] In step 9, the combined biological sample/hydrogenation buffer solution is aspirated through reservoir opening 723A, through flow channel 744, through feed opening 722, and into a reaction chamber (not shown). Biological samples can be immobilized on the surfaces that form the reaction chamber. For example, clusters may be formed that include biological samples. Steps 10-13 are a sequencing run. In step 10, rotary valve 704 may be in a third angle position so that a solution of nucleotides may be drawn through flow channel 744 into the reaction chamber. At this time, the base can be incorporated into appropriate biological samples (eg, template nucleic acids). In step 11, the rotary valve 704 may be in a fourth angle position so that the wash solution can flow through the reaction chamber and carry the nucleotide solution out of the reaction chamber. After step 11, an image of the reaction chamber can be taken with an image detector. The color of the light emitted by the clusters can be used to identify the bases that make up the clusters. In step 12, the rotary valve 704 may be in a fourth angle position so that a cleavage solution can flow through the reaction chamber and fluorophores (and, if present, reversible terminator groups) can be removed from the clusters. In step 13, the rotary valve 704 may again be in the third angle position and the wash solution may flow through the reaction chamber in order to remove the fissile solution. Steps 10-13 may be repeated until sequencing is complete and/or until reagents are depleted.

Table 1 Hole Type of fluid in the flow channel Current direction Stage 1 721 1st biological sample direct Stage 2 723A 1st biological sample Reverse Stage 3 721 2nd biological sample direct Stage 4 723A 2nd biological sample Reverse Stage 5 721 3rd biological sample direct Stage 6 723A 3rd biological sample Reverse Stage 7 721 4th biological sample direct Stage 8 723A 4th biological sample Reverse Stage 9 723A Pooled biological samples + hydrogenation buffer direct Stage 10 723B Nucleotide solution direct Stage 11 723C Wash solution direct Stage 12 723D splitting solution direct Stage 13 723C Wash solution direct Steps 10-13 are repeated until discovery is complete

[00146] The above embodiments may be used in conjunction with the subject matter of U.S. Provisional Application No. 61/951462 (Attorney Case No. IP-1210-PRV_296PRV2) (hereinafter "the '462 application"), which is incorporated herein by reference in its entirety. At least a portion of the '462 application is reproduced below.

[00147] The methods described herein can be used in conjunction with a variety of nucleic acid sequencing methods. In particular, applicable methods are those in which the nucleic acids are attached at fixed positions in the array (chip) in such a way that their relative positions do not change, and the chip is repeatedly detected or visualized. Embodiments in which images are obtained in different color channels, eg matching different labels used to distinguish one parent nucleotide type from another, are particularly useful. 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") methods.

[00148] "Sequencing by synthesis ("SBS") methods generally involve enzymatic extension of the resulting nucleic acid strand by iterative addition of nucleotides to the template strand. In traditional SBS methods, a single nucleotide monomer can be applied 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 may be applied to a target nucleic acid in the presence of a delivery polymerase.

[00149] In SBS, nucleotide monomers that have a terminator group or that do not have any terminator groups can be used. Methods using nucleotide monomers without terminators include, for example, pyrosequencing and sequencing using gamma-phosphate labeled nucleotides, as described in more detail below. In methods using nucleotide monomers without terminators, the number of nucleotides added in each round is usually variable and depends on the template sequence and mode of nucleotide delivery. For SBS methods that use nucleotide monomers with a terminator group, the terminator may be virtually irreversible under the sequencing conditions used, as is the case with traditional Sanger sequencing, which uses dideoxynucleotides, or the terminator may be reversible, as is the case in case of sequencing methods developed by Solexa (currently Illumina, Inc.).

[00150] SBS methods can use nucleotide monomers that have a tag group or nucleotide monomers that do not contain a tag group. Accordingly, turn-on events can be detected based on a characteristic of the label, such as the fluorescence of the label; based on a characteristic of the nucleotide monomer, such as molecular weight or charge; based on a nucleotide incorporation by-product, such as proton or pyrophosphate release; or 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, the two or more different labels may be indistinguishable by the detection methods used. For example, different nucleotides present in a sequencing reagent may have different labels and be distinguished using suitable optics, as illustrated by the sequencing methods developed by Solexa (now Illumina, Inc.).

[00151] In another exemplary type of SBS, cycle sequencing is achieved by incrementally adding reversible terminator nucleotides containing, for example, an easily cleavable or photobleaching dye tag, as described, for example, in International Patent Publication WO 04/018497 and US Pat. No. 7,057,026 , the disclosures of which are incorporated here by reference. This approach is commercialized by Illumina Inc. and is also described in International Patent Publication WO 91/06678 and International Patent Publication WO 07/123744, each of which is incorporated here by reference. The availability of fluorescently labeled terminators, in which both ends can be interchanged and the fluorescent label can be cleaved, facilitates efficient reverse-terminated cycle sequencing (CRT). Polymerases can also be co-engineered so that they are efficiently incorporated into and extended from these modified nucleotides.

[00152] Preferably, in reversible terminator sequencing embodiments, the labels do not substantially inhibit elongation under SBS reaction conditions. However, the detection marks may be removable, for example by cleavage or degradation. Images can be captured after incorporating labels into ordered nucleic acid fragments. In specific embodiments, each cycle includes the simultaneous delivery of four different types of nucleotides to the chip, and each type of nucleotide has a spectrally different label. Four images can then be obtained, each using a detection channel that is selective for one of four different marks. Alternatively, different types of nucleotide can be added sequentially and an image of the chip can be obtained between each addition step. In such embodiments, each image will show nucleic acid fragments that include nucleotides of one particular type. Different fragments will be present or absent in different images due to the different content of each fragment in the sequence. However, the relative position of the fragments in the images will remain unchanged. Images resulting from such reversible terminator SBS methods may be stored, processed and/or analyzed as set forth herein. After the image capture step, the labels can be removed and the reversible terminator groups can be removed for subsequent rounds of nucleotide addition and detection. Removing marks after they have been detected in one particular cycle and before a subsequent cycle can provide the benefit of reducing background signal and crosstalk between cycles. Examples of useful labels and removal methods are listed below.

[00153] In specific embodiments, some or all of the nucleotide monomers may include reversible terminators. In such embodiments, reversible chain terminators/cleavable fluorophores may include a fluorophore linked to the ribose group via a 3' ester bond (see Metzker, Genome Res. 15:1767-1776 (2005), which is incorporated here by reference). Other approaches separate terminator chemistry from fluorescent label cleavage (see Ruparel et al., Proc Natl Acad Sci USA 102: 5932-7 (2005), which is incorporated herein by reference in its entirety). Ruparel et al. described the development of reversible terminators that used small 3'-allyl groups to block extension but could be easily deblocked by a short treatment with a palladium catalyst. The fluorophore was attached to the base via a photo-cleavable linker, which could be easily cleaved off with a 30 second exposure to long wavelength ultraviolet (UV) light. Thus, either disulfide reduction or photocleavage can be used as the cleavable linker. Another approach to reversible termination is to use the natural termination that occurs after bulk dye is placed on the dNTP. The presence of a charged bulk dye on dNTP can act as an effective terminator through steric and/or electrostatic hindrance. The presence of one turn-on event prevents further turn-ons until that dye is removed. Cleavage of this dye removes the fluorophore and effectively reverses termination. Examples of modified nucleotides are also described in US Pat. No. 7,427,673 and US Pat. No. 7,057,026, the disclosures of which are incorporated herein by reference in their entirety.

[00154] Additional exemplary SBS systems and methods that can be used with the methods and systems described herein are described in US Patent Publication No. 2007/0166705, US Patent Publication No. 2006/0188901, US Patent No. 7057026, US Patent Publication No. 2006/ 0240439, US Patent Publication No. 2006/0281109, International Patent Publication WO 05/065814, US Patent Publication No. 2005/0100900, International Patent Publication WO 06/064199, International Patent Publication WO 07/010251, US Patent Publication No. 2012/0270305 and US Patent Publication No. 2013/0260372, the disclosures of which are hereby incorporated by reference in their entirety.

[00155] Some embodiments may use the 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 materials included in US Patent Publication No. 2013/0079232. As a first example, a pair of nucleotide types may be detected at the same wavelength but differ based on a difference in intensity for one member of the pair compared to the other, or based on a change in one member of the pair (e.g., via chemical modification, photochemical modification or physical modification) that causes the appearance or disappearance of an obvious signal compared to the signal detected for another element of the pair. As a second example, three of the four different types of nucleotides can be detected under specific conditions, while the fourth "optically inactive" type of nucleotide does not contain a label that is detectable under these conditions, or minimally detectable under these conditions (for example, minimal detection due to background fluorescence, etc.). The inclusion of the first three nucleotide types in the nucleic acid can be determined based on the presence of their respective signals, and the inclusion of the fourth nucleotide type in the nucleic acid can be determined based on the absence or minimal detection of any signal. As a third example, one type of nucleotide may include label(s) that are detected on two different channels, while other types of nucleotides are detected on no more than one of the channels. The above three exemplary configurations are not considered to be mutually exclusive and may be used in various combinations. One exemplary embodiment that combines all three examples is a fluorescence-based SBS method that uses a first type of nucleotide that is detectable through a first channel (e.g., deoxyadenosine triphosphate (dATP) having a label that is detectable through a first channel at excitation with light of the first wavelength), a second type of nucleotide that is detected by the second channel (for example, deoxycytidine triphosphate (dGTP) having a label that is detected by the second channel when excited by light with a second wavelength), a third type of nucleotide that is also detected by the first and second channels (e.g., deoxythymidine triphosphate (dTTP) having at least one label that is detectable through both channels when excited by light of the first and/or second wavelength), and a fourth type of nucleotide that does not have a label that not detected or minimally detected on any channel (eg dGTP not having a label).

[00156] In addition, as described in the materials included in US Patent Publication No. 2013/0079232, sequencing data can be obtained using a single channel. In such so-called "single dye" sequencing approaches, the first type of nucleotide is labeled, but the label is removed after the first image is obtained, and the second type of nucleotide is labeled only after the first image is obtained. The third type of nucleotide retains its label in both the first and second images, while the fourth type of nucleotide remains unlabeled in both images.

[00157] Some embodiments may use ligation sequencing techniques. Such methods use DNA ligase to incorporate oligonucleotides and identify the inclusion of such oligonucleotides. Oligonucleotides typically have different labels that correlate with the type of particular nucleotide in the sequence to which the oligonucleotides are attached. As with other SBS methods, images can be obtained after processing an array of nucleic acid fragments with labeled sequencing reagents. Each image will show nucleic acid fragments that have one particular type of tag embedded. Different fragments will be present or absent in different images due to the different content of each fragment in the sequence, but the relative position of the fragments in the images will remain the same. Images resulting from ligation-based sequencing methods can be stored, processed and analyzed as described here. Exemplary sequencing systems and methods that can be used with the methods and systems described herein are described in US Pat. No. 6,969,488, US Pat. No. 6,172,218, and US Pat. No. 6,306,597, the disclosures of which are incorporated herein by reference in their entirety.

[00158] Some embodiments may use nanopore sequencing (see Deamer, D.W. & Akeson, M. "Nanopores and nucleic acids: prospects for ultrarapid sequencing". Trends Biotechnol. 18, 147-151 (2000); Deamer, D. and D. Branton, "Characterization of nucleic acids by nanopore analysis", Acc. Chem. Res. 35:817-825 (2002), Li, J., M. Gershow, D. Stein, E. Brandin, and J. A. Golovchenko, "DNA molecules and configurations in a solid-state nanopore microscope" Nat Mater 2:611-615 (2003, the disclosures of which are hereby incorporated by reference in their entirety). In such embodiments, the target nucleic acid is passed through the nanopore. The nanopore can be a synthetic pore or a biological membrane protein such as alpha-hemolysin. As the target nucleic acid passes through the nanopore, each nitrogenous base pair can be identified by measuring fluctuations in the electrical conductivity of the pore. (See U.S. Patent No. 7001792; Soni, G. V. & Meller, "A. Progress towards ultrafast DNA sequencing using solid-state nanopores". Clin. Chem. 53, 1996-2001 (2007); Healy, K. "Nanopore-based single-molecule DNA analysis". Nanomed. 2, 459-481 (2007); Cockroft, S. L., Chu, J., Amorin, M. & Ghadiri, M. R. "A single-molecule nanopore device detects DNA polymerase activity with single-nucleotide resolution". J. Am. Coll. Soc. 130, 818-820 (2008), the disclosures of which are hereby incorporated by reference in their entirety). In other embodiments, an endonuclease can be coupled to the nanopore such that nucleotides released sequentially from the end of the nucleic acid by the endonuclease are detected as they pass through the nanopore. Each nucleotide can be distinguished on the basis of different base groups or on the basis of added groups. The data obtained from nanopore sequencing can be stored, processed and analyzed as described here. In particular, this data can be processed as an image in accordance with the exemplary processing of optical images and other images, which is set forth here.

[00159] Some embodiments may use methods involving real-time monitoring of DNA polymerase activity. Nucleotide inclusions can be detected through fluorescence resonance energy transfer (FRET) interactions between a fluorophore-bearing polymerase and gamma-phosphate-labeled nucleotides, as described, for example, in US Pat. No. 7,329,492 and US Pat. , or nucleotide inclusions can be detected using zero-mode waveguides as described, for example, in US Pat. No. 7405281 and US patent publication No. 2008/0108082 (all of which are incorporated here by reference). Illumination can be limited to a zeptoliter scale volume around the limited polymerase surface so that incorporation of fluorescently labeled nucleotides can be observed with low background (see Levene, M.J. et al. "Zero-mode waveguides for single-molecule analysis at high concentrations". Science 299 , 682-686 (2003); Lundquist, P. M. et al. "Parallel confocal detection of single molecules in real time", Opt. Lett. 33, 1026-1028 (2008), Korlach, J. et al. "Selective aluminum passivation for targeted immobilization of single DNA polymerase molecules in zero-mode waveguide nano structures". Proc. Natl. Acad. Sci. USA 105, 1176-1181 (2008), the disclosures of which are hereby incorporated by reference in their entirety). Images resulting from such methods may be stored, processed and analyzed as set forth here.

[00160] Some embodiments of the SBS include detecting a proton released upon incorporation of a nucleotide into the extension product. For example, sequencing based on the detection of released protons may use an electrical detector and related techniques that are commercially available from Ion Torrent (Gilford, Conn., a subsidiary of Life Technologies), or the sequencing methods and systems described in US Patent Publication No. 2009/0026082; US Patent Publication No. 2009/0127589; US Patent Publication No. 2010/0137143; or US Patent Publication No. 2010/0282617, each of which is incorporated here by reference.

[00161] The above SBS methods can advantageously be implemented in multiplex formats such that many different target nucleic acids are processed simultaneously. In specific embodiments, different target nucleic acids can be processed in a common reaction vessel or on the surface of a particular substrate. This allows convenient feeding of sequencing reagents, removal of unreacted reagents, and detection of turn-on events in a multiplexed manner. In embodiments using surface-bound target nucleic acids, these target nucleic acids may be in array format. In an array format, target nucleic acids can typically be associated with a surface in a spatially distinct manner. Target nucleic acids can be linked by direct covalent attachment, attachment to a bead or other particle, or attachment to a polymerase or other molecule that is attached to a surface. The array (also referred to as a chip) may include a single copy of the target nucleic acid in each region (also referred to as an element), or multiple copies of the same sequence may be present in each region or element. Multiple copies can be obtained by amplification methods such as bridge amplification or emulsion PCR, as described in more detail below.

[00162] The methods set forth herein may use chips having cells with any of a variety of different densities, including, for example, at least about 10 cells/cm ² , 100 cells/cm ² , 500 cells/cm ² , 1000 cells/cm ² , 5,000 cells/cm ² , 10,000 cells/cm ² , 50,000 cells/cm ² , 100,000 cells/cm ² , 1,000,000 cells/cm ² , 5,000,000 cells/cm ² or more. The methods and apparatus set forth herein may include detection components or devices with a resolution that is at least sufficient to distinguish individual features at one or more of these exemplary densities.

[00163] The advantage of the methods described here is that they provide fast and efficient detection of multiple target nucleic acids in parallel. Accordingly, the present invention provides integrated systems capable of preparing and detecting nucleic acids using methods known in the art, such as those exemplified above. Thus, the integrated system of the present invention may include hydraulic (also referred to as fluid or jet) components capable of delivering amplification reagents and/or sequencing reagents to one or more immobilized DNA fragments, and this system contains components such as pumps, valves , tanks, hydraulic lines, etc. A flow cell may be provided and/or used in an integrated system for detecting target nucleic acids. Exemplary flow cells are described, for example, in US Patent Publication No. 2010/0111768 A1 and US Patent Application No. 13/273666, each of which is incorporated here by reference. As illustrated by way of example for flow cells, one or more of the hydraulic components of the integrated system can be used for the amplification method and for the detection method. Using the embodiment with nucleic acid sequencing as an example, one or more of the hydraulic components of the integrated system can be used for the amplification method set forth herein and for supplying sequencing reagents in a sequencing method such as the examples of sequencing methods illustrated above. Alternatively, the integrated system may include separate hydraulic systems for performing the amplification methods and for performing the detection methods. Examples of integrated sequencing systems that are capable of generating amplified nucleic acids as well as determining nucleic acid sequences include, without limitation, the MiSeq™ or NextSeq™ platform (manufactured by Illumina, Inc., San Diego, California). , USA) or the devices described in US Patent Application Publications No. 2012/0270305 A1 or 2013/0260372 A1, each of which is incorporated here by reference.

[00164] "Activity detector" means any device or component that is capable of detecting (detecting) activity indicative of a particular reaction or process. An activity detector may be capable of detecting predetermined events, properties, quantities, or characteristics within a predetermined volume or area. For example, an activity detector may be capable of capturing an image of a given volume or area. The activity detector may be capable of detecting the concentration of ions within a given volume of solution or over a given area. Exemplary activity detectors include charge-coupled devices (CCDs) (eg, CCD cameras); photomultiplier tubes (PMT); molecular research devices or detectors such as those used with nanopores; chip layouts such as those described in US Pat. No. 7,595,883, which is incorporated herein by reference in its entirety; and CMOS field effect transistor (FET) sensors, including chemically sensitive field effect transistors (chemFET), ion sensitive field effect transistors (ISFET) and/or metal-oxide-semiconductor field effect transistors, i. MOSFETs. Exemplary activity detectors are described, for example, in International Patent Publication No. WO2012/058095.

[00165] The term "biosensor" embraces any design having multiple reaction zones or sites. The biosensor may include a solid state imaging device (eg, a CCD or CMOS imager) and optionally a flow cell mounted thereon. The flow cell may include at least one flow channel that is in fluid communication with the reaction zones. As one specific example, the biosensor is configured to be hydraulically and electrically coupled to the bioassay system. The bioassay system can supply reagents to the reaction zones according to a predetermined protocol (eg, sequencing by synthesis) and perform multiple imaging events. For example, the bioassay system may direct solutions to flow along the reaction zones. At least one of the solutions may include four types of nucleotides with the same or different fluorescent labels. These nucleotides can bind to the corresponding oligonucleotides located in the reaction zones. The bioassay system may then illuminate the reaction zones using an excitation light source (eg, solid state light sources such as light emitting diodes or LEDs). The excitation light may have a predetermined wavelength or wavelengths, including a range of wavelengths. Excited fluorescent labels produce emission signals that can be detected by light detectors.

[00166] In one aspect, the solid state imager includes a CMOS image sensor containing light detectors (photodetectors) configured to detect emission signals. In some embodiments, each of the light detectors has only one pixel, and the ratio of the number of pixels to the number of detection paths determined by the filter walls may be substantially 1:1. Exemplary biosensors are described, for example, in US Patent Application No. 13/833619.

[00167] A "detection surface" means any surface that includes an optical detector. The detector may be based on any suitable technology, such as those involving charge-coupled devices (CCDs) or complementary metal-oxide-semiconductor (CMOS) structures. In particular embodiments, a single photon avalanche photodiode (CMOS-SPAD) CMOS imager can be used, for example, to discriminate fluorophores using fluorescence lifetime imaging (FLIM). Exemplary CMOS based systems that can be used for FLIM are described in US Patent Publication No. 2008/0037008 A1; in Giraud et al., Biomedical Optics Express 1: 1302-1308 (2010); or Stoppa et al., IEEE European Solid-State Device Conference (ESSCIRC), Athens, Greece, IEEE, pp. 204-207 (2009), each of which is incorporated here by reference in its entirety. Other useful detection devices that may be used include, for example, those described in US Pat. No. 7,329,860 as well as in US Patent Publication No. 2010/0111768, all of which are incorporated herein by reference in their entirety.

[00168] In addition, it should be understood that other signal detecting devices known in the art can be used to detect the signals produced in the method described here. Particularly useful are, for example, detectors used to detect pyrophosphate or protons. Pyrophosphate can be detected using detectors such as those commercially available from 454 Life Sciences (Branford, CT, USA, Roche) or those described in US Patent Publication No. 2005/0244870, which is incorporated here by reference in in all its fullness. Exemplary systems for detecting primer extension based on proton release include those commercially available from Ion Torrent (Gilford, CT, USA, a subsidiary of ThermoFisher) or described in US Patent Publication Nos. 2009/0026082; 2009/0127589; 2010/0137143 and 2010/0282617, each of which is incorporated here by reference in its entirety. Exemplary detection surfaces and detectors are described, for example, in US Patent Publication No. 2013/0116128 A1, which is incorporated here by reference.

[00169] "Sequencing module" means a CMOS chip that has been adapted for sequencing applications. This module may comprise a surface containing a substrate of hydrophilic regions for nucleic acid attachment and amplification surrounded by hydrophobic regions. For example, dynamic pads having a hydrophilic patch, such as those described above, can be used. Alternatively or additionally, a set of dynamic pads, including some that are in a hydrophilic state while the surrounding pads are in a hydrophobic state, may form hydrophilic regions surrounded by a hydrophobic region. The nucleic acid attachment surface may optionally comprise a plurality of isolated regions, such that each isolated region contains a plurality of nucleic acid molecules, which is preferably derived from a single nucleic acid molecule to be sequenced. For example, the hydrophilic region may include a gel. The hydrophilic regions could be smooth, textured, porous, non-porous, and so on. The hydrophobic regions are preferably located between the hydrophilic regions. Reagents move along the surface due to any number of forces.

[00170] The subject matter described herein includes, in one or more embodiments, a disposable, integrated microfluidic cartridge and methods for making and using the same. The method for creating a disposable, integrated microfluidic cartridge optionally uses a flexible printed circuit board (FPB) and roll-to-roll (R2R) printed electronics to seamlessly integrate CMOS technology and digital inkjet technology. Namely, the disposable, integrated microfluidic cartridge includes an inkjet layer stack into which a CMOS sensor is integrated, and the whole is mounted in a housing. Accordingly, conventional injection molded inkjet technology can be integrated with flexible PCB technology. The inkjet layers are formed using materials suitable for use in the R2R printed electronics fabrication process. In addition, the inkjet technology layers include a polymerase chain reaction (PCR) area and a reagent mixing and distribution area. The ink jet layers also include a series of membrane valves that can completely seal the PCR area.

[00171] The method of using a disposable, integrated microfluidic cartridge includes performing multilocus PCR and subsequent mixing required for sequencing.

[00172] The embodiments set forth herein include a CMOS flow cell in which most or up to about 100% of the biosensor active area is available for reagent delivery and illumination.

[00173] FIG. 14 illustrates a flowchart of one example of a method 100 for using flexible printed circuit board (PCB) and roll-to-roll (R2R) printed electronics to seamlessly integrate CMOS and digital inkjet technology. Namely, using method 100, multilayer laminated inkjet technology can be integrated with flexible PCB technology (see Fig. 15). In addition, using the structure formed using method 100, conventional injection molded inkjet technology can be integrated with flexible PCB technology (see FIGS. 26-45). Method 100 may include, but is not limited to, the following steps.

[00174] In step 110, the inkjet layers are formed and then laminated and bonded together. For example, Fig. 15 illustrates an exploded view of the stack of inkjet layers 200 that can be laminated and bonded together at this stage. In this example, the inkjet layers 200 comprise, in that order, an inlet/outlet layer 210, a hydraulic channel layer 220, a flexible printed circuit (PCB) layer 260, a sequencing chamber bottom layer 280, a sequencing chamber layer 250, and a membrane layer 240 that is coplanar. with the top layer 290 of the sequencing chamber. The inlet/outlet layer 210, fluid passage layer 220, flexible PP layer 260, sequencing chamber bottom layer 280, sequencing chamber layer 250, membrane layer 240, and sequencing chamber top layer 290 are suitable for molding using the R2R printed electronics fabrication process.

[00175] The inlet/outlet layer 210 may be made of, for example, polycarbonate, poly(methyl methacrylate) (PMMA), cyclic olefin copolymer (COC), and/or polyimide. The inlet/outlet layer 210 may be about 25 microns to about 1000 microns thick in one example, or about 250 microns thick in another example. The inlet/outlet layer 210 is provided with an arrangement of holes (or holes). These openings (or holes) provide flow paths that can serve as inlets and/or outlets to, for example, various fluid supply reservoirs (not shown). In more detail, the inlet/outlet layer 210 is shown and described herein below with reference to FIG. 55A and 55B.

[00176] The hydraulic channel layer 220 may be made of, for example, polycarbonate, PMMA, COC, and/or polyimide. The hydraulic channel layer 220 may be about 25 microns to about 1000 microns thick in one example, or about 250 microns thick in another example. In the hydraulic channel layer 220, an arrangement of hydraulic channels is provided. These hydraulic channels provide flow paths from one destination to another along the layers 200 of the inkjet technique. Because the fluid passage layer 220 is sandwiched between the inlet/outlet layer 210 and the flexible PP layer 260, fluid can be trapped within the fluid channels by the inlet/outlet layer 210 on the bottom and the flexible PP layer 260 on top. In one example, the hydraulic channel layer 220 is used to perform the PCR and subsequent mixing required for sequencing. The hydraulic channel layer 220 is shown and described in more detail hereinafter with reference to FIGS. 56A and 56B.

[00177] The flexible PP layer 260 may be made of, for example, polycarbonate, PMMA, COC, and/or polyimide. The flexible PP layer 260 may have a thickness of about 30 microns to about 300 microns in one example, or a thickness of about 200 microns in another example. In the flexible PP layer 260, an arrangement of holes (or holes) is provided. These openings (or holes) provide flow paths that can serve as inlets and/or outlets for diaphragm valves that are used to control fluid flow in the hydraulic channels of the hydraulic channel layer 220. More details of the flexible PP layer 260 are shown and described here below with reference to FIG. 57A and 57B.

[00178] The bottom layer 280 of the sequencing chamber can be made of, for example, polycarbonate, PMMA, COC, and/or polyimide. The bottom layer 280 of the sequencing chamber may be about 25 microns to about 1000 microns thick in one example, or about 250 microns thick in another example. In the lower layer 280 of the sequencing chamber, an arrangement of holes is provided for forming membrane valves within the stack of layers 200 of the inkjet technique. The bottom layer 280 of the sequencing chamber also includes a CMOS device, such as a CMOS image sensor 262, which is located in close proximity to the sequencing chamber of the layer 250 of the sequencing chamber. The bottom layer 280 of the sequencing chamber is coplanar with the CMOS device and acts as a fluid connection layer to the inlet/outlet of the sequencing chamber of the sequencing chamber layer 250. In more detail, the lower layer 280 of the sequencing chamber is shown and described here below with reference to FIG. 58A and 58B.

[00179] The sequencing chamber layer 250 may be made of, for example, polycarbonate, PMMA, COC, and/or polyimide. The sequencing chamber layer 250 may be about 50 µm to about 300 µm thick in one example, or about 100 µm thick in another example. The layer 250 of the sequencing chamber is provided with an arrangement of holes for forming membrane valves within the stack of layers 200 of the fluid technique. The sequencing chamber layer 250 also includes a sequencing chamber. More details of the sequencing chamber layer 250 are shown and described hereinafter with reference to FIG. 59A and 59B.

[00180] The membrane layer 240 may be made of a silicone elastomer, for example. The membrane layer 240 may have a thickness of about 25 microns to about 1000 microns in one example, or a thickness of about 250 microns in another example. Membrane layer 240 serves as a flexible membrane for opening and closing membrane valves within a stack of inkjet layers 200, the membrane valves being created by combining flexible PP layer 260, bottom sequencing chamber layer 280, sequencing chamber layer 250, and membrane layer 240 in that order. Diaphragm valves are shown and described in more detail herein below with reference to FIGS. 22A, 22B, 23A and 23B. Membrane layer 240 is shown and described in more detail hereinafter with reference to FIGS. 60A and 60B.

[00181] The top layer 290 of the sequencing chamber is made of a low autofluorescence material with good optical properties, such as COC. The top layer 290 of the sequencing chamber may be about 25 microns to about 1000 microns thick in one example, or about 250 microns thick in another example. The top layer 290 of the sequencing chamber is used to cover the sequencing chamber located in the layer 250 of the sequencing chamber. More detail on top layer 290 of the sequencing chamber is shown and described herein below with reference to FIG. 60A and 60B.

[00182] Referring now again to FIG. 14, in step 115, the CMOS device is attached to the flexible PWB. For example, a CMOS image sensor 262 (see FIG. 15) is attached to the bottom layer 280 of the inkjet layer 200 sequencing chamber. Fig. 16 illustrates a perspective view of one example of a CMOS image sensor 262. In one example, the CMOS image sensor 262 is about 9200 microns long, about 8000 microns wide, and about 800-1000 microns thick, and may have about 50 I/O pads. The CMOS image sensor 262 may comprise an array of pixels. In one example, the pixel array is 4384×3292 pixels, with overall dimensions of 7272 µm×5761 µm. It should be understood that a CMOS chip can have a wide range of sizes and number of I/O pads. For example, with digital inkjet technology, a rectangular crystal (eg, non-square, narrow, and long) can be used, using only a portion of the crystal in any given analysis protocol.

[00183] Continuing with step 115, FIG. 17A, 17B, 18, 19, and 20 illustrate side views of a structure 400 that shows one example of a process for attaching a CMOS device to a flexible PWB. Structure 400 is a multilayer structure. Turning now to FIG. 17A, the initial formation of the structure 400 begins with a flexible PCB. For example, the flexible PP includes, in that order, a polyimide layer 410, a PP heater layer 412, a polyimide layer 414, a PP wiring layer 416, and a polyimide layer 418. Namely, Fig. 17 shows a flexible PCB with a PCB heater layer and PCB wiring layer, also known as "coupon foil".

[00184] Next, referring now to FIG. 17B, a low temperature isotropic conductive adhesive (low temperature IPC) 420 is applied over the polyimide layer 418.

[00185] Next, referring now to FIG. 18, a CMOS device such as a CMOS image sensor 262 is placed on the coupon foil, namely on top of the low-temperature PKI 420. In one example, the CMOS image sensor 262 is placed on top of the low temperature PKI 420 using a well-known overlay process. Fig. 18 shows the input/output pads 422 of the CMOS image sensor 262 in contact with the low temperature FID 420 and thereby electrically connected to the PCB wiring layer 416 . Other attachment options are also available, including, but not limited to, controlled-slump/flip-chip mounting, solder-pin mounting, and the like. Fig. 18 also shows that the CMOS image sensor 262 includes a biolayer 424 that faces away from the polyimide layer 418. A protective film 426 may be placed over the biolayer 424 until ready for use.

[00186] Next, referring now to FIG. 19, a set of hydraulic layers 428 is provided over the flexible PP polyimide layer 418. Namely, a laminated polycarbonate film is provided that is coplanar to the CMOS surface. An example of hydraulic layers 428 are the jetting layers 200 shown in FIG. fifteen.

[00187] Next, referring now to FIG. 20, the flip-chip mounting of the CMOS image sensor 262 on the coupon foil is completed by spreading the epoxy "underfill" adhesive 430 in the gaps around the CMOS image sensor 262.

[00188] Referring now again to FIG. 14, at step 120, the final assembly of the microfluidic cartridge is performed, which includes together the hydraulic layers and the integrated CMOS device(s). For example, Fig. 21 illustrates a side view of one example of a microfluidic cartridge 800. The microfluidic cartridge 800 includes an inkjet portion 810 and a CMOS portion 812 that is based on the structure 400 shown in FIG. 20. The final assembly steps may include, for example, spreading (printing) the 430 "underfill" epoxy bond adhesive, removing the protective film 426, applying the low temperature non-conductive adhesive 814 (e.g., UV curable or thermally non-conductive adhesive) to the CMOS portion 812 , depositing a low autofluorescence cyclic olefin (COC) copolymer layer 816 on the CMOS portion 812 of the microfluidic cartridge 800, and laminating the flexible PP heater 818 on both sides of the inkjet portion 810. During the formation of the microfluidic cartridge 800, it is critical to use a self-aligning process so that the surfaces of the CMOS device and the hydraulic layers are flush with each other.

[00189] A flow path is formed through the microfluidic cartridge 800. Namely, a sample inlet 820 is provided at the inlet of the inkjet portion 810, and an outlet 822 is provided downstream of the CMOS portion 812. The inlet 820 feeds the sample into the PCR chamber 824. The PCR chamber 824 then delivers the sample to the reagent distribution area 826. The reagent distribution area 826 then supplies the sample to the sequencing chamber 828. The biolayer 424 of the CMOS image sensor 262 is oriented toward the sequencing chamber 828. The sequencing chamber 828 then supplies the sample to the outlet 822. In addition, the microfluidic cartridge 800 includes some membrane valves 830 that control the flow of fluid into and out of the PCR chamber 824.

[00190] FIG. 22A and 22B illustrate perspective views of one example of a membrane valve 830, where the membrane valves may be integrated into fluid technology layers 200, for example. Turning now to FIG. 22A, there is a perspective view of the diaphragm valve 830. In this example, the diaphragm valve 830 includes, in that order, a backing layer 910, a hydraulic channel layer 912, and a reservoir layer 914. Base layer 910, hydraulic channel layer 912, and reservoir layer 914 may be made of, for example, polycarbonate, PMMA, COC, and/or polyimide. The reservoir layer 914 has a recessed area that creates a small reservoir 916 in the reservoir layer 914. The membrane layer 918 is stretched over the reservoir 916. The reservoir 916 has an inlet 920 and an outlet 922 which provide a flow path to the respective hydraulic channels 924. In order to better show the features of the reservoir 916 and the inlet 920 and outlet 922, FIG. . 22B shows a diaphragm valve 830 without a membrane layer 918 covering reservoir 916. The membrane layer 918 is made of an elastomeric membrane material (eg, silicone elastomer) that is flexible and stretchable.

[00191] FIG. 23A and 23B show a sectional view of the diaphragm valve 830 along line A-A of FIG. 22A. An actuator, such as actuator 1010, may be used to open and close diaphragm valve 830. For example, FIG. 23A shows the diaphragm valve 830 in the open state, in which the actuator 1010 is not in contact with the membrane layer 918. In contrast, FIG. 23B shows the diaphragm valve 830 in a closed state, in which the actuator 1010 is in contact with the membrane layer 918. Namely, the end of the actuator 1010 is used to press the central part of the membrane layer 918 against the outlet 922 and thereby block the flow of liquid through it. Diaphragm valve 830 (ie, diaphragm valves 242, 244, and 246) may be actuated using, for example, mechanical or pneumatic actuators such as solenoids or pneumatic pumps.

[00192] FIG. 24 illustrates a schematic of one example of a microfluidic cartridge 1100 that includes both CMOS technology and digital inkjet technology integrated together. Namely, the microfluidic cartridge 1100 includes inkjet layers 200 that are hydraulically and operably connected to four sample stores 1110 (e.g., sample stores 1110a, 1110b, 1110c, 1110d), thirteen reagent stores 1112 (e.g., reagent stores 1112a-1112m ), as well as with an exhaust pump 1114. The fluid technology layers 200 include a PCR region 270 and a reagent mixing and distribution region 275. The PCR region 270 includes, for example, four PCR channels 222 (eg, PCR channels 222a, 222b, 222c, 222d). The inlets of the channels 222a, 222b, 222c and 222d PCR served respectively supplies 1110a, 1110b, 1110c, 1110d sample. Because the microfluidic cartridge 1100 includes four PCR channels 222 that are fed with four sample supplies 1110, the microfluidic cartridge 1100 is configured to quadruple sample multiplexing.

[00193] The inputs of the four PCR channels 222 are controlled using the four membrane valves 242. Namely, the inputs of the PCR channels 222a, 222b, 222c, and 222d are controlled using the membrane valves 242a, 242b, 242c, and 242d, respectively. Similarly, the outputs of the four PCR channels 222 are controlled using the four membrane valves 244. Namely, the outputs of the PCR channels 222a, 222b, 222c, and 222d are controlled using the membrane valves 244a, 244b, 244c, and 244d, respectively. The outputs of the four PCR channels 222 feed the common PCR output channel 224, which then feeds the reagent mixing and distribution area 275. The presence of membrane valves 242 and membrane valves 244 in the layers 200 inkjet technology allows you to completely seal the area 270 PCR.

[00194] The reagent mixing and distribution area 275 includes an arrangement of thirteen reagent channels 226 (eg, reagent channels 226a-226m). In addition, thirteen reagent channels 226a-226m are fed by thirteen reagent stores 1112a-1112m, respectively. A rotary valve assembly (not shown) is used to hydraulically connect some PCR channel 222 to some reagent supply 1112. In this way, some mixture of PCR can be created. A rotary valve assembly (not shown) is also used to hydraulically connect some PCR mix to the sequencing feed channel 228, which feeds it into the inlet of the sequencing chamber 258. In addition, the sequencing camera 258 has a CMOS image sensor 262.

[00195] At the outlet of the sequencing chamber 258, a sequencing outlet 230 is provided. The outlet pump 1114 is hydraulically and operably connected to the sequencing outlet 230. An outlet pump 1114 is used to provide positive or negative pressure to move fluid in either direction along the flow paths in the fluidized layers 200 . In addition, a series of three membrane valves 246 are provided along the length of the sequencing outlet 230. The membrane valves 242, 244, and 246 may be implemented in accordance with the membrane valve 830 shown and described in FIG. 22A, 22B, 23A and 23C.

[00196] The three membrane valves 246 on the sequencing outlet 230 may be used as pumps instead of or in combination with the outlet pump 1114. Therefore, in one embodiment, the microfluidic cartridge 1100 includes only the outlet pump 1114 and the three membrane valves 246 are omitted. In another embodiment, the microfluidic cartridge 1100 includes only three diaphragm valves 246 and the exhaust pump 1114 is omitted. In yet another embodiment, the microfluidic cartridge 1100 includes both an exhaust pump 1114 and three diaphragm valves 246. In yet another embodiment, the microfluidic cartridge 1100 includes any other type of pumping mechanism in place of the exhaust pump 1114 and/or three diaphragm valves 246. In more detail, one embodiment of the microfluidic cartridge 1100 is shown and described below with reference to FIG. 25-60B.

[00197] FIG. 25 and 26 illustrate perspective views of an assembled microfluidic cartridge 1200, which is one physical embodiment of the integrated microfluidic cartridge 1100 shown in FIG. 24. The microfluidic cartridge assembly 1200 is an example of a conventional injection molded inkjet technique that integrates with flexible printed circuit (FPB) technology. In this example, the microfluidic cartridge assembly 1200 is a multi-chamber microfluidic cartridge that includes a housing 1210 secured on top of a base plate 1212. The housing 1210 and base plate 1212 may be made of molded plastic, for example, and fastened together with screws ( see Fig. 32). The overall height of the microfluidic cartridge 1200 assembly may be, for example, from about 12 mm to about 100 mm. The overall length of the microfluidic cartridge 1200 assembly may be, for example, from about 100 mm to about 200 mm. The overall width of the microfluidic cartridge 1200 assembly may be, for example, from about 100 mm to about 200 mm.

[00198] Within housing 1210 is an inkjet assembly 1400 as shown in FIG. 27A and 27B. Namely, Fig. 27A and 27B illustrate perspective views of one example of an inkjet assembly 1400 that fits into the microfluidic cartridge assembly 1200 shown in FIG. 25 and 28. The inkjet assembly 1400 is based on the integrated microfluidic cartridge 1100 shown in FIG. 24. Namely, the inkjet assembly 1400 includes the inkjet layers 200 as shown and described in FIG. 15 and 24. The inkjet assembly 1400 also includes a rotary valve assembly 1410 that is located relative to thirteen reagent channels 226a-226m in the reagent mixing and distribution region 275 of the inkjet layers 200. The length of the inkjet layers 200 may be, for example, from about 100 mm to about 200 mm. The width of the inkjet layers 200 may be, for example, from about 100 mm to about 200 mm.

[00199] In addition, the node 1400 fluid technology includes a heater 1412 flexible PP, which is wrapped around both sides of the area 270 PCR layers 200 fluid technology. The flex PCR heater 1412 is provided with two individually controllable heater lanes such that there is one heater lane on one side of the PCR area 270 and another heater lane on the other side of the PCR area 270. The flexible PV heater 1412 is one example of the flexible PV heater 818 of the microfluidic cartridge 800 shown in FIG. 21. In more detail, one example of heater tracks is shown and described herein below with reference to FIG. 28A and 28B. In more detail, one example of a flexible PWB heater 1412 is shown and described herein below with reference to FIG. 54A, 54B and 54C.

[00200] Referring now again to FIG. 25 and 26, the housing 1210 of the microfluidic cartridge 1200 assembly also includes four sample loading holes 1214 (e.g., sample loading holes 1214a, 1214b, 1214c, 1214d) that are substantially aligned with the inputs of the four PCR channels 222 (e.g., channels 222a, 222b, 222c, 222d PCR) layers 200 of the inkjet technique. The housing 1210 of the microfluidic cartridge 1200 assembly also includes thirteen reagent reservoirs 1216 that "feed" the thirteen reagent channels 226 (eg, reagent channels 226a-226m) of the fluid technology layers 200. The thirteen reagent tanks 1216 may be the same or different sizes. For example, reagent reservoirs 1216 may contain liquid volumes ranging from about 0.001 ml to about 0.150 ml.

[00201] The body 1210 of the microfluidic cartridge 1200 assembly also includes a waste reservoir 1218 that is fed by the sequencing outlet 230. The waste reservoir 1218 may contain a volume of liquid in the range, for example, from about 25 ml to about 100 ml. Fig. 26 shows that the reagent tanks 1216 and the waste tank 1218 can be closed and sealed, for example, with a foil seal 1220.

[00202] FIG. 28A and 28B illustrate, respectively, a plan view and a sectional view of one example of a heater track 1500 that may be installed in the ink jet assembly 1400 shown in FIG. 27A and 27B. Namely, Fig. 28A shows a plan view of an example of a heater track 1500 that has a zigzag pattern. Fig. 28B show a sectional view of one side of the flexible PP heater 1412 of the inkjet assembly 1400, which includes a heater track 1500. The flexible PCB heater 1412 is a multilayer structure that includes, for example, in that order, a one-sided flexible copper layer 1510, an adhesive layer 1512, a dielectric layer 1514, a copper heater layer 1516 in which the heater track 1500 is patterned, and a layer 1518 Kapton® material. Copper heater layer 1516 shows a section of heater track 1500 along line A-A shown in FIG. 28A.

[00203] FIG. 29, 30, 31, 32, 33A, and 33B illustrate various other views of the microfluidic cartridge 1200 assembly of FIG. 25 showing it in more detail. Namely, Fig. 29 shows a perspective view, and FIG. 30 is a top side view of the housing 1210 of the microfluidic cartridge 1200 assembly, both showing the configuration of the thirteen reagent tanks 1216 and the waste tank 1218 in more detail. Fig. 31 shows a top side view of the housing 1210 of the microfluidic cartridge 1200 assembly with the foil seal 1220 installed. The foil seal 1220 has an opening so that the four sample loading holes 1214 remain open and accessible.

[00204] FIG. 32 shows a perspective view of a side of the base plate 1212 of the microfluidic cartridge 1200 assembly. Fig. 33A shows a top side view of the base plate 1212 of the microfluidic cartridge 1200 assembly. Fig. 33B shows a side view of the microfluidic cartridge 1200 assembly. Fig. 32, 33A and 33B show the base plate 1212 in more detail. Namely, the base plate 1212 includes an opening 1222 and an opening 1224 in order to expose portions of the region 270 of the PCR layers 200 of the ink jet assembly 1400 of the ink jet. Through the hole 1224 is visible a set of pads 1226 input/output for bringing into contact with the heater 1412 flexible PP node 1400 inkjet technology.

[00205] Along one edge of opening 1222 are four openings 1228 for accessing and actuating four diaphragm valves 242 of the inkjet layers 200 of the inkjet assembly 1400. Namely, opening 1228a is substantially aligned with diaphragm valve 242a. Orifice 1228b is substantially aligned with diaphragm valve 242b. Orifice 1228c is substantially aligned with diaphragm valve 242c. Orifice 1228d is substantially aligned with diaphragm valve 242d.

[00206] Along the opposite edge of the hole 1222 are four holes 1230 to access and actuate the four membrane valves 244 layers 200 jet technology node 1400 jet technology. Namely, opening 1230a is substantially aligned with diaphragm valve 244a. Orifice 1230b is substantially aligned with diaphragm valve 244b. Orifice 1230c is substantially aligned with diaphragm valve 244c. Orifice 1230d is substantially aligned with diaphragm valve 244d.

[00207] In addition, the base plate 1212 includes an opening 1232 for accessing and actuating the diaphragm valves 246 of the inkjet layers 200 of the inkjet assembly 1400. The base plate 1212 also includes an opening 1234 on the sequencing chamber 258. One corner of the baseplate 1212 has a bevel 1236 that is used to orient the microfluidic cartridge assembly 1200, for example, in a microfluidic instrument seat (not shown). Fig. 32 and 33A also show four screws 1238 that are used to secure the base plate 1212 to the housing 1210. In addition, the rotary valve assembly 1410 is shown in relation to the mixing and dispensing region 275 of the fluid technology layers 200 of the fluid technology assembly 1400. The rotary valve assembly 1410 includes a head having a gripping portion 1240 with which a user or apparatus can rotate the flow regulator 1242 (see FIG. 35).

[00208] Starting with the microfluidic cartridge assembly 1200 with baseplate 1212 side up, FIG. 34-42 essentially show a step-by-step disassembly of the microfluidic cartridge assembly 1200 as a means of showing the placement and installation of its internal components. First Fig. 34 shows the microfluidic cartridge 1200 assembled with the base plate 1212 removed in order to expose the inkjet assembly 1400. In this way, the side of the layer 260 of the flexible PP layers 200 of the inkjet technique becomes visible. In addition, one side of the flexible PP heater 1412 is visible. Also visible is the spacer 1244 between the jetting layers 200 and the base plate 1212. FIG. Diaphragm valves 242, 244 and 246 are visible in 34.

[00209] Turning now to FIG. 35, there the gripping portion 1240 of the rotary valve assembly 1410 has been removed to expose the flow controller 1242. The underside (not shown) of the gripper portion 1240 is designed to mate with the flow controller 1242 so that the flow controller 1242 can be rotated to direct fluid flow through one of the thirteen reagent channels 226.

[00210] Turning now to FIG. 36, the flow regulator 1242 of the rotary valve assembly 1410 has been removed to expose the flow paths associated with the PCR output channel 224, the reagent channels 226, and the sequencing feed channel 228 of the fluid layers 200.

[00211] Turning now to FIG. 37, the inkjet layers 200 are shown transparent in order to show the flow paths within the microfluidic cartridge 1200 assembly.

[00212] Turning now to FIG. 38, the inkjet layers 200 have been removed and only the flexible PWB heater 1412 within the housing 1210 is shown. Referring now to FIG. 39, the flexible PP heater 1412 has been removed and only the inkjet layers 200 within the housing 1210 are shown.

[00213] Turning now to FIG. 40, both the inkjet technique layers 200 and the flexible PCB heater 1412 have been removed from the housing 1210. This drawing shows the flow paths in the housing 1210, which are associated with holes 1214 sample loading, thirteen tanks 1216 reagent and tank 1218 for waste. For example, housing 1210 includes openings 1246 leading to sample loading openings 1214, openings 1248 leading to thirteen reagent tanks 1216, and opening 1250 leading to waste tank 1218. Fig. 40 also shows four threaded holes 1252 for screwing screws 1238 into them. In addition, FIG. 40 shows the CMOS image sensor 262 and part of the protective cap 1254 that covers the CMOS image sensor 262. Turning now to FIG. 41, the CMOS image sensor 262 has been removed so that the protective cap 1254 is fully visible. Referring now to FIG. 42, the protective cap 1254 has been removed to expose a free area 1256 in the housing 1210 that is related to the CMOS image sensor 262.

[00214] FIG. 43 shows a transparent perspective view of the housing 1210 of the microfluidic cartridge 1200 assembly to show the positions of the access holes relative to the sample loading holes 1214, the reagent reservoirs 1216, and the waste reservoir 1218. Namely, in this drawing, you can see the position of the holes 1246 relative to the holes 1214 sample loading, the position of the holes 1248 relative to the tanks 1216 of the reagent, and the position of the hole 1250 relative to the tank 1218 for waste.

[00215] FIG. 44 shows a transparent perspective view of the housing 1210 of the microfluidic cartridge 1200 assembled with various inkjet channels superimposed thereon. Namely, in this drawing, you can see the positions of the various channels of inkjet technology with respect to the openings 1214 sample loading, tanks 1216 reagent and tank 1218 for waste. Fig. 45 shows a sectional view of the microfluidic cartridge 1200 assembly of FIG. 25, which shows it in more detail.

[00216] FIG. 46A, 46B, 47A, 47B, and 48 show various views of the body 1210 of the microfluidic cartridge 1200 assembly of FIG. 25, which show it in more detail. Namely, Fig. 46A and 46B show top and side views of housing 1210, respectively. 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. While Fig. 46A, 46B, 47A and 47B show the exterior of the housing 1210, FIG. 48 shows a plan view of the inside of the case 1210.

[00217] FIG. 46A, 46B, 51A, 51B, and 52 show various views of the base plate 1212 of the microfluidic cartridge 1200 assembly of FIG. 25, which show it in more detail. Namely, Fig. 49 and 50 show perspective views of the outside and inside of the base plate 1212, respectively. FIG. 41A shows a plan view of the outside of the base plate 1212, while FIG. 41B shows a side view of the base plate 1212. FIG. 49, 50, 51A, 38B and 39 show that the base plate 1212 further includes four holes 1258 for screws 1238, a recessed area 1260 with a hole 1262 in its center for receiving the grip portion 1240 and the flow regulator 1242 of the rotary valve assembly 1410.

[00218] FIG. 53A and 53B illustrate other perspective views of the inkjet assembly 1400 of the microfluidic cartridge 1200 assembly, showing it in more detail. Namely, each of FIG. 53A and 53B show a perspective view of the inkjet assembly 1400. Fig. 53A show the inkjet assembly 1400 without the flexible PV heater 1412, while FIG. 53B shows the inkjet assembly 1400 with the flexible PV heater 1412 installed. In addition, there is a notch 1414 at one end of the fluid technology layers 200 and within the PCR region 270. The notch 1414 is designed to receive the flexible PP heater 1412.

[00219] FIG. 54A, 54B and 54C illustrate various views showing in more detail the flexible PP heater 1412 of the inkjet assembly 1400 of the microfluidic cartridge 1200 assembly. Namely, Fig. 54A and 54B show perspective views, respectively, of each side of the flexible PTFE heater 1412, while FIG. 54C shows a side view of the flex CB heater 1412. The flexible PWB heater 1412 includes a U-shaped wraparound panel 1416 and a side protruding panel 1418, which are made using flexible PWB technology. The U-shaped female panel 1416 includes a panel 1420 and a panel 1422, each having a heater track 1500 patterned thereon, such as heater tracks 1500a and 1500b. One example of heater track 1500 is shown in FIG. 28A and 28B. The space between the panel 1420 and the panel 1422 is defined such that the flexible PP heater 1412 can be pressed onto the region 270 of the PCR fluid layers 200 and extend into the cutout 1414 as shown in FIG. 53b. Fig. 54B and 41C also show I/O pads 1226 that provide electrical connections to the two heater tracks 1500 as well as to the CMOS image sensor 262.

[00220] A side-projecting panel 1418 exits panel 1420 near a bend in the U-shaped female panel 1416. The side-protruding panel 1418 is designed to extend to the CMOS image sensor 262. As shown in FIG. 53B, the end of the side-projecting panel 1418 farthest from the U-shaped wrapping panel 1416 is shaped to fit on the CMOS image sensor 262. The purpose of the side-protruding panel 1418 is to provide electrical connection to the CMOS image sensor 262, which is assembled on a rigid or flexible PCB.

[00221] FIG. 55A and 55B show respectively a perspective view and a plan view of the inlet/outlet layer 210 of the inkjet layers 200 shown in FIG. 15 and FIG. 27. Again, the inlet/outlet layer 210 may be made of, for example, polycarbonate or any other materials that are suitable for use in the R2R process. The inlet/outlet layer 210 provides an interface between the inkjet layers 200 and the housing 1210 of the microfluidic cartridge 1200 assembly. Namely, the inlet/outlet layer 210 provides flow paths from the sample loading holes 1214, the thirteen reagent tanks 1216, and the waste tank 1218 of the body 1210 to the fluid channel layer 220 of the fluidic layers 200. For example, inlet/outlet layer 210 includes a set of holes 212 that are substantially aligned with holes 1246 to sample loading holes 1214 in housing 1210. Inlet/outlet layer 210 includes a set of holes 214 that are substantially aligned with holes 1248 reagent reservoirs 1216 in housing 1210. The inlet/outlet layer 210 also includes an aperture 216 that is substantially aligned with the opening 1250 of the waste reservoir 1218 in housing 1210.

[00222] FIG. 56A and 56B show respectively a perspective view and a plan view of the hydraulic channel layer 220 of the inkjet layers 200 shown in FIG. 15 and FIG. 27. Again, the hydraulic channel layer 220 may be made of, for example, polycarbonate or any other materials that are suitable for use in the R2R process. The hydraulic channel layer 220 is that layer of the fluidic layers 200 in which all fluids flow. Namely, all PCR and sequencing operations take place in the hydraulic channel layer 220. The PCR operations take place in the PCR channels 222 in the PCR region 270 . The PCR output channel 224 feeds the reagent mixing and distribution area 275 . Reagent distribution takes place using the reagent channels 226 in the reagent mixing and distribution area 275 . Thirteen reagent channels 226 are patterned to feed the rotary valve assembly 1410. The sequencing feeder 228 feeds the inlet of the sequencing chamber 258 of the sequencing chamber layer 250 shown in FIG. 58A and 58B. Next, the sequencing outlet 230 is fluidly connected to the outlet of the sequencing chamber 258.

[00223] FIG. 57A and 57B show respectively a perspective view and a plan view of the flexible PP layer 260 of the inkjet layers 200 shown in FIG. 15 and FIG. 27. Again, the flexible PP layer 260 may be made of, for example, polyimide or any other materials that are suitable for use in the R2R process. The flexible PP layer 260 includes a set of holes (or holes) 264 that correspond to the inlet/outlet holes of the diaphragm valves 242. The flexible PP layer 260 also includes a set of holes (or holes) 266 that correspond to the inlet/outlet holes of the diaphragm valves 244 If membrane valves 246 are present, then the flexible PP layer 260 includes a set of holes (or holes) 267 that correspond to the inlet/outlet holes of the membrane valves 246. In addition, the flexible PP layer 260 includes a set of holes 268 that essentially are aligned with the rotary valve assembly 1410 and provide flow paths to it.

[00224] FIG. 58A and 58B show respectively a perspective view and a plan view of the bottom layer 280 of the sequencing chamber of the layers 200 of the inkjet technique shown in FIG. 15 and FIG. 27. Again, the bottom layer 280 of the sequencing chamber can be made of, for example, polycarbonate or any other materials that are suitable for use in the R2R process. The bottom layer 280 of the sequencing chamber includes a set of holes 282 for forming membrane valves 242 within the fluid layer stack 200. The bottom layer 280 of the sequencing chamber also includes a set of holes 284 for forming membrane valves 244 within the stack of fluid layers 200. If membrane valves 246 are present, then the bottom layer 280 of the sequencing chamber includes a set of holes 286 for forming membrane valves 246 within the stack of fluid layers 200. In addition, the bottom layer 280 of the sequencing chamber includes a set of holes 288 that substantially align with and provide flow paths to the rotary valve assembly 1410. Additionally, the bottom layer 280 of the sequencing chamber includes a pair of holes 289 that are fluidly connected to the sequencing chamber 258 of the layer 250 of the sequencing chamber.

[00225] The bottom layer 280 of the sequencing chamber is that layer of the inkjet layers 200 on which the CMOS technology is integrated. Namely, the CMOS image sensor 262 is mounted on the bottom layer 280 of the sequencing chamber. The position of the CMOS image sensor 262 essentially corresponds to the position of the sequencing camera 258 of the sequencing camera layer 250 .

[00226] FIG. 59A and 59B show respectively a perspective view and a plan view of the sequencing chamber layer 250 of the inkjet layers 200 shown in FIG. 15 and FIG. 27. Again, sequencing chamber layer 250 may be made of, for example, polycarbonate or any other materials that are suitable for use in the R2R process. The sequencing chamber layer 250 is that layer of the inkjet layers 200 in which sequencing operations take place, namely using the sequencing chamber 258.

[00227] The layer 250 of the sequencing chamber includes a set of holes 252 for forming membrane valves 242 within the stack of layers 200 of the fluid technique. The sequencing chamber layer 250 also includes a set of holes 254 for forming membrane valves 244 within the stack of fluidized layers 200. If membrane valves 246 are present, then the sequencing chamber layer 250 includes a set of holes 255 for forming membrane valves 246 within the stack of fluid layers 200. In addition, the sequencing chamber layer 250 includes a set of holes 256 that substantially align with and provide flow paths to the rotary valve assembly 1410.

[00228] FIG. 60A and 60B show, respectively, a perspective view and a plan view of the membrane layer 240 and the top layer 290 of the sequencing chamber of the inkjet layers 200 shown in FIG. 15 and FIG. 27. The membrane layer 240 may be made of, for example, silicone elastomer, while the top layer 290 of the sequencing chamber may be made of, for example, COC. Membrane layer 240 serves as an elastic membrane to open and close membrane valves 242, 244, and 246 within a stack of fluidic layers 200, membrane valves 242, 244, and 246 being created by combining, in that order, flexible PP layer 260, bottom layer 280 of the sequencing chamber, layer 250 of the sequencing chamber; and layer 240 of the membrane. Fig. 60A and 60B also show the top sequencing chamber layer 290 that is used to cover the sequencing chamber 258 of the sequencing chamber layer 250.

[00229] FIG. 61A and 61B illustrate a flowchart of one example of a method 4800 for using the microfluidic cartridge 1200 assembly to perform multilocus PCR and subsequent mixing required for sequencing. Since the microfluidic cartridge 1200 assembly is based on the microfluidic cartridge 1100 shown in FIG. 24, the microfluidic cartridge assembly 1200 is configured to quadruple sample multiplexing. In addition, in method 4800, thirteen reagent reservoirs 1216 are designated as reagent reservoirs 1216a, 1216b, 1216c, 1216d, 1216e, 1216f, 1216g, 1216h, 1216i, 1216j, 1216k, 1216l, and 1216m. In addition, method 4800 utilizes an exhaust pump 1114 that is fluidly coupled to the microfluidic cartridge assembly 1200. The outlet pump 1114 is located downstream of the chamber 258 sequencing. The outlet pump 1114 is capable of providing both positive pressure and negative pressure (ie, vacuum). Method 4800 includes, but is not limited to, the following steps.

[00230] At 4810, the microfluidic cartridge 1200 assembly that has been prepared for use is provided. Namely, the microfluidic cartridge 1200 is assembled with one or more of its reservoirs loaded with the desired fluids. For example, the reagent reservoirs 1216 may be filled with the same or different reagent fluids. In one example, all reagent tanks 1216a-m are filled with hydrogenation buffer (HT1). Next, method 4800 proceeds to block 4815.

[00231] In step 4815, all membrane valves are closed and then the PCR samples/mixture is loaded. "PCR mix" means a PCR master mix (i.e. a mixture of all necessary components except template and primers) that has been optimized for use in conventional PCR to amplify template DNA. At this point, diaphragm valves 242a and 244a are closed, diaphragm valves 242b and 244b are closed, diaphragm valves 242c and 244c are closed, and diaphragm valves 242d and 244d are closed. Thus, all PCR channels 222a, 222b, 222c and 222d are completely sealed. Then, the liquid of the first sample is mixed with the PCR mixture (hereinafter referred to as sample/PCR_MIX1) and loaded into the sample loading hole 1214a. The second sample liquid is mixed with the PCR mixture (hereinafter referred to as sample/PCR_MIX2) and loaded into the sample loading hole 1214b. The liquid of the third sample is mixed with the PCR mixture (hereinafter referred to as sample/PCR_MIX3) and loaded into the sample loading hole 1214c. The fourth sample liquid is mixed with the PCR mixture (hereinafter referred to as sample/PCR_MIX4) and loaded into the sample loading hole 1214d. Upon completion of this step, a volume of sample/PCR_MIX is in each of the sample loading holes 1214 and is ready for processing. Next, method 4800 proceeds to block 4820.

[00232] At 4820, the membrane valves are opened for the first sample. The first sample is then pulled into the PCR region. The diaphragm valves for the first sample are then closed. Open, for example, membrane valves 242a and 244a for PCR channel 222a. Then, using the outlet pump 1114, the sample/PCR_MIX1 is drawn into the PCR channel 222a. The membrane valves 242a and 244a for the PCR channel 222a are then closed, with some volume of sample/PCR_MIX1 now sealed in the PCR channel 222a. Next, method 4800 proceeds to block 4825.

[00233] At decision block 4825, it is determined whether another sample is waiting to be loaded into the PCR region, ie. to area 270 PCR. If yes, then method 4800 proceeds to block 4830. If not, then method 4800 proceeds to block 4835.

[00234] In step 4830, the diaphragm valves are opened for the next sample. The next sample is then pulled into the PCR area. The membrane valves are then closed for the next sample. In one example, membrane valves 242b and 244b are opened for PCR channel 222b. Then, using the outlet pump 1114, pull the sample/PCR_MIX2 into the PCR channel 222b. The membrane valves 242b and 244b for PCR channel 222b are then closed, with some volume of sample/PCR_MIX2 now sealed in PCR channel 222b.

[00235] In another example, membrane valves 242c and 244c are opened for PCR channel 222c. Then, using the outlet pump 1114, draw the sample/PCR_MIX3 into the PCR channel 222c. The membrane valves 242c and 244c for the PCR channel 222c are then closed, with some volume of sample/PCR_MIX3 now sealed in the PCR channel 222c.

[00236] In another example, membrane valves 242d and 244d are opened for PCR channel 222d. Then, using the outlet pump 1114, pull the sample/PCR_MIX4 into the PCR channel 222d. The membrane valves 242d and 244d for the PCR channel 222d are then closed, with some volume of sample/PCR_MIX4 now sealed in the PCR channel 222d.

[00237] Next, method 4800 returns to block 4825.

[00238] In step 4835, with sample/PCR_MIX1 in PCR channel 222a, sample/PCR_MIX2 in PCR channel 222b, sample/PCR_MIX3 in PCR channel 222c, and sample/PCR_MIX4 in PCR channel 222d, PCR operations are performed. After completing PCR operations, sample/PCR_MIX1 is now PCR_MIX1, sample/PCR_MIX2 is now PCR_MIX2, sample/PCR_MIX3 is now PCR_MIX3, and sample/PCR_MIX4 is now PCR_MIX4. Next, method 4800 proceeds to block 4840.

[00239] At 4840, the rotary valve is rotated to the first PRC mixture position. For example, by rotating the gripping portion 1240 of the rotary valve assembly 1410, the position of the rotary valve assembly 1410 is set to the PCR channel 222a containing PCR_MIX1. Next, method 4800 proceeds to block 4845.

[00240] In step 4845, the diaphragm valves are opened for the first PCR mixture. The first PCR mixture is then drawn through the rotary valve to the CMOS device. The membrane valves for the first PCR mixture are then closed. Open, for example, membrane valves 242a and 244a for PCR channel 222a. Then, using the outlet pump 1114, PCR_MIX1 is pulled out of the PCR channel 222a into the PCR outlet channel 224 through the rotary valve assembly 1410. The diaphragm valves 242a and 244a are then closed. Next, method 4800 proceeds to block 4850.

[00241] At step 4850, the rotary valve is rotated to the position of the hydrogenation buffer (HT1), that is, to the reservoir 1216 of the reagent, which contains HT1. In method 4800, at least one reagent reservoir 1216 contains a volume of HT1. As an example, this volume of HT1 is contained in the reagent tank 1216k. Therefore, by rotating the gripping portion 1240 of the rotary valve assembly 1410, the position of the rotary valve assembly 1410 is now set to the reagent tank 1216k containing HT1. Next, method 4800 proceeds to block 4855.

[00242] In step 4855, the first PCR mix is pushed into the HT1 tank. For example, using outlet pump 1114, PCR_MIX1 is pushed through rotary valve assembly 1410 into reagent reservoir 1216k and mixed with HT1 there. Next, method 4800 proceeds to block 4860.

[00243] At decision block 4860, it is determined whether another PCR mixture is expected to be mixed with HT1. If yes, then method 4800 proceeds to block 4865. If not, then method 4800 proceeds to block 4885.

[00244] In step 4865, the rotary valve is rotated to the position of the next PCR mixture. In one example, by rotating the gripping portion 1240 of the rotary valve assembly 1410, the position of the rotary valve assembly 1410 is set to the PCR channel 222b containing PCR_MIX2. In another example, by rotating the grip portion 1240 of the rotary valve assembly 1410, the position of the rotary valve assembly 1410 is set to the PCR channel 222c containing PCR_MIX3. In yet another example, by rotating the grip portion 1240 of the rotary valve assembly 1410, the position of the rotary valve assembly 1410 is set to the PCR channel 222d containing PCR_MIX4. Next, method 4800 proceeds to block 4870.

[00245] In step 4870, the membrane valves are opened for the next PCR mixture. The next PCR mixture is then drawn through the rotary valve to the CMOS device. The membrane valves for the next PCR mixture are then closed. In one example, membrane valves 242b and 244b are opened for PCR channel 222b. Then, using the outlet pump 1114, PCR_MIX2 is drawn from the PCR channel 222b to the PCR outlet channel 224 through the rotary valve assembly 1410. The diaphragm valves 242b and 244b are then closed. In another example, diaphragm valves 242c and 244c are opened for PCR channel 222c. Then, using the outlet pump 1114, PCR_MIX3 is pulled out of the PCR channel 222c into the PCR outlet channel 224 through the rotary valve assembly 1410. The diaphragm valves 242c and 244c are then closed. In another example, membrane valves 242d and 244d are opened for PCR channel 222d. Then, using the outlet pump 1114, PCR_MIX4 is pulled out of the PCR channel 222d into the PCR outlet channel 224 through the rotary valve assembly 1410. The diaphragm valves 242d and 244d are then closed. Next, method 4800 proceeds to block 4875.

[00246] In step 4875, the rotary valve is rotated to position HT1. For example, by rotating the gripping portion 1240 of the rotary valve assembly 1410, the position of the rotary valve assembly 1410 is returned to the reagent reservoir 1216k containing HT1. Next, method 4800 proceeds to block 4880.

[00247] In step 4880, the next PCR mix is pushed into the HT1 tank. In one example, using outlet pump 1114, PCR_MIX2 is pushed through rotary valve assembly 1410 into reagent reservoir 1216k and mixed with HT1 there. In another example, using outlet pump 1114, PCR_MIX3 is pushed through rotary valve assembly 1410 into reagent reservoir 1216k and mixed with HT1 there. In yet another example, using outlet pump 1114, PCR_MIX4 is pushed through rotary valve assembly 1410 into reagent reservoir 1216k and mixed with HT1 there. Next, method 4800 returns to block 4860.

[00248] In step 4885, the mixture from the HT1 reservoir is drawn into the sequencing chamber and the clustering/sequencing recipe is performed. For example, with the reagent reservoir 1216k now containing a mixture of HT1, PCR_MIX1, PCR_MIX2, PCR_MIX3, and PCR_MIX4, this mixture is drawn out of the reagent reservoir 1216k, then pushed along the sequencing feed channel 228 and fed into the sequencing chamber 258. Then, using the CMOS image sensor 262, the clustering/sequencing recipe is performed. This completes the method 4800.

[00249] One or more embodiments may include a CMOS flow cell having an accessible biosensor active area. For example, a CMOS flow cell can be designed as a disposable consumable. Accordingly, it may be advantageous for the CMOS flow cell to be a small and inexpensive device. In a small CMOS flow cell, it is important to use as much biosensor active area as possible. However, current designs of CMOS flow cells do not allow 100 percent utilization of the biosensor active area. Therefore, new approaches are needed in order to provide increased utilization of the biosensor core in a CMOS flow cell. Embodiments set forth herein may include a CMOS flow cell in which most or up to about 100% of the biosensor active area is available for reagent delivery and illumination, as shown and described herein below with reference to FIG. 62-75.

[00250] FIG. 62 illustrates a side view of one example of a 4900 CMOS flow cell in which most or up to about 100% of the biosensor active area is available for reagent delivery and illumination. The CMOS flow cell 4900 includes a PP substrate 4910, which is, for example, a flexible PP substrate. On top of the PP substrate 4910 is a CMOS biosensor device 4920. The CMOS biosensor device 4920 is a CMOS image sensor with a biolayer located thereon. Also on top of the PP substrate 4910 and around the CMOS biosensor device 4920 is a laminate film 4930. The laminate film 4930 may be made of, for example, epoxy, polyimide or other thermoplastic film, silicone, Kapton® material, bismaleimide triazine (BT), and the like. The PP substrate 4910 and the laminate film 4930 may be formed using flexible PP technology. A planarization surface can also be created by machining a cavity in a PCB substrate.

[00251] The purpose of the laminate film 4930 is to provide an expanded surface around the perimeter of the CMOS biosensor device 4920 that is substantially flat flush with the top of the CMOS biosensor device 4920. In one example, if the chip thickness of the CMOS biosensor device 4920 is about 100 µm, then the thickness of the laminate film 4930 is about 100 µm ± about 5 µm.

[00252] A small gap between the PP substrate 4910 and the laminate film 4930 forms a groove or channel 4950 around the perimeter of the CMOS biosensor device 4920. The width of the groove or channel 4950 may be, for example, from about 100 microns to about 1000 microns. The groove or channel 4950 is filled with filler 4952 to form a substantially continuous flat surface of the CMOS biosensor device 4920 and laminate film 4930. Filler 4952 is a material that does not interfere with reactions taking place on top of the CMOS biosensor device 4920. Aggregate 4952 may be, for example, an ultraviolet (UV) curable epoxy resin, a thermoset epoxy resin, or the like.

[00253] On top of the CMOS biosensor assembly 4920 and laminate film 4930 is a flow cell cover 4940 into which a flow channel 4942 is embedded. In addition, the flow cell cover 4940 includes a first port 4944 and a second port 4946 that provide inlet/outlet ports. to the flow channel 4942. The flow cell cap 4940 is formed from a material that is optically clear and has little or no autofluorescence in the part of the spectrum that will be used for analytical detection, such as, but not limited to, a cyclic olefin (COC) copolymer. The total thickness of the flow cell cap 4940 may be, for example, from about 300 microns to about 1000 microns. Outside the flow channel 4942, there is a bonding area for connecting the flow cell cap 4940 to the laminate film 4930. The bonding may be carried out with a low autofluorescence adhesive.

[00254] Because there is a substantially continuous flat surface across the CMOS biosensor device 4920 and throughout the laminate film 4930, the flow channel area 4942 within the flow cell cap 4940 can be sized to encompass the entire CMOS biosensor device 4920; namely, it may cover approximately 100% of the active area of the biosensor. In one example, if the die size of the CMOS biosensor device 4920 is about 8 mm x 9 mm, then the core size is about 7 mm x 8 mm. However, the die size of the CMOS biosensor device 4920 may be, for example, up to about 25 mm×25 mm, with a proportionally larger active area.

[00255] FIG. 62 shows, for example, the reagent fluid 4954 filling the flow channel 4942. Chemical reactions take place in the reagent fluid 4954 in the flow channel 4942, which is located on top of the CMOS biosensor device 4920. When illuminated through the flow cell cover 4940, the CMOS biosensor device 4920 is used to sense the chemical reactions that take place in the flow channel 4942. Electrical connections (not shown) are made through the PP substrate 4910 to receive signals from the CMOS biosensor device 4920. In the 4900 CMOS flow cell, approximately 100% of the biosensor active area of the CMOS biosensor device 4920 is available for reagent delivery and illumination.

[00256] FIG. 63 illustrates an exploded view of an example of one embodiment of the CMOS flow cell 4900 shown in FIG. 62. FIG. 63 shows that the CMOS biosensor device 4920 includes an active area 4922. Any portion of the CMOS biosensor device 4920 outside the active area 4922 is an inactive area 4924. The CMOS biosensor device 4920 may be attached to the PP substrate 4910 using, for example, flip-chip mounting technology. In addition, the laminate film 4930 includes an aperture or window 4932 that is sized to receive the CMOS biosensor device 4920 when applied to the PP substrate 4910. An opening or window 4932 is provided in the laminate film 4930 in advance before laminating the laminate film 4930 onto the PP substrate 4910. When the flow cell cap 4940 is connected to the laminate film 4930, the flow channel 4942 is substantially aligned with the CMOS biosensor device 4920 and extends beyond the area of the CMOS biosensor device 4920. On FIG. 63 the flow cell cap 4940 is shown transparent. Fig. 64 and 65 illustrate respectively a perspective view and a side view of the 4900 CMOS flow cell shown in FIG. 63 fully assembled.

[00257] FIG. 66 illustrates perspective views of one example of a lid 4940 of the CMOS flow cell 4900 shown in FIG. 63, 64 and 65. Namely, FIG. 66 shows a top and bottom perspective view of the cover 4940 of the 4900 CMOS flow cell shown in FIG. 63, 64 and 65. In this example, the diameter of the first hole 4944 and the second hole 4946 may be about 750 microns. In addition, the depth or height of the flow channel 4942 may be about 100 microns.

[00258] FIG. 67, 68, 69, and 70 illustrate one example of a process for providing an extended flat surface in a CMOS flow cell onto which the flow cell cover can be placed.

[00259] In the first step, referring now to FIG. 67 provide a laminate film 4930 and a CMOS biosensor device 4920 over the PP substrate 4910. There is a groove or channel 4950 around the perimeter of the CMOS biosensor device 4920. The groove or channel 4950 exists because the opening or window 4932 in the laminate film 4930 is slightly larger than the CMOS biosensor device 4920.

[00260] In the next step, referring now to FIG. 68, the top side of the groove or channel 4950 is sealed with, for example, an optically clear elastomer 4960 that has surface features to tightly fit the groove or channel 4950. The elastomer 4960 is optically clear so that UV light can pass through. The purpose of the 4960 elastomer is to block the top of the trough or channel 4950 in preparation for filling.

[00261] In the next step, referring now to FIG. 69, using, for example, a pair of through holes 4916 in the PP substrate 4910, the groove or channel 4950 is filled with filler 4952, such as a UV curable epoxy resin, for which the elastomer 4960 must be optically clear.

[00262] In the next step, referring now to FIG. 70, once the filler 4952 has cured, the elastomer 4960 is removed and the flow cell now has a substantially continuous flat surface for receiving a flow cell cap, such as a flow cell cap 4940.

[00263] FIG. 71A, 71B, 71C, and 71D illustrate another example of a process for providing an extended flat surface in a CMOS flow cell onto which a flow cell cover can be placed.

[00264] In a first step, referring now to FIG. 71A provide a CMOS biosensor device 4920 on top of a PP substrate 4910.

[00265] In the next step, referring now to FIG. 71B provide a mold 5510 (eg, a clamshell type) around the CMOS biosensor device 4920 and the PP substrate 4910. The mold 5510 provides a space or void 5512 over the PP substrate 4910 and around the perimeter of the CMOS biosensor device 4920.

[00266] In the next step, referring now to FIG. 71C, using, for example, a low pressure injection molding process or a reaction injection molding process, the space or void 5512 in the mold 5510 is filled with filler 4952, such as a UV or thermoset epoxy resin.

[00267] In the next step, referring now to FIG. 71D, once the filler 4952 is cured, the mold 5510 is removed and the flow cell now has a substantially continuous flat surface for receiving a flow cell cap, such as a flow cell cap 4940.

[00268] FIG. 72, 73, 74, and 75 illustrate yet another example of a process for providing an extended flat surface in a CMOS flow cell onto which the flow cell cover can be placed.

[00269] In a first step, referring now to FIG. 72 provide a CMOS biosensor device 4920 on top of the PP substrate 4910. In addition, a mechanical piece 5910 is provided on a PP substrate 4910 at one end of the CMOS biosensor device 4920. Similarly, a mechanical piece 5912 is provided on a PP substrate 4910 at the other end of the CMOS biosensor device 4920. Mechanical parts 5910 and 5912 may be pure silicone, glass or plastic, for example. There is a groove or channel 5914 between the mechanical part 5910 and the CMOS biosensor device 4920. Another groove or channel 5914 is between the mechanical part 5912 and the CMOS biosensor device 4920.

[00270] In the next step, referring now to FIG. 73 provide a set of barriers 5916 at the ends of channels or channels 5914. For example, barriers 5916a and 5916b block the ends of one channel or channel 5914, and barriers 5916c and 5916d block the ends of another channel or channel 5914 in preparation for filling.

[00271] In the next step, referring now to FIG. 74, the grooves or channels 5914 are filled with filler 4952, such as a UV or thermoset epoxy. Filler 4952 is retained between barriers 5916a and 5916b and between barriers 5916c and 5916d.

[00272] In the next step, referring now to FIG. 75, once the filler 4952 is cured, a substantially continuous flat surface is now present in the flow cell for receiving the flow cell cap, such as the flow cell cap 4940.

[00273] It should be appreciated that various aspects of the present invention may be embodied as a method, system, computer readable medium, and/or computer program product. Aspects of the present invention may take the form of hardware implementations, software implementations (including firmware, resident software, microcode, etc.), or embodiments combining software and hardware aspects, which may be collectively referred to herein as "circuit", "module" or "system". In addition, the methods of the present invention may take the form of a computer program product on a computer-usable storage medium with computer-usable program code embodied in the storage medium.

[00274] Any suitable computer usable media can be used for the software aspects of the present invention. The computer or machine-readable medium used can 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 transient and/or non-transitory embodiments. More specific examples (non-exhaustive list) of a computer-readable medium include some or all of the following: an electrical connection with one or more wires, a portable computer floppy disk, a hard disk drive, a random access memory (RAM), a read only memory (ROM), an erasable programmable ROM (EPROM). or flash memory), optical fiber, compact disc portable read-only memory (CD-ROM), optical storage, transmission media such as those media that support the Internet or an intranet, or a magnetic storage device. It should be noted that the computer-used or machine-readable medium may even be paper or other suitable medium on which the program is printed, as long as the program can be read electronically, for example by optically scanning the paper or other medium, then compiled, interpreted, or otherwise processed in a suitable manner. , if necessary, and then saved to the computer's storage device. As used herein, a computer usable or machine-readable medium can be any medium that can contain, store, transmit, distribute, or carry a program for use on or in connection with a system, apparatus, or device for executing instructions.

[00275] The software code for carrying out the operations of the methods and operation of the apparatuses set forth herein may be written in an object-oriented programming language such as Java, Smalltalk, C++, and the like. However, the program code for carrying out the 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 that executes the program code. Program code may be referred to simply as a software application that is stored in memory (such as the computer-readable medium discussed above). Program code may cause the processor (or any device controlled by the processor) to create a graphical user interface ("GUI"). The graphical user interface may be visually created on a display device, although the graphical user interface may also have audio elements. The software code, however, may run on any processor-controlled device such as a computer, server, personal digital assistant, telephone, television, or any processor-controlled device using a processor and/or digital signal processor.

[00276] The software code may be executed locally and/or remotely. The software code, for example, may be wholly or partially stored in the local memory of a processor-controlled device. The software code, however, may also be at least partially remotely stored, accessed, and downloaded to a processor-controlled device. The user's computer, for example, may completely execute the program code or only partially execute the program code. The program code may be a stand-alone software package that resides at least partially on the user's computer and/or partially runs on a remote computer, or runs entirely on a remote computer or server. In the latter scenario, the remote computer may be connected to the user's computer via a communications network.

[00277] The methods and apparatus set forth herein can be applied regardless of the network environment. The communication network may be a cable network operating in the radio frequency band and/or based on the Internet Protocol (IP). The communications network, however, may also include a distributed computing network such as the Internet (sometimes alternatively referred to as the "World Wide Web"), an intranet, a local area network (LAN), and/or a wide area network (WAN). The communication network may include coaxial cables, copper wires, fiber optic lines, and/or hybrid coax lines. The communication network may even include wireless sections using any portion of the electromagnetic spectrum and any signaling standard (such as the IEEE 802 family of standards, GSM/CDMA/TDMA or any cellular standard and/or ISM band). The communication network may even include portions of power lines in which signals are transmitted via electrical wiring. The methods and apparatus set forth herein can be applied to any wireless/wired communications network, regardless of physical component base, physical configuration, or communications standard(s).

[00278] Some aspects of the present invention are described with reference to various methods and method steps. It should be understood that each step of the method may be implemented in program code and/or machine instructions. Program code and/or machine instructions may create the means to implement the functions/actions described in these methods.

[00279] Program code may also be stored in computer readable memory, which may instruct a processor, computer, or other programmable data processing device to operate in a particular manner, such that the program code stored in computer readable memory produces or transforms an article, including instructions that perform various aspects of the steps of the method.

[00280] Program code may also be loaded onto a computer or other programmable data processing device to cause a series of operational steps to be performed to create a processor/computer implemented process such that the program code provides steps to implement the various functions/actions described in methods of the present invention.

[00281] In one embodiment, a system is provided that includes a removable cartridge having a cartridge body. The removable cartridge also includes a hydraulic network that is located inside the cartridge body. This hydraulic network is configured to receive and hydraulically guide a biological sample to perform at least one of sample analysis or sample preparation. The removable cartridge also includes a flow control valve that is operatively connected to the hydraulic network and is movable relative to the hydraulic network to control the flow of the biological sample through it. The cartridge body includes a body side that defines the appearance of the removable cartridge and provides functional (operational) access to the flow control valve. The system also includes a host device having a control side that is configured to co-operate in a releasable manner with a body side of the removable cartridge. Said body side and the control side together form a system interface. The main instrument includes a valve actuator that communicates with the flow control valve via a system interface. The removable cartridge also includes a sensing assembly that is held by at least one of the removable cartridge or main device. The detection unit includes an image detector and a reaction chamber that is in fluid communication with the hydraulic network. The image detector is configured to detect intended reactions within the reaction chamber.

[00282] In one aspect, the control side of the host device described here and the body side of the removable cartridge described here are generally flat and facing each other. The system interface may be a one-way interface in which the main instrument and the removable cartridge are operatively connected to each other only through the body side and the control side. Optionally, the main device and the removable cartridge may be operatively coupled such that the main device and the removable cartridge are attached to each other at the system interface in at least one of fluid communication, electrical communication, or thermal communication through the system interface.

[00283] In another aspect, the control side of the main device described here may be the top of the main device due to gravity, so that the removable cartridge sits on and is supported by the main device.

[00284] In another aspect, the valve actuator of the host device described herein may include an elongated actuator body that extends through the side of the housing into the cartridge housing.

[00285] In another aspect, the flow control valve of the removable cartridge described herein may include an elongated actuator body that extends through the control side into the main instrument.

[00286] In another aspect, the main instrument described here may have a side of the instrument that faces in the opposite direction relative to the control side. The main instrument may have an instrument size that extends between said control side and instrument side. The main unit and the removable cartridge may have a larger overall size than the size of the unit.

[00287] In another aspect, each of the removable cartridge and the main device may include a contact set of electrical contacts. These contact sets can be electrically connected to each other at the system interface.

[00288] In another aspect, the body side of the removable cartridge described herein may be a first body side, and the cartridge body may also include a second body side. The first and second sides of the housing face different directions. The system interface is a multi-sided interface in which the main instrument and the removable cartridge are operatively connected to each other along each of the first and second sides of the housing.

[00289] Optionally, the first and second sides of the body of the removable cartridge described herein may be generally perpendicular to each other. The main device may have a body of the device, which includes first and second control sides that face in perpendicular directions and form an open recess of the main device. At least a portion of the removable cartridge may be located within this open recess in such a way that the first and second sides of the housing interact with the first and second control sides.

[00290] In one aspect, the valve actuator of the host device described herein may include an elongated body that extends through the system interface between the first body side and the first control side. The second side of the housing and the second control side may include respective contact sets of electrical contacts. These contact sets may be electrically connected to each other along the system interface.

[00291] In another aspect, the first and second sides of the body of the removable cartridge described here face opposite directions. The main instrument may have an instrument side and a cartridge receiving socket that extends to that side of the instrument. A removable cartridge may be placed inside the cartridge receiving slot.

[00292] In another aspect, the removable cartridge and the main device are fluidly connected along the first side of the housing and electrically connected along the second side of the housing. Optionally, the host includes a locking mechanism that engages at least one of the first side of the housing or the second side of the housing in order to hold the removable cartridge within the host.

[00293] In another aspect, each of the removable cartridge and the main device may include a flow port. These flow ports are hydraulically connected to each other at the system interface.

[00294] In another aspect, the system described herein may include a locking mechanism that is attached to at least one of the removable cartridge or host device. This locking mechanism is configured to removably secure the cartridge body to the main apparatus.

[00295] In another aspect, the image detector of the system described here may be held by the main instrument and the reaction chamber may be held by a removable cartridge.

[00296] In another aspect, the flow control valve of the removable cartridge described herein may include a flexible membrane that is configured to control the flow of a biological sample through a hydraulic network. This flexible membrane can be bent between the first and second states of the valve actuator.

[00297] In another aspect, a body side of a removable cartridge described herein may include an access port through it that receives a valve actuator.

[00298] In another aspect, the flow control valve of the host device described herein may include a rotary valve configured to control the flow of fluid through a hydraulic network. This rotary valve can be rotated by the valve actuator.

[00299] In another aspect, the host device described herein may include a thermal block, and the fluid circuit of the cartridge body may include a sample passage where the intended reactions with the biological sample take place. The docking side may include an access port that extends along the sample path and is configured to receive a fuser for changing the temperature of the sample path.

[00300] In another aspect, the hydraulic network of the removable cartridge described herein may include a plurality of channels and a storage module. This storage module may include a plurality of reagent storage tanks that are used for at least one of sample preparation or sample analysis.

[00301] In another aspect, the host device described herein includes a system controller having a valve control module configured to control the operation of a valve actuator to control the flow of a biological sample through a hydraulic network.

[00302] In one embodiment, a method for sequencing nucleic acids is provided. This method includes providing a removable cartridge having a cartridge body, a hydraulic network located within the cartridge body, and a flow control valve that is operatively connected to the hydraulic network and movable with respect to the hydraulic network. The cartridge body includes a body side that defines the appearance of the removable cartridge. This method also includes bringing the removable cartridge into contact with the main apparatus. The body side of the removable cartridge is releasably mated with the control side of the main instrument to form a system interface together. The main instrument includes a valve actuator that communicates with the flow control valve via a system interface. The method also includes hydraulically guiding the biological sample through the hydraulic 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, the flow of the biological sample being controlled by a valve actuator on the flow control valve. The method also includes detecting a biological sample using an image detector directed at the reaction chamber, wherein the detection assembly is held by at least one of the removable cartridge or main instrument.

[00303] In one aspect, the method described here may also include removing the removable cartridge from the host device. The removable cartridge can be replaced by functional pairing of the second removable cartridge with the main device. Several removable cartridges can be paired with the main instrument in series, used for sample preparation and/or analysis when paired with the main instrument and then removed from the main instrument.

[00304] Accordingly, the method may include bringing the second removable cartridge into contact with the host device, wherein the body side of the second removable cartridge releasably interacts with the control side of the host device, collectively forming a system interface.

[00305] In another aspect, the method described herein includes removing a removable cartridge from a host device. Optionally, this method includes bringing the second removable cartridge into contact with the main device, and the body side of the second removable cartridge releasably interacts with the control side of the main device, together forming a system interface.

[00306] In another aspect of the method described here, the hydraulic guidance of the biological sample and the imaging of the biological sample are repeated multiple times and sequentially in a single removable cartridge.

[00307] In another aspect, the method described herein includes sealing a biological sample within a sample preparation area of a hydraulic network and amplifying the biological sample while the biological sample is sealed within the sample preparation area.

[00308] In another aspect, a flow control valve used in the method described herein includes a movable valve having at least one flow passage that extends between valve openings, as well as a valve actuator configured to move the movable valve between different provisions.

[00309] In another aspect, the movable valve used in the method described herein is at 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 through the flow channel into the reaction chamber. a reagent chamber that reacts with the biological sample in the reaction chamber.

[00310] In another aspect of the method described herein, a component position includes a plurality of component positions, the method further comprising moving a movable valve between component positions in a predetermined sequence to flow various reactants into the reaction chamber.

[00311] In another aspect, the biological sample used in the method described here includes nucleic acids, and the given sequence is performed in accordance with the sequencing by synthesis (SBS) protocol.

[00312] In another aspect, the flow cell used in the method described here includes a reaction chamber. The biological sample is immobilized on one or more surfaces of the flow cell.

[00313] In one embodiment, a removable cartridge is provided that includes a cartridge body having a sample opening that extends outside the cartridge body and is configured to receive a biological sample. The cartridge body has a set of electrical contacts and a mechanical interface that are accessible from the outside. The cartridge body is designed to be detachably attached to the main device. The removable cartridge may also include a hydraulic network having a plurality of channels, a reaction chamber, and a storage module. The storage module includes a plurality of reagent storage tanks. The hydraulic network is configured to direct the reactants from the reservoirs to the reaction chamber, wherein the mechanical interface is movable relative to the hydraulic network to control the flow of fluid through the hydraulic network. The system also includes an imaging device located within the cartridge body and positioned to detect intended reactions within the reaction chamber. The imaging device is electrically connected to a set of electrical contacts for communicating with the main instrument. The mechanical interface may be movable by the host when the removable cartridge is attached to the host.

[00314] In one aspect, the mechanical interface of the removable cartridge described herein may include a channel valve that is configured to control the flow of fluid through one of the channels of the hydraulic network.

[00315] In another aspect, the body of the removable cartridge described herein may include an access port that provides access to a mechanical interface. Optionally, the mechanical interface includes a rotary valve.

[00316] In another aspect, the body of the removable cartridge described herein may include an access port open to the outside and the channels include a sample port that is in fluid communication with the sample port. The access port may extend along the sample path and may be configured to receive a thermal block to control the temperature of the sample path.

[00317] In another aspect, the body of the removable cartridge described herein may include a fluid communication opening that is externally accessible and in fluid communication with a fluid network. The fluid communication opening is configured to interact with the opening of the device to receive fluid through it.

[00318] In another aspect, the housing of the removable cartridge described herein may include first and second sides of the housing facing in opposite directions. The first side of the housing may include a set of electrical contacts. The second side of the housing may include a mechanical interface.

[00319] In another aspect, the removable cartridge also includes a locking mechanism that can be attached to the cartridge body. This locking mechanism may be configured to releasably attach the cartridge body to the main apparatus.

[00320] In one embodiment, a removable cartridge is provided that includes a cartridge body having a sample opening that extends outside the cartridge body and is configured to receive a biological sample. The removable cartridge may also include a rotary valve located within the cartridge body. A rotary valve has a hydraulic side and a plurality of valve openings that open onto that hydraulic side. The rotary valve has at least one flow channel extending between the openings of the valve, and the rotary valve is able to rotate between different angular positions. The removable cartridge may also include a microfluidic body having a body side that is slidably mated with the hydraulic side of the rotary valve. This microfluidic body may at least partially form a hydraulic network that includes a sample conduit in fluid communication with the sample orifice. The sample channel has a network opening that opens onto said first side of the microfluidic body. The hydraulic network may also include a reservoir configured to hold the reagent. This reservoir is in fluid communication with a reservoir opening that opens onto the hydraulic side of the microfluidic body. The hydraulic network also includes a supply channel in fluid communication with the reaction chamber of the hydraulic network. The feed channel has a feed opening that opens onto said first side of the microfluidic body. The rotary valve is rotatable between the first and second angular positions. The network orifice is hydraulically connected to the supply orifice via the rotary valve when the rotary valve is in the first angular position. The reservoir opening is in fluid communication with the supply opening via the rotary valve when the rotary valve is in the second angular position.

[00321] In one aspect, the housing of the removable cartridge described herein may have an exterior that is configured to interact with a host device. The rotary valve may include a mechanical interface that is externally accessible and configured to interact with the main instrument.

[00322] In another aspect, the rotary valve in the first angle position may be configured, while in the detachable cartridge described herein, to receive sample liquid when suction force draws sample liquid towards the supply port. The rotary valve in the second angle position may be configured to allow the sample liquid to be expelled into the reservoir when the displacement force pushes the sample liquid out of the supply port into the reservoir.

[00323] In another aspect, the rotary valve of the removable cartridge described here is rotatable about an axis. The feed hole can be aligned with this axis.

[00324] In one embodiment, a removable cartridge is provided that includes a cartridge body having a sample opening that extends outside the cartridge body and is configured to receive a biological sample. The cartridge body may include a docking side that is designed to face the main device and is configured to be removably connected to it. The removable cartridge also includes a hydraulic network that is housed inside the housing. This hydraulic network includes a sample conduit that is in fluid communication with the sample port. The removable cartridge also includes a channel valve having a flexible member that is movable between first and second positions. The flexible member blocks flow through the sample port when it is in the first position and allows flow through the sample port when it is in the second position. The mating side of the cartridge body includes an access port that allows access to the channel valve from the outside of the cartridge body. The access hole is configured to receive the drive of the main device to move the flexible part between the first and second positions.

[00325] In another aspect, the flexible part of the removable cartridge described herein may include a flexible layer that covers the internal cavity of the hydraulic network. This flexible layer may be configured to be pressed into the cavity to block flow through it.

[00326] In another aspect, the removable cartridge also includes a rotary valve that is located within the cartridge body. This rotary valve is rotatable between different positions to change the flow path in the hydraulic network. The rotary valve may include a mechanical interface accessible along the mating side.

[00327] In another aspect, the fluid network of the removable cartridge described herein may include a network opening in fluid communication with a sample passage, a supply opening in fluid communication with a reaction chamber, and a reservoir opening in fluid communication with a reservoir that is configured to store a reagent. . The removable cartridge may also include a rotary valve located within the cartridge housing. The rotary valve can hydraulically communicate the supply port and the network port when it is in the first corner position, and can hydraulically link the supply port and the reservoir port when it is in the second corner position.

[00328] In another aspect, the mating side of the removable cartridge described herein may be a first mating side, and the removable cartridge may have a second mating side. The first and second docking sides face opposite directions. The second docking side is configured to interact mechanically, hydraulically or thermally with the device.

[00329] In one embodiment, a host device is provided that includes a system housing having a control side that is configured to interact with a removable cartridge. The main instrument also includes a rotary motor that is configured to interact with the rotary valve of the removable cartridge. The main device also includes an actuator that is configured to interact with the removable cartridge channel valve, and a set of electrical contacts configured to be electrically connected to the removable cartridge. The main instrument also includes a system controller that is configured to control a rotating motor and drive to execute the assay protocol within the removable cartridge. The system controller is configured to receive imaging data from a removable cartridge through a set of electrical contacts. Optionally, the main apparatus includes a thermal block for heating a portion of the removable cartridge.

[00330] In one embodiment, a removable cartridge is provided that includes a cartridge body having a sample opening that extends outside the cartridge body and is configured to receive a biological sample. The cartridge body includes a docking side that is designed to face the main instrument and is configured to be removably attached to it. The removable cartridge also includes a microfluidic body located within the cartridge body. The microfluidic body has a body side and includes a hydraulic network. The hydraulic network has a plurality of discrete channels and corresponding openings that extend to this side of the body at the valve receiving area. The removable cartridge also includes a rotary valve located within the cartridge body. The rotary valve has a hydraulic side and at least one flow channel that extends between a plurality of valve openings. The valve ports face the hydraulic side. The hydraulic side rotatably mates with the valve-receiving area of said side of the microfluidic body, wherein the rotary valve is movable between different angular positions to fluidly couple the discrete channels. The rotary valve has a mechanical interface that is accessible along the docking side and is configured to interact with the main instrument such that the rotary valve is controlled by the main instrument.

[00331] In one embodiment, a removable cartridge is provided that includes a cartridge body having a sample opening that extends outside the cartridge body and is configured to receive a biological sample. The cartridge body has a docking side, which is configured to be detachably attached to the main device. The removable cartridge also includes a microfluidic structure that resides within the cartridge body and includes a plurality of stacked printed circuit board (PCB) layers. The PP layers include hydraulic layers that form channels and a reaction chamber when the PP layers are stacked on top of each other. The PP layers also include a wiring layer. The removable cartridge also includes a CMOS imager that is configured to be mounted on the microfluidic structure and electrically connected to the wiring layer. The CMOS imager is oriented to detect intended reactions within the reaction chamber.

[00332] In one aspect, the removable cartridge includes input/output (I/O) contacts that are accessible from the outside of the cartridge housing. The I/O pins may be electrically connected to the CMOS imager.

[00333] In one aspect, the microfluidic structure of the removable cartridge described herein includes a channel valve, wherein at least a portion of the channel valve is formed by layers of PP. The channel valve is configured to be actuated to block and allow flow through one of the channels.

[00334] An element or step mentioned here in the singular should be understood as not excluding the plural of said elements or steps, unless such an exception is explicitly stated. In addition, references to "one embodiment" should not be interpreted as excluding the existence of additional embodiments that also include these features. In addition, unless explicitly stated otherwise, embodiments "comprising", "comprising", or "having" an element or sets of elements with a particular property may include additional elements, whether or not they have that property.

[00335] It should be noted that the specific arrangement of components (eg, number, types, placement, etc.) of the illustrated embodiments may be modified in various alternative embodiments. In various embodiments, different numbers of a given module or block may be used, a different type or types of a given module or block may be used, a given module or block may be added, or a given module or block may be omitted.

[00336] It should be understood that the above description is intended to be illustrative and not limiting. For example, the embodiments described above (and/or aspects thereof) may be used in combination with each other. In addition, numerous modifications may be made to adapt a particular situation or material to the knowledge of various embodiments of the invention without departing from its scope. The dimensions, types of materials, orientations of the various components, and the number and positions of the various components described herein are intended to characterize the parameters of some embodiments and are by no means limiting, but merely exemplary embodiments. Numerous other embodiments and modifications within the spirit and scope of the claims will become apparent to those skilled in the art upon consideration of the above description. Therefore, the patentable scope of protection must be determined by reference to the appended claims along with the full scope of the equivalents to which those claims entitle.

[00337] In the present description, the phrase "in one exemplary embodiment", etc. means that the described embodiment is just one example. This phrase is not intended to limit the subject matter of the invention to this embodiment. Other embodiments of the subject matter may not include the described feature or structure. In the appended claims, the terms "comprising" and "in which" are used as language equivalents of the respective terms "comprising" and "wherein". In addition, in the appended claims, the terms "first", "second" and "third", etc. are used simply as markers, without imposing any numerical restrictions on enumerable objects. In addition, the limitations of the following claims are not written in the "means plus function" format and are not intended to be interpreted under 35 U.S.C. § 112(f) unless and until such limitations expressly use the phrase "means for” followed by the formulation of the function without additional construction.

1. System containing:

a removable cartridge having a cartridge body and including a hydraulic network disposed within the cartridge body configured to receive and hydraulically guide a biological sample to perform at least one of sample analysis or sample preparation, the removable cartridge also including a flow control valve , which is operatively connected to and movable with respect to the hydraulic network to control the flow of a biological sample through it, and the cartridge body has a body side that defines the appearance of the removable cartridge and provides functional access to the flow control valve;

a host having a control side configured to releasably interact with a body side of the removable cartridge, said housing side and the control side together forming a system interface, the host including a valve actuator that interacts with a flow control valve through the system interface; and

a detection unit held by at least one of the removable cartridge or the main device, including an image detector and a reaction chamber that is in fluid communication with the hydraulic network, and the image detector is configured to detect the provided reactions inside the reaction chamber.

2. The system of claim. 1, in which the control side and the side of the case are generally flat and facing each other, while the system interface is a one-way interface, in which the main device and the removable cartridge are operatively connected to each other only through the side of the case and the managing party.

3. The system of claim. 2, in which the main device and the removable cartridge are operatively connected in such a way that the main device and the removable cartridge are attached to each other at the system interface with the establishment of at least one of hydraulic communication, electrical communication or thermal communication through the system interface.

4. The system according to any one of paragraphs. 1-3, in which the control side is the top of the main device subject to gravity so that the removable cartridge sits on and is supported by the main device.

5. The system according to any one of paragraphs. 1-4, wherein the valve actuator includes an elongated actuator body that extends through the body side into the cartridge body.

6. The system according to any one of paragraphs. 1-5, wherein the flow control valve includes an elongated actuator body that extends through the control side into the main instrument.

7. The system according to any one of paragraphs. 1-6, in which the main device has a side of the device that faces in the opposite direction from the control side, and the main device has a device size extending between the control side and this side of the device, and the main device and the removable cartridge have a larger combined size than this instrument size.

8. The system according to any one of paragraphs. 1-7, in which each of the removable cartridge and the main device includes a contact set of electrical contacts, and these contact sets are electrically connected to each other at the system interface.

9. The system according to any one of paragraphs. 1-8, wherein the body side is a first body side, and the cartridge body further has a second body side, with the first and second body sides facing different directions, wherein the system interface is a multi-sided interface in which the main device and the removable cartridge are functionally connected to each other along each of the first and second sides of the body.

10. The system of claim. 9, in which the first and second sides of the housing are generally perpendicular to each other, the main device has a device body having first and second control sides that face in perpendicular directions and form an open recess of the main device, and a removable cartridge is located inside the open recess so that the first and second sides of the housing interact with the first and second control sides.

11. The system of claim 10, wherein the valve actuator includes an elongated body that extends through the system interface between the first side of the housing and the first control side, the second side of the housing and the second control side including respective contact sets of electrical contacts, and these contact sets are electrically connected to each other along the system interface.

12. The system of claim. 9, wherein the first and second sides of the housing face generally opposite directions, and the main device has a device side and a cartridge receiving socket extending to this side of the device, while the removable cartridge is located inside the cartridge receiving socket.

13. The system of claim. 12, wherein the removable cartridge and the main device are hydraulically connected along the first side of the housing and electrically connected along the second side of the housing.

14. The system of claim 12, wherein the host includes a locking mechanism that engages at least one of the first side of the housing or the second side of the housing to hold the removable cartridge within the host.

15. The system according to any one of paragraphs. 1-14, wherein the removable cartridge and the host device each include a flow port, the flow ports being fluidly coupled to each other at a system interface.

16. The system according to any one of paragraphs. 1-15, further comprising a locking mechanism attached to at least one of the removable cartridge or main device and configured to removably attach the cartridge body to the main device.

17. The system according to any one of paragraphs. 1-16, in which the image detector is held by the main instrument and the reaction chamber is held by a removable cartridge.

18. The system according to any one of paragraphs. 1-17, wherein the flow control valve includes a flexible membrane configured to control the flow of a biological sample through a hydraulic network, the flexible membrane being flexed between first and second actuating states of the valve.

19. The system according to any one of paragraphs. 1-18 wherein said side of the cartridge body includes an access port through it that receives a valve actuator.

20. The system according to any one of paragraphs. 1-19, wherein the flow control valve includes a rotary valve configured to control the flow of fluid through a hydraulic network, the rotary valve being rotated by a valve actuator.

21. The system according to any one of paragraphs. 1-20, wherein the main instrument includes a thermal block and the cartridge housing hydraulic network includes a sample passage where the intended reactions with the biological sample take place, the housing side including an access port extending along the sample passage and formed with the ability to accept a thermal block to change the temperature of the sample channel.

22. The system according to any one of paragraphs. 1-21, wherein the hydraulic network includes a plurality of channels and a storage module including a plurality of reagent storage tanks that are used for at least one of sample preparation or sample analysis.

23. The system according to any one of paragraphs. 1-22, wherein the main instrument includes a system controller having a valve control module configured to control the operation of a valve actuator to control the flow of a biological sample through a hydraulic network.

24. The system of claim 23, wherein the valve control module is configured to control the operation of a valve actuator to conduct a sequencing by synthesis (SBS) protocol.

25. A method for sequencing nucleic acids, comprising:

providing a removable cartridge having a cartridge body, a fluid network located within the cartridge body, and a flow control valve that is operatively connected to the fluid network and movable relative to the fluid network, the cartridge body having a body side that defines the appearance of the removable cartridge;

bringing the removable cartridge into contact with a host device, wherein a housing side of the removable cartridge releasably interacts with a control side of the host device, collectively forming a system interface, and the host device includes a valve actuator that is operatively interacts with a flow control valve through the system interface;

hydraulically directing the biological sample to flow through the hydraulic network of the cartridge to conduct at least one of sample analysis or sample preparation in the cartridge, directing the biological sample to flow into the reaction chamber, this flow of the biological sample being controlled by actuating a valve actuator on the flow control valve; and

visualization of the biological sample in the reaction chamber using the detection node, which is held by at least one of the removable cartridge or main device.

26. The method of claim 25, further comprising removing the removable cartridge from the host device.

27. The method of claim 26, further comprising bringing the second removable cartridge into contact with the host device, wherein the body side of the second removable cartridge releasably interacts with the control side of the host device, collectively forming a system interface.

28. The method of claim 27, wherein the hydraulic guidance of the biological sample and the visualization of the biological sample are repeated multiple times and sequentially.

29. The method according to any one of paragraphs. 25-28 further comprising sealing the biological sample within the sample preparation area of the hydraulic network and amplifying the biological sample while the biological sample is sealed within the sample preparation area.

30. The method according to any one of paragraphs. 25-29, wherein the flow control valve includes a movable valve having at least one flow passage that extends between valve openings, the valve actuator being configured to move the movable valve between different positions.

31. The method of claim 30 wherein the movable valve is at the sample position when the biological sample flows through the flow channel and is directed to the reaction chamber, the method further comprising moving the movable valve to the component position and flowing a reagent through the flow channel into the reaction chamber, moreover, this reagent reacts with a biological sample in the reaction chamber.

32. The method of claim 31, wherein the component position includes a plurality of component positions, the method further comprising moving a movable valve between component positions in a predetermined sequence to flow various reactants into the reaction chamber.

33. The method according to any one of paragraphs. 25-32, in which the biological sample includes nucleic acids, and the given sequence is performed in accordance with the sequencing by synthesis (SBS) protocol.

34. The method according to any one of paragraphs. 25-33, wherein the reaction chamber includes a flow cell, wherein the biological sample is immobilized on one or more surfaces of the flow cell.

35. Removable cartridge containing:

a cartridge body having a sample opening that extends outside the cartridge body and is configured to receive a biological sample, the cartridge body having a set of electrical contacts and a mechanical interface that are accessible from the outside, the cartridge body being removably attachable to the main instrument;

a hydraulic network containing a plurality of channels, a reaction chamber and a storage module, wherein the storage module includes a plurality of reservoirs for storing reagents, the hydraulic network is configured to direct reagents from the reservoirs to the reaction chamber, while the mechanical interface is movable relative to the hydraulic network to control the flow fluid medium through a hydraulic network; and

an imaging device located inside the cartridge body and positioned to detect intended reactions within the reaction chamber, wherein the imaging device is electrically connected to a set of electrical contacts for communication with the main device, and the mechanical interface is configured to be moved by the main device when the removable cartridge is attached to the main device.

36. The removable cartridge of claim 35, wherein the mechanical interface includes a channel valve configured to control the flow of fluid through one of the hydraulic network channels.

37. The removable cartridge of claim 35 or claim 36, wherein the cartridge body includes an access port allowing access to the mechanical interface.

38. The removable cartridge of claim 35, wherein the mechanical interface includes a rotary valve.

39. Removable cartridge according to any one of paragraphs. 35-38, wherein the cartridge body includes an access port that is open to the outside and the channels include a sample port in fluid communication with the sample port, the access port extending along the sample port and configured to Adopt a thermal block to control the temperature of the sample channel.

40. Removable cartridge according to any one of paragraphs. 35-39, wherein the cartridge body includes a fluid communication opening that is externally accessible and in fluid communication with the hydraulic network, the fluid communication opening being configured to interact with the device opening to receive fluid through it.

41. Removable cartridge according to any one of paragraphs. 35-40, wherein the cartridge body includes first and second body sides that face in opposite directions, the first side of the body including a set of electrical contacts and the second side including a mechanical interface.

42. Removable cartridge according to any one of paragraphs. 35-41 further comprising a locking mechanism attached to the cartridge body and configured to removably attach the cartridge body to the main appliance.

43. Removable cartridge according to any one of paragraphs. 35-42, in which the storage module includes reagents for conducting a sequencing by synthesis (SBS) protocol.

44. Removable cartridge containing:

a cartridge body having a sample opening that extends outside the cartridge body and is configured to receive a biological sample;

a rotary valve located inside the cartridge body and having a fluid side and a plurality of fluid side openings of the valve, and the rotary valve has at least one flow channel passing between the holes of the valve, and the rotary valve is able to rotate between different angular positions;

a microfluidic body having a body side that is slidably coupled to the hydraulic side of the rotary valve, the microfluidic body at least partially forming a hydraulic network that includes:

a sample passage in fluid communication with the sample hole and having a network hole that opens to said first side of the microfluidic body;

a reservoir configured to hold a reagent and in fluid communication with a reservoir opening that opens onto a hydraulic side of the microfluidic body; and

a supply channel in fluid communication with the reaction chamber of the hydraulic network and having a supply opening that exits to said first side of the microfluidic body;

wherein the rotary valve is rotatable between the first and second angular positions, wherein the network opening is hydraulically connected to the supply opening through the rotary valve when the rotary valve is in the first angular position, and when the rotary valve is in the second angular position, the tank opening is hydraulically connected with a supply port through a rotary valve.

45. The removable cartridge of claim 44, wherein the cartridge body has an exterior configured to interact with the host, the rotary valve including a mechanical interface that is accessible from the outside and configured to interact with the host.

46. The removable cartridge of claim 44 or claim 45, wherein the storage module includes reagents for conducting a sequencing by synthesis (SBS) protocol.

47. Removable cartridge according to any one of paragraphs. 44-46, wherein the rotary valve at the first angle position is configured to receive the sample fluid when a force acting on the fluid moves the sample fluid towards the supply port, wherein the rotary valve at the second angle position is configured to allow the sample fluid to be expelled into the reservoir, when the displacement force pushes the sample fluid out of the supply port into the reservoir.

48. Removable cartridge according to any one of paragraphs. 44-47, in which the rotary valve rotates about an axis, and the supply hole is aligned with this axis.

49. Removable cartridge containing:

a cartridge body having a sample opening that extends outside the cartridge body and is configured to receive a biological sample, the cartridge body including a docking side that is designed to face and is configured to be removably attached to the main instrument;

a hydraulic network located inside the housing and including a sample channel in fluid communication with the sample hole;

a conduit valve including a flexible member movable between first and second positions and blocking flow through the sample conduit when in the first position and allowing flow through the sample conduit when in the second position, the docking the side of the cartridge body includes an access port that exposes a channel valve outside the cartridge body and is configured to receive a main instrument valve actuator to move the flexible member between first and second positions.

50. The removable cartridge of claim 49, wherein the flexible member comprises a flexible layer that covers and is configured to press into that cavity to block flow through it.

51. A removable cartridge according to claim 49 or claim 50, further comprising a rotary valve located within the cartridge body and configured to rotate between different positions to change the flow path in the hydraulic network, and the rotary valve includes a mechanical interface that is functionally accessible along the docking side.

52. Removable cartridge according to any one of paragraphs. 49-51, in which the hydraulic network includes a network opening in flow communication with the sample channel, a supply opening in flow communication with the reaction chamber, and a tank opening in flow communication with the reservoir configured to store the reagent. , wherein the removable cartridge further comprises a rotary valve located inside the cartridge body, wherein the rotary valve hydraulically connects the supply hole and the network opening, being in the first angular position, and hydraulically connects the supply opening and the tank opening, being in the second angular position.

53. Removable cartridge according to any one of paragraphs. 49-52, in which the docking side is the first docking side, and the removable cartridge has a second docking side, the first and second docking sides facing opposite directions, and the second docking side is configured to interact with the device mechanically, hydraulically or thermally.

54. The main device, containing:

a system case having a control side configured to interact with the removable cartridge;

a rotating motor configured to interact with the rotary valve of the removable cartridge;

a valve actuator configured to interact with the removable cartridge channel valve;

a set of electrical contacts configured to electrically connect to the removable cartridge; and

a system controller configured to control a rotating motor and valve actuator to conduct an assay protocol within the removable cartridge, the system controller configured to receive imaging data from the removable cartridge via a set of electrical contacts.

55. The main device according to claim 54, further comprising a thermal block for heating part of the removable cartridge.

56. Removable cartridge containing:

a cartridge body having a sample opening that extends outside the cartridge body and is configured to receive a biological sample, the cartridge body having a mating side designed to face and removably attachable to the main instrument;

a microfluidic body disposed within the cartridge body, the microfluidic body having a body side and including a hydraulic network including a plurality of discrete channels and corresponding orifices that extend to that side of the body in a valve receiving region; and

a rotary valve disposed within the cartridge body, the rotary valve having a hydraulic side and at least one flow passage extending between a plurality of valve openings extending to its hydraulic side which rotatably mates with the valve receiving area of the body side, wherein the rotary valve is movable between different angle positions to hydraulically couple the discrete channels, wherein the rotary valve has a mechanical interface that is accessible along the mating side and is configured to interact with the main instrument so that the rotary valve is controlled by the main instrument.

57. The removable cartridge of claim 56 wherein the rotary valve rotates about an axis and the valve openings include a supply port and the axis extends through the supply port.

58. Removable cartridge containing:

a cartridge body having a sample opening that extends outside the cartridge body and is configured to receive a biological sample, the cartridge body having a docking side configured to be detachably attached to the main instrument;

a microfluidic structure located inside the cartridge body and containing a plurality of stacked printed circuit board (PCB) layers, and the PP layers include hydraulic layers that form channels and a reaction chamber when these PP layers are stacked on top of each other, while the PP layers also include a layer of wiring;

an imager configured to be mounted on the microfluidic structure and electrically connected to the conductive wiring layer, the imager being oriented so as to detect intended reactions within the reaction chamber.

59. The removable cartridge of claim 58, further comprising input/output (I/O) contacts that are accessible from the outside of the cartridge body and are electrically connected to the imager.

60. The removable cartridge of claim 58 or claim 59 wherein the microfluidic structure includes a channel valve wherein at least a portion of the channel valve is formed by layers of PP, the channel valve being actuable to block and allow flow through one of the channels.

Claims (16)

1. Replaceable cartridge for carrying out biochemical reactions, containing: a housing including a plurality of reagent tanks and a sample loading port; reaction chamber; jet technology unit, which includes a hydraulic network and a rotary valve, wherein the hydraulic network includes a sample channel, a plurality of tank channels and a supply channel, wherein the sample channel is in flow communication with a sample loading hole at the first end and a network hole at the second end, each of the plurality of tank channels is in flow communication with a corresponding reagent tank from a plurality of reagent tanks at the first end and a corresponding tank opening from a plurality of tank openings at the second end, and the feed channel is in fluid communication with the feed hole at the first end and the reaction chamber at the second end, and wherein the rotary valve includes a valve body containing a flow channel, a first opening and a second opening, the second opening being in flow communication with the supply opening, the valve body being able to rotate around an axis from the first position, in which the first opening is in flow communication with the supply opening. a network opening to a second position in which the first opening is in fluid communication with a corresponding tank opening of the plurality of tank openings; and a seal sealing against the housing, wherein the seal closes and seals a plurality of reagent reservoirs in the housing. 2. The cartridge of claim. 1, further comprising a CMOS image sensor located adjacent to the reaction chamber, and the CMOS image sensor is designed to detect light signals from the reaction chamber. 3. The cartridge of claim 2 further comprising a contact set of electrical contacts, wherein the CMOS image sensor is in electrical communication with the contact set. 4. The cartridge of claim 2, wherein the reaction chamber includes a flow cell lid comprising a flow channel, a first port, and a second port, the first port being in fluid communication with the supply port and the second port being in fluid communication with the outlet port. 5. The cartridge of claim 4, wherein the CMOS image sensor includes an active area for light detection, wherein the entire active area of the CMOS image sensor is used for light detection within the flow path of the flow cell lid. 6. The cartridge according to claim 1, further comprising an optical path directing illumination light from a light source external to the cartridge of the main device into the reaction chamber. 7. The cartridge of claim. 1, in which the reaction chamber includes an optically transparent window. 8. The cartridge according to claim. 1, in which the jet technology assembly further includes a plurality of membrane valves and an outlet channel, while the plurality of membrane valves are in fluid communication with the outlet channel, which is in fluid communication with the reaction chamber, and the plurality of membrane valves are capable of function as a pump. 9. The cartridge of claim 1, further comprising a waste reservoir, wherein the ink jet assembly further includes an outlet channel in fluid communication with the reaction chamber and the waste reservoir. 10. The cartridge of claim 7, wherein the waste reservoir is contained within the body.

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