RU2791650C2 - Cartridge for carrying out biochemical reactions - Google Patents

Abstract

FIELD: creation of samples for biochemical analysis and/or carrying out biochemical reactions.

SUBSTANCE: cartridge for conducting biochemical reactions contains: a microfluidic body containing a sample hole configured to receive a biological sample, a sample channel in fluid communication with the sample hole, an intermediate channel, a feeding channel and a reagent reservoir; the first rotary valve rotatably mounted on the microfluidic body and comprising the first flow channel and an intermediate orifice, the first rotary valve hydraulically connecting the first flow channel to another orifice at the second position of the first rotary valve; the second rotary valve rotatably mounted on the microfluidic body and comprising the second flow channel hydraulically connected to the supply channel, with the second rotary valve hydraulically connecting the second flow channel to the first rotary valve via an intermediate channel in the first position of the second rotary valve and hydraulically connects the second flow channel with the reagent reservoir in the second position of the second rotary valve.

EFFECT: ensuring the performance of genome sequencing with improved quality of analyzes.

12 cl, 29 dwg, 1 tab

Description

BACKGROUND OF THE INVENTION

[ 0001 ] Embodiments of the present application relate generally to systems and methods for generating samples for biochemical analysis and/or conducting biochemical reactions, and more specifically to systems and methods using a rotary valve.

[ 0002 ] Various biochemical protocols involve conducting a large number of controlled reactions on substrate surfaces or inside 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 cycle analysis of a nucleotide sequence (eg, sequencing by synthesis (from the English sequencing-by-synthesis - SBS)) a dense array of DNA fragments (eg, template nucleic acids) is sequenced during repeated cycles of enzymatic processing. After each cycle, an image can be captured and subsequently analyzed along with other images to determine the sequence of the DNA fragments. In another biochemical, an unknown analyte with an identifiable label (eg, a fluorescent label) can be exposed to an array of known probes having predetermined addresses in the array. Observation of the chemical reactions that occur between probes and an unknown analyte can help identify or reveal the properties of the analyte.

[ 0003 ] There is a general need for systems that automatically perform assays such as those described above, which would reduce labor or user involvement. Currently, most systems require separate preparation of a biological sample by the user before loading that biological sample into the system for analysis. It may be desirable for a user to upload one or more biological samples to the system, select the type of analysis to be performed by the system, and receive the results after a predetermined period of time, such as one day or less. At least some of the systems currently in use 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 certain price range.

BRIEF DESCRIPTION OF THE INVENTION

[ 0004 ] In accordance with one embodiment, a system is provided that includes a hydraulic network with a sample passage, a reaction chamber, and a reservoir. The sample passage is in fluid communication with a sample port for receiving a biological sample. The system also includes a pumping unit designed to be in fluid communication with the hydraulic network. The system also includes a rotary valve that has a flow path and is rotatable between first and second valve positions. The flow passage fluidly couples the reaction chamber and the sample passage when the rotary valve is in the first valve position, and fluidly couples the reservoir and the reaction chamber when the rotary valve is in the second valve position. The pump assembly causes the biological sample to flow to the reaction chamber when the rotary valve is in the first valve position and causes the reaction component to flow from the reservoir to the reaction chamber when the rotary valve is in the second valve position.

[ 0005 ] In one embodiment, a method is provided including rotating a rotary valve having a flow path to a first valve position. When the valve is in its first position, the flow channel is in fluid communication with the reaction chamber. The method may also include flowing the biological sample from the sample channel or first reservoir through the flow channel into the reaction chamber when the rotary valve is in the first valve position. This method may also include turning the rotary valve to the second valve position. In the second position of the valve, the flow channel can hydraulically connect the second reservoir and the reaction chamber. The method may also include flowing the reaction component from the second reservoir into the reaction chamber. In the reaction chamber, the reaction component interacts with the biological sample.

[ 0006 ] In one embodiment, a system is provided that includes a flow control system provided with a hydraulic network and a pumping unit in fluid communication with the hydraulic network. The hydraulic network includes a sample channel for receiving a biological sample, a plurality of reservoirs, and a reaction chamber. The system also includes a rotary valve that has a flow channel. The rotary valve is rotatable to its various positions to establish hydraulic connection between the reaction chamber and the sample channel or one of the reservoirs. The system also includes a detection device that is configured to detect light signals from the reaction chamber during an analysis protocol. The system also includes a system controller that is configured to control a rotary valve and a pump assembly to flow a biological sample from the sample channel into the reaction chamber. The system controller is also configured to control the rotary valve, the pump assembly, and the detection device during a plurality of protocol cycles, wherein each of the protocol cycles includes: (a) turning the rotary valve to a valve position corresponding to the first tank, in which the reaction chamber is in flow communication with the first reservoir of the plurality of reservoirs; (b) controlling the pump assembly to cause a flow of fluid from the first reservoir into the reaction chamber; (c) turning the rotary valve to a valve position corresponding to the second tank, in which the reaction chamber is in fluid communication with the second tank of the plurality of tanks; (d) controlling the pump assembly to cause a flow of fluid from the second reservoir into the reaction chamber; and (e) operating the detection device to detect light signals from the reaction chamber while fluid from the second reservoir is flowing through the reaction chamber, or after fluid from the second reservoir has flowed through the reaction chamber.

[ 0007 ] In accordance with one embodiment, a method is provided, including providing a microfluidic body and a rotary valve. The microfluidic body has a body side and a hydraulic network including a supply port and a supply port. The feed opening goes to the side of the body. The rotary valve is rotatably mounted on the side of the body. The rotary valve has a first channel opening, a second channel opening and a flow channel extending between the first channel opening and the second channel opening. The method also includes turning the rotary valve to the first valve position, in which the first opening of the channel is in fluid communication with the supply opening of the microfluidic body. The method also includes flowing the biological sample through the first channel opening into the flow channel when the rotary valve is in the first valve position. The method also includes turning the rotary valve to a second valve position with a biological sample inside the flow channel so that the first opening of the channel is sealed by the side of the body. The method also includes performing a thermal cycling operation with changing the temperature of the biological sample in the flow channel to a selected temperature.

[ 0008 ] According to one embodiment, a system is provided including a microfluidic body having a body side and a hydraulic network including a feed port and a feed port. The feed opening goes to the side of the body. The system also includes a rotary valve rotatably mounted on the side of the body. The rotary valve has a first channel opening, a second channel opening and a flow channel extending between the first channel opening and the second channel opening. The rotary valve is rotatable between the first and second valve positions. The first opening of the channel is in fluid communication with the supply opening of the microfluidic body when the rotary valve is in the first valve position. The first opening of the channel is sealed by the microfluidic body when the rotary valve is in the second valve position. The system also includes a pump assembly that is configured to cause fluid to flow through the supply opening into the flow channel when the rotary valve is in the first valve position. The system also includes a thermal cycler that is positioned relative to the rotary valve and configured to control the temperature experienced by the fluid within the flow channel when the rotary valve is in the second valve position.

[ 0009 ] According to one embodiment, a system is provided including a microfluidic body having a hydraulic network having an inlet, an outlet, and a sample reservoir. The system also includes a rotary valve rotatably connected to the microfluidic body. The rotary valve has a first channel section and a second channel section. The first section of the channel hydraulically connects the inlet and the sample reservoir when the rotary valve is in the first valve position. The second section of the channel hydraulically connects the outlet and the sample reservoir when the rotary valve is in the first valve position. The system also includes a pumping assembly configured to provide fluid flow through the inlet and the first section of the channel into the sample reservoir when the rotary valve is in the first valve position. The rotary valve is movable to a second valve position in which the sample reservoir is sealed by the rotary valve. The system may also include a thermal cycler that is positioned relative to the microfluidic body to provide thermal energy to the sample reservoir when the rotary valve is in the second valve position.

[ 0010 ] According to one embodiment, a system is provided that includes a microfluidic body that has a hydraulic network including a sample reservoir and a separate assay channel. The analytical channel passes between the first and second holes. The hydraulic network also includes a supply port. The system may also include a thermal cycler that is positioned relative to the thermoregulatory zone of the microfluidic reservoir. The analytical channel passes through the thermoregulation zone. The thermal cycler is configured to supply thermal energy to the thermoregulation zone. The system also includes a rotary valve that is pivotally connected to the microfluidic body and is movable between first and second valve positions. The rotary valve has a connection channel and a separate flow channel. The connecting channel hydraulically connects the sample reservoir and the first opening of the analytical channel, and the flow channel hydraulically connects the second opening of the analytical channel and the supply hole when the rotary valve is in the first valve position. The rotary valve is movable to the second position of the valve to seal the first and second holes of the analytical channel.

BRIEF DESCRIPTION OF THE DRAWINGS

[ 0011 ] FIG. 1 is a schematic representation of a system in accordance with one embodiment that is designed to perform at least one of biochemical analysis or sample preparation.

[ 0012 ] FIG. 2 is a plan view of a flow control system in accordance with one embodiment that may be used with the system of FIG. 1.

[ 0013 ] FIG. 3 is a sectional view of a valve train in a first state or position that can be used with the flow control system of FIG. 2.

[ 0014 ] FIG. 4 is a section of the valve mechanism according to FIG. 3 in the second state or position.

[ 0015 ] FIG. 5 is a sectional view of a valve train in a first state or position that can be used with the flow control system of FIG. 2.

[ 0016 ] FIG. 6 is a section of the valve mechanism according to FIG. 5 in the second state or position.

[ 0017 ] FIG. 7 is a sectional view of a valve train in a first state or position that can be used with the flow control system of FIG. 2.

[ 0018 ] FIG. 8 is a section of the valve mechanism according to FIG. 7 in the second state or position.

[ 0019 ] FIG. 9 is a sectional view of a rotary valve installed in a microfluidic body in accordance with one embodiment.

[ 0020 ] FIG. 10 is a top view of the microfluidic body of FIG. 9.

[ 0021 ] FIG. 11 is a section of the detection unit, which can be used to detect the intended reactions in the reaction chamber.

[ 0022 ] FIG. 12 is a flow diagram of a method in accordance with one embodiment.

[ 0023 ] FIG. 13 is a plan view of a rotary valve made in accordance with one embodiment that is pivotally mounted on a microfluidic body.

[ 0024 ] FIG. 14 is a section of the rotary valve according to FIG. 13 rotatably mounted on the microfluidic body.

[ 0025 ] FIG. 15A-15L show different rotary valve angles at different stages of the assay protocol.

[ 0026 ] FIG. 16 is a plan view of a rotary valve in accordance with one embodiment.

[ 0027 ] FIG. 17 is a plan view of the rotary valve of FIG. 16 during the amplification protocol.

[ 0028 ] FIG. 18 is a plan view of a rotary valve made in accordance with one embodiment.

[ 0029 ] FIG. 19 shows a method in accordance with one embodiment.

[ 0030 ] FIG. 20 is a perspective view of a flow control system according to one embodiment that includes a rotary valve and a microfluidic body.

[ 0031 ] FIG. 21 is a perspective view of the flow control system of FIG. 20 when the rotary valve is in the position intended for the amplification protocol.

[ 0032 ] FIG. 22 is a sectional view of the flow control system of FIG. 20.

[ 0033 ] FIG. 23 is a schematic representation of a system in accordance with one embodiment that is designed to perform at least one of biochemical analysis or sample preparation.

[ 0034 ] FIG. 24 is a plan view of a flow control system according to one embodiment that uses connecting channels.

[ 0035 ] FIG. 25 is a partially exploded perspective view of the flow control system of FIG. 24.

[ 0036 ] FIG. 26 is a bottom perspective view of a rotary valve according to one embodiment.

[ 0037 ] FIG. 27 is a side perspective view of the rotary valve of FIG. 26.

[ 0038 ] FIG. 28 is a section of the rotary valve according to FIG. 26.

[ 0039 ] FIG. 29 is an enlarged section of the rotary valve of FIG. 26.

DETAILED DESCRIPTION OF THE INVENTION

[ 0040 ] The embodiments described herein may be used to carry out the reactions envisaged for sample preparation and/or biochemical assays. As used herein, the term "biochemical analysis" may include at least one of biological analysis or chemical analysis. Fig. 1 is a schematic representation of a system 100 for biochemical analysis and/or sample preparation. The system 100 includes a main device 102 and a removable cartridge 104 that is configured to be mounted in a removable manner on the main device 102. The main device 102 and the removable cartridge 104 can be configured to cooperate with each other to transfer a biological sample to various locations within the system. 100 to carry out the intended reactions involving the biological sample, to prepare the biological sample for subsequent analysis, and optionally to detect one or more events in the biological sample. These events may indicate intended reactions in the biological sample. The removable cartridge 104 may be similar to an integrated microfluidic cartridge, such as the cartridges shown and described in US Provisional Application No. 62/003264, filed May 27, 2014, which is incorporated herein by reference in its entirety. However, the embodiments described herein are not limited to integrated devices and may also be used in larger systems.

[ 0041 ] Although the following will be described with reference to the main apparatus 102 and the removable cartridge 104 shown in FIG. 1, it should be understood that the main apparatus 102 and the removable cartridge 104 illustrate only one exemplary embodiment of the system 100 and that other embodiments 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 performing analysis of a biological sample. In the illustrated embodiment, both the main device 102 and the removable cartridge 104 are capable of performing certain functions. However, it should be understood 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 designed to detect intended responses using a detection unit (eg, an imaging device). In alternative embodiments, the detection assembly may be included with the host device 102. As another example, in the illustrated embodiment, the host device 102 is a "dry" device that does not supply, receive, or exchange fluids with a removable cartridge 104. In alternative embodiments, implementation the main apparatus 102 may, for example, supply the removable cartridge 104 with reagents or other liquids subsequently consumed (eg, used in the intended reactions) by the removable cartridge 104.

[ 0042 ] 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 analogues or mimetics of the above 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 samples, 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.

[ 0043 ] In some embodiments, the biological sample may include added material such as water, deionized water, saline solutions, acidic solutions, basic solutions, detergent solutions, and/or pH buffers. The added material may also include reagents that will be used in a prescribed 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.

[ 0044 ] However, it should be understood 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 in further processed (eg, through separation or amplification procedures) to obtain prepared nucleic acids. The prepared nucleic acids can then be analyzed (e.g., 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 is again used 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) may 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.

[ 0045 ] In some embodiments, system 100 may 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 have been partially or previously prepared for analysis by a user. For example, the user may be provided with a solution containing nucleic acids that have already been isolated from whole blood and/or amplified.

[ 0046 ] As used herein, the term "intended reaction" encompasses a change in at least one of the chemical, electrical, physical, or optical properties (or quantities) of the analyte of interest. In specific embodiments, the intended reaction is an association event (eg, insertion 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 consist in changing the 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; binding 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 reaction envisaged can also be proton addition or removal, for example detectable as a change in the pH of the surrounding solution or environment. An additional reaction provided may be to detect the flow of ions across the membrane (eg, natural or synthetic bilayer membrane), for example, as ions pass through the membrane, the current is interrupted and this 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.

[ 0047 ] In specific embodiments, the implementation of the envisaged reaction includes the incorporation of a molecule with a fluorescent label in the analyte. The analyte may be an oligonucleotide and the fluorescently labeled molecule may be a nucleotide. The intended response can be detected when excitation radiation is directed to the labeled nucleotide oligonucleotide and the fluorophore emits a detectable (detectable) fluorescent signal. In alternative embodiments, the detected 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 fluorophore or reduce fluorescence by bringing the quencher and fluorophore closer together.

[ 0048 ] As used herein, the term "reaction component" encompasses 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 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 components may interact with the biological sample directly or indirectly. In some embodiments, the removable cartridge 104 is preloaded with one or more reaction components 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).

[ 0049 ] 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 the implementation of at least one of the 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 so 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 carried out until (a) the prescribed number of reactions is carried out; (b) the required number of events is not detected; (c) the stipulated period of system time has not expired; (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 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 then terminating the session until at least one of (a)-(g) occurs.

[ 0050 ] The assay protocol may include a sequence of operations for performing the intended reactions, detecting the intended reactions, and/or analyzing the intended reactions. Collectively, the removable cartridge 104 and the main apparatus 102 may include the components 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 main instrument 102 and/or removable cartridge 104. For example, fluid operation may include controlling a pump to cause a flow of a biological sample or reaction component into a reaction chamber. . The temperature control operation may include controlling the temperature of a provided portion of the system 100. For example, the temperature control operation may include raising or lowering the temperature of a polymerase chain reaction (PCR) zone where a fluid containing a biological sample is stored. The discovery 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, which 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. Mechanical operation may include controlling the movement or position of a 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 operatively coupled to a movable valve in the removable cartridge 104. In some cases, a combination of different operations may occur simultaneously. For example, the detector may capture images of the reaction chamber as the pump controls the flow of fluid through the reaction chamber. 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.

[ 0051 ] Similar or identical hydraulic (fluidic) elements (eg, channels, openings, reservoirs, etc.) may be designated differently to more clearly distinguish between these hydraulic elements. For example, holes may be referred to as tank holes, supply holes, mains holes, feed holes, and so on. It should be understood that if two or more hydraulic elements are designated differently (eg, reservoir channel, sample channel, flow channel, connection channel), these hydraulic elements need not be structurally different. In addition, the claims may be supplemented with such designations to more clearly distinguish between such hydraulic elements in the claims.

[ 0052 ] 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 in which this substance is held. The liquid may be water-based and 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 intended that references to a liquid in this application may cover a liquid that has been formed by combining two or more liquids. For example, separate reagent solutions can be combined later to carry out the intended reactions.

[ 0053 ] Removable cartridge 104 is configured to be removably interoperable or releasably coupled with host device 102. In this context, when the terms "removably cooperating" or "removably connected" (or the like) are used to describe the relationship between the removable cartridge and the main appliance, 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. Therefore, 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 detachably connected to the main device mechanically so that the parts of the main device holding the removable cartridge are not destroyed. The removable cartridge can be detachably connected to the main device hydraulically so that the openings of the main device are not destroyed. The main instrument 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 readily detachable, where the components can be separated from each other without undue effort or a significant amount of time spent separating the components. In some embodiments, the removable cartridge 104 and the main device 102 may be readily detachable without destroying either the removable cartridge 104 or the main device 102.

[ 0054 ] In some embodiments, the removable cartridge 104 may be permanently modified or partially damaged during a session with the main instrument 102. For example, containers that contain liquids may include foil lids that are pierced to allow liquid to flow through the system. 100. In such embodiments, the foil closures may break, so 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.

[ 0055 ] 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 to perform additional prescribed reactions. Therefore, the removable cartridge 104 can in some cases be remanufactured so that the same removable cartridge 104 can be used with different consumables (eg, reaction components and biological samples). Refurbishment can be done at the manufacturing facility after the cartridge has been removed from the main instrument at the customer's facility.

[ 0056 ] As shown in FIG. 1, 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 tanks, reaction chambers, waste tanks, detection chambers, multipurpose reaction and detection chambers, and so on. and so on. The hydraulic elements may be fluidly coupled to one another in a manner provided so that the system 100 is capable of performing sample preparation and/or analysis.

[ 0057 ] As used herein, the term "fluidly coupled" (or the like) refers to two spatial regions connected to each other such that a liquid or gas can be directed between the two spatial regions. In some cases, fluid communication allows you to direct the fluid between two spatial regions back and forth. In other cases, the hydraulic connection is unidirectional, so that there is only one direction of flow between two spatial regions. For example, the assay reservoir may be in fluid communication with the channel such that fluid can be transferred into the channel from the assay reservoir. However, in some embodiments, it may not be possible to direct the fluid through the channel back to the assay reservoir. In specific embodiments, hydraulic network 106 is configured to receive a biological sample and direct the 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.

[ 0058 ] 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 "held" when used in relation to a biological sample, includes essentially attaching a biological sample to a surface or enclosing a biological sample in a space provided for this. The term "immobilized" as used herein, when used in relation to a biological sample, includes essentially attaching the biological sample to some 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 on the surface of a substrate using adsorption techniques, including non-covalent interactions (e.g., electrostatic forces, van der Waals forces, and dehydration of hydrophobic interfaces), and covalent bonding techniques, in which functional groups or linkers facilitate attachment. biological sample to the surface. The immobilization of a biological sample on the surface of a substrate can be based on the properties of the surface of the substrate, the liquid medium carrying the biological sample, and the properties of the biological sample itself. In some cases, the surface of the substrate may be functionalized (eg, modified chemically or physically) to facilitate 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 be bound to the biological sample to immobilize the biological sample on it. In some cases, the biological sample can be immobilized on the surface using a gel, for example, as described in US patent publications No. 2011/0059865 A1 and 2014/0079923 A1, each of which is incorporated here in its entirety by reference.

[ 0059 ] In some embodiments, the implementation of nucleic acids can be immobilized on the surface and amplified using bridged amplification. Suitable bridge amplification methods are described, for example, in US Patent No. 5641658, 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, each of which is incorporated herein in its entirety. Another suitable method for amplifying nucleic acids on 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). For example, a nucleic acid molecule can hybridize to one of the primers on the surface, followed by extension of the immobilized primer to produce 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 obtaining the first copy of the nucleic acid, the original nucleic acid molecule can hybridize with the second immobilized primer on the surface and can be extended at the same time or after the primer in solution is extended. In any of the embodiments, 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 implementation of the biological sample can be held in a given space with the reaction components intended for use during amplification of the biological sample (eg, PCR).

[ 0060 ] One or more of the embodiments set forth herein may be for implementing an assay protocol that is or includes an amplification (or PCR) protocol. During the amplification protocol, the temperature of the biological sample in the reservoir or channel may be changed in order to amplify the biological sample (eg, the DNA of the biological sample). For example, a biological sample may go through (1) a preheating step to about 95° C. in about 75 seconds; (2) a denaturation step at about 95° C. for about 15 seconds; (3) a renaturation-elongation step at about 59° C. for about 45 seconds; and (4) a step of maintaining the temperature at about 72° C. for about 60 seconds. Embodiments may involve multiple rounds of amplification. It is noted that the above cycle describes only one particular embodiment and that alternative embodiments may include modifications to this amplification protocol.

[ 0061 ] The methods and systems set forth herein may use chips having any of a variety of different cell densities, including, for example, at least about 10 cells/cm ² , 100 cells/cm ² , 500 cells/cm ² , 1000 cells/ cm ² , 5000 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 at least sufficient resolution to distinguish individual elements at one or more of these exemplary densities.

[ 0062 ] In the illustrated embodiment, the removable cartridge 104 includes a cartridge body 110 having a plurality of body sides 111-114. Body sides 111-114 include non-mating sides 111-113 and a docking side 114. Docking side 114 is designed to interface 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 formed by one or more sub-components that are combined by a user of the system 100. The sub-components may be combined before the removable cartridge 104 is removably mounted to the main apparatus 102, or after one of the sub-components is removably mounted. on the main instrument 102. For example, the storage module 150 may be held by a first sub-body (not shown) and the remaining components of the removable cartridge 104 (eg, hydraulic network and imaging device) may include a second sub-body (not shown). The first and second sub-hulls may be combined to form the cartridge body 110.

[ 0063 ] The hydraulic network 106 is held in the cartridge body 110 and includes a plurality of sample holes 116 extending onto 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 for receiving a biological sample. By way of example only, the biological sample may be whole blood or saliva. In some embodiments, the biological sample may be nucleic acids and other materials (eg, reagents, buffers, etc.) for PCR. Although in FIG. 1 shows three sample holes 116, embodiments may include one sample hole, two sample holes, or more than three sample holes.

[ 0064 ] The fluid network 106 also includes a fluid connection hole 118 extending to the mating side 114 and opening outward of the cartridge body 110. The fluid connection port 118 is designed to be in fluid communication with the system pump 119 of the host 102. The fluid connection port 118 is in fluid communication with the pump channel 133, which is part of the hydraulic network 106. During operation of the system 110, the system pump 119 is configured to provide a negative pressure causing fluid to flow through pump passage 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 where 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 into the reaction chamber 126 where 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 performing detection operations, system pump 119 may cause fluid to flow to waste reservoir 128.

[ 0065 ] In addition to hydraulic network 106, removable cartridge 104 may include one or more mechanical interfaces 117 that can be controlled by host 102. For example, removable cartridge 104 may include a valve assembly 120 that has a plurality of flow control valves 121-123, functionally connected to the hydraulic network 106. Each of the flow control valves 121-123 may be a mechanical interface 117 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 system pump 119, to control the flow of fluid in the hydraulic network 106.

[ 0066 ] For example, in the illustrated embodiment, the fluid network 106 includes a sample conduit 131 located downstream immediately after and in fluid communication with the sample holes 116. In FIG. 1, only one sample channel 131 is shown, but alternative embodiments may include multiple sample channels 131. The sample channel 131 may include a sample preparation area 132. Valve assembly 120 has a pair of channel valves 121, 122, which may also be referred to as flow control valves. The channel valves 121, 122 can be selectively actuated by the main instrument 102 to block or block the flow of fluid through the sample channel 131. In particular embodiments, the channel valves 121, 122 may be actuated to create a seal that retains a prescribed volume of liquid in the sample preparation region 132 of the sample channel 131. The provided volume in the sample preparation area 132 may contain a biological sample.

[ 0067 ] The valve assembly 120 may also include a movable valve 123. The movable valve 123 has a valve body 138 that may include at least one flow passage 140 extending between the respective orifices. The valve body 138 is able to move between different positions, aligning its openings with the corresponding openings of the hydraulic network 106. For example, one of the positions of the movable valve 123 may determine the type of fluid that enters the reaction chamber 126. In the first position, the movable valve 123 may be aligned with the corresponding opening of the sample channel 131 and allow the biological sample to enter the reaction chamber 126. In the second position, the movable valve 123 may be aligned with one or more corresponding openings of the reservoir channels 161, 162, which are in fluid communication with the reservoirs 151, 152 of the storage module 150, respectively. Each of the tanks 151, 152 is designed to store a reaction component that can be used to carry out the envisaged reactions. The reservoir channels 161, 162 are downstream of and in fluid communication with the reservoirs 151, 152, respectively. In some embodiments, the implementation of the movable valve 123 can move separately to various positions to align with the corresponding openings of the reservoir channels.

[ 0068 ] In the illustrated embodiment, mobile valve 123 is a rotary valve (or rotary valve) rotatable about axis 142. Mobile valve 123 may be similar to rotary valve 216 (shown in FIG. 2). However, it should be understood that alternative embodiments may include movable valves that do not pivot to different positions. In such embodiments, the movable valve can be slid in one or more linear directions to align with the respective holes. The rotary and linear motion valves described herein may be similar to those described in International Application No. PCT/US2013/032309, filed March 15, 2013, which is incorporated herein by reference in its entirety.

[ 0069 ] 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 cause light to be emitted when excited by light. with the correct 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 be applied to the biological sample located in 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 into reaction chamber 126. In one exemplary embodiment, light source 158 may be a light emitting diode ( LED, LED). However, in alternative embodiments, light source 158 may include other types of light generating devices such as lasers or lamps.

[ 0070 ] In some embodiments, the detection assembly 108 includes an image detector 109 and a reaction chamber 126. The image detector 109 is configured to detect intended reactions in the reaction chamber 126. In some embodiments, the image detector 109 may be positioned relative to the reaction chamber 126 to detect light signals ( for example, absorption, reflection/refraction, or emission) from the reaction chamber 126. The imaging detector 109 may include one or more imaging devices such as a charge-coupled device (CCD) camera or a complementary metal-oxide-semiconductor imager ( CMOS). In some embodiments, the image detector 109 can detect light signals emitted by chemiluminescence. In other embodiments, discovery node 108 may not be limited to rendering assignments. For example, detection assembly 108 may be one or more electrodes that detect an electrical property of a fluid.

[ 0071 ] As indicated here, the main instrument 102 is configured to operably interact with the removable cartridge 104 and control various operations in the removable cartridge 104 to perform the intended reactions and/or obtain data on the biological sample. To this end, the docking side 114 is designed to allow or enable the host 102 to control the operation of one or more components of the removable cartridge 104. For example, the docking side 114 may include a plurality of access holes 171-173 that allow the host 102 to control the valves 121-123. Docking side 114 may also include an access port 174 for receiving a thermal cycler 186 (eg, a thermal or heat transfer unit) of host 102. In the illustrated embodiment, thermal cycler 186 is a thermal block. The access hole 174 extends along the sample channel 131. As shown, access holes 171-174 open onto docking side 114.

[ 0072 ] In some embodiments, the hydraulic network 106 and the valve assembly 123 may form a flow control system 164. Flow control system 164 may include components that work together to control the flow of one or more fluids through system 100 or, more specifically, removable cartridge 104 to perform one or more intended operations. In other embodiments, flow control system 164 may include additional components such as system pump 119. Flow control system 164 may be similar or identical to flow control system 200 (shown in FIG. 2).

[ 0073 ] The host device 102 has a control side 198 configured to releasably mate with the docking side 114 of the removable cartridge 104. The docking side 114 of the removable cartridge 104 and the control side 198 of the host device 102 may collectively form a system interface 195. The system interface 195 is a common the boundary between the removable cartridge 104 and the main device 102, along which the removable cartridge 104 and the main device 102 are operatively interacted. cartridge 104 through docking side 114. For example, host device 102 may have one or more control components that control respective components of removable cartridge 104.

[ 0074 ] 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 195 in at least one of electrical communication, thermal communication, optical communication, valve communication or hydraulic communication via system interface 195. In the illustrated embodiment, the host device 102 and 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 transmit data and/or power. Through thermal communication, the main device 102 and removable cartridge 104 can transmit and/or receive thermal energy from each other, and through optical communication, the main device 102 and removable cartridge 104 can transmit light signals (eg, illumination light).

[ 0075 ] In the illustrated embodiment, system interface 195 is a one-way interface 195. For example, control side 198 and body side 114 are generally flat and face in opposite directions. The system interface 195 is one-way, so that the removable cartridge 104 and the main device 102 are operatively connected to each other only through the docking side 114 and the control side 198. 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. The multiple sides may be planar and may be perpendicular or opposite to each other (eg, enclosing all or part of a rectangular volume).

[ 0076 ] To control the operations of the removable cartridge 104, the host device 102 may include valve actuators 181-183 operable with flow control valves 121-123, a thermal cycler 186 configured to supply and/or remove thermal energy to/from the area 132 sample preparation, and a contact set of 188 electrical contacts. The main instrument 102 may also include a light source 158 located along the control side 198. The main instrument 102 may also include a system pump 119 with a control hole 199 located on the control side 198.

[ 0077 ] The system 100 may also include a locking mechanism 176. In the illustrated embodiment, the locking mechanism 176 includes a rotary shutter 177 that is configured to engage the latch engaging member 178 of the removable cartridge 104. Alternatively, the removable cartridge 104 may include a rotary shutter 177 , and the main appliance 102 may include a latch engaging member 178. When the removable cartridge 104 is installed on the main appliance 102, the latch 177 can be rotated and pushed behind the latch engaging member 178. The camming effect created by the locking mechanism 176 may push or drive the removable cartridge 104 the main device 102 to securely fasten the removable cartridge 104 thereon.

[ 0078 ] The main instrument 102 may include a user interface 125 configured to be input by the user to perform the intended analysis protocol and/or configured to communicate the analysis information to the user. The user interface 125 may be embedded in the host device 102. For example, the user interface 125 may include a touch screen attached to the body of the main device 102 for detecting the user's touch and the location of the touch relative to the information displayed on the touch screen. Alternatively, the user interface 125 may be located remotely from the main instrument 102.

[ 0079 ] The host device 102 may also include a system controller 180 configured to control the operation of at least one of the valve actuators 181-183, the thermal cycler 186, the contact set 188, the light source 158, or the system pump 119. The system controller 180 is shown conceptually as a collection of electronic modules, however, it can be implemented using any combination of dedicated hardware boards, processors, digital signal processors (DSPs), etc. Alternatively, system controller 180 may be implemented using a commercially available personal computer (PC) with a single processor or multiple processors with functional operations distributed among these processors. In addition, the electronic modules described below can be implemented using a hybrid configuration in which certain modular functions are performed by dedicated hardware, while the remaining modular functions are performed by a commercial PC, and the like.

[ 0080 ] The system controller 180 may include a plurality of electronic modules 190-193 configured to control the operation of certain components of the main apparatus 102 and/or the removable cartridge 104. For example, the electronic module 190 may be a flow control module 190 for controlling the flow of fluids. flow control module 190 may be operatively coupled to valve actuators 181-183 and system pump 119. Flow control module 190 may selectively actuate valve actuators 181-183 and system pump 119 to cause fluid to flow through one or more paths and/or block the flow of fluid along one or more paths.

[ 0081 ] By way of example only, the valve actuator 183 may rotate into contact with the movable valve 123. The valve actuator 183 may include a rotary motor 189 configured to drive (eg, rotate) the valve actuator 183. The flow control module 190 may actuate the valve actuator 183 to move the movable valve 123 to the first angular position. When the movable valve 123 is in the first angular position, the flow control module 190 may actuate the system pump 119, thereby drawing the biological sample from the sample preparation area 132 into the reaction chamber 126. The flow control module 190 may then actuate the valve actuator 183 to moving the movable valve 123 to the second angular position. When the movable valve 123 is in the second angular position, the flow control module 190 may actuate the system pump 119, thereby drawing one or more reaction components from the respective reservoir(s) into the reaction chamber 126. In some embodiments, the system pump 119 may be configured to provide positive pressure so as to actively pump fluid in the opposite direction. Similar operations can be used to add multiple liquids to one common tank, thereby mixing these liquids in the tank. Therefore, fluid connection opening 118 may allow fluid (eg, gas) to exit cartridge body 110 or may receive fluid into cartridge body 110.

[ 0082 ] The system controller 180 may also include a temperature control module 191. Thermoregulation module 191 may control thermal cycler 186 to supply and/or remove thermal energy from sample preparation area 132. In one specific example, thermal cycler 186 can raise and/or lower the temperature experienced by the biological sample in sample channel 131 in accordance with a PCR protocol. Although not shown, system 100 may include additional thermal devices that are placed adjacent to sample preparation area 132.

[ 0083 ] The system controller 180 may also include a detection module 192 configured to control the detection node 108 to obtain data about a biological sample. The detection module 192 can control the operation of the detection node 108 through the contact set 188. For example, the detection node 108 can be connected with the possibility of communication to the contact set 188 of the electrical contacts 196 along the docking side 114. In some embodiments, the implementation of the electrical contacts 196 may be flexible contacts (for example , pogo contacts, or contact rods) capable of being repositioned to and from docking side 114. Electrical contacts 196 extend 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 coupled, the detection module 192 can control the detection node 108 to acquire data at predetermined times or for predetermined periods of time. For example, detection module 192 may control detection assembly 108 to capture an image of reaction chamber 126 when a fluorophore is attached to a biological sample. A number of images may be taken.

[ 0084 ] Optionally, system controller 180 includes an analysis module 193 configured to analyze data to provide a user of system 100 with at least partial results. For example, the analysis module 193 may analyze the image data provided by the image detector 109 . Such analysis may include identification of the nucleic acid sequence of the biological sample.

[ 0085 ] System controller 180 and/or electronic modules 190-193 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 180 and/or electronic modules 190-193 execute a set of instructions stored therein to perform one or more analysis protocols. The storage elements may be in the form of information sources or physical storage elements within the main instrument 102 and/or the removable cartridge 104. The protocols performed by the assay system 100 may be designed to perform, for example, DNA or RNA quantitation, protein analysis, DNA sequencing (e.g., sequencing by synthesis (SBS)), sample preparation and/or preparation of a library of fragments for sequencing.

[ 0086 ] The instruction set may include various instructions that direct system 100 to perform specific operations, such as the methods and processes of the various embodiments described herein. The instruction set may be in the form of a software system program. As used herein, the terms "software" and "embedded software" are used interchangeably and include any computer program designed to be implemented by a computer and stored in memory, 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 types of memory listed are examples only and thus do not limit the types of memory suitable for storing a computer program.

[ 0087 ] The software may take various forms such as system software or application software. In addition, the software may be in the form of a collection of individual programs or a software module within a larger program or part of a software module. In addition, the software may include modular programming in the form of object-oriented programming. Once the discovery data is received, the discovery data may be automatically processed by the system 100, processed in response to user input, or processed in response to a request made by another information processing engine (eg, a remote request over a communication channel).

[ 0088 ] System controller 180 may be connected to other components or subsystems of system 100 via communication channels, which may be hardwired or wireless. The system controller 180 may also be communicatively connected to external systems or servers. System controller 180 may receive user inputs or commands from a user interface (not shown). The user interface may include a keyboard, mouse, touch screen panel and/or voice recognition system, and the like.

[ 0089 ] System controller 180 may also serve to provide processing capabilities such as storing, interpreting and/or executing software instructions, and controlling the overall operation of system 100. System controller 180 may be configured and programmed to manage data and/or features of the power supply of various components. Although the system controller 180 is shown in FIG. 1 as a single construct, it is understood that system controller 180 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 combined (integrated) with the main device, and one or more components may be located remotely from the main device.

[ 0090 ] FIG. 2 is a plan view of a flow control system 200 implemented in accordance with one embodiment. Flow control system 200 may be part of a sample preparation and/or sample analysis system (not shown), such as system 100 (shown in FIG. 1). In some embodiments, the entire flow control system 200 resides in an integrated device, such as a removable cartridge 104 (FIG. 1). In other embodiments, flow control system 200 may be part of a standard system (eg, a desktop system). In FIG. 2 components of the flow control system 200 are located in a localized area. In other embodiments, the implementation of the components of the system 200 flow control may be remote from each other and distributed in different areas.

[ 0091 ] In the illustrated embodiment, the flow control system 200 includes a hydraulic network 202 configured to arrange one or more fluid (eg, gas or liquid) flows through it. The hydraulic network 202 has some arrangement of mutually connected hydraulic elements. The fluid elements may be configured to direct fluid to predetermined regions within fluid network 202 where, for example, the fluid may be subjected to predetermined conditions and/or undergo predetermined reactions. The hydraulic elements may be selectively coupled to each other by one or more valves such that during operation, one or more hydraulic elements may be disconnected from one or more other hydraulic elements.

[ 0092 ] In the illustrated embodiment, fluid network 202 includes sample ports 204A-204D and sample conduits 206A-206D in fluid communication with sample ports 204A-204D, respectively. Sample conduits 206A-206D extend from respective sample ports 204A-204D to a common junction or intersection 209. Hydraulic network 202 also includes a combined sample conduit 208 extending from junction 209 to supply port 210 (shown in FIG. 9). A rotary valve 216 is located above the supply port 210.

[ 0093 ] The hydraulic network 202 also includes a supply port 226 (shown in FIG. 9) and a supply conduit 224 extending from the supply port 226. The supply conduit 224 extends between the supply port 226 and the flow cell 320 of the hydraulic network 202. The flow cell 320 has an inlet 322, an outlet 324, and a reaction chamber 326 extending therebetween. flow into the waste tank 330 of the hydraulic network 202. In FIG. 2, the waste container 330 is shown as a small rectangle, however, it should be understood that the volume of the waste container 330 may be larger than, for example, the containers 240-244.

[ 0094 ] While fluid flows through reaction chamber 326, the fluid may interact with material (eg, analytes) present within reaction chamber 326. In the reaction chamber 326, the intended reactions can be detected. For example, a detection assembly (not shown) may be adjacent to reaction chamber 326 and detect light signals from reaction chamber 326.

[ 0095 ] In the illustrated embodiment, the sample holes 204A-204D extend to the side or surface 214 of the microfluidic body 212 such that the sample holes 204A-204D are exposed to the outside of the microfluidic body 212. The sample channels 206A-206D and the combined sample channel 208 extend through (eg, within) the microfluidic body 212. The feed opening 210 may extend to the side 214 of the body. Alternatively, feed port 210 may extend to the bottom (not shown) or side of microfluidic body 212. Therefore, sample channels 206A-206D are in fluid communication with a single port, such as feed port 210. However, in alternative embodiments, the sample passages 206A-206D may be in fluid communication with the individual supply openings facing the body side 214. In such alternative embodiments, each sample passage may extend between a respective sample port and a respective feed port.

[ 0096 ] In the illustrated embodiment, hydraulic network 202 also includes a plurality of reservoir channels 220. Each of the reservoir channels 220 is fluidly positioned between reservoir opening 222 (shown in FIG. 10) and reservoir 240. The reservoir apertures 222 extend to body side 214. Similar to supply port 210, reservoir ports 222 may be closed by rotary valve 216. Optionally, fluid network 202 may include a reservoir conduit 228 that is hydraulically positioned between common sample conduit 208 and reservoir 230.

[ 0097 ] In the illustrated embodiment, the flow control system 200 includes a microfluidic body 212. The microfluidic body 212 may be a physical structure forming the hydraulic elements of the hydraulic network 202. For example, the microfluidic body 212 may include stacked layers of a printed circuit board (PCB), of which one or more layers are etched or molded to form one or more channels (e.g., sample channels 206A-206D, common sample channel 208, reservoir channels 220, 228, and feed channel 224) and one or more holes (e.g., holes 204A-204D for sample, reservoir ports 222, supply ports 210, and supply ports 226) of the hydraulic network 202. The flow cell 320 may be attached to a microfluidic body 212. Such microfluidic bodies are shown and described in U.S. Provisional Application No. 62/003264 and U.S. Provisional Application No. 61/951462. Each of these provisional applications is incorporated herein by reference in its entirety. Alternatively or additionally, other materials such as glass or plastic may be used with the PP layers. In alternative embodiments, microfluidic body 212 may be collectively formed from multiple body components. In some cases, the hydraulic network 202 is at least partially formed by a system of pipes.

[ 0098 ] Rotary valve 216 is rotatable about axis 299 to various valve positions (e.g., angular positions) to fluidly couple various channels of hydraulic network 202. Rotary valve 216 can be slidably engaged with body side 214 and can be positioned to close the row body side 214 openings, such as tank openings 222, supply opening 210, and supply opening 226. Rotary valve 216 includes at least one flow passage 218 (shown in FIG. 9) for fluidly connecting the various passages. For example, when rotary valve 216 is in the first valve position, flow passage 218 may fluidly connect sample passage 208 to supply passage 224. channel 224.

[ 0099 ] Each of the sample holes 204A-204D is configured to receive a respective biological sample. For example, a user of flow control system 200, such as a technician or laboratory assistant, may load (eg, pipette) a biological sample into one or more sample ports 204A-204D. Biological samples may be taken from the same individual (eg, a human) or may be from different multiple individuals from a population. It should be understood that the biological sample may be taken from other species such as animals, plants, bacteria or fungi. In the illustrated embodiment, the sample holes 204A-204D are accessible from outside the flow control system 200. In alternative embodiments, sample ports 204A-204D may be part of a larger hydraulic network such that biological samples are delivered to sample ports 204A-204D through this larger hydraulic network.

[ 00100 ] As shown in FIG. 2, each of the sample channels 206A-206D may include a sample preparation area 232. In the illustrated embodiment, the sample channels 206A-206D have corresponding undulating or tortuous paths along the respective sample preparation area 232. These corresponding undulating or tortuous trajectories may provide a larger volume of biological sample in the thermoregulatory region 234 . In alternative embodiments, the sample preparation area 232 may have different dimensions than other portions of the corresponding sample channel. For example, sample preparation area 232 may form a wide chamber or well with increased depth.

[ 00101 ] In sample preparation area 232, the biological sample may undergo a process of preparing the biological sample for subsequent reactions and/or analysis. For example, a biological sample may be affected by changes in pressure and/or temperature. Alternatively, or in addition, the biological sample may be mixed with one or more reaction components within the sample preparation area 232. In some embodiments, flow control system 200 may include a thermoregulation band or zone 236 (shown in dotted line) along a thermoregulatory region 234 that is adjacent to sample preparation regions 232 of sample channels 206A-206D. In some embodiments, the thermoregulatory band 236 may be a flexible PP heater, such as the flexible PP heater described in US Provisional Application No. 61/951,462, which is incorporated herein by reference in its entirety. The flexible heater PCB may extend along the thermoregulation region 234 and include electrically conductive paths that generate heat when electrical current is passed through them.

[ 00102 ] The thermoregulatory band 236 is designed to control the temperature of biological samples in the respective sample channels 206A-206D along the thermoregulatory region 234. Temperature control can be carried out during the amplification protocol, in which biological samples experience an increase/decrease in temperature in accordance with a given schedule (plan) in order to amplify the biological sample. In such embodiments, biological samples may be loaded into sample ports 204A-204D along with a mixture of amplification reagents (eg, PCR). Alternatively, the amplification mixture may be delivered separately to the sample preparation areas 232 via hydraulic network 202. For example, the sample preparation areas 232 may be in fluid communication with another conduit (not shown) through which the amplification mixture may be delivered.

[ 00103 ] In some embodiments, flow control system 200 includes a storage node or module 238 . As shown, storage node 238 includes a plurality of tanks 240-244. Each of the tanks 240-244 is designed to contain a reaction component that can be used in a given assay protocol (eg, SBS protocol). Each of the tanks 240-244 may be in fluid communication with the corresponding opening through one of the channels 220 of the tanks. As described here, the rotary valve 216 is configured to rotate to various valve positions in accordance with a predetermined schedule (plan) to establish a hydraulic connection of the supply channel 224 with other channels of the hydraulic network 202.

[ 00104 ] In some embodiments, the flow control system 200 may also include channel valves 246, 248. As shown, each of the sample channels 206A-206D is connected to a pair of channel valves 246, 248. Corresponding to each sample channel 206A-206D region 232 sample preparation passes between a respective pair of channel valves 246, 248. Each pair of channel valves 246, 248 is designed to seal a respective biological sample within the sample preparation region 232 when the biological sample is exposed to different conditions. For example, channel valves 246, 248 may seal an appropriate biological sample between themselves during thermal cycling of the biological sample in accordance with the PCR protocol.

[ 00105 ] To cause flow through the hydraulic network 202, the flow control system 200 may include a pump assembly 332. In the illustrated embodiment, the flow control system 200 includes only one pump that is located downstream of the reaction chamber 326 and pumps or sucks fluid through the hydraulic network 202. In alternative embodiments, one or more pumps may be used to move fluid through hydraulic network 202. For example, one or more pumps may be fluidly coupled upstream of reservoirs 240-243 and/or reservoir 244. Sample ports 204A-204D may also be fluidly coupled to an upstream pump causing a biological sample to flow into conduit 208 for sample.

[ 00106 ] FIG. 3-8 show the various valve mechanisms by which the flow control system 200 (FIG. 2) can control (eg, regulate) the flow through the hydraulic network 202 (FIG. 2). More specifically, in FIG. 3 and 4 show a cross-section of valve mechanism 250 that includes channel valve 246. Although the following description applies to channel valve 246, channel valve 248 (FIG. 2) and other valves may have similar or analogous features. As shown, the microfluidic body 212 includes a plurality of layers 252-254 stacked on top of each other. Layers 252-254 may be printed circuit board (PCB) layers. One or more of the layers 252-254 may be etched such that when the layers 252-254 are stacked, the microfluidic body 212 forms a sample channel 206. The sample channel 206 includes a valve or internal cavity 256.

[ 00107 ] Channel valve 246 is configured to control the flow of fluid through sample channel 206. For example, channel valve 246 may provide maximum clearance so that fluid can flow unhindered. Channel valve 246 may also prevent fluid from flowing through it. As used herein, the term "impede" may encompass slowing down fluid flow or completely blocking fluid flow. As shown, the sample passage 206 has first and second openings 258, 260 in fluid communication with the valve cavity 256. The passage is configured to allow fluid to flow into the valve cavity 256 through the first opening 258 and out of the valve cavity 256 through the second opening 260. In the illustrated embodiment, channel valve 246 is a flexible membrane capable of bending between the first and second states. In FIG. 3 the flexible membrane is shown in the first state, and in FIG. 4 - in the second state. In particular embodiments, the flexible membrane is a flexible layer. The flexible layer is configured to be pressed into the valve cavity 256 and overlap the first hole 258 to block the flow of fluid through it. In alternative embodiments, channel valve 246 may be another physical element capable of moving between different states or positions to adjust fluid flow.

[ 00108 ] The flow control system 200 (FIG. 2) may also include a valve actuator 262 configured to actuate the channel valve 246. For example, the valve actuator 262 may flex the flexible membrane between the first and second states. The valve actuator 262 includes an elongated body 264, such as a pin or rod, extending through the opening or opening 266 for access. Access port 266 provides direct access for valve actuator 262 to port valve 246, which in the illustrated embodiment is a flexible membrane. In FIG. 5, valve actuator 262 is shown in a first state or position. In FIG. 6, valve actuator 262 is shown in a second state or position. In the second position, the valve actuator 262 is coupled to the port valve 246 while being advanced some distance towards the first port 258. The valve actuator 262 may deform the port valve 246 so that the port valve 246 closes the first port 258. For this reason, fluid flow through the first port 258 is blocked by the channel valve 246.

[ 00109 ] FIG. 5 and 6 show a sectional view of valve mechanism 270 including channel valve 272. In some embodiments, channel valve 246 (FIG. 2) may be replaced by channel valve 272. Valve mechanism 270 may be similar to valve mechanism 250. For example, valve mechanism includes channel valve 272 and valve actuator 274. The valve actuator 274 has an elongated body 276, such as a nozzle, that extends through the opening or opening 278 for access. The access opening 278 may form a closed or sealed chamber. In an exemplary embodiment, the channel valve 272, which may be a flexible membrane, is driven pneumatically by a valve actuator 274. More specifically, the valve actuator 274 is configured to supply a fluid (e.g., air) to pressurize the sealed chamber, thereby causing the channel valve 272 to deform. thereby blocking the flow through the sample channel 279.

[ 00110 ] FIG. 7 and 8 show a valve mechanism 280 including a channel valve 282. The valve mechanism 280 may have similar features as the valve mechanisms 250 (FIG. 3), 270 (FIG. 5). Channel valve 282 is rotatably connected to valve actuator 284. Channel valve 282 is a flat body shaped to allow flow through sample port 286 at a first angle position (shown in FIG. 7) and block flow through sample port 286 at a second angle position (shown in FIG. 8). More specifically, while in the second angular position, the channel valve 282 may close the opening 288.

[ 00111 ] FIG. 9 is a sectional view of a rotary valve 216 operatively coupled to a valve actuator 290. Rotary valve 216 is slidably coupled to side 214 of microfluidic body 212. Valve actuator 290 is configured to rotate rotary valve 216 about axis 299 to predetermined valve positions (or angular positions) to establish fluid communication between various channels of hydraulic network 202 (FIG. 1) . The rotary valve 216 includes a valve body 292 having a liquid side 294 and a working side 296. The working side 296 may include a mechanical interface 298 configured to interact with the valve actuator 290. In the illustrated embodiment, the mechanical interface 298 includes a flat body or rib coinciding with the axis 299. The valve actuator 290 includes a slot 300 configured to receive the mechanical interface 298 such that the valve actuator 290 is operably coupled to the rotary valve 216. More specifically, the valve actuator 290 valve may engage with rotary valve 216 such that valve actuator 290 is capable of rotating rotary valve 216 about axis 299.

[ 00112 ] The body side 214 has a supply port 210 and a supply port 226. The body side 214 also has reservoir ports 222A-222E (shown in FIG. 10). The flow channel 218 extends between the first and second openings 306, 308 of the channel. The first and second openings 306, 308 of the channel open onto the liquid side 294 of the valve body 292. In an exemplary embodiment, rotary valve 216 includes only two channel openings 306, 308 and only one flow channel 218. However, in other embodiments, rotary valve 216 may include more than two channel openings and/or more than one flow channel. Such embodiments may provide for fluid connection of more than two passages at the same angular position of the rotary valve 216.

[ 00113 ] As shown in FIG. 9, the supply port 226 is aligned with and in fluid communication with the channel port 308, and the supply port 210 is aligned with and in fluid communication with the port port 306. Depending on the angular position of the rotary valve 216, the channel opening 306 may also be in fluid communication with one of the tank openings 222A-222E. As noted above, rotary valve 216 is rotatable about axis 299. In some embodiments, supply port 226 and channel port 308 are positioned such that supply port 226 and port port 308 are aligned with axis 299. More specifically, axis 299 extends through each from the supply hole 226 and the hole 308 of the channel.

[ 00114 ] When valve actuator 290 is operatively coupled to rotary valve 216, valve actuator 290 may apply actuator force 310 in a direction against body side 214. In such embodiments, the actuator force 310 may be sufficient to seal the flow channel 218 between the channel openings 306, 308 and to seal the reservoir openings 222 and/or the supply opening 210.

[ 00115 ] Accordingly, the rotary valve 216 at the first angle position can fluidly communicate the supply port 226 and the supply port 210, and at the second angle position can hydraulically communicate the supply port 226 and the corresponding reservoir port 222. As the rotary valve 216 rotates between different angle positions, the rotary valve 216 actually changes the flow path in the hydraulic network.

[ 00116 ] The fluid may flow through the flow channel 218 in any direction. For example, a system pump (not shown), such as system pump 119 (FIG. 1), may be in fluid communication with supply port 226. The system pump may provide suction force to force fluid through supply port 210 (or a corresponding reservoir port 222). ) into the flow channel 218 and through the supply port 226. Alternatively, the system pump can provide positive pressure to move the fluid in the flow channel 218, causing the fluid to pass through the supply port 226 into the flow channel 218 and through the supply port 210 ( or corresponding reservoir opening 222).

[ 00117 ] FIG. 10 is a plan view of body side 214 illustrating supply port 210, feed port 226, and reservoir ports 222A-222E. In FIG. 10, the flow channel 218 is shown in two different angular positions, however, it will be understood that the flow channel 218 may have other angular positions. The angular positions of the flow channel 218 correspond to the positions of the rotary valve 216 (FIG. 2). The reservoir openings 222A-222E are fluidly connected to the respective reservoirs via the respective reservoir channels. For example, reservoir opening 222A is in fluid communication with reservoir 243; reservoir opening 222B is in fluid communication with reservoir 242; reservoir opening 222C is in fluid communication with reservoir 241; reservoir opening 222D is fluidly coupled to reservoir 240; and reservoir port 222E is fluidly coupled to reservoir 244. As described above, depending on the angular position of rotary valve 216 (FIG. 2), flow channel 218 may fluidly couple supply port 226 to supply port 210 or to one of the corresponding ports 222A-222E. tanks.

[ 00118 ] Table 1 shows the various steps in the sequencing by synthesis (SBS) protocol. In an exemplary embodiment, reservoir 244 contains a hydrogenation buffer, reservoir 243 contains a nucleotide solution, reservoir 242 contains a wash solution, and reservoir 241 contains a digest solution. While Table 1 provides a schedule for the SBS protocol, it should be understood that different schedules are possible depending on the desired assay protocol. In the following example, biological samples were amplified within the respective sample preparation area 232 (FIG. 2) according to the PCR protocol.

[ 00119 ] In stage 1, flow channel 218 has a valve position that hydraulically couples supply port 210 and supply port 226. In stage 1, channel valves 246, 248 (FIG. 2) connected to sample channel 206A are deactivated (e.g., in the first condition), allowing the first biological sample to flow through the sample port 206A and the sample port 208. However, the channel valves 246, 248 connected to the sample channels 206B-206D are activated, sealing the second, third, and fourth biological samples in the respective sample preparation area 232. Therefore, in step 1, the pump assembly 332 (FIG. 2) can cause the first biological sample to flow into the flow channel 218. In step 2, the rotary valve 216 rotates to a second valve position while the first biological sample is held in the flow channel 218 so that the flow passage 218 fluidly couples reservoir opening 222E and supply opening 226. In a second valve position, pump assembly 332 may cause fluid to flow through flow passage 218 such that the first biological sample flows through reservoir opening 222E into the hydrogenation buffer.

[ 00120 ] In step 3, the rotary valve 216 is rotated back to the first valve position, the channel valves 246, 248 are selectively actuated to allow the second biological sample to flow into the flow channel 218 while the third and fourth biological samples are sealed in the areas 232 sample preparation. In step 4, rotary valve 216 is rotated back to the second valve position, while flow channel 218 retains a second biological sample, which is added to the hydrogenation buffer with the first biological sample. In steps 5-8, the third and fourth biological samples are removed from the respective sample preparation areas and added to the hydrogenation buffer. Therefore, four biological samples can be stored in one hydrogenation buffer tank. While in reservoir 243, biological samples and hydrogenation buffer can undergo reactions to prepare biological samples for SBS sequencing.

[ 00121 ] In step 9, pump assembly 332 draws the combined biological samples/hydrogenation buffer through reservoir opening 222E, through flow channel 218, through supply opening 226, and into reaction chamber 326 (FIG. 2). Biological samples can be immobilized on the surfaces that form the reaction chamber. For example, clusters can be formed that include biological samples. Steps 10-13 constitute a sequencing run. In step 10, rotary valve 216 may be in a third valve position such that a solution of nucleotides may be drawn through flow channel 218 into the reaction chamber. At this time, nucleotides can be inserted into appropriate biological samples (eg, renatured primers into template nucleic acids). In step 11, the rotary valve 216 can be in the fourth valve position so that a wash solution can flow through the reaction chamber, entraining a solution of nucleotides out of the reaction chamber. After step 11, an image of the reaction chamber can be taken with an image detector, such as a detector 404 (FIG. 11). The color of the light emitted by the clusters can be used to identify the bases embedded in the clusters. In step 12, rotary valve 216 can be in the fourth valve position so that a cleavage solution can flow through the reaction chamber and fluorophores (and, if present, reversible terminator fragments) can be removed from the clusters. In step 13, the rotary valve 216 may again be in the third valve position and a wash solution may flow through the reaction chamber to remove the fissile solution. Steps 10-13 may be repeated until sequencing is complete and/or until reagents are used up.

Table 1

Hole Type of fluid flowing through the flow channel Current direction Stage 1 210 1st biological sample To feed port 226 Stage 2 222E 1st biological sample From supply port 226 Stage 3 210 2nd biological sample To feed port 226 Stage 4 222E 2nd biological sample From supply port 226 Stage 5 210 3rd biological sample To feed port 226 Stage 6 222E 3rd biological sample From supply port 226 Stage 7 210 4th biological sample To feed port 226 Stage 8 222E 4th biological sample From supply port 226 Stage 9 222E Pooled biological samples + hydrogenation buffer To feed port 226 Stage 10 222A Nucleotide solution To feed port 226 Stage 11 222V Wash solution To feed port 226 Stage 12 222C splitting solution To feed port 226 Stage 13 222V Wash solution To feed port 226 Repeat steps 10-13 until discovery is complete

[ 00122 ] FIG. 11 shows a sectional view of a portion of the detection assembly 400. In the illustrated embodiment, the detection assembly 400 is integral with the flow cell 320. More specifically, the detection assembly includes a detection device 404 that is located adjacent to the flow cell 320 and the reaction chamber 326. The flow cell 320 may be mounted on the detection device 404. In the illustrated embodiment, the flow cell 320 is attached directly to the detection device 404 using one or more attachment mechanisms (eg, adhesive, tie, fasteners, and the like). In some embodiments, flow cell 320 may be releasably coupled to detection device 404. In particular embodiments, the detection device 404 is configured to detect light signals from the reaction chamber 326. Therefore, the detection device 404 may be referred to as an image detector in some embodiments.

[ 00123 ] In the illustrated embodiment, the detection device 404 includes a base 425 of the device. In particular embodiments, the device base 425 includes a plurality of superimposed layers (eg, silicon layer, dielectric layer, dielectric metal layer, etc.). Device base 425 may include a sensor array 424 of light sensors 440, a light guide array 426 of light guides 462, and a reaction array 428 of reaction recesses 408 with corresponding reaction sites 414. In certain embodiments, these components are arranged such that each light sensor 440 is aligned with one light guide 462 and one reaction site 414. However, in other embodiments, one light sensor 440 may receive photons from more than one light guide 462 and/or from more than one reaction site 414. As used herein, one light sensor may include one pixel or more than one pixel . The detection device 404 may be fabricated using complementary metal-oxide-semiconductor (CMOS) technology. In specific embodiments, the detection device 404 is a CMOS image detector.

[ 00124 ] It is noted that the term "array" or "subarray" does not necessarily cover any and every element of a particular type that a detector may have. For example, sensor array 424 may not include any and every light sensor in detector 404. In contrast, the detection device 404 may include other photosensitive sensors (eg, another array(s) of photosensitive sensors). As another example, the light guide array 426 may not include any and every detector light guide. On the contrary, there may be other fibers that are made differently than the fibers 462, or that have other relationships with other elements of the device 404 detection. For this reason, unless expressly stated otherwise, the term "array" may or may not include all such elements of the detection device.

[ 00125 ] In the illustrated embodiment, flow cell 320 includes a side wall 406 and a flow cover 410 supported by side wall 406 and other side walls (not shown). The side walls are connected to the detector surface 412 and extend between the flow cover 410 and the detector surface 412. In some embodiments, the sidewalls are formed by a layer of curable adhesive that holds the flow cap 410 to the detection device 404.

[ 00126 ] The flow cell 320 is sized and shaped such that a reaction chamber 326 exists between the flow cover 410 and the detection device 404. As shown, the reaction chamber 326 may have a height H ₁ . By way of example only, the height H ₁ may be about 50-400 microns (microns), or more particularly about 80-200 microns. In the illustrated embodiment, the height of H ₁ is about 100 µm. The flow cover 410 may include a material that is transparent to the excitation light 401 entering the reaction chamber 326 from outside the detection unit 400. As shown in FIG. 7, the excitation light 401 reaches the flow cover 410 at an oblique angle. However, this is only an illustration, since the excitation light 401 may reach the flow cover 410 at different angles. The reaction chamber 326 is sized and shaped so that the fluid is directed along the surface 412 of the detector. The height H ₁ and other dimensions of the reaction chamber 326 can be selected to maintain a substantially uniform flow of fluid along the surface 412 of the detector. Reaction chamber 326 may also be sized to control bubble formation.

[ 00127 ] The sidewalls 406 and the flow cover 410 may be separate components connected to each other. In other embodiments, the side walls 406 and the flow cover 410 may be integrally fabricated such that the side walls 406 and the flow cover 410 are formed from a single piece of material. As an example, flow cap 410 (or flow cell 320) may contain a transparent material such as glass or plastic. The flow cap 410 may form a substantially rectangular block with a flat outer surface and a flat inner surface that defines the reaction chamber 326. This block may be mounted on the side walls 406. Alternatively, the flow cell 320 may be etched to form the flow cap 410 and sidewalls 406. For example, a recess may be etched into the transparent material. When the etched material is mounted on the detection device 404, the recess may become the reaction chamber 326.

[ 00128 ] The detection device 404 has a detector surface 412 that can be functionalized (eg, chemically or physically modified as appropriate to carry out the intended reactions). For example, the detector surface 412 may be functionalized and may include a plurality of reaction sites 414 with one or more biomolecules immobilized thereon. The surface 412 of the detector is provided with an array of reaction depressions or reaction depressions 408 with open sides. Each of the reaction recesses 408 may include one or more reaction centers 414. The reaction recesses 408 may be formed, for example, by a recess or a change in depth along the surface 412 of the detector. In other embodiments, the detector surface 412 may be substantially flat.

[ 00129 ] As shown in FIG. 11, reaction centers 414 may be patterned along detector surface 412. For example, reaction centers 414 may be arranged on detector surface 412 in rows and columns in a microarray-like manner. However, it will be appreciated that different patterns of reaction center arrangements may be used. Reaction centers may include biological or chemical substances that emit light signals. For example, biological or chemical substances of reaction centers may emit radiation in response to excitation light 401. In particular embodiments, reaction centers 414 include clusters or colonies of biomolecules (eg, nucleic acids) immobilized on detector surface 412.

[ 00130 ] FIG. 12 is a flowchart of a method 470. In some embodiments, the method 470 may include preparing a biological sample and/or detecting intended reactions of a biological sample for analysis. For example, method 470 may involve constructs or aspects of various embodiments (eg, systems and/or methods) described herein. 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.

[ 00131 ] Method 470 may be implemented or implemented using a flow control system similar or identical to flow control system 200 (FIG. 2). Method 470 includes rotating (at 472) the rotary valve to the first valve position. The rotary valve has at least one flow channel. In the first valve position, the flow passage may be in fluid communication with the sample passage (or other reservoir of the flow control system) and in fluid communication with the reaction chamber such that the flow passage hydraulically couples the sample passage and the reaction chamber. For example, a rotary valve may have first and second port openings. The first opening of the conduit may align with an opening (eg, a supply opening or a reservoir opening), and the second opening of the conduit may align with a supply opening. When the rotary valve is in the first valve position, the other openings may be sealed by the rotary valve, blocking the flow of fluids through these other openings.

[ 00132 ] The method 470 may also include flowing (at 474) the biological sample from the sample conduit (or first reservoir) into the flow conduit when the rotary valve is in the first valve position. For example, the biological sample may flow through the supply port into the flow path of the rotary valve. As another example, the biological sample may be placed in a reservoir, such as a reservoir containing a hydrogenation buffer. The biological sample (together with the hydrogenation buffer) can flow through the reservoir opening into the flow channel.

[ 00133 ] Optionally, the biological sample may continue to flow (at 476) into the reaction chamber. Alternatively, method 470 may include rotating (at 478) a rotary valve to a second valve position while the biological sample is in the flow channel. In the second valve position, the flow path may be in fluid communication with another reservoir, such as a reservoir containing a hydrogenation buffer. At 480, the biological sample within the flow channel may be forced to flow (eg, by a pump assembly) into the reservoir. Method 470 may include repeating steps 472, 474, 478, and 480 until each of the biological samples is in a common reservoir. At step 482, the biological samples along with the hydrogenation buffer may simultaneously flow through the flow channel into the reaction chamber.

[ 00134 ] Thus, one or more biological samples can be directed to the reaction chamber using a rotary valve. In alternative embodiments, the biological sample (or samples) has a straight channel leading to the reaction chamber and the sample does not flow through the rotary valve. Optionally, the method 470 may include cycling the provided steps to carry out the intended reactions, for example, the steps described with reference to Table 1. reservoir. At 486, the reaction component may flow into the reaction chamber to interact with the biological sample(s) therein. Optionally, at step 488, method 470 includes detecting intended reactions in the reaction chamber. Method 470 may then return to block 484.

[ 00135 ] FIG. 13 is a plan view of a rotary valve 500, according to one embodiment, rotatably mounted on the side 502 of the microfluidic body 504. The rotary valve 500 may have features similar to those of the rotary valve 216 (FIG. 2). The microfluidic body 504 includes a plurality of reservoirs 506-510 configured to contain reaction components and/or biological samples. More specifically, reservoirs 506-509 contain first, second, third, and fourth biological samples (or sample fluids). Tank 510 contains a hydrogenation buffer. Each of the reservoirs 506-510 is in fluid communication with a respective opening through a respective reservoir channel 516-520, which are represented by the lines in FIG. 13. As shown, these openings include tank openings or supply openings 526-530 extending to body side 502 and in fluid communication with tanks 506-510. The microfluidic body 504 also has a feed port 524 (shown in FIG. 13) extending to the side 502 of the body.

[ 00136 ] The rotary valve 500 includes a valve body 512 with a fluid side 513 (shown in FIG. 14) contacting the body side 502 and an opposite operating side 514. The valve body 512 has first, second, third, and fourth flow channels 536- 539. Each of the flow channels 536-539 is designed to hold a biological sample during an amplification or PCR protocol. Each of the flow channels 536-539 has a common channel opening (or outlet) 544 located in the center. In other embodiments, the flow channels 536-539 do not have a common channel opening. The common channel opening 544 is located on an axis 542 about which the rotary valve 500 pivots. The flow channels 536-539 have corresponding first channel openings (or inlets) 546-549. Thus, each of the flow channels 536-539 extends from a respective first channel opening 546-549 to a common channel opening 544. Similar to rotary valve 216 (FIG. 2), rotary valve 500 is rotatable to various valve positions to establish fluid communication between reservoirs and channels. However, unlike rotary valve 216, rotary valve 500 can be used during an amplification protocol. More specifically, valve body 512 may contact thermal cycler 570 (shown in FIG. 14) while biological samples are held in flow channels 536-539.

[ 00137 ] In some embodiments, the flow channels 536-539 may be provided with anti-diffusion segments 545. The anti-diffusion segments 545 are configured to reduce the likelihood of diffusion occurring when a biological sample within the flow channels 536-539 is subjected to a PCR protocol. For example, flow channels 536-539 shown in FIG. 13 have non-linear paths and dimensions that vary along the path. More specifically, the flow channels 536-539 have tortuous or undulating paths that deviate back and forth as the flow channel progresses from the respective first channel opening to the common channel opening 544. The first openings 546-549 of the channel are located radially outside. In addition to the shape of the flow channels 536-539, the flow channels 536-539 are sized to decrease as the flow channel 536-539 travels from the respective first channel opening to the common channel opening 544. In other embodiments, the anti-diffusion segments 545 do not have a tortuous path. The flow channel segments 536-539 that do not include the anti-diffusion segments 545 may be referred to as the sample preparation area 543, which is that portion of the respective flow channel where the biological sample may be exposed to various conditions such as temperature changes. However, it should be understood that, in at least some embodiments, a biological sample may also be present in the anti-diffusion segments 545.

[ 00138 ] FIG. 14 shows a side section of a rotary valve 500 when thermal cycler 570 is mounted on operating side 514. In some embodiments, thermal cycler 570 may exert mounting force 572 pressing valve body 512 against side 502 of microfluidic body 504. Although FIG. 14 not shown, the valve body 512 may include one or more mechanical interfaces (eg, non-planar surface features such as pins) that come into contact with the thermal cycler 570. The thermal cycler 570 is configured to control the temperature of the flow channels 536-539. In particular embodiments, thermal cycler 570 may simultaneously control the temperature of each of the flow channels 536-539. In other embodiments, thermal cycler 570 may not selectively contact all of the flow channels at one time.

[ 00139 ] As shown in FIG. 14, the common port 544 is in fluid communication with the supply port 524. The shaft 542 passes through the common port 544 and the supply port 524. The first port 547 of the flow port 537 is in fluid communication with the reservoir port 527. However, the first opening 549 of the flow channel 539 is sealed by the side 502 of the body. Therefore, in the valve position shown in FIG. 14, fluid (e.g., fluid containing a biological sample) may flow from reservoir 507 (FIG. 13) into flow channel 537.

[ 00140 ] FIG. 15A-15L, a plan view of rotary valve 500 shows various valve positions in which different operations may occur. In preparation for the amplification protocol, a system controller, such as system controller 180 (FIG. 1), is configured to selectively control a pump assembly (not shown) and rotary valve 500. The pump assembly may be similar to pump assembly 332 and include one or more flow pumps. . In some embodiments, one pump may be located downstream of the rotary valve 500 and be dedicated to pushing fluids through the common orifice 544 of the channels (FIG. 15A).

[ 00141 ] Optionally, flow channels 536-539 (FIG. 15A) may be primed with fluid prior to receiving a biological sample. For example, in FIG. 15A-15D show the first opening of a respective flow passage fluidly connected to a reservoir opening 530 in fluid communication with the reservoir 510. Thus, the first opening of each flow passage may be individually connected to the reservoir 510. When the flow passage is in fluid communication with the reservoir 510, the system controller may selectively actuate the pump assembly to create a flow of the reaction component in the tank 510, causing the reaction component to flow into the corresponding flow channel.

[ 00142 ] Therefore, after FIG. 15D each of the flow channels 536-539 is loaded with a reaction component. Although the reaction component is associated with reservoir 510, other reaction components may be used to prime the flow channels 536-539. For example, the flow channels 536-539 may be in fluid communication with a separate reservoir (not shown) containing, for example, water or a buffer solution. In the illustrated embodiment, each of the flow channels 536-539 is individually connected to the tank 510. In alternative embodiments, one or more flow channels 536-539 can be connected to the tank 510 or to separate tanks at the same time.

[ 00143 ] Once flow channels 536-539 have been primed, flow channels 536-539 (FIG. 15E) can be loaded with biological sample from reservoirs 506-509 (FIG. 15E), respectively. For example, as shown in FIG. 15E, flow channel 538 is in fluid communication with reservoir opening 528 and is therefore in fluid communication with reservoir 508. At this time, flow channels 536, 537, and 539 are closed by body side 502. The pump assembly may cause the biological sample to flow into the reservoir 508 so that the biological sample flows into the flow channel 538. The amount of flow may be determined by the amount of fluid in the reservoir 508. After the biological sample is loaded into the flow channel 538, the rotary valve 500 may be is selectively rotated, and the pump assembly can likewise be selectively actuated to load the biological samples in reservoirs 506, 507, and 509 into flow channels 536, 537, and 539, respectively, as shown in FIG. 15F-15H.

[ 00144 ] With biological samples loaded into the respective flow channels 536-539, the rotary valve 500 can be selectively rotated so that each of the first holes 546-549 of the channels is closed (or sealed) by the side 502 of the microfluidic body 504. The position of the valve in which the first openings 546-549 of the channels are sealed, shown in FIG. 15I. The thermal cycler 570 (FIG. 14) can then be commanded to cycle the temperature in accordance with the intended amplification protocol. Although the flow channels 536-539 are only sealed at one end, the pump assembly and anti-diffusion segments 545 can prevent movement and/or diffusion of a biological sample (eg, PCR gate) into or through the feed port 524 (FIG. 14).

[ 00145 ] After the amplification protocol, biological samples can be loaded into a common tank. For example, as shown in FIG. 15J-15L, the biological samples in the flow channels 536-538 may be fluidly coupled to the reservoir 510. The pump assembly may be selectively actuated to cause the biological sample to flow into the reservoir 510. Although not shown, the flow channel 536 is also may be hydraulically connected to the reservoir 510 so that the biological sample located in the flow channel 536 can be loaded into the reservoir 510. Therefore, each of the biological samples from the reservoir 506-509 can be loaded into the common reservoir 510 after the amplification protocol. The pump assembly can then be selectively actuated to create a flow of mixed biological samples through the feed port 524. Biological samples can be hydraulically delivered to a reaction chamber, such as reaction chamber 326 (FIG. 2). The biological samples can then undergo the intended reactions as described herein. In specific embodiments, biological samples may be used during the SBS protocol.

[ 00146 ] FIG. 16 is a plan view of a rotary valve 600, according to one embodiment, mounted on side 616 of microfluidic body 618. Rotary valve 600 may have similar features as rotary valve 216 (FIG. 2) and rotary valve 500 (FIG. 13). Rotary valve 600 includes a valve body 602 with flow channels 604-606. Each of the flow channels 604-606 extends between the first opening (or inlet) 608 of the channel and the second opening (or outlet) 610 of the channel. Unlike valve 500, flow channels 604-606 are not in fluid communication with a common orifice of the channels.

[ 00147 ] In the illustrated embodiment, each of the flow channels 604-606 is in flow communication with the preceding channel 612 and the following channel 614. In FIG. 16, rotary valve 600 is in such a position that each of the flow channels 604-606 can receive a biological sample from a respective prior channel 612. For example, flow channels 604-606 can simultaneously receive respective biological samples. The flow of biological samples into the flow channels 604-606 can be created by a common pump. For example, subsequent channels 614 may merge and be in fluid communication with a single pump. Alternatively, separate pumps may be hydraulically connected to the flow channels 604-606.

[ 00148 ] FIG. 17 is a plan view of the rotary valve 600 after the rotary valve has been rotated to a position where the first and second openings 608, 610 of each of the flow channels 604-606 are sealed by the side 616 of the microfluidic body 618. In the position of the valve shown in FIG. 17, a thermal cycler (not shown) may contact the valve body 602 to control the temperature experienced within the flow channels 604-606. For this reason, biological samples may be subjected to an amplification protocol as described here. Unlike the embodiment shown in FIG. 13-15, flow channels 604-606 are sealed at both ends to reduce the chance of diffusion and PCR gate movement.

[ 00149 ] FIG. 18 is a plan view of a rotary valve 620 according to one embodiment. Rotary valve 620 may be similar or identical to rotary valve 600 (FIG. 16) and include a plurality of flow channels 624-626. As shown, the rotary valve 620 is divided into three areas or zones 634-636 thermoregulation. Areas 634-636 thermoregulation in the form of sectors are shown in dotted lines. Each thermoregulation region 634-636 has a different temperature range controlled by one or more thermal cyclers (not shown). More specifically, after the biological samples are loaded into the flow channels 624-626, the rotary valve 620 can be selectively rotated to various positions. The flow channel in the thermoregulatory region 634 may be affected by the temperature provided for the denaturation of nucleic acids. The flow channel in the thermoregulation region 635 may experience the temperature provided for the renaturation-elongation step, and the flow channel in the thermoregulation region 636 may experience the temperature provided for the preheat and/or temperature holding step. The system controller can selectively rotate the rotary valve 620 to three different valve positions to cycle biological samples through multiple PCR amplification steps. Thus, unlike the flow channels of the rotary valve 600, the flow channels 624-626 are maintained at different temperatures.

[ 00150 ] FIG. 19 is a flowchart of a method 650. In some embodiments, the method 650 may include preparing a biological sample and optionally detecting intended reactions of the biological sample for analysis. For example, method 650 may utilize constructs or aspects of various embodiments (eg, systems and/or methods) described herein, such as those described with reference to FIGS. 13-18. 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.

[ 00151 ] Method 650 may include providing (at 652) a microfluidic body and a rotary valve. The microfluidic body may have a body side and a fluid network that includes a supply port, such as reservoir ports 526-529, and a supply port. The supply opening may extend to the side of the body. The rotary valve may be rotatably mounted on the side of the body and have a first channel opening, a second channel opening, and a flow channel extending between the first channel opening and the second channel opening. In some embodiments, multiple flow channels may be used, these flow channels having separate second channel openings, such as in the embodiments described with reference to FIGS. 16-18, or have a common second channel opening, such as in the embodiment described with reference to FIG. 13-15. In such embodiments with one common second channel opening, the second channel opening may be referred to as a common channel opening.

[ 00152 ] The method 650 may include rotating (at 654) the rotary valve to a first valve position in which the first orifice of the conduit is in fluid communication with the supply orifice of the microfluidic body. The method 650 may also include flowing (at 656) the biological sample through the first channel opening into the flow channel when the rotary valve is in the first valve position. The biological sample may flow in the direction from the first channel opening to the second channel opening. Leakage (at 656) may include selectively adjusting flow rate and/or duration of flow so that the biological sample does not substantially leak past the second channel opening or feed opening. In embodiments involving multiple flow channels, as in rotary valve 500, steps 654 and 656 may be repeated until each of the flow channels contains a corresponding biological sample. However, rotation (at 654) for each flow channel does not occur to the same valve position. More specifically, when the biological sample is forced to flow into the corresponding flow channel, the other flow channels may be sealed at one end or at both ends.

[ 00153 ] The method 650 may also include rotating (at 658) the rotary valve to a second valve position with the biological sample within the flow channel so that the first opening of the channel is sealed by the side of the body, and performing (at 660) a thermal cycling operation to change the temperature of the biological sample in the flow channel to the selected temperature. Implementation (at 660) may occur in accordance with the provided schedule (plan). For example, the schedule may involve performing a PCR operation to amplify a biological sample for later analysis.

[ 00154 ] Optionally, after implementation (at 660), the biological sample or biological samples may be loaded (at 662) into the reservoir. For example, the reservoir may contain a hydrogenation buffer solution that prepares the biological sample for subsequent analysis. At step 664, the biological sample (or combined biological samples) may be fed into the reaction chamber for subsequent analysis at step 666.

[ 00155 ] FIG. 20 is a perspective view of a flow control system 700 according to one embodiment that includes a microfluidic body 702 and a rotary valve 704. Unlike rotary valves 500 (FIG. 13), 600 (FIG. 16), and 620 ( Fig. 18) amplification occurs not only within the rotary valve 704. On the contrary, the amplification occurs at least partially within the microfluidic body 702. More specifically, the microfluidic body 702 has a first body side 706 (shown in Fig. 22) and a second side 708 bodies (shown in Fig. 22) facing in opposite directions. The microfluidic body 702 has a hydraulic network 705 including a plurality of sample tanks 711-714, a plurality of supply channels 721-724 with respective inlets 731-734 and a common outlet 736. The inlets 731-734 and the outlet 736 open to the first side 706 body (FIG. 22).

[ 00156 ] Rotary valve 704 is rotatably mounted on microfluidic body 702 along first side 706 of the body. In the illustrated embodiment, rotary valve 704 has a first channel section 726 and a second channel section 728. The first and second channel portions 726, 728 may extend to the fluid side 709 (shown in FIG. 22) of the rotary valve 704. Alternatively, the first and second channel portions 726, 728 may extend between respective channel openings to the fluid side 709. The first and second channel sections 726, 728 are separate from each other and extend only along a portion of the liquid side 709. In an exemplary embodiment, the second channel section 728 is in fluid communication with the outlet 736 at any angle position of the rotary valve 704.

[ 00157 ] FIG. 20, the rotary valve 704 is shown in the intended position in which the sample reservoir 711 is in fluid communication with the pump assembly. More specifically, the first section 726 of the channel is hydraulically placed between the inlet 731 and the reservoir 711 for the sample. The second channel section 728 is hydraulically placed between the sample reservoir 711 and the outlet 736. For this reason, the supply channel 721 is in fluid communication with the supply channel 756 through the first channel section 726, the sample reservoir 711 and the second channel section 728.

[ 00158 ] Although not shown, the flow control system 700 may include a pumping assembly designed to create a fluid flow through the inlet 731 and the first channel section 726 into the sample reservoir 711. The fluid may include a biological sample that is loaded, for example, into a remote reservoir (not shown) in fluid communication with the supply channel 721. Fluid flow and dimensions of the sample reservoir may be designed such that the biological sample is substantially exited the sample reservoir 711 through the second channel portion 728. After the biological sample is loaded into the sample reservoir 711, the rotary valve 704 can be selectively rotated so that the first channel section 726 and the second channel section 728 are in fluid communication with the sample reservoir 712. The sample reservoir 712 may be loaded with a biological sample coming from the supply channel 722. Similarly, the respective biological samples may be loaded into the sample reservoirs 713 and 714.

[ 00159 ] FIG. 21 is a perspective view of the flow control system 700 after the respective biological samples have been loaded into the tanks 711-714. In some embodiments, rotary valve 704 includes gas reservoirs 741-744, which are shown in FIG. 20. During the amplification protocol, the gas reservoirs 741-744 are designed to be compatible with the sample reservoirs 711-714. For example, as shown in FIG. 22, the sample tank 711 and the gas tank 741 are combined to form a sample preparation chamber 751. The gas in the gas reservoir 741 may function as a gas ballast in the sample preparation chamber 751. After thermal cycling, the biological samples may flow through a feed conduit 756 (FIG. 20) in fluid communication with an outlet 736. As described here, the biological samples may be directed to a reaction chamber, such as reaction chamber 326 (FIG. 2), where the intended reactions can occur and be detected.

[ 00160 ] FIG. 23 is a schematic view of a system 800 implemented in accordance with one embodiment. System 800 may have similar features as system 100 (FIG. 1). For example, system 800 includes a flow control system 802 having a fluid network 804. The fluid network 804 may include a number of interconnected channels, openings, reservoirs, and other spatial areas designed to contain or move fluid through them. For example, hydraulic network 804 includes rotary valves 806, 808. Rotary valve 806 is intended for use in the sample preparation step, and rotary valve 808 is intended for use in the sample analysis step. Rotary valves 806, 808 are hydraulically connected by an intermediate channel 810 of the hydraulic network 804. The hydraulic network 804 also includes a supply channel 812, a reaction chamber 814 and a waste reservoir 816. Flow control system 802 includes a pump assembly 818 in fluid communication with hydraulic network 804. In the illustrated embodiment, pump assembly 818 includes a single pump, however, in other embodiments, it may include multiple pumps. System 800 may include a microfluidic body (not shown) with body side 819. Rotary valves 806, 808 are rotatably mounted on the side 819 of the body. The microfluidic body may also include or form an intermediate channel 810 and a supply channel 812.

[ 00161 ] The hydraulic network 804 also includes a plurality of analytical channels 821-824 and a plurality of sample reservoirs 831-834. Each of the assay channels 821-824 extends between respective first and second ports 826, 828 and is intended to fluidly communicate the respective sample reservoir with the intermediate channel 810. As shown, the assay channels 821-824 extend through the thermoregulation region 825. In the illustrated embodiment, the analytical channels 821-824 have undulating trajectories as they pass through the thermoregulatory region 825. Part of the analytical channels 821-824, passing through the area 825 thermoregulation, may be areas 827 sample preparation. As an alternative to, or in addition to, non-linear paths, the assay channels 821-824 may be of various sizes to accommodate the intended volume of the respective biological samples.

[ 00162 ] Rotary valve 806 is movable between multiple valve positions. The rotary valve 806 includes a connection channel 840 and a flow channel 842. The connection channel 840 and the flow channel 842 are configured to fluidly couple one of the sample reservoirs 831-834 to the intermediate channel 810. For example, as shown in FIG. 23, the connecting passage 840 hydraulically connects the opening 853 (or supply opening) of the sample reservoir 833 with the first opening 826 of the analysis channel 823. Simultaneously, the flow passage 842 hydraulically connects the second opening 828 with the intermediate opening 856. The intermediate opening 856 extends to the side 819 of the body and is in flow communication with intermediate channel 810.

[ 00163 ] Accordingly, the system controller (not shown) can selectively rotate the rotary valve 806 to fluidly couple the sample reservoirs 831-834 to the respective assay channels 831-824. The system controller can selectively control the pump assembly 818 to create a flow of biological samples in the sample tanks so that the biological samples are located in the sample preparation areas 827 of the analytical channels 821-824.

[ 00164 ] When the biological sample (or samples) is in the respective assay channel, the rotary valve 806 can be rotated by the system controller to another valve position in which the first and second ports 826, 828 of each of the assay channels 821-824 are closed or sealed by the side 819 body. With sealed analytical channel(s), the biological sample(s) may be subjected to an amplification protocol. For example, a thermal cycler (not shown) may be located adjacent to the thermoregulatory region 825 to provide thermal energy in accordance with an amplification protocol. In specific embodiments, each of the biological samples may be placed in the thermoregulation region 825 at the same time. In alternative embodiments, the implementation of biological samples can be placed in the area 825 thermoregulation at different times.

[ 00165 ] After the biological samples are amplified, the rotary valve 806 can be returned to the proper valve positions to load the appropriate biological sample into the flow channel 842. With the biological sample located in the flow channel 842, the rotary valve can be rotated to another position, in which the flow channel 842 is in fluid communication with the reservoir 835. The reservoir 835 may contain, for example, a hydrogenation buffer solution. The biological sample may be loaded into reservoir 835. Optionally, rotary valve 806 and pump assembly 818 may be operated in a similar manner to load biological samples from other assay channels into reservoir 835. Biological samples may then be directed to another stage. For example, biological samples may flow through flow channel 842, through intermediate opening 856, and into intermediate channel 810. In alternative embodiments, biological samples may be directed to rotary valve 808 without first being loaded into reservoir 835.

[ 00166 ] As shown in FIG. 23, rotary valve 808 includes a flow path 870 and a plurality of reagent reservoirs 871-878. After the biological samples have been prepared using the rotary valve 806, the biological samples may be transferred to the reaction chamber 814. Optionally, before the biological samples are delivered to the reaction chamber 814, the rotary valve 808 may be rotated so as to hydraulically connect one or more reservoirs. 871-878 for reagents with reaction chamber 814. More specifically, flow channel 870 can be rotated into a position provided for fluid communication of one of the reagent tanks 871-878 with reaction chamber 814. For this reason, rotary valve 808 can be used to prepare reaction chamber 814 to receive biological samples. For example, reagent reservoirs 871-878 may contain clustering reagents, enzymes, and/or capture probes.

[ 00167 ] After the biological samples are delivered to the reaction chamber 814, the rotary valve 808 can be selectively rotated to various valve positions. For example, rotary valve 808 may be rotated according to a predetermined cycle to re-deliver reaction components for the SBS protocol. This cycle may be similar to the cycle shown in Table 1 above. Thus, the rotary valve 808 can be used to prepare the reaction chamber for receiving a biological sample and/or for conducting an assay protocol.

[ 00168 ] FIG. 24 and 25 illustrate another embodiment that uses a rotary valve provided with a connection channel. Fig. 24 is a plan view of the flow control system 900, and FIG. 25 is a partially exploded perspective view of the flow control system 900. As shown, the flow control system 900 includes a microfluidic body 902 with opposite first and second body sides 904, 906 (FIG. 25). The microfluidic body 902 includes a plurality of flow channels 908 and a plurality of sample reservoirs 910. Each of the flow channels 908 is designed to be in fluid communication with a respective sample reservoir 910. The flow channels 908 may be of the same shape and size as the flow channels 536-539 (FIG. 13).

[ 00169 ] The flow control system 900 also includes a rotary valve 912. The rotary valve 912 includes a plurality of connecting passages 913-916 for fluid communication between respective flow passages 908 and sample reservoirs 910. In particular embodiments, each of the connection channels 913-916 is an open-side groove on the outside of the rotary valve 912. In alternative embodiments, the connection channels 913-916 are not "open-sided", but instead extend between the first and second outward opening holes. . For example, connection channel 916 in FIG. 24 hydraulically connects the respective sample reservoir 910 to the respective flow channel 908 depending on the angular position of the rotary valve 912. When the biological sample flows through the connection channel 913, the other connection channels 914-916 are not hydraulically connected to the respective sample reservoir 910. More specifically, other connecting channels 914-916 are sealed by a rotary valve 912.

[ 00170 ] Similar to that described in other embodiments, a thermal cycler (not shown) can change the temperature experienced by biological samples within the flow channels 908. For example, one or both of the sides 904, 906 of the body can be in contact with the thermal cycler. After the amplification protocol has been completed, biological samples can be transported to another spatial region for further modification/preparation and/or analysis.

[ 00171 ] As described above in various embodiments, the rotary valve and the microfluidic body may include various hydraulic elements that co-operate in controlling the flow of one or more fluids in a provided manner. It should be understood that the above embodiments are illustrative and not restrictive. 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 accommodate a particular situation or material with the knowledge of the various embodiments without departing from their scope.

[ 00172 ] FIG. 26-29 are various views of a rotary valve 950 in accordance with one embodiment. Rotary valve 950 can be used as a rotary valve in various embodiments, such as rotary valve 216 (FIG. 2). In FIG. 26 and 27 are bottom and side perspective views of the rotary valve 950, respectively. The rotary valve 950 has a liquid side 952 and a working side 954. The working side 954 includes a mechanical interface 956 for interacting with a valve actuator (not shown).

[ 00173 ] FIG. 28 is a sectional view of a rotary valve 950. As shown, the rotary valve 950 is provided with a body cover 960 and a side cover 964 that are fixed in fixed positions relative to each other. The body cover 960 defines the valve cavity 962, the side cover 964 closes one end of the valve cavity 962 on the liquid side 952. The rotary valve 950 also includes a rotating shaft 966 provided with a mechanical interface 956, a valve spring 968, a manifold 970 and a compressible diaphragm 972, which are located in valve cavity 962. Rotating shaft 966 is provided to rotate manifold 970 and compressible membrane 972 along side cover 964 about axis 978.

[ 00174 ] Manifold 970 is attached to a rotating shaft 966 on one side and to a compressible membrane 972 on the opposite side. Valve spring 968 may deflect or press manifold 970 and compressible membrane 972 against the inner surface of side cover 964. In particular embodiments, compressible membrane 972 may be made of polypropylene or the like. As shown in FIG. 26, the side cover 964 has a central flow port 980, a drain port 981, and outer flow ports 982. In FIG. 26, side cover 964 is partially transparent to show central flow port 980, drain port 981, and outer flow ports 982. A total of nine outer flow ports 982 are shown, but other embodiments may include a different number of ports.

[ 00175 ] FIG. 29 is an enlarged sectional view of a rotary valve 950 showing the interaction of a manifold 970, a compressible diaphragm 972, and a side cover 964. As shown, the side cover 964 and the compressible diaphragm 972 may form a lubricant reservoir 990 therebetween. In some embodiments, an external lubricant reservoir 991 may also be provided. 26. Drain hole 981 is in fluid communication with lubricant reservoir 990 so that lubricant can be loaded into reservoir 990. As shown, manifold 970 and compressible membrane 972 form a flow channel 984 therebetween. As rotating shaft 966 rotates manifold 970 with compressible membrane 972, flow channel 984 rotates with them. Anti-rotation friction forces can be reduced by the compressible diaphragm 972 and lubricant reservoir 980. Thus, the life of the rotary valve 950 can be longer than other known valves.

[ 00176 ] In accordance with one embodiment, a system is provided that includes a hydraulic network having a sample passage, a reaction chamber, and a reservoir. The sample channel is in fluid communication with the sample port configured to receive a biological sample. This system also includes a pump assembly configured to be in fluid communication with the hydraulic network. The system also includes a rotary valve that has a flow path and is rotatable between first and second valve positions. The flow passage fluidly couples the reaction chamber and the sample passage when the rotary valve is in the first valve position, and fluidly couples the reservoir and the reaction chamber when the rotary valve is in the second valve position. The pump assembly causes the biological sample to flow to the reaction chamber when the rotary valve is in the first valve position and causes the reaction component to flow from the reservoir to the reaction chamber when the rotary valve is in the second valve position.

[ 00177 ] In one aspect, the pump assembly may include a system pump in fluid communication with the reaction chamber and located downstream of the reaction chamber.

[ 00178 ] In another aspect, the rotary valve may be configured to retain a biological sample in the flow channel while rotating the rotary valve from a first valve position to a second valve position. The pump assembly may be configured to induce a flow of the biological sample into the reservoir when the rotary valve is in the second valve position.

[ 00179 ] Optionally, the sample channel may be the first sample channel, and the biological sample may be the first biological sample. The hydraulic network may include a second sample conduit with a second biological sample. The rotary valve may be rotatable to a third valve position such that the flow passage is in fluid communication with the second sample passage. The pump assembly may be configured to cause the flow of the second biological sample in the second sample channel into the flow channel, while the rotary valve is configured to hold the second biological sample in the flow channel when the rotary valve is rotated from the third valve position to the second valve position. The pump assembly may be configured to cause the flow of the second biological sample in it into the reservoir when the rotary valve is in the second valve position. In some embodiments, the pump assembly may be configured to cause the first and second biological samples to flow from the reservoir to the reaction chamber.

[ 00180 ] In another aspect, the reservoir may be the first reservoir. The fluid network may further include a second reservoir, wherein the rotary valve may be movable to a third valve position such that the flow path hydraulically couples the second reservoir and the reaction chamber.

[ 00181 ] In another aspect, the sample conduit may be a first sample conduit and the hydraulic network includes a second sample conduit. Optionally, each of the first and second sample passages may be in fluid communication with the rotary valve through a common supply port. Optionally, the system may also include a channel valve connected to the sample channel. The channel valve may be movable between first and second positions, respectively, to block flow and allow flow through the sample channel.

[ 00182 ] In another aspect, the rotary valve is rotatable about an axis. The hydraulic network may include a supply opening that is aligned with this axis and hydraulically connects the flow channel and the reaction chamber.

[ 00183 ] In another aspect, the hydraulic network may also include a reagent conduit. The sample conduit and the reagent conduit may be in fluid communication with a common supply port located upstream of the flow conduit. The feed port can hydraulically couple the sample path and the reagent path to the flow path.

[ 00184 ] In another aspect, the system may include a detection unit configured to detect intended reactions in the reaction chamber. Optionally, the detection node includes an image detector, which can be positioned to detect light signals from the reaction chamber. Optionally, the image detector may have a fixed location relative to the hydraulic network.

[ 00185 ] In another aspect, the system includes a system controller that can be configured to automatically control a rotary valve and a pumping assembly to conduct repeated cycles of the sequencing by synthesis (SBS) protocol.

[ 00186 ] In one embodiment, a method is provided that includes rotating a rotary valve with a flow path to a first valve position. In the first position of the valve, the flow channel is in fluid communication with the reaction chamber. The method may also include flowing the biological sample from the sample passage or first reservoir through the flow passage into the reaction chamber when the rotary valve is in the first valve position. The method may also include rotating the rotary valve to a second valve position. In the second position of the valve, the flow channel can hydraulically connect the second reservoir and the reaction chamber. The method may also include flowing the reaction component from the second reservoir into the reaction chamber. In the reaction chamber, the reaction component interacts with the biological sample.

[ 00187 ] In one aspect, the method may include detecting intended reactions between a reaction component and a biological sample in a reaction chamber. Optionally, detection of intended reactions may include detection of light signals from the reaction chamber. Light signals may indicate intended reactions.

[ 00188 ] In another aspect, the method also includes separately flowing a plurality of biological samples into a reservoir and thereby combining the biological samples therein. Biological samples can simultaneously flow through the flow channel into the reaction chamber when the rotary valve is in the first valve position.

[ 00189 ] In another aspect, the method also includes turning the rotary valve to the third valve position and flowing the wash solution from the third reservoir into the reaction chamber. The method may also include turning the rotary valve to the second valve position and flowing the reaction component from the second reservoir into the reaction chamber. Optionally, the method includes performing repeated cycles of the sequencing by synthesis (SBS) protocol.

[ 00190 ] In another aspect, the method also includes amplifying the biological sample in a sample channel or reservoir prior to feeding the biological sample through the flow channel into the reaction chamber.

[ 00191 ] In one embodiment, a system is provided that includes a flow control system with a hydraulic network and a pumping assembly in fluid communication with the hydraulic network. The hydraulic network includes a sample channel configured to receive a biological sample, a plurality of reservoirs, and a reaction chamber. The system also includes a rotary valve with a flow channel. The rotary valve is rotatable to different positions of the valve for hydraulic connection of the reaction chamber with the sample channel or with one of the reservoirs. The system also includes a detection device configured to detect light signals from the reaction chamber during the analysis protocol. The system also includes a system controller configured to control a rotary valve and a pump assembly to create a biological sample flow from the sample channel to the reaction chamber. The system controller is also configured to control the rotary valve, the pump assembly, and the detection device during a plurality of protocol cycles, wherein each of the protocol cycles includes: (a) turning the rotary valve to a valve position corresponding to the first reservoir so that the reaction chamber is in flow communication with the first reservoir of the plurality of reservoirs; (b) controlling the pump assembly to cause a flow of fluid from the first reservoir into the reaction chamber; (c) turning the rotary valve to a valve position corresponding to the second tank so that the reaction chamber is in fluid communication with the second tank of the plurality of tanks; (d) controlling the pump assembly to cause a flow of fluid from the second reservoir into the reaction chamber; and (e) operating the detection device to detect light signals from the reaction chamber when fluid from the second reservoir flows through the reaction chamber or after fluid from the second reservoir has flowed through the reaction chamber.

[ 00192 ] In another aspect, the sample channel may include a sample preparation area. The system may also include a thermal cycler configured to control the temperature of the biological sample in the sample preparation area. The system controller may operate a thermal cycler to amplify the biological sample in the sample preparation area before the biological sample flows from the sample channel into the reaction chamber.

[ 00193 ] Optionally, each of the protocol cycles includes turning the rotary valve to the valve position corresponding to the third tank so that the reaction chamber is in fluid communication with the third tank of the plurality of tanks, and operating the pump assembly to cause a flow of fluid from the third tank into the reaction chamber .

[ 00194 ] In another aspect, the detection device includes a CMOS image detector. In another aspect, a flow cell is connected to the detection device. The flow cell may form a reaction chamber. Optionally, the flow cell is fixed in a fixed position relative to the detection device.

[ 00195 ] In another aspect, the flow control system includes a microfluidic body that has a body side. The side of the body may include a plurality of openings extending to that side of the body, wherein the rotary valve seals the plurality of openings when the flow passage is in fluid communication with at least one of the other openings. In particular embodiments, the system is designed to perform a sequencing by synthesis (SBS) protocol.

[ 00196 ] In accordance with one embodiment, a method is provided that includes providing a microfluidic body and a rotary valve. The microfluidic body has a body side and a hydraulic network including a supply port and a supply port. The feed opening goes to the side of the body. The rotary valve is rotatably mounted on the side of the body. The rotary valve has a first channel opening, a second channel opening and a flow channel extending between the first channel opening and the second channel opening. The method also includes rotating the rotary valve to a first valve position in which the first opening of the channel is in fluid communication with the supply opening of the microfluidic body. The method also includes flowing the biological sample through the first channel opening into the flow channel when the rotary valve is in the first valve position. The method also includes rotating the rotary valve to a second valve position with the biological sample within the flow channel such that the first opening of the channel is sealed by the side of the body. The method also includes performing a thermal cycling operation with changing the temperature of the biological sample in the flow channel to a selected temperature.

[ 00197 ] In another aspect, the microfluidic body may include a reservoir opening extending to the side of the body and in fluid communication with the reservoir. The method may also include turning a rotary valve to align the first channel opening and the reservoir opening and causing the biological sample to flow in the flow channel through the first channel opening into the reservoir. Optionally, the method includes causing a biological sample to flow from the reservoir through a flow channel and through a feed orifice of the microfluidic body.

[ 00198 ] In another aspect, the second port opening may be aligned with the supply port when the rotary valve is in the second valve position.

[ 00199 ] In another aspect, the second port opening may be sealed by the side of the body when the rotary valve is in the second valve position.

[ 00200 ] In another aspect, the first channel opening is the first inlet and the flow channel is the first flow channel. The rotary valve may include a second inlet and a second flow channel. The second flow channel may extend between the second inlet and the second channel opening.

[ 00201 ] In another aspect, the first channel opening is the first inlet and the second channel opening is the first outlet. The rotary valve may include a second inlet and a second outlet with a flow channel extending therebetween.

[ 00202 ] In another aspect, the rotary valve may have a liquid side and a working side facing in opposite directions. The thermal cycler may be in contact with the working side to control the temperature of the biological sample.

[ 00203 ] In another aspect, the method may include causing a biological sample to flow from the reservoir through the flow channel and through the microfluidic body feed port into the reaction chamber. The method may also include detecting light signals from the reaction chamber. Optionally, the reaction chamber has a remote location relative to the rotary valve.

[ 00204 ] In another aspect, the reaction chamber includes a flow cell. Detection of light signals from the reaction chamber may include detection of light signals using an image detector coupled to the flow cell. Optionally, the image detector and flow cell are attached to each other.

[ 00205 ] In accordance with one embodiment, a system is provided that includes a microfluidic body having a body side and a hydraulic network that includes a feed port and a feed port. The feed opening goes to the side of the body. The system also includes a rotary valve rotatably mounted on the side of the body. The rotary valve has a first channel opening, a second channel opening and a flow channel extending between the first and second channel openings. The rotary valve is rotatable between the first and second valve positions. The first opening of the channel is in fluid communication with the supply opening of the microfluidic body when the rotary valve is in the first valve position. The first opening of the channel is sealed by the microfluidic body when the rotary valve is in the second valve position. The system also includes a pump assembly configured to cause fluid to flow through the supply opening into the flow channel when the rotary valve is in the first valve position. The system also includes a thermal cycler that is positioned relative to the rotary valve and configured to control the temperature experienced by the fluid in the flow path when the rotary valve is in the second valve position.

[ 00206 ] In one aspect, the microfluidic body may include a reservoir opening that extends to the side of the body and is in fluid communication with the reservoir. The rotary valve may be pivotable to a third valve position in which the first opening of the channel and the opening of the reservoir are aligned. The pump assembly may be configured to cause fluid to flow in a flow channel through a reservoir opening into the reservoir. Optionally, the pump assembly is configured to cause fluid to flow from the reservoir through the flow channel and the microfluidic body feed port.

[ 00207 ] In another aspect, the rotary valve is rotatable about an axis. The second hole of the channel and the feed hole can be aligned with this axis.

[ 00208 ] In another aspect, the flow channel may be the first flow channel. The rotary valve may include a second flow channel extending between respective channel openings.

[ 00209 ] In another aspect, the system includes a reaction chamber in fluid communication with the feed port and a detection device positioned to detect intended reactions in the reaction chamber. Optionally, the reaction chamber has a remote location relative to the rotary valve. Optionally, the system includes a flow cell with a reaction chamber. The detector may be an image detector that is positioned adjacent to the flow cell. In some embodiments, the image detector and the flow cell may be attached to each other.

[ 00210 ] In accordance with one embodiment, a system is provided that includes a microfluidic body with a hydraulic network that has an inlet, an outlet, and a sample reservoir. The system also includes a rotary valve rotatably connected to the microfluidic body. The rotary valve has a first channel section and a second channel section. The first section of the channel hydraulically connects the inlet and the sample reservoir when the rotary valve is in the first valve position. The second section of the channel hydraulically connects the outlet and the sample reservoir when the rotary valve is in the first valve position. The system also includes a pump assembly configured to create a fluid flow through the inlet and the first section of the channel into the sample reservoir when the rotary valve is in the first valve position. The rotary valve is movable to a second valve position in which the sample reservoir is sealed by the rotary valve. The system may also include a thermal cycler that is positioned relative to the microfluidic body to provide thermal energy to the sample reservoir when the rotary valve is in the second valve position.

[ 00211 ] In one aspect, the rotary valve may include a closed gas reservoir. The closed gas reservoir can be aligned with the sample reservoir when the rotary valve is in the second valve position. The closed gas reservoir and the sample reservoir may be combined to form a reaction chamber.

[ 00212 ] In another aspect, the system also includes a feed conduit in fluid communication with the outlet. The feed channel can hydraulically connect the outlet to the reaction chamber. The system includes a reaction chamber and a detection device that is positioned to detect intended reactions in the reaction chamber.

[ 00213 ] In another aspect, the reaction chamber may have a remote location relative to the rotary valve. Optionally, the system may include a flow cell with a reaction chamber. The detection device may be an image detector that is positioned adjacent to the flow cell.

[ 00214 ] In accordance with one embodiment, a system is provided that includes a fluidized microfluidic body that has a sample reservoir and a separate assay channel. The analytical channel passes between the first and second holes. The hydraulic network also includes a supply port. The system may also include a thermal cycler that is positioned adjacent to the thermoregulatory region of the microfluidic body. The analytical channel passes through the area of thermoregulation. The thermal cycler is configured to supply thermal energy to the thermoregulation area. The system also includes a rotary valve rotatably connected to the microfluidic body and rotatable between first and second valve positions. The rotary valve has a connecting channel and a separate flow channel. The connecting channel hydraulically connects the sample reservoir and the first opening of the analytical channel, and the flow channel hydraulically connects the second opening of the analytical channel and the supply hole when the rotary valve is in the first valve position. The rotary valve is movable to the second position of the valve to seal the first and second holes of the analytical channel.

[ 00215 ] In one aspect, the flow channel may be configured to receive a biological sample from the assay channel. The rotary valve may be rotatable to a third valve position in which the flow channel is in fluid communication with the reservoir. The biological sample may be allowed to flow through the flow channel into the reservoir.

[ 00216 ] In another aspect, the system includes a reaction chamber that is in fluid communication with a feed port and a detection device that is positioned to detect intended reactions in the reaction chamber. Optionally, the reaction chamber may have a remote location relative to the rotary valve. Optionally, the system also includes a flow cell with a reaction chamber. The detection device may be an image detector that is positioned adjacent to the flow cell.

[ 00217 ] As used herein, reference to any element or step 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 construed as excluding the existence of additional embodiments that also include these features. In addition, unless explicitly stated to the contrary, embodiments "comprising" or "having" any element or a plurality of elements having a particular property may include additional elements, whether or not they have that property.

[ 00218 ] It should be noted that the specific arrangement of components (eg, number, types, placement, etc.) in the illustrated embodiments may be modified in various alternative embodiments. In various embodiments, a different number 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.

[ 00219 ] It should be understood that the above description is intended to be illustrative and not restrictive. For example, the embodiments (and/or aspects thereof) described above may be used in combination with each other. In addition, many modifications can be made to adapt a particular situation or material to the knowledge of various embodiments and without going beyond their 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 define the parameters of certain embodiments, which are not restrictive in any way, but rather are only exemplary embodiments. Many other embodiments and modifications within the spirit and scope of the claims will become apparent to those skilled in the art upon reading the foregoing description. Therefore, the patentable scope of an invention is determined by reference to the appended claims, along with the full scope of the equivalents to which those claims entitle.

[ 00220 ] As used in the description, the phrase "in one exemplary embodiment" and the like means that the described embodiment is only an example. This phrase is not intended to limit the subject matter of the invention to this embodiment. Other embodiments of the object of the invention may not include the specified feature or design. In the appended claims, the terms "comprising" and "in which" are used as language equivalents of the respective terms "comprising", "including" and "wherein". In addition, in the appended claims, the terms "first", "second" and "third", etc. are used only as markers and are not intended to impose numerical restrictions on enumerated objects. In addition, the limitations of the appended claims are not set forth in a "means plus function" format and are not intended to be interpreted under 35 U.S.C. ”, followed by a statement of function without further construction.

Claims (20)

1. Cartridge for carrying out biochemical reactions, containing: microfluidic body containing: a sample port configured to receive a biological sample, a sample channel in fluid communication with the sample port, intermediate channel, feed channel and reagent tank; a first rotary valve rotatably mounted on the microfluidic body and containing a first flow channel and an intermediate opening, wherein the first rotary valve hydraulically connects the first flow channel with the sample channel in the first position of the first rotary valve and hydraulically connects the first flow channel with another hole in the second position of the first rotary valve; And a second rotary valve rotatably mounted on the microfluidic body and containing a second flow channel hydraulically connected to the supply channel, wherein the second rotary valve hydraulically connects the second flow channel with the first rotary valve through the intermediate channel in the first position of the second rotary valve and hydraulically connects the second flow channel a channel with a reservoir for reagents in the second position of the second rotary valve. 2. The cartridge of claim 1, wherein the reagent reservoir is a first reagent reservoir and the microfluidic body further comprises a second reagent reservoir, wherein the second rotary valve hydraulically couples the second flow path to the second reagent reservoir at the third position of the second rotary valve . 3. The cartridge of claim. 1, in which the microfluidic body further comprises another reservoir, while the first rotary valve hydraulically connects the first flow channel with this reservoir in the third position of the first rotary valve. 4. The cartridge of claim 1, wherein the microfluidic body further comprises a waste reservoir. 5. Cartridge according to claim. 1, additionally containing a pump assembly. 6. Cartridge according to claim. 1, in which the supply channel is hydraulically connected to the reaction chamber. 7. The cartridge of claim. 1, in which the microfluidic body contains a region of thermoregulation. 8. The cartridge of claim. 1, in which the first rotary valve contains a liquid side and a working side, while the working side contains a mechanical interface. 9. Cartridge according to claim 1, additionally containing a flow cell with a reaction chamber hydraulically connected to the feed channel. 10. The cartridge of claim 9, wherein the flow cell comprises a flow cap including a material that is transparent to excitation light. 11. The cartridge of claim 1, further comprising a detection assembly, the detection assembly comprising an image detector and a reaction chamber, wherein the image detector detects light signals generated by one or more reactions from an array of reaction depressions in the reaction chamber. 12. The cartridge of claim 11, wherein the image detector is a metal-oxide-semiconductor imager.

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