Universal assay cartridge with integrated valve body and syringe tube use

Abstract

Integrated valve body and syringe tube components to facilitate controlled fluid flow in assay cartridges are provided herein. Such components can include a valve body and syringe tube that are integrally formed as a single component. Components can include a syringe tube having a conduit in fluid communication with a valve body having a lysing chamber and a set of ports to facilitate direct fluid flow between chambers and fluid flow through the lysing chamber of the valve body. Valve body can be sealed by a cap that is welded to the valve body. Such components can be configured for use within sample cartridges configured for mechanical lysing, or for both mechanical and chemical lysing.

Description

UNIVERSAL ASSAY CARTRIDGE WITH INTEGRATED VALVE BODY AND SYRINGE TUBE USECROSS-REFERENCES TO RELATED APPLICATIONS

[0001] This application is a Non-Provisional of and claims priority to U.S. Provisional Application No. 63 / 626,351 titled “Universal Assay Cartridge with Integrated Valve Body and Syringe Tube Use,’’ filed January 29, 2024, the entire contents of which are incorporated herein by reference.BACKGROUND OF THE INVENTION

[0002] The present invention relates generally to the field of biochemical analysis, and in particular to sample cartridges for analyzing a fluid sample.

[0003] The analysis of fluids such as clinical or environmental fluids generally involves a series of processing steps, which may include chemical, optical, electrical, mechanical, thermal, or acoustical processing of the fluid samples. Whether incorporated into a bench-top instrument, a disposable cartridge, or a combination of the two, such processing typically involves complex fluidic assemblies and processing algorithms.

[0004] Conventional systems for processing fluid samples employ a series of chambers each configured for subjecting the fluid sample to a specific processing step. As the fluid sample flows through the system sequentially from chamber to chamber, the fluid sample undergoes the processing steps according to a specific protocol. Because different protocols require different configurations, conventional systems employing such sequential processing arrangements are not versatile or easily adaptable to different protocols.

[0005] In recent years, there has been considerable development in the field of biological testing devices that facilitate the manipulation of a fluid sample within a sample cartridge to prepare the sample for biological testing by polymerase chain reaction (PCR). One notabledevelopment in this field is the GeneXpert® sample cartridge by Cepheid®. The configuration and operation of these types of cartridges can be further understood by referring to U.S. Patent No. 6.374,684 entitled “Fluid Control and Processing System,” and U.S. Patent No. 8,048,386 entitled “Fluid Processing and Control,” which are herein incorporated by reference in their entirety. The fluid sample is transported through the system sequentially from chamber to chamber by pressurized fluid flow facilitated by actuation of a plunger through a syringe tube and through one or more flow channels of a rotatable valve body that is rotated to align the flow channels between particular chambers of the cartridge. While these sample cartridges represent a considerable advancement in the state of the art when developed, as with any precision instrument, there are certain challenges in regard to performance and use of such systems and processes. Moreover, the precise requirements of different target types (e.g. bacterial or viral) typically necessitate the development of specialized devices and cartridges for each type or class of target, such that testing for a panel of differing targets associated with multiple suspected diseases or conditions, multiple samples must be obtained and multiple cartridges utilized, which quickly becomes costly, cumbersome and time-consuming. While such sample processing cartridges have been developed, including cartridges that can process both viral and bacterial targets, and these cartridges represent a considerable advancement in the state of the art, there are certain challenges in regard to manufacturing, assembly, performance of such cartridges, systems and processes. For example, the sample cartridge is a precision instrument requiring interaction between various components and sub-assemblies. Defects in any of these components or subassemblies can result in a defective cartridge and impact reliability of manufacturing processes, overall manufacturing and product reliability'. Further, the cartridge includes a number of subassemblies, including a syringe tube and valve assembly and the flow paths through the valve assembly can be such that carryover or contamination can be a concern.

[0006] Thus, there is a need for sample cartridges that overcome various challenges observed with regard to performance. There is further need for sample cartridges that provide greater versatility in performing assays for range of differing targets. There is further need for such devices that perform a wide range of sample processing steps in a robust and consistent manner and that are compatible with existing technologies to reduce costs and improve patient access.BRIEF SUMMARY OF THE INVENTION

[0007] The present invention pertains to an integrated valve body and syringe tube (VBST), which improves ease of manufacture and assembly, and which can include one or more fluid flow features that improve performance. The integrated valve body and syringe tube can be formed as a single component, such as by injection molding. The valve body defines a lysing chamber to facilitate mechanical lysing and flow paths on an underside, sealed by a cap or a thin film, and optionally a gasket.

[0008] In one aspect, the integrated valve body and syringe tube includes a valve body with a lysing chamber that holds a filter, which allows clearance for fluid flow paths to improve fluid flow and reduce possible contamination between flow paths. The valve assembly is sealed by a cap. In some embodiments, this design can provide suitable sealing such that no gasket is needed. The substrate can be formed of polycarbonate, copolyester, polypropylene or any suitable material. The integrated valve body and syringe tube component is particularly advantageous when used with a unitary cartridge body. A unitary cartridge body can be understood by referring to U.S. Patent Application No. 18 / 184,326 filed March 15, entitled “Unitary Cartridge Body and Associated Components and Methods of Manufacture,'’ the entire contents of which are incorporated herein by reference for all purposes.

[0009] In one aspect, the invention provides a sample cartridge for separating a desired analyte from the sample and for holding the analyte for chemical reaction and optical detection. The invention also pertains to an instrument module that receives the cartridge for sample processing and operates the cartridge to perform sample preparation and analytical testing. The desired analyte is typically intracellular material (e.g.. nucleic acid, proteins, carbohydrates, or lipids). In a preferred use, the analyte is nucleic acid which the cartridge separates from the fluid sample and holds for amplification (e.g., using PCR or an isothermal amplification method) and optical detection.

[0010] In another aspect, the invention pertains to a sample cartridge that utilizes a valve body platform that allows for detection of enveloped and free nucleic acid targets. In some embodiments, the valve body includes a sample processing region or lysing chamber that provides for heat, mechanical, and / or chemical lysis. This allows a single cartridge to providelysing for a multitude of differing types of target, thus, can be considered a “universal assay cartridge.” In some embodiments, the sample cartridge can perform processing and detection of both bacterial targets requiring mechanical lysing and viral targets suited for chemical lysing, such as the cartridges described in U.S. Application No. 17 / 855,578, which is incorporated herein by reference in its entirety for all purposes. In some embodiments, the improved valve assembly provides for a sample cartridge capable of combined capture and detection of targets that do not require heat and / or mechanical lysis as well as targets that do require heat and / or mechanical lysis. In some embodiments, such valve assemblies are compatible with existing instrument modules that currently operate conventional sample cartridges directed to only one type of lysing.

[0011] In some embodiments, the valve assembly interfaces with the existing cartridge body such that operation of the cartridge by the instrument module is substantially the same or similar as a conventional sample cartridge. In some embodiments, the instrument module includes updates in modified operating instructions to perform workflows of sample preparation that perform multiple operations, such as chemical lysing, heat lysing, and mechanical lysing, of a single fluid sample with the same sample cartridge. In some embodiments, the instrument module reads or obtains the information regarding a panel of assays being performed, then operates according to a workflow that corresponds to one or all of heat lysing, mechanical lysing and chemical lysing depending on the assays being performed.

[0012] In another aspect, the invention pertains to a valve assembly that improves performance in regard to any of: consistency of fluid flow and filtering, distribution of forces, and distribution of in-fill (e.g. glass beads) for mechanical lysing. The valve assembly can include additional features that improve upon performance and functionality of the valve assembly as compared to conventional valves assemblies of sample cartridges.

[0013] In some embodiments, the valve assembly includes a valve body that interfaces with a valve cap to define an interior sample processing region or lysing chamber therebetween, the valve cap and valve body securing a filter therebetween, a fluid inlet in the valve cap and a fluid outlet in the valve body. In some embodiments, the cap includes a boss feature that interfaces with the valve body and is reduced in height, as compared to the conventional design, so as toaccommodate a thicker filter material. In some embodiments, the fluid flow path through the inlets and outlet and lysing chamber or sample processing region have been improved to smooth transitions and eliminate any sharp angles to improve fluid flow therethrough and reduce residual buffer carryover.

[0014] In some embodiments, support features are added to the valve cap and valve body within the sample processing region or lysing chamber defined therebetween so as to improve infill ability and to reduce filter stress.

[0015] In some embodiments, the improvements include utilizing one or more protrusions in the cap adjacent an inlet port so as to increase clearance between any filter and the cap to improve fluid flow of sample and improve flow of in-fill, such as glass beads, for mechanical lysing. The one or more protrusions can include one or more posts on either or both sides of the inlet or outlet ports. In some embodiments, the posts are oval shaped with a major axis extending in a direction of flow. In some embodiments, the cap comprises lysing chamber protrusions contoured to be received within and enclose the lysing chamber. In some embodiments, the cap comprises one or more flow path protrusions contoured to be received the one or more fluid flow paths.

[0016] In other embodiments, the improvements include one or more protrusions or posts extending from the valve body adjacent an outlet so as to improve fluid flow across the filter region by maintaining a suitable gap between the filter and the valve body. This feature can reduce maximum pressure, for example by 5 psi, during assay testing and avoid clogging of the filter. In some embodiments, the posts are oval shaped with a major axis extending in a direction of flow. In some embodiments, the valve body includes a series of ridges extending in the direction of the fluid flow. In embodiments, having one or more posts near the outlet, the ridges extend only partly across the chamber, for example, about % or less across the chamber and the posts are disposed between the series of ridges and the outlet.

[0017] In some embodiments, the valve assembly utilizes filters that are laser cut, which studies have shown to have reduced tears by up to 10%. Conventional approaches typically utilize mechanical cutting means, such as die cutting.

[0018] In a preferred embodiment, the cartridge has a sample port for introducing a sample into the cartridge, and a sample flow path extending from the sample port. The cartridge also has a lysing chamber in the sample flow path. The lysing chamber contains at least one filter for capturing cells or viruses from the sample as the sample flows through the lysing chamber. The lysing chamber is defined by at least one wall having an external surface for contacting the transducer to sonicate the lysing chamber. Beads may optionally be disposed in the lysing chamber for rupturing the cells or viruses as the chamber is sonicated. The cartridge can also include a waste chamber in fluid communication with the lysing chamber via the sample flow path for receiving the remaining sample fluid after the sample flows through the lysing chamber. The cartridge can further include a third chamber connected to the lysing chamber via an analyte flow path for receiving the analyte separated from the sample. The third chamber is preferably a reaction chamber for chemically reacting and optically detecting the analyte. The cartridge also includes at least one flow controller (e.g., valves) for directing the sample into the waste chamber after the sample flows through the lysing chamber and for directing the analyte separated from the sample into the third chamber. The design of the cartridge permits the efficient processing of large sample volumes to enable the accurate detection of low concentration analytes.

[0019] In some embodiments, the sample cartridge employs a rotary valve configuration that allows fluidic communication between a fluid processing region selectively with a plurality of chambers including, for example, a sample chamber, a waste chamber, a wash chamber, a lysis chamber, and a master mix or reagent chamber. The fluid flow among the fluid processing region and the chambers is controlled by adjusting the position of the rotary valve. In this way, the metering and distribution of fluids in the apparatus can be varied depending on the specific protocol.

[0020] In accordance with some aspects of the present invention, a fluid control and processing system comprises a housing having a plurality of chambers, and a valve body including a first fluid processing region continuously coupled fluidicly with a fluid displacement region. The fluid displacement region is depressurizable to draw fluid into the fluid displacement region and pressurizable to expel fluid from the fluid displacement region. The valve body includes a plurality of external ports. The first fluid processing region is fluidicly coupled with at least two of the external ports. The fluid displacement region is fluidicly coupled with at leastone of the external ports of the valve body. The valve body is adjustable with respect to the housing to allow the external ports to be placed selectively in fluidic communication with the plurality of chambers. At least one of the plurality of chambers is a processing chamber including at least one port for selectively communicating with at least one of the external ports of the valve body. The processing chamber provides an additional fluid processing region.

[0021] In some embodiments, at least one of the fluid processing regions in the valve body or in the processing chamber contains a fluid processing material which is an enrichment material or a depletion material. The fluid processing material may comprise at least one solid phase material. The solid phase material may comprise at least one of beads, fibers, membranes, filter paper, glass wool, polymers, cellulose fibers, and gels. In some embodiments, the filter is formed of glass fibers to facilitate affinity binding with the nucleic acids. In some embodiments, the filter has a nominal pore size of 0.2 to 2 pm, preferably 0.5 to 1 pm, typically about 0.7 pm. In some embodiments, the cartridge includes glass beads for mechanical lysing, the glass beads having a nominal diameter of about 200 pm or less, typically about 100 pm. In some embodiments, the filter is a glass fiber disk without acrylic binder. In some embodiments, the filter material has a nominal thickness between 400 pm and 450 pm, typically about 420 pm. In some embodiments, the cut filter is anywhere between 0.375”- 0.400” in diameter, with the nominal diameter being around 0.385” or 9779 pm. The fluid processing material may comprise a filter and beads, and in some embodiments comprises at least two types of beads. In some embodiments, a single type of solid phase material is used to perform at least two different functions which are selected from the group consisting of cell capture, cell lysis, binding of analyte, and binding of unwanted material. In some embodiments, the processing chamber includes a receiving area for receiving a processing module containing an enrichment material or a depletion material. In a specific embodiment, at least one of the chambers is a reagent chamber containing dried or lyophilized reagents. In some embodiments, the fluid processing material comprises at least one liquid phase material, such as ficoll, dextran, polyethylene glycol, and sucrose. The fluid processing material is contained in the fluid processing region by one or more frits. In a specific embodiment, the external ports are disposed on a generally planar external port surface of the valve body.

[0022] In some embodiments, the filter materials (e.g. glass beads, glass fibers) can be chemically treated to enhance performance. In some embodiments, the filter materials are chemically treated to improve binding and / or separation for isolation and purification of nucleic acids from nucleic-acid containing samples passed through the filter material. In some embodiments, the chemical treatment can include bonding of a compound to the filter material. In some embodiments, the compound comprises a DNA binding ligand, such as an amino-containing compound and can be used as a separating material for nucleic acid isolation. Particularly, the DNA binding ligand on the surface of the glass support provides high nucleic acid binding capacity for isolating the nucleic acid from a sample. In some embodiments, the compound is chemically bonded to the glass material via a linker, such as by an oligoethylene linker or a PEG oligomer. Suitable chemical treatments include, but are not limited to. those described in PCT Publication No. WO2023 / 212336A1 published November 2, 2023, entitled “Nucleic Acid Extraction and Isolation with Heat Labile Silanes and Chemically Modified Solid Supports,” the entire contents of which are incorporated herein by reference for all purposes.

[0023] In accordance with another aspect of the invention, a fluid control and processing system comprises a housing having a plurality of chambers, and a valve body including a fluid processing region continuously coupled fluidicly with a fluid displacement region. The fluid displacement region is depressurizable to draw fluid into the fluid displacement region and pressurizable to expel fluid from the fluid displacement region. The valve body includes at least one external port, the fluid processing region is fluidicly coupled with at least one external port, and the fluid displacement region is fluidicly coupled with at least one external port of the valve body. The valve body is adjustable with respect to the housing to allow the at least one external port to be placed selectively in fluidic communication with the plurality of chambers.

[0024] In some embodiments, the sample cartridge employs a rotary valve configuration to control fluidic movement within the cartridge that allows for selective fluidic communication between a fluid sample processing region and a plurality of chambers in the cartridge. Nonlimiting exemplary chambers can include, a sample chamber, a reagent chamber, a waste chamber, a wash chamber, a lysate chamber, an amplification chamber, and a reaction chamber. The fluid flow among the fluid sample processing region and the chambers is controlled byadjusting the position of the rotary valve. In this way, the metering and distribution of fluids in the cartridge can be varied depending on the specific protocol, which allows sample preparation to be adaptable to different protocols such as may be associated with a particular sample type for different types of analysis or different types of samples. For example, the sample cartridge can include a means for cell lysis, e.g., a sonication means so that bacteria and cells in a fluid sample to be analyzed can be lysed. Additional lysis means suitable for use with the instant invention are well known to persons of skill in the art, and can include, chemical lysis, mechanical lysis, and thermal lysis. In some embodiments, the sample includes bacteria, eukaryotic cells, prokaryotic cells, parasites, or viral particles.

[0025] In some embodiments, sample processing comprises sample processing steps that are performed from initial sample preparation steps, intermediate processing steps, and further processing steps to facilitate a detection of a target analyte in the biological sample with an attached reaction vessel. For example, sample processing can include preliminary preparation steps, such as filtering, grinding, mincing, concentrating, trapping debris or purifying a rough sample, or steps for fragmenting of DNA or RNA of the target analyte, such as by sonication or other mechanical or chemical means. Sample processing can include various intermediate processing steps, such as filtering, chromatography, or further processing of nucleic acids in the sample, including but not limited to chromatography, bisulfite treatment, reverse transcription, amplification, hybridization, ligation, or fragmentation of DNA or RNA. Sample processing may further include final processing steps, such as final amplification, hybridization, sequencing, chromatographic analysis, filtering and mixing with reagents for a reaction to detect the target analyte, which detection can include optical, chemical and / or electrical detection. While the sample cartridge typically performs analytical testing in an attached reaction tube or reaction vessel, it is appreciated that the sample cartridge can utilize various other means as well (e.g. semiconductor chip).

[0026] In some embodiments, the sample processing device can be a fluid control and processing system for controlling fluid flow among a plurality of chambers within a cartridge, the cartridge comprising a housing including a valve body having a fluid sample processing region continuously coupled fluidically with a fluid displacement chamber. The fluid displacement chamber is depressurizable to draw fluid into the fluid displacement chamber andpressurizable to expel fluid from the fluid displacement chamber. The fluid sample processing region includes a plurality of fluid transfer ports each fluidically coupled with one of a plurality of external ports of the valve body. The fluid displacement chamber is fluidically coupled with at least one of the external ports. The valve body is adjustable with respect to the plurality of chambers within the housing to allow the external ports to be placed selectively in fluidic communication with the plurality of chambers. In some embodiments, the valve body is adjustable with respect to the housing having multiple chambers, to place one external port at a time in fluidic communication with one of the chambers.

[0027] In some embodiments of the cartridge, the fluid sample processing region can be disposed between the fluid displacement chamber and at least one fluid transfer port. The term “fluid processing region” refers to a region in which a fluid sample is subject to processing including, without limitation, chemical, optical, electrical, mechanical, thermal, or acoustical processing. For example, chemical processing may include a chemical treatment, a change in pH, or an enzymatic treatment; optical processing may include exposure to UV or 1R light; electrical processing may include electroporation, electrophoresis, or isoelectric focusing; mechanical processing may include mixing, filtering, pressurization, grinding or cell disruption; thermal processing may include heating or cooling from ambient temperature; and acoustical processing may include the use of ultrasound (e.g. ultrasonic lysis). In some embodiments, the fluid processing region may include an active member, such as a filter, to facilitate processing of the fluid. Additional active members suitable for use with the instant invention are well known to persons of skill in the art. In some embodiments, an energy transmitting member is operatively coupled with the fluid sample processing region for transmitting energy thereto to process fluid contained therein. In some embodiments, the valve body includes a crossover channel, and the valve body is adjustable with respect to the plurality of chambers to place the crossover channel in fluidic communication with two of the chambers concurrently.

[0028] The cartridge housing includes one or more branches that extend to one or more transfer ports to which a reaction vessel can be attached so as to facilitate transfer of fluid sample from a chamber of the cartridge into the reaction vessel. In some embodiments, the reaction vessel extends from the housing of the cartridge. These aspects can be understood further by referring to U.S. Patent No. 8,048,386, which is herein incorporated by reference in its entiretyfor all purposes. It is understood that fluid may flow in either direction into or out of the transfer ports, in various embodiments, fluid flow is not limited in any particular direction. For example, in an embodiment having a pair of transfer ports, air may be pumped into or evacuated from one of the pair of transfer ports to facilitate flow of the fluid sample into a conduit of the reaction vessel through the fluid transfer port.

[0029] In some embodiments, methods for processing an unprepared sample can include steps of: receiving a sample cartridge in a cartridge receiver of a module, the sample cartridge including a biological fluid sample to be analyzed, a plurality of processing chambers fluidically interconnected by a moveable valve body; receiving an electronic instruction to process the biological sample into a prepared sample from the module; performing a sample preparation method in the sample cartridge to process the biological fluid sample into the prepared sample; transporting the prepared sample into a reaction vessel fluidically coupled with the sample cartridge; and performing analysis of the biological fluid sample within the reaction vessel. In some embodiments, transporting the sample may include steps of: moving a cartridge interface unit to move the valve body to change fluidic interconnections between the plurality of sample processing chambers; applying pressure to a pressure interface unit to move fluid between the plurality of processing chambers according to position of the valve body; and fluidically moving the prepared sample into the reaction vessel, and performing analysis of the fluid sample within the reaction vessel with the module. Any result of the analysis can be obtained by the module and communicated to various other devices as desired. In some embodiments, the sample cartridge can be coupled to various other diagnostic components, such as a silicon chip, or may transport the prepared fluid sample to other external diagnostic equipment.BRIEF DESCRIPTION OF THE DRAWINGS

[0030] FIGS. 1A-1B show a sample cartridge having a valve assembly configured for performing differing sample processes, such as mechanical lysing of targets and / or chemical lysing of targets, which is configured for PCR and optional integrated nucleic acid analysis of the target assay panel in accordance with some embodiments. FIG. 1 A shows the sample cartridge body with reaction vessel attached, FIG. IB shows an exploded view of the sample cartridge.

[0031] FIG. 1C shows a conventional sample cartridge having a sub-assembly with separable syringe tube and valve assembly components.

[0032] FIG. ID shows a sample cartridge having an integrated valve body and syringe tube component, in accordance with aspects of the invention.

[0033] FIGS. 1E-1G shows illustrative, but non-limiting embodiments of the modules, and systems (e.g., processing units) for the PCR detection and / or quantification of polypeptide(s) and optional integrated nucleic acid analysis for the targeted assay panel. FIG. IE illustrates a module configured to receive and interact with the valve assembly of the cartridge to operate the cartridge to facilitate sample preparation and analysis. FIG. IF illustrates a processing unit (e.g. analytical testing unit) of the module that interacts with the fluid sample in the reaction vessel to facilitate sample processing and analytical testing (e.g. PCR and, optionally, nucleic acid analysis) for the targeted assay panel. FIG. 1G illustrates an analytical system having multiple such modules within an enclosure so as to receive multiple sample cartridges therein for testing of the targeted assay panel and / or various other targets or panels.

[0034] FIG. 1H shows a non-limiting workflow for PCR and optional nucleic acid analysis (e.g., nucleic acid amplification) of the targeted assay using a sample cartridge, in accordance with some embodiments.

[0035] FIG. 2 illustrates ultrasonic mechanical lysing techniques as performed by one type of conventional sample cartridge and chemical lysing techniques, as performed by conventional sample cartridges, both techniques can be performed by a universal sample cartridge in accordance with some embodiments.

[0036] FIGS. 3A-3B illustrate a comparison of integrated valve body and syringe tube components used in differing types of cartridges in accordance with some embodiments.

[0037] FIGS. 4A-4B illustrate differing types of cartridges that utilize filters with glass beads suited for mechanical lysis of certain types of targets, in accordance with some embodiments.

[0038] FIGS. 5A-5C illustrate components of an exemplary valve body and associated filter, in accordance with some embodiments.

[0039] FIGS. 6A-6B illustrate an exemplary valve cap in accordance with some embodiments.

[0040] FIGS. 7A-7B illustrate an exemplary valve body in accordance with some embodiments.

[0041] FIGS. 8A-8B illustrate detail views of a filter pocket for a conventional valve body and for a valve body of a universal cartridge, respectively, in accordance with some embodiments.

[0042] FIGS. 9A-9E illustrate various views of a valve body for a sample cartridge utilizing mechanical lysing, in accordance with some embodiments.

[0043] FIGS. 10A-10C illustrate various views of an exemplary valve cap of a sample cartridge utilizing mechanical lysing, in accordance with some embodiments.

[0044] FIGS. 11 A-l IB illustrate various views of an exemplary valve cap in accordance with some embodiments.

[0045] FIGS. 12A-12B illustrate various views of the exemplary valve cap in accordance with some embodiments.

[0046] FIGS. 13A-13D illustrate various views of an exemplary valve body of a universal sample cartridge, in accordance with some embodiments.

[0047] FIG. 14A illustrates cross-sectional views of a valve assembly for a universal cartridge in accordance with some embodiments.

[0048] FIG. 14B illustrates cross-sectional views of a valve assembly for a sample cartridge utilizing mechanical lysing in accordance with some embodiments.

[0049] FIG. 14C illustrates a cross-sectional view of a valve assembly of a universal sample cartridge before addition of support posts.

[0050] FIG. 14D illustrates a cross-sectional view of a valve assembly of a universal sample cartridge after addition of support posts.

[0051] FIGS. 15A-15B illustrate various models of washflow analysis for a valve assembly of a universal sample cartridge, in accordance with some embodiments.

[0052] FIGS. 16A-16B illustrate stress distribution of a valve assembly of a universal sample cartridge FIG. 16B, in accordance with some embodiments, as compared to a conventional valve design, FIG. 16 A.

[0053] FIGS. 17A-17D illustrate filter displacement of a valve assembly of a universal sample cartridge, FIGS. 17C-17D, in accordance with some embodiments, as compared to a conventional valve design, FIGS. 17A-17B.

[0054] FIG. 18 illustrates washout of residual buffer of a valve assembly of a universal sample cartridge, in accordance with some embodiments, as compared to a conventional valve design.

[0055] FIG. 19 illustrates buffer carryover of a valve assembly of a universal sample cartridge, in accordance with some embodiments, as compared to a conventional valve design.

[0056] FIG. 20 depicts various assay workflows, in accordance with some embodiments.

[0057] FIGS. 21 A-21D depict an integrated valve body and syringe tube component for use with a sample cartridge configured for mechanical lysing, in accordance with some embodiments.

[0058] FIGS. 22A-22E depict an integrated valve body and syringe tube component for use with a universal sample cartridge configured for both mechanical lysing and chemical lysing, in accordance with some embodiments.

[0059] FIGS. 23A-23C depict valve body sealing caps for use with a sample cartridge configured for mechanical lysing, in accordance with some embodiments.

[0060] FIGS. 24A-24C depict valve body sealing caps for use with a universal sample cartridge configured for both mechanical lysing and chemical lysing, in accordance with some embodiments.

[0061] FIGS. 25A-25D depict flowpaths associated with the integrated valve body and syringe tube as compared to the conventional three-part design, in accordance with some embodiments.

[0062] FIGS. 26A-26B depict the valve body weld joint areas and defined flowpaths of the integrated valve body and syringe tube, in accordance with some embodiments.

[0063] FIGS. 27A-27B depict the fluid volumes of the various flow paths defined in the integrated valve body as compared to a conventional valve body design, in accordance with some embodiments.DETAILED DESCRIPTION OF THE INVENTION

[0064] The present invention relates generally to a system, device and methods for fluid sample manipulation and analysis, in particular, sample cartridges that facilitate processing and analytical testing of biological samples. In particular, the present invention pertains to an integrated valve body and syringe tube (VBST), which improves ease of manufacture and assembly, and which can include one or more fluid flow features that improve performance and ease in manufacturing. The integrated valve body and syringe tube can be formed as a single component, such as by injection molding. The valve body defines a lysing chamber to facilitate mechanical lysing and flow paths on an underside, sealed by a cap, and optionally a gasket. Such components are particularly advantageous when used in sample cartridges configured for mechanical lysing or universal sample cartridges that perform both mechanical and chemical lysing, especially such cartridges having a unitary cartridge body design.

[0065] In one aspect, the integrated valve body and syringe tube concept results in fewer parts which results in reduced tolerance stackup, and improves ease of manufacture and assembly. This concept also allows for improved joint types and technologies which ultimately enable improved manufacturability as well as improved reliability for the end-user due to fewer nonconformances and less failures as a result of manufacturing processes. Furthermore, these integrated components can allow for redesign of the fluid channel to further optimize fluid flow while minimizing cross-contamination whilst balancing other manufacturing and functional requirements of the product.I. System Overview

[0066] In one aspect, the invention pertains to a sample cartridge that utilizes a valve body platform that allows for detection of enveloped and free nucleic acid targets. In some embodiments, the valve body includes a sample processing region or lysing chamber that provides for either or both mechanical and chemical lysis. This allows a single cartridge to provide lysing for a multitude of differing types of targets, thus, can be considered a “universal assay cartridge.” In some embodiments, the sample cartridge can perform processing and detection of both bacterial targets requiring mechanical lysing and viral targets suited for chemical lysing.

[0067] The sample cartridge device can be any device configured to perform one or more process steps relating to preparation and / or analysis of a biological fluid sample according to any of the methods described herein. In some embodiments, the sample cartridge device is configured to perform at least sample preparation. The sample cartridge can further be configured to perform additional processes, such as detection of a target nucleic acid in a nucleic acid amplification test (NAAT), e.g., Polymerase Chain Reaction (PCR) assay, by use of a reaction tube attached to the sample cartridge. In some embodiments, the reaction tube extends from the body of the cartridge. Preparation of a fluid sample generally involves a series of processing steps, which can include chemical, electrical, mechanical, thermal, optical or acoustical processing steps according to a specific protocol. Such steps can be used to perform various sample preparation functions, such as cell capture, cell lysis, binding of analyte, and binding of unwanted material.

[0068] A sample cartridge suitable for use with the invention, includes one or more transfer ports through which the prepared fluid sample can be transported into an attached reaction vessel for analysis. FIGS. 1A-1B and ID illustrate an exemplary universal sample cartridge 100 with integrated VBST 130, the cartridge configured for sample preparation and analytics testing by PCR when received in an instrument module in accordance with some embodiments. The sample cartridge 100 is attached to a reaction vessel 110 (“PCR tube”) adapted for analysis of a fluid sample processed within the sample cartridge 100. In some embodiments the reaction tube extends form the cartridge body. Such a sample cartridge 100 includes various components including a cartridge body main housing 102 having one or more chambers 105 for processing of the fluid sample, which typically require sample preparation before analysis. The instrument module facilitates the processing steps needed to perform sample preparation and the prepared sample is transported through one of a pair of transfer ports into fluid conduit of the reaction vessel 110 attached to the housing of the sample cartridge 100. The prepared biological fluid sample is then transported into a chamber of the reaction vessel 110 where the biological fluid sample undergoes nucleic acid amplification. In some embodiments, the amplification is a polymerase chain reaction. In some embodiments, concurrent with the amplification of the biological fluid sample, an excitation means and an optical detection means of the module are used to detect optical emissions that indicate the presence or absence of a target nucleic acid analyte of interest, e.g., a bacteria, a virus, a pathogen, a toxin, or other target analyte. It isappreciated that such a reaction vessel could include various differing chambers, conduits, or micro-well arrays for use in detecting the target analyte. In some embodiments, the reaction tube can comprise multiple separate reaction chambers isolated from each other for the detection of different analytes. In some embodiments, the reaction tube can comprise 2, 3, 4, 5, 6, 7, 8, 9, 10 or more separate isolated reaction chambers. The sample cartridge can be provided with means to perform preparation of the biological fluid sample before transport into the reaction vessel. Any chemical reagent required for viral or cell lysis, or means for binding or detecting an analyte of interest (e.g. reagent beads) can be contained within one or more chambers of the sample cartridge, and as such can be used for sample preparation.

[0069] An exemplary use of a reaction vessel for analyzing a biological fluid sample is described in commonly assigned U.S. Patent Application No. 6,818,185, entitled “Cartridge for Conducting a Chemical Reaction,” filed May 30, 2000, the entire contents of which are incorporated herein by reference for all purposes. Examples of the sample cartridge and associated module are shown and described in U.S. Patent No. 6,374,684, entitled “Fluid Control and Processing System” filed August 25, 2000, and U.S. Patent No, 8,048,386, entitled “Fluid Processing and Control,” filed February 25, 2002, incorporated herein by reference in their entirety for all purposes.

[0070] Various aspects of the sample cartridge 100 can be further understood by referring to U.S. Patent No. 6,374,684, which is herein incorporated by reference in its entirety for all purposes, and which describes certain aspects of a sample cartridge in greater detail. Such sample cartridges can include a fluid control mechanism, such as a rotary fluid control valve, that is connected to the chambers of the sample cartridge. Rotation of the rotary fluid control valve permits fluidic communication between chambers and the valve so as to control flow of a biological fluid sample deposited in the cartridge into different chambers in which various reagents can be provided according to a particular protocol as needed to prepare the biological fluid sample for analysis. To operate the rotary valve, the cartridge processing module comprises a motor such as a stepper motor that is typically coupled to a drive train that engages with a feature of the valve in the sample cartridge to control movement of the valve in coordination with movement of the syringe, thereby resulting in movement of the fluid sample according to the desired sample preparation protocol. The fluid metering and distributionfunction of the rotary valve according to a particular sample preparation protocol is demonstrated in U.S. Patent No. 6,374,684, which is herein incorporated by reference in its entirety for all purposes.H. Example Universal Assay Cartridge and Valve AssembliesA. Overview

[0071] As shown in FIGS. 1A-1B and ID, the assay panel cartridge 100 comprises a cartridge body 102 having a sample chamber 104 for receiving a biological sample, a plurality of chambers 105 for reagents or buffers and sample processing, and the integrated valve body and syringe tube 130. The chambers are sealed by lid 106 that secures atop the cartridge body by a snap-fit connection or any suitable means. The cartridge body includes a base 108, which can be integrally formed with the cartridge body or a separate component. The integrated valve body and syringe tube 130 includes a syringe tube 12 extends to a valve body 30 that is sealed on an underside by a cap 50. The chambers 105 are disposed around a central syringe barrel conduit 103 that is in fluid communication with the valve body 20 of the integrated valve body 30 through the syringe tube 12. The valve body typically contains one or more channels and / or a chamber that contains a filter 40 to bind and elute a nucleic acid. The filter is sealed in the chamber by a cap 50 attached to the underside of the valve body as described herein. In some embodiments the cartridge further comprises one or more temperature controlled channels or chambers that can, in certain embodiments, function as thermocycling chambers. A “plunger” not shown can be operated to draw fluid into the syringe barrel 103 and rotation of the valve body 30 aligns the ports on a top side thereof with corresponding ports in the respective chambers to provide selective fluid communication between the various reagent chambers and channels, reaction chamber(s), mixing chambers, and optionally, any temperature controlled regions. Thus, the various reagent chambers 105, reaction chambers, filter material(s), and temperature controlled chambers or channels are selectively in fluid communication by rotation of the valve body and syringe tube and reagent movement (e.g., chamber loading or unloading) is operated by the “syringe” action of the plunger within the syringe tube. By comparison, FIG. 1C shows the various separate components of a conventional cartridge 1 having a lid apparatus 2, a plunger barrel 3, cartridge body 4, syringe tube 5, valve body 6, sealing cap 7 and cartridge foot 8. While a unitary cartridge body is shown here, it is appreciated that the integrated valve bodyand syringe tube can be used with various other cartridges includes legacy cartridges having separately attached lid assemblies.B. Examples Assays

[0072] In one aspect, the universal sample cartridge described herein can perform sample preparation and analytical testing for assays that are currently performed by conventional sample cartridges. For example, a sample cartridge (e.g. Cartridge A design) can be configured with an integrated valve body and syringe tube, the valve body being configured to perform analytical detections for targets including bacteria, spores, and hardy cells, which require mechanical lysis (e.g. ultrasonics, sonication) so that the released nucleic acids can be physically captured and detected. A universal sample cartridge (e.g. Cartridge A+) can be configured with an integrated valve body and syringe tube, the valve body being configured to perform chemical lysing for target including virus, free DNA, and fragile cells, and to perform mechanical lysing (e.g. ultrasonics, sonication) for targets including bacteria, spores, and hardy cells, as shown in FIG. 2.

[0073] Advantageously, the universal sample cartridge described herein can perform both of these sample preparation tasks, individually, sequentially or in combination, such that the sample cartridge can replace the conventional cartridges as well as perform complicated assay panels that would otherwise not be achievable with a single sample cartridge. For example, the universal sample cartridge can be used for the simultaneous detection of the major viral, parasitic and bacterial causes of undifferentiated febrile illness (UFI) in a Tropical Fever Assay panel, all of which can be performed by a sample cartridge utilizing the improved valve assembly described herein. Lysis requirements of possible target organisms responsible for UFI include both viral targets that require chemical lysis, parasitic and bacterial targets may require mechanical lysis. Additional multi-target assay panels that can be developed for use with the universal sample cartridge may include a Gastrointestinal (GI) Panel, Breast Cancer Panel, and Bacterial Agents or any mixed-target panel. As shown, the differing targets within a single panel can include any of viral targets, fungal targets, parasitic targets, and bacterial targets, or any combination thereof.C. Reaction Modules

[0074] In certain embodiments the cartridge 100 is configured for insertion into a reaction module 300, e.g., as shown in FIG. IE. As illustrated in FIG. IF the module is configured to receive the cartridge 100 therein. In certain embodiments the reaction module provides heating plates 308 to heat the temperature controlled chamber or channel. The module can optionally additionally include a fan 304 to provide cooling where the temperature controlled channel or chamber is a thermocycling channel or chamber. Electronic circuitry 302 can be provided to pass information (e.g., optical information) to a computer for analysis. In certain embodiments the module can contain optical blocks 306 to provide excitation and / or detection of one or more (e.g., 1, 2, 3, 4, or more) optical signals representing, e.g., signal DNAs amplified for various PCR targets. In various embodiments an electrical connector 312 can be provided for interfacing the module with a system (e.g. system controller or with a discrete analysis / controller unit). As illustrated, in FIG. IF the sample can be introduced into the cartridge using a pipette 310. In certain embodiments, the module also contains a controller that operates a plunger in the syringe barrel and the rotation of the valve body.D. Analytical System

[0075] In certain embodiments a system (e.g., a processing unit) is provided. One illustrative, but non-limiting embodiment is shown in FIG. 1G. System 400 includes an enclosure 401 that is configured to support and power multiple sample processing modules 300, where each processing module is configured to hold and operate a removable cartridge 100. In some embodiments, the system is configured to operate the sample processing modules to perform a PCR assay for one or more target analytes and optionally to determine the level of one or more target RNA / DNA sequences within a corresponding removable sample cartridge. Typically, the processing on a sample within the corresponding removable sample cartridge involves operating the cartridge to perform a method as described herein. In certain embodiments the system is configured to contain one sample processing module. In certain embodiments the system is configured to contain at least two or more sample processing modules (e.g., at least 4, 8, 12, 16, 20, 24, 28, 32, 48, 80, or more sample processing modules). In some embodiments, the system provides a user interface that allows the user input operational instructions and / or to monitor operation of the cartridges to determine the presence or quantity of one or more nucleic acids.

[0076] While the methods described herein are described primarily with reference to the GENEXPERT® cartridge by Cepheid Inc. (Sunnyvale, Calif.) (see, e.g., FIG. 1 A), it will be recognized, that in view of the teachings provided herein the methods can be implemented on other cartridge / microfluidic systems, including alternative cartridge designs having valve assemblies that involve multiple interfacing components, as well as cartridge body defined by multiple interfacing components to form the multiple chambers of the cartridges, for example, those described in Korean Application No. 102293717B1 and KR102362853B1, cartridges that utilizes ultrasonic waves to lyse cells in a biological sample, for example, those described in International Application No. WO2021 / 245390A1, cartridges and systems that utilizes an electrowetting grid for microdroplet manipulation and electrosensor arrays configured to detect analytes of interest, for example, those described in International Application No.WO2016 / 077341A2, cartridges that facilitate movement of nucleic acid from one chamber to the next chamber by opening a vent pocket, for example, those described in International Application No. WO2012 / 145730A2, multiplexed assay systems comprising a plurality of thermocycling units such that individual chambers can be heated, cooled, and / or compressed to mix fluid within the chamber or to propel fluid in the chamber into another chamber, for example, those described in International Application No. WO2015 / 138343A1, and as well as systems for rapid amplification of nucleic acids facilitated by flexible portions of the sample cartridge aligned to accomplish temperature cycling for nucleic acid amplification, for example, those described in International Application No. WO2017 / 147085A1. Such cartridge systems can include, for example microfluidic systems implemented using soft lithography, micro / nano- fabricated microfluidic systems implemented using hard lithography, and the like.

[0077] FIG. 1H shows a non-limiting workflow for PCR and optional nucleic acid analysis (e.g., nucleic acid amplification) of the targeted assay with a sample cartridge. In some embodiments, PCR and nucleic acid analysis when performed, are both performed on the same sample. Thus, a single sample can be introduced into one sample chamber. In other embodiments the sample may be processed differently for PCR and nucleic acid analysis for target assay panels.E. Example Valve Assemblies

[0078] FIGS. 3A-3B illustrates example valve bodies of integrated valve body and syringe tube components for differing types of cartridges, in accordance with aspects of the invention. Cartridge A performs only mechanical lysing for more hardy targets, and Cartridge A+ (also referred to as the universal cartridge) can perform either mechanical lysing or chemical lysing for viruses, free NA or more fragile targets. Both cartridges can include a syringe tube 12 and valve body component 30’, 30”, which can be integrally formed, and which interfaces with valve cap 50’, 50” to seal the lysing chamber. In one aspect, the additional capabilities of the valve assembly of the universal sample cartridge rely in part on a filter, features of the valve body and cap, as well as the particular workflow sequence performed by the instrument interface of the module.

[0079] In some embodiments, the filter is configured to accommodate glass beads 140 to further facilitate mechanical lysis of hardy targets. In this embodiment, the filter 40 is formed of glass fibers and has, e.g, a 0.7 pm pore size. In contrast, embodiments of Cartridge A utilize a filter formed as a disk of a polymer film (i.e., PCTE) 40’ and has, e.g, a 0.8 pm pore size, which while suitable for mechanical lysing, is not suited for chemical lysing. By utilizing a filter having a pore size of 0.7 pm, the filter is suitable for receiving suitably sized glass beads for mechanical lysing. Utilizing glass fibers to form the filter facilitates affinity bonding with the free nucleic acid released by chemical lysing. Thus, this glass filter is suited for both mechanical and chemical lysing. FIGS. 4A-4B illustrate that these types of glass filters can be used in both the Cartridge A and Cartridge A+ designs.

[0080] FIGS. 5A-5C illustrate the valve cap 50, valve body 30 and filter 40 of a valve assembly of a universal sample cartridge, in accordance with some embodiments. The valve cap 50 and valve body 30 include interlocking portions on their peripheries that engage with each other so that interior circular regions interface to form a sample processing region or lysing chamber 32. The cap includes interior circular region 51 that interfaces with the valve body to enclose an interior sample processing region or lysing chamber 32 defined in the valve body. The filter 40 is sized to be secured between the valve cap and valve body. It is appreciated these designs can be incorporated into an integrated valve body and syringe tube component.

[0081] FIGS. 6A-6B illustrate valve caps in accordance with some embodiments. FIG. 6 A shows a valve cap 50’ in accordance with some embodiments, and FIG. 6B shows a valve cap 50” of a similar design that further includes a pair of posts. As shown, the valve caps include an interior circular region 51 that encloses the lysing chamber 32 when interfaced with the valve body (see, for example, FIGS. 7A and 7B) and a fluidic inlet 52 through which fluid sample and any glass bead infill enters the lysing chamber. In the design of FIG. 6B, the valve cap further includes a pair of protrusions or posts 53, which presses the filter away from the inlet so as to improve consistency of glass infill across the filter so as to improve mechanical lysing.

[0082] FIGS. 7A-7B illustrate valve bodies in accordance with some embodiments. FIG. 7A shows a valve body 30’ in accordance with some embodiments, and FIG. 7B shows a valve body 30” of a similar design that further includes a pair of posts. As shown, the valve bodies include the interior circular region that defines the lysing chamber 32 when interfaced with the valve cap and a fluidic outlet channel 32b to filter port 32c (not shown in FIGS 7A-7B, see, e.g. FIGS, 21 A-21D) through which fluid sample exits the lysing chamber 32. In the design of FIG. 7B, the valve body further includes a pair of protrusions or posts 33, which inhibits deflection of the filter toward the outlet so as to reduce pressure peaks on the filter and reduce tearing. It is appreciated these designs can be incorporated into an integrated valve body and syringe tube component. Various aspects of the lysing chamber and associated flowpaths can be further undersood by referring to FIGS. 8A-19.

[0083] FIGS. 8A and 8B illustrate cross-sectional views of a valve body of a conventional valve assembly (FIG. 8A) and a valve body of an improved valve assembly (FIG. 8B) in accordance with some embodiments. As can be seen, the edges 132 of the chamber or filter pocket have been smoothed such that sharp edges are eliminated, which reduces uneven flow and pressure distributions and promote uniform flow through the lysing chamber.

[0084] FIGS. 9A-9E illustrate various views of a valve body of a conventional sample cartridge utilizing mechanical lysing and FIGS. 10 A- 10C illustrate various views of a valve cap of a conventional sample cartridge utilizing mechanical lysing, for comparison with the improved valve assemblies described herein.

[0085] FIGS. 11A-12B illustrate various views of a valve cap 50” for a universal sample cartridge, in accordance with some embodiments. The two posts 53 can be seen adjacent the inlet52. FIG. 11 A illustrating the side of the valve cap facing the valve body when assembled, and FIG. 1 IB the other side. FIGS. 12A and 12B provide close up views of the fluidic inlet 52 and protrusions or posts 53 in an interior circular region 51. FIGS. 13A-13D illustrate various views of a valve body 30 for a universal sample cartridge, in accordance with some embodiments. The two posts 33 can be seen adjacent the inlet of the outlet channel 32b and a series of support ridges 34 support the filter upstream of the outlet. Certain differences between the conventional sample cartridge and the universal sample cartridge can be seen by comparing FIG. 9E and FIG. 13D.

[0086] FIG. 14A illustrates a cross-sectional view of a valve assembly (Cart A+), in accordance with some embodiments, illustrating the valve body 30”, and the gap 45” between the filter 40 and cap 50” where any glass beads for mechanical lysing are filled, and where the filter 40 gets “pinched” or “sandwiched” 145” between the valve body 30” and valve cap 50”, for comparison with the conventional valve assembly (Cart A) shown in FIG. 14B. In the design of FIG. 14 A, the cap 50” and valve body 30” have been modified, as described previously, for improved fill in the void region between the filter 40 and the cap 50”. In FIG. 14B, a thinner filter material has been used to create a larger void 45’ to facilitate glass fill, and also illustrated is and where the filter 40 gets “pinched” or “sandwiched” 145’ between the valve body 30’ and valve cap 50’. In the improved design in FIG. 14A, a thicker filter material has been used such that the void 45” between the filter 40 and cap 50” is smaller, while including the posts on the gap maintain a suitable gap to facilitate the glass fill process.

[0087] FIGS 14C and 14D further ilustrate the gap between the filter and valve cap, respectively without and with posts. FIG. 14C illustrates a cross-sectional view of a valve assembly of a sample cartridge, before addition of the posts, demonstrating an inconsistent, minimal gap (e.g., a gap less than 0.003”) or no gap between the filter and the valve cap under some conditions. FIG. 14D shows the valve assembly with the addition of posts 53, which provides a more consistent, enlarged gap (e.g., a gap greater than 0.003”) 145” to facilitate, for example, glass infill.III. Experimental Results as to Performance Characteristics

[0088] FIGS. 15A-15B illustrate various models of a valve assembly for a universal sample cartridge, in accordance with some embodiments. Modeling of this design demonstratedimproved flow through the fluid channels and washing of residual buffer through the flow path of the valve assembly. In some embodiments, improved wash flow of buffer and reduction of buffer carryover is achieved by streamlining the fluid flow path through the inlet and outlet of the valve assembly. In this embodiment, the fluid flow path is streamlined by smoothing any transitions and eliminating any sharp (e.g. 90 degree) corners. In the inlet-filter-outlet models of FIGS 15A and 15B, a no-slip boundary condition was used on all walls and the glass beads and filter were modeled as porous media with the resistance of the porous media obtained from the scientific literature. The total volume of the inlet plus outlet manifolds was 40 pL. In the simulation process, the fluid domain was originally filled with buffer 1, and at time t=0, buffer 2 was introduced at the inlet 152 at a rate of 10 pL / sec for 200 pL of total inflow. FIG. 15A illustrates the model in the context of a portion of the valve assembly, and FIG. 15B results of the model for the buffer 1 concentration at t=20 secs. In FIG. 15B residual buffer 1 can be seen at the inlet elbow 153, filter entry 154, outlet manifold 155, outlet turn 156, and wall 157 near the outlet 158.

[0089] FIGS. 16A and B demonstrate the marked advantages of the improved valve design support protrusions, FIG. 16B, over conventional designs, FIG. 16A, in regard to filter stress. As shown by the finite element analysis in FIGS. 16A-16B, the peak stress in the valve body with the pair of support posts adjacent the outlet 162” was 8% lower than in the conventional design 162’ without any support posts.

[0090] FIGS. 17A-17D demonstrate the marked advantages of the improved valve design having support protrusions over conventional designs in regard to vertical displacement of the filter. FIG. 17A shows the peak filter displacement along a cross-section through the middle of the filter, and FIG. 17B along a cross-section at the rib for a conventional design. FIG. 17C shows the peak filter displacement along a cross-section through the middle of the filter, and FIG. 17D along a cross-section at the rib for a improved valve design having support protrusions. As shown by the finite element analysis in FIGS. 17A-17D, the peak displacement in the filter supported by the pair of support posts adjacent the outlet was 10% lower at regions of maximum displacement 172 than in the conventional design without any support posts.

[0091] FIG. 18 demonstrates that the use of support posts in the improved valve design did not significantly impact wash out of residual buffer. As can be seen in computational fluid dynamicsanalysis of FIG. 18, there was 0.28% buffer 1 remaining and an outlet buffer 1 concentration of 0.15% in the conventional design 180, and in the improved valve body design 182, there was 0.38% buffer 1 remaining and 0.28% outlet buffer 1 concentration. These results demonstrated comparable wash out and residual buffer between the two designs.

[0092] FIG. 19 illustrates experimental results as to buffer carryover of a valve assembly of a multi-assay sample cartridge, in accordance with some embodiments, as compared to a conventional valve designs. As shown, the results demonstrate that the addition of the posts, cap supports 192 and valve body supports 194, did not significantly impact buffer carryover.IV. Assay Workflows

[0093] In one aspect, the sample cartridge having an improved valve assembly, as described herein, is capable of a variety of workflows that perform: chemical lysing of targets, mechanical lysing of targets, or both. Accordingly, the sample cartridge can perform an existing workflow associated with conventional specialized cartridges, or can perform entirely new workflows that perform both.

[0094] Exemplary assay workflows that can be performed with a single universal cartridge, in accordance with some embodiments, are shown in FIG. 20. In any of these embodiments, the filter can be formed of glass filter to promote affiniting binding of the nucleic acids (NA) to the glass fibers and a pore size suited for chemical lysing as well. In any of these workflows, the nucleic acid amplification can be PCR, real-time PCR, isothermal amplification (including but not limited to nucleic acid sequence-based amplification, loop-mediated isothermal amplification, helicase-dependent amplification, rolling circle amplification, multiple displacement amplification, whole genome amplification or recombinase polymerase amplification) or other nucleic acid amplification methods known to persons of skill in the art.

[0095] In Workflow A, the sample is optionally exposed to a sample treatment or chemically lysed, then the treated or lysed fluid sample is flowed through the filter where targets are captured. In some embodiments, the sample treatment is used to either weaken the cell wall or to inactivate the sample or make it less viscous to facilitate being processed through the filter. The filter is then washed, leaving the targets on the filter. Next, the targets are mechanially lysed, such as by sonication, to release nucleic acid (NA). In some embodiments, mechanical lysingincludes in-filling glass beads along the filter to aid in mechanical lysing of the target. Next, the NA is eluted from the filter and then nucleic acid amplification is performed is performed.

[0096] In Workflow B, the sample is chemically lysed to obtain the NA targets. In some embodiments, after chemicaly lysing, the NA is bound to the filter by the presence of precipitating and binding reagent. Next, the filter is washed with a rinse reagent while the NA remains bound to the filter. Typically, the wash reagents have some amount of salt which still promotes the binding of the NA to the filter, while allowing removal of non-target materials. Next, the filter is eluted to remove the NA targets. In some embodiments, the elution is performed with a pH neutral buffer or basic buffer fluid. The target NA is then delivered to an attached reaction vessel to perform nucleic acid amplification.

[0097] In Workflow C, the fluid sample is exposed to sample treatment and / or chemically lyse the targets. Next, the NA freed by chemical lysing is bound to the filter. This step may utilize precipitating and binding reagent. Next, the filter is washed with a rinse reagent while the NA remains bound to the filter. Typically, the wash reagents have some amount of salt which still promotes the binding of the NA to the filter, while allowing removal of non-target materials. Next, the targets captured in the filter are heat and / or mechanically lysed. This may utilize sonication, and may further utilize glass beads to facilitate mechanical lysing of select targets. Then, the lysed target NA is eluted from the filter. In some embodiments, the elution is performed with a pH neutral buffer or basic buffer fluid. The target NA is then delivered to an attached reaction vessel to perform nucleic acid amplification. Thus, in this workflow, the workflow allows for lysing of multiple differing targets, some requiring only chemicaly lysing (e.g. viral targets), and others requiring mechanical lysing (e.g. bacteria, spores, etc.), such that all these target NAs can be released from a single sample and tested by the same sample cartridge. While the above workflow desribed mechanical lysing after chemical lysing, it is appreciated that other workflows may be utilized in which chemical lysing occurs after mechanical lysing, while in other workflows, the chemical and mechanical lysing can occur concurrently. In some embodiments, the sample cartridge includes an identifier with information as to the appropriate workflow needed for a particular panel of assays, so that an instrument module receiving the sample cartridge operates according to the specified workflow.IV. Examples of VBST Components and Assemblies

[0098] FIGS. 21 A-24C depict examples of integrated VBST component and associated sealing caps, as developed for cartridges that utilize mechanical lysing (e.g. Cartridge A) as well as for universal cartridges (e.g. Cartridge A+) that can perform either mechanical lysing or chemical lysing, in accordance with some embodiments. Various differing approaches can be used to secure the cap to the valve body, such as by welding, including laser welding, ultrasonic welding, a shear welding design, or any combination thereof.

[0099] FIGS. 21 A-21D depict an integrated VBST having a valve body 30’ configured for a sample cartridge configured to perform mechanical lysing of select targets, such as bacteria, spores or other hardy cells, in accordance with some embodiments. Such cartridges include the Cartridge A design shown and described herein. FIG. 21 A shows the entire integrated VBST component. FIG. 21B shows a detail view of the valve body. As shown, the valve body defines a plunger port 31a through which fluid enters the fluid channels upon movement of the plunger through the syringe tube. The plunger path 31b extends from the plunger port 3 la to inlet 36a from which, based on a rotation position of the valve body and alignment of the ports with corresponding ports in the cartridge body, the fluid can be advanced along a filter path through the lysing chamber 32 or along a direct path 36b to direct port 36c into another chamber of the cartridge body. The filter path extends from the inlet 36a, through the lysing chamber 32 and exits the lysing chamber along outlet channel 32b and out through filter port 32c. The direct path extends from the inlet 36a to the direct port 36c. As shown, the fluid channel along the direct path include an expanded notch area, which is a result of how the valve body is formed. The notch can remain open (as shown), or can be filled with a plug of the cap (see plug 56 in FIG. 23C). FIG. 21C-21D show underside view of alternative designs of valve bodies, which can be configured to attach to the cap by a laser weld lap joint 37, as shown in FIG. 21C, or by an ultrasonic butt weld joint 38, as in the design shown in FIG. 21D. It is noted that any open surface area on the surface interfacing with the cap can be used for the laser weld joint.

[0100] FIGS. 22A-22D depict an integrated VBST having valve body 30” configured for a universal sample cartridge configured to perform either mechanical lysing or chemical lysing of select targets, in accordance with some embodiments. Such cartridges includes the Cartridge A+ design shown and described herein. FIG. 22A shows the entire integrated VBST component.FIG. 22B shows a detail view of the valve body. As shown, the valve body defines a plunger port 31a through which fluid enters the fluid channels upon movement of the plunger through the syringe tube. The plunger path 31b extends from the plunger port 31 a to inlet 36a from which, based on a rotation position of the valve body and alignment of the ports with corresponding ports in the cartridge body, the fluid can be advanced along a filter path through the lysing chamber or along a direct path 36b to direct port 36c into another chamber of the cartridge body. The filter path extends from the inlet 36a, through the lysing chamber and exits the lysing chamber 32 along outlet channel 32b and out through filter port 32c. The direct path extends from the inlet 36a to the direct port 36c. As shown, the fluid channel along the direct path includes an expanded notch area, which is a result of how the valve body is formed. The notch can remove open (as shown), or can be filled with a plug of the cap (see plug 56 in FIG. 23C). FIG. 22C-22D show underside view of alternative designs of valve bodies, which can be configured to attach to the cap by a laser weld lap joint 37, as shown in FIG. 22C, or by an ultrasonic butt weld joint 38, as in the design shown in FIG. 22D. It is noted that any open surface area on the surface interfacing with the cap can be used for the laser weld joint. FIG. 22E shows additional details regarding the entrance channel 32a and the outlet channel 32b from the lysing chamber 32. As shown, in this integrated valve body design the entrance channel 32a after inlet 36a has a meander that includes an acute angle a from a midplane M joining both filter and direct ports. In this embodiment, angle « is 19 degrees from the midplane joining both filter and direct ports. It is appreciated that in other embodimens, angle a can be an angle between 10- 30 degrees, typically between 15-25 degrees, depending on the geometry of the valve body and desired flow characteristics. This feature is designed to inhibit contamination from potential inflow of fluid sample into the lysing chamber when using the direct flowpath and direct port to transfer fluid between chambers during processing. As shown in the cross-section, the outlet channel 32b extending from the lysing chamber 32 to the filter port 32c extends upwards in the valve body at an incline angle of about 25 degrees. In other embodiments, this angle can be an angle between 20-30 degrees depending on the geometry of the valve body and desired flow characteristics. In another aspect, the plunger channel 31b is formed by an elliptical, angled slide, and the filter outlet channel 32b is formed by an elliptical, angled slide, thereby ensuring forming of all fluid channels with all slides terminating either on the cap-side or cartridge-side eliminating the formation of any holes on other surfaces of the valve body which in turneliminates need for additional joints. Also, the elliptical angled slides can ensure that all ports on the terminating faces (cap-side or cartridge-side) are circular in shape.

[0101] FIGS. 23A-24C depict cap designs that can be used with the differing type of cartridge designs, such as the Cartridge A and Cartridge A+ designs shown and described herein. It is appreciated that these are exemplary and variations can be realized. In some embodiments, a thin film can be used to seal the chambers rather than a cap.

[0102] FIGS. 23A-23C show a cap 50’ configured for use with a cartridge configured for mechanical lysing, specifically the Cartridge A design. FIG. 23A shows the valve body facing side having an inner circular region that defines the lysing chamber and FIG. 23B shows the module facing side. In the design of FIGS. 23A-23B, there is no plug component for the expanded region of the direct flow channel in the valve body. FIG. 23 C shows an alternative design of cap 50’ that includes a plug 56 that fills the extended region fluid channel of the valve body to improve flow through the channels and avoid areas where residual fluid may accumulate and contribute to cross-contamination.

[0103] FIGS. 24A-24C show alternative designs of cap 50”configured for use with the universal cartridge, specifically the Cartridge A+ design. FIG. 24 A shows cap 50”, specifically, the valve body facing side having an inner circular region that defines the lysing chamber and FIG. 24B shows the module facing side of cap 50”. In the design of FIGS. 24A-24B, there is no plug component for the expanded region of the direct flow channel in the valve body. FIG. 24C shows an alternative design of cap 50’ that further includes a plug 56 that fills the extended region fluid channel of the valve body to improve flow through the channels and avoid areas where residual fluid may accumulate and contribute to cross-contamination.

[0104] It is appreciated that variations of the valve body and cap designs can be realized that utilize differing features, such as differing types of joints between the valve body, the use of a plug in the cap to partially fill excess portions of the fluid channels of the valve body, and / or the use of a gasket. Table 1 below depicts variations of the designs having differing combinations of features that have been developed. It is appreciated that the integrated VBST and cap could be formed of various materials. In some embodiments, the substrate is formed of polycabronate (PC). Other materials such as copolyester (CP) or polyproplyene (PP) could be used if desired. It is appreciated that none of the designs require a gasket, which is one advantage of thesedesigns over conventional cartridge designs. However, it is considered that variations could be realized that utilize a gasket as well. In some embodiments, the joint between the cap and the valve body is a laser weld lap joint. In some embodiments, the joint between the cap and the valve body is an ultrasonic butt weld. In some embodiments, the joint between the cap and the valve body is a laser weld and an ultrasonic butt weld. In some embodiments, the joint between the cap and the valve body is a shear weld joint. Any suitable joint design could be used.Table 1: Summary of VBST and Cap Variations

[0105] In some embodiments, the valve body can further include a gasket. The gasket can be formed of an elastomer or any suitable material. In some embodiments, the gasket is an overmold that is injection molded into the valve body, where the valve body can be formed of polypropylene, copolyester, polycarbonate, or any suitable material.

[0106] FIGS. 25A-25D show a comparison between the integrated VBST component (i.e. integrated syringe tube 12 and valve body 30”) and cap (2-part design) shown in FIGS. 25C- 25D, and a conventional design having separate valve body 3000, syringe tube 1200 and cap 5000 (3 part design), shown in FIGS. 25A-25B. FIGS. 25A and 25D show exploded assembly views, while FIGS. 25B and 25 C illustrate the flowpath design. The structures shown in both FIGS. 25B and 25C seal the cap using ultrasonic energy director welding. Experimental studiesdemonstrated that a 2-part design enables more precise control of filter capture when used with the Cartridge A and Cartridge A+ designs. Furthermore, the fluid channel can be optimized to reduce cross-contamination and improve ability to fill with glass beads. One approach by which to optimize flow and reduce cross-contamination is the inclusion of a plug feature, as discussed above.

[0107] FIGS. 26A-26B show additional aspects of the VBST sub-assembly suitable for both the Cartridge A and Cartridge A+ designs. FIG. 26A shows a valve body design that includes an energy director ridge 38 around the inner cavity to facilitate ultrasonic welding for sealing of the cap with the valve body. FIG. 26B shows the fluid flow paths and fluid ports defined within the valve body, which include the direct port 36c, the plunger port 31a and the filter port 32c.

[0108] FIGS. 27A-27B depict the volumes associated with the differing flow paths (total path 701, direct path 703, and filter path 705) and fluid domain volumes in the Cartridge A+ design when using a conventional valve body and syringe tube, FIG. 27A, and when using an integrated VBST, FIG. 27B. The total volume 701 is slightly less for the integrated VBST configuration (53.9 pl as compared to 55.5 pl), with the direct path 703 being greater (9.2 pl to 6.5 pl) for the integrated VBST configuration and the filter path 705 being slightly less (48.7 pl to 51.7 pl) integrated VBST configuration. It is appreciated that in a version using a cap with a plug, that the volume of the direct flow path may be further reduced due to the plug. Accordingly, it is considered that the integrated VBST can be incorporated into the existing Cartridge A+ with minimal, if any, modifications to the operation and control. Thus, the integrated VBST designs described herein for both the Cartridge A and Cartridge A+ designs have demonstrated compatibility with existing modules and control sequences to allow seamless integration with existing modules and systems.

[0109] It is to be understood that other variations and configurations not explicitly illustrated in the figures are included in various aspects and embodiments of the present inventions, including, but not limited to, additional sample processing regions or lysing chambers, additional flow channels, additional ports, flow valves, various sizes of filter geometry, angles of slide geometries, etc.

[0110] In some aspects and embodiments, the valve body and syringe tube can include various additional components or modifications to components that allow for additional functionality.Such components or modifications can include any of: (i) variations of the valve body that include one or more additional fluid domains (e.g. sample processing regions or lysing chambers), which can optionally include a gasket port and optionally a flow valve; (ii) variations of the valve cap having additional protrusion features that match the additional fluid domain(s) as well as housing for additional flow valve(s) and corresponding flow channel(s); and (iii) additional valve mechanisms, such as a one-way or two-way valves, for example, an insertable stopper that fits within a flow valve housing that defines a one-way valve.[01U] For example, in some aspects and embodiments, the valve body comprises with two off-center sample processing regions or lysing chambers. In some embodiments, these can be used for two filters, which can allow for additional capacity or allow for use of different types to perform differing types of processing or detection of differing targets. In some embodiments, the additional sample processing region or lysing chamber can be used with a valve as discussed further below.

[0112] The valve body can include one or more valves along any of the flow channels and / or ports defined therein or within an sample processing region or lysing chamber. The one or more valves can include one-way valves, two-way valves or any combination thereof. In some embodiments, the one or more valves can lead to one or more ports to facilitate fluid flow into and / or out from the cartridge. In other embodiments, the one or more valves can be configured to selectively facilitate or inhibit flow through certain ports or through flow channels so as to improve fluid flow control and further inhibit cross-contamination and improve efficiency. The one or more valves can include deformable membranes, stoppers, mechanical valves or any suitable means of controlling fluid flow

[0113] It is appreciated that geometries and dimensions of fluid channels, fluid domains, filter pockets, sample processing region or lysing chamber, flow valves, ports, weld joints can vary based on further analysis, testing, and experimentation throughout the product life cycle including depth, width, length, diameter of ports, angles of channel / filter pocket openings and intersections on both the valve body and valve cap.

[0114] In the foregoing specification, the invention is described with reference to specific embodiments thereof, but those skilled in the art will recognize that the invention is not limited thereto. Various features, embodiments and aspects of the above-described invention can beused individually or jointly. Further, the invention can be utilized in any number of environments and applications beyond those described herein without departing from the broader spirit and scope of the specification. The specification and drawings are, accordingly, to be regarded as illustrative rather than restrictive. It will be recognized that the terms “comprising,” “including,” and “having,” as used herein, are specifically intended to be read as open-ended terms of art. It is appreciated that the term “about” refers to + / - 10%, or + / - 20% of the recited value. Any references to publication, patents, or patent applications are incorporated herein by reference in their entirety for all purposes.

Claims

WHAT IS CLAIMED IS:

1. A component to facilitate fluid processing in an assay cartridge having multiple processing chambers, the component comprising: a syringe tube having an upper opening and a conduit extending therethrough to a distal plunger port; a valve body that is coupled to a distal end of the syringe tube and in fluid communication with the plunger port, wherein the valve body further comprises: a lysing chamber having a fluid inlet and a fluid outlet; one or more fluid flow paths in fluid communication with the lysing chamber and the plunger port; a direct port extending from the one or more fluid flow paths to an upper side of the valve body; and a filter port extending from the outlet of the lysing chamber to an upper side of the valve body; wherein the syringe tube and the valve body are integrally formed.

2. The component of claim 1, wherein the one or more flow paths include an inlet in fluid communication with a direct path extending to the direct port and a filter path extending to the lysing chamber by an entrance channel that has a meander that includes an acute angle between 10-25 degrees from a midplane extending between the filter and direct ports.

3. The component of claim 1, wherein the syringe tube and valve body are formed of polycarbonate, copolyester or polypropylene.

4. The component of claim 1, wherein the lysing chamber includes a filter.

5. The component of claim 4, wherein the filter is formed of glass fibers to facilitate affinity binding with free nucleic acids, wherein the filter has a pore size selected to accommodate correspondingly sized glass beads to facilitate mechanical lysing.

6. The component of claim 5, wherein the assay cartridge includes a plurality of glass beads in a chamber of the plurality, the glass beads correspondingly sized to the pore size of the filter.

7. The component of claim 6, wherein the pore size is between 0.2 um and 2 um.

8. The component of claim 4, wherein the lysing chamber includes a plurality of ribs to support the filter.

9. The component of claim 8, wherein the lysing chamber includes one or more posts adjacent the fluid outlet to facilitate outflow from the lysing chamber.

10. The component of claim 4, wherein the filter is sealed within the lysing chamber by a cap securely attached and sealed to a side of the valve body opposite the syringe tube.

11. The component of claim 10, wherein the cap is securely attached and sealed to the valve body by a weld.

12. The component of claim 11, wherein the cap is securely attached and sealed to the valve body by a laser weld lap joint.

13. The component of claim 11, wherein the cap is securely attached and sealed to the valve body by an ultrasonic butt weld.

14. The component of claim 11, wherein the cap is securely attached and sealed to the valve body by both a laser weld lap joint and an ultrasonic butt weld.

15. The component of claim 11, wherein the cap is securely attached and sealed to the valve body by a shear weld joint design.

16. The component of claim 10, wherein the cap includes one or more posts extending into the lysing chamber adjacent the fluid outlet of the lysing chamber to facilitate more uniform outflow from the lysing chamber.

12. The component of claim 10, wherein the cap includes a plug disposed within the one or more fluid flow paths within a direct flow path between the plunger port and the direct port so as to reduce a volume of an area prone to accumulation of residual sample fluid.

13. The component of claim 10, wherein the cap is without any plug extending into the one or more fluid flow paths.

19. The component of claim 10, wherein the cap is sealed to the valve body by a gasket that is an overmold formed by being injection molded into the valve body.

20. The component of claim 10, wherein the cap includes a lysing chamber protrusion contoured to be received within and enclose the lysing chamber.

21. The component of claim 20, wherein the further includes one or more flow path protrusion contoured to be received within and enclose the one or more fluid flow paths.

22. The component of claim Error! Reference source not found., wherein the lysing chamber is offset from center and the valve body includes an additional lysing chamber offset from center.

23. The component of claim 22, wherein the additional lysing chamber is fluidically connected to the direct port at one end and includes an additional outlet port at or near an opposite end.

24. The component of claim 22, wherein the filter is sealed within the lysing chamber by a cap securely attached and sealed to a side of the valve body opposite the syringe tube, andwherein the cap includes additional protrusions that correspond to the additional lysing chamber and any additional flow channels associated therewith.

25. The component of claim 24, wherein the cap further includes one or more valves.

26. The component of claim 24, wherein the one or more valves includes a one-way valve.

27. The component of claim 26, wherein the one-way valve includes a valve housing that protrudes into the additional lysing chamber and includes a corresponding flowpath and a movable stopper that moves between an open position to allow fluid flow into and / or out from the additional lysing chamber and a closed position that seals the additional lysing chamber.

28. An assay cartridge for processing and / or analytical testing of a biological fluid sample, the assay cartridge comprising: a cartridge body having a plurality of chambers therein, wherein at least one chamber is configured to receive a fluid sample, wherein the cartridge body is a unitary component; the valve body and syringe tube of claim 1; and a filter disposed within the lysing chamber, wherein the filter is formed of glass fibers to facilitate affinity binding with free nucleic acids, wherein the filter has a pore size selected to accommodate correspondingly sized glass beads to facilitate mechanical lysing.

29. The assay cartridge of claim 28, further comprising a cap securely attached and sealed to the valve body thereby sealing the one or more fluid flow paths and lysing chamber.

30. The assay cartridge of claim 29, wherein the cap is securely attached and seals the valve body by any of: an ultrasonic butt weld, a laser weld lab joint, a shear weld joint, or any combination thereof.

31. The assay cartridge of claim 29, wherein the cap includes a plug disposed within the one or more fluid channels adjacent a direct flow path between the plunger port and the direct port so as to reduce a volume of an area prone to accumulation of residual sample fluid.

32. The assay cartridge of claim 29, wherein the cartridge is configured such that the flow paths of the valve body are fluidically sealed to the plurality of chambers of the cartridge without requiring any gasket.

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