System and method for target detection with applications in characterizing food quality and improving food safety

Abstract

To provide a method, system and apparatus for target detection.SOLUTION: A method for target detection is provided, the method comprising: loading a sample into a plurality of microwells on a plate; providing an oil layer that seals the plurality of microwells; and imaging the plurality of microwells using an imaging subsystem.SELECTED DRAWING: Figure 1-1

Description

[Technical Field]

[0001] The present invention relates generally to the field of food safety, and more particularly to novel and useful systems and methods for target detection applied to characterizing food quality and improving food safety.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 091,101, filed October 13, 2020, which is incorporated herein by reference in its entirety. [Background technology]

[0003] Microbial identification and / or quantification is relevant in many fields, including those related to the quality and safety of consumable products (e.g., foods, beverages, supplements, topical consumables, etc.). Culturing samples of such consumable products to detect the presence or absence of microorganisms is typically time-consuming, manual, and cost-constrained, providing motivation for developing new technologies to address these and other drawbacks.

[0004] The food industry is subject to numerous requirements for monitoring numerous food safety and hygiene parameters, including contaminants, pathogens, and quality indicators, throughout various stages, from raw material production, procurement, and handling to finished product manufacturing, distribution, and consumption. One aspect of these requirements is the enumeration of microbial food quality indicators (QIs). Characterization of such QIs provides indicators of product quality (e.g., spoilage, shelf life) and process hygiene and helps predict pathogen occurrence. Several methods exist for enumerating QIs in food and environmental samples, the most widely used being colony counting (e.g., agar plate implementations, ready-to-use pad implementations containing dried reagents, etc.) and most-probable-number (MPN) techniques. However, such methods are prone to one or more of the following: human error, inherent assay variability, poor technical reproducibility, long time-to-results, lack of automation, detection-related inhibition, limited enumeration range, low sensitivity, analog as opposed to digital readout, high cost, high waste, complex workflow, low throughput, and food matrix incompatibility. Furthermore, this technique may require specialized reagents for enumeration and characterization of various target microorganisms.

[0005] In clinical settings, pathogenic microorganisms may exhibit varying degrees of susceptibility to antimicrobial agents. For this reason, clinicians often benefit from identifying both the pathogen's species or strain and its susceptibility to various classes of antimicrobial agents and combinations thereof. However, clinical evaluation methods for microbial infections used in the art typically require at least 16 to 48 hours to identify antimicrobial susceptibility and are prone to the same deficiencies described above.

[0006] Therefore, there is a need for new and useful systems and methods for target detection that can be applied to characterizing food quality and improving food safety. [Brief explanation of the drawings]

[0007] [Figure 1]1A and 1B show schematic diagrams of embodiments of microwell assemblies for target detection. [Figure 2] FIG. 2 shows a schematic diagram of one embodiment of a microwell assembly for target detection. [Figure 3] 3A and 3B show schematic diagrams of specific examples of microwell assemblies for target detection. [Figure 4] FIG. 4 shows a side view of a microwell assembly with a cover component for target detection. [Figure 5] FIG. 5 shows a side view of an operational mode of a microwell assembly having an elastomeric cover component. [Figure 6] 6A and 6B show embodiments of vent channels in a microwell assembly for target detection. [Figure 7] 7A and 7B show an embodiment of a porous film layer of a microwell assembly for target detection. [Figure 8] FIG. 8 illustrates an embodiment of a microwell assembly in which the base substrate and / or cover substrate are comprised of a porous material. [Figure 9] FIG. 9 shows one embodiment of a platform for automating sample processing using units of microwell assemblies. [Figure 10] Figure 10A shows a flow chart of one embodiment of a method for target detection. Figure 10B shows a flow chart of one aspect of a method for target detection. DETAILED DESCRIPTION OF THE INVENTION

[0008] The following description of preferred embodiments of the invention is not intended to limit the invention to those preferred embodiments, but rather to enable any person skilled in the art to make and use the invention.

[0009] 1. Advantages The present invention may provide several advantages over conventional systems and methods.

[0010] In particular, the present invention can provide the advantage of providing an innovative solution for target detection assays involving sample dispensing (e.g., for MPN assays) in a streamlined, efficient, and / or automated manner with respect to minimizing manual steps associated with dispensing. The present invention also includes innovative designs for sample processing consumables that enable parallel processing of multiple samples in a cost-effective, high-throughput manner in various industries. Such designs optionally include structures and features that function to provide automated sample application and dispensing, controlled liquid spreading, controlled sample containment, variable-volume sample dispensing, humidity control, evaporation prevention, and / or cross-talk prevention, as described in more detail below.

[0011] The present invention also provides the advantage of providing a system and method for target detection in a manner that significantly reduces human error, workflow complexity, inherent assay variability, and the time required for target characterization and enumeration.

[0012] The present invention also provides the advantage of automating and / or simplifying processing steps, and in some embodiments, can automatically dispense samples (e.g., samples of 1 mL or less in volume, samples of greater than 1 mL in volume) for MPN assays. By efficiently processing small volumes, the present invention can also significantly reduce waste associated with assay operations, optimize run success, and optimize consistency between runs and / or between different users.

[0013] In embodiments, the present invention also provides the advantage of performing small volumes, significantly reducing the time required for the converted enzyme substrate, such as a chromogenic substrate, to reach the detection threshold, thereby significantly reducing the time to result and test turnaround time. Also, by dividing the sample into smaller partitions in a rapid manner, the counting range is increased, improving the accuracy of the MPN estimation.

[0014] The present invention also offers the advantage of mitigating inhibition problems associated with the generation of converted enzyme substrates and / or detection by colorimetric and / or fluorometric (eg, multi-channel fluorometric) methods.

[0015] Aspects of the present invention also offer the advantage of providing a structure and environment for growing microorganisms, as well as kits, compositions, methods and devices for rapidly analyzing the growth and / or numbers of microorganisms in a cost-effective and time-efficient manner.

[0016] Additionally, the present invention provides a closed system for the growth and detection of microorganisms, preventing laboratory contamination with potentially harmful pathogens.

[0017] Additionally, with software and workflow improvements, the system and / or method can minimize the number of manual operations performed by the user and provide relevant system status reports to ensure smooth operation and sample processing.

[0018] Additionally or alternatively, the present systems and / or methods may provide any other suitable advantages.

[0019] 2. Microwell Plates 1A and 1B, one embodiment of a microwell assembly 100 for target detection includes a base substrate 110 and a set of sample processing regions (including sample processing region 120) defined on a broad surface of substrate 110, each of the set of sample processing regions including a set of microwell subarrays 130 arranged in a gradient between an upstream end 10 and a downstream end 90 of sample processing region 120, with a boundary 190 separating sample processing region 120 from an adjacent sample processing region. With respect to the gradient of the microwell subarrays for each sample processing region, a first microwell subarray 130 containing wells having a first characteristic dimension (e.g., smallest characteristic dimension) can be arranged at the upstream end 10, and a terminal microwell subarray 170 containing wells having a second characteristic dimension (e.g., largest characteristic dimension) can be arranged at the downstream end 90 of sample processing region 120, with other aspects described in more detail below.

[0020] 1B, the microwell assembly 100 can include a cover substrate 210 configured to mate with the base substrate 110 in a mated mode, the cover substrate 210 including a network of vent channels 220 facing the base substrate 110 (in the mated mode), the network of vent channels aligning with the set of sample processing regions when the base substrate 110 and cover substrate 210 are mated, and providing gas exchange between the base substrate 110 and the environment 50 (such as the environment surrounding the microwell assembly, the local environment between the base substrate 110 and the cover substrate 210, etc.). The cover substrate 210 can also be separated from the base substrate 110 by one or more functional layers, as described in more detail below.

[0021] The microwell assembly 100 functions to provide a mechanism for rapid, low-cost sample distribution and processing, with particular application in most probable number (MPN) determination. In particular, the microwell assembly can accept a sample volume and distribute it (e.g., with minimal user manual intervention) across multiple subarrays of microwells, each with characteristic dimensions, thereby facilitating the rapid manipulation and execution of serial dilution tests across multiple samples in parallel (e.g., for food safety / food quality applications, among others) to measure the concentration of one or more target microorganisms in the samples. The microwell assembly 100 can allow gas exchange between the contents of the microwells and / or the environment surrounding the microwell assembly 100 while preventing liquid exchange, thereby providing humidity control and preventing evaporation. The microwell assembly 100 can also prevent crosstalk of liquids, pathogens, and / or conversion substrates between different samples being processed. Additional features of the microwell assembly 100 are described in further detail below with respect to the individual elements of the microwell assembly 110.

[0022] 2.1 Sample Processing Area with Base Substrate and Microwell Subarray 2.1.1 Base substrate 1A, microwell assembly 100 includes a base substrate 110 that supports a set of sample processing regions, described in more detail below. That is, base substrate 110 functions to support a set of samples and to facilitate a process for detecting the presence, estimating the concentration, and / or characterizing microorganisms in the set of samples. In some embodiments, base substrate 110 can prevent liquid exchange (e.g., by structural features and / or material properties) while also functioning to allow gas exchange between the contents of the microwells and the environment surrounding microwell assembly 100, thereby providing humidity control, preventing evaporation, and preventing crosstalk between different samples being processed.

[0023] In terms of material composition, the base substrate 110 can be composed of one or more of a polymer (e.g., polypropylene, polydimethylsiloxane, polystyrene, polyvinyl chloride, polymethyl methacrylate, cyclic olefin copolymer, polycarbonate), a silicon-derived material, glass, a metallic material, a ceramic material, a natural material, a synthetic material, and / or any other suitable material. In particular, the selection of materials can be based on one or more of manufacturing considerations, desired surface properties for sample processing, optical properties, bulk properties (e.g., with respect to porosity, with respect to density, etc.), surface properties, thermal properties, mechanical properties, and / or any other suitable properties. Furthermore, all portions of the base substrate 110 can be constructed using one or more of the same materials, different materials (e.g., if each portion of the base substrate 110 has different design constraints), and / or any combination of materials. Furthermore, the base substrate 110 can be a unitary body or a base substrate 110 having separate portions that are bonded together (e.g., during manufacturing).

[0024] With respect to optical properties, one or more materials of the base substrate 110 can have any degree of transparency, reflectivity, or other optical properties. For example, a material can be transparent to allow for optical analysis, investigation, or observation (e.g., from the bottom surface of the base substrate 110, from the top surface of the base substrate 110, etc.), but may also be opaque, transparent, translucent, and / or of any suitable opacity. For example, if a high degree of porosity is desired in connection with bulk properties such as porosity (e.g., to provide gas exchange functionality), the base substrate 110 need not be transparent. Furthermore, aspects of the material and / or construction can be configured to promote containment of a detectable signal (e.g., with respect to containment of fluorescence, with respect to containment of other conversion substrates) within the individual microwells of the base substrate 110. Furthermore, aspects of the substrate material can be configured and / or treated to prevent absorption of sample processing materials (e.g., fluorescent substrates, colorimetric substrates, other conversion substrates, samples, etc.).

[0025] With respect to bulk properties, the material(s) of the base substrate 110 can be configured to have a level of porosity that allows gas exchange between the contents of the microwells and the environment (e.g., the environment of the system) through the base substrate 110 while preventing liquid exchange, thereby providing humidity control, preventing evaporation, and preventing crosstalk between different samples during processing. Additionally or alternatively, with respect to bulk properties, the material(s) of the base substrate 110 can be configured to have a level of density or other bulk properties appropriate for sample processing and / or incubation purposes necessary to maintain microbial viability. In embodiments, the base substrate 110 can be constructed from or otherwise incorporate a polymer (e.g., polytetrafluoroethylene (PTFE), polyethylene (PE), polyvinyl alcohol (PVA), etc.), ceramic, or another suitable material (e.g., natural material, synthetic material) with an appropriate ingress rating (e.g., according to the IP scale, according to another rating scale) or particle retention properties (e.g., rated retention for particles less than 1 micron, rated retention for particles 1 micron or greater). In one example, the base substrate 110 can be constructed of a PTFE-based material having an IP rating (e.g., IP65-69), although the base substrate 110 can alternatively be constructed of a non-porous material that allows gas exchange through other elements of the microwell assembly 100 (e.g., vents, microscale channels, nanoscale channels, etc.).

[0026] With respect to surface properties, the base substrate 110 material(s) can be configured to have desired hydrophilic / hydrophobic properties (e.g., highly hydrophilic), determined, for example, by contact angle and wettability. With respect to other electrical and physical properties, the base substrate 110 material(s) can be configured to have desired charge (e.g., related to the properties of the sample fluid and / or sample processing fluid used), electric field characteristics, conductivity, resistance, and / or any other suitable surface or physical property. Additionally or alternatively, the base substrate 110 material(s) is / are preferably configured to be non-reactive with the fluids and microorganisms used during sample processing. Additionally or alternatively, the base substrate 110 material(s) can be configured to absorb inhibitors that interfere with the viability of the microorganisms and / or the conversion of the detectable enzyme substrate. Additionally or alternatively, the surface of the base substrate 110 exposed to the receiving fluid can have a desired surface finish.

[0027] Regarding thermal properties, one or more materials of the base substrate 110 can be configured to have desired thermal properties with respect to heat transfer and / or heat retention properties. In particular, the base substrate 110 can be configured to have desired thermal conductivity and / or heat capacity properties (e.g., as appropriate for sample incubation steps). In one embodiment, the base substrate 110 can be configured to have thermal properties that allow efficient heat transfer to or from fluids in contact with the base substrate 110. For example, in embodiments in which the base substrate 110 is coupled to heating or cooling elements, the base substrate 110 can be configured to facilitate heat transfer to and / or from the contents of the microwells during incubation. However, the base substrate 110 can have other appropriate thermal properties based on the intended use. For example, the base substrate 110 can be configured to have low thermal conductivity (e.g., as an insulating material) so that the material does not significantly affect the temperature of the fluids it contacts during operation.

[0028] With respect to mechanical properties, the material or materials of the base substrate 110 can be configured to have desired mechanical properties, including one or more of stiffness, strength, elastic behavior, hardness, and other properties. Additionally or alternatively, the base substrate can be configured to be compatible with an automated plate arm robotic subsystem.

[0029] In terms of dimensions, the base substrate 110 can have the format of an SBS microwell plate (e.g., a footprint of 127.76 mm x 85.48 mm), although the base substrate 110 can alternatively have other suitable dimensions. The plates of the microwell assembly can additionally or alternatively be designed to be easily stackable for packaging or use during sample runs.

[0030] Additionally or alternatively, the base substrate 110 can be configured to be sterilizable (eg, using an autoclave, using other sterilization methods, etc.).

[0031] 2.1.2 Sample Processing Area with Microwell Subarray As shown in FIG. 1A , the base substrate 110 defines a set of sample processing regions (including the illustrated sample processing region 120), each of which includes a set of microwell subarrays 130 arranged in a gradient (e.g., volume, size, surface area, footprint, cross-sectional area, etc.) between the upstream end 10 and downstream end 90 of the sample processing region 120. The set of sample processing regions functions to accept a set of samples and facilitate distribution of the set of samples across the set of microwell subarrays to enable the operation of serial dilution tests on each sample for detection of one or more targets (e.g., targets related to food safety and / or food quality). In some embodiments, the set of sample processing regions can be configured to store dried sample processing materials (e.g., media, fluorescent substrates, colorimetric substrates, other dyes, etc.) prior to accepting the samples to enhance the efficiency of sample processing. Additionally or alternatively, in other embodiments, the set of sample processing regions can include other suitable components (e.g., pre-packaged components). In some embodiments, a set of sample processing areas can be configured to be hydrophilic, hydrated, treated, and / or blocked with a non-specific absorbent.

[0032] In embodiments, the set of sample processing regions can be arranged as a set of lanes across the broad surface of the base substrate 110, with each region configured to accept a separate sample, allowing for high-throughput parallel processing of the samples. In embodiments in which the broad surface of the base substrate 110 has a major axis and a minor axis, the set of sample processing regions can be arranged parallel to the major axis or parallel to the minor axis (e.g., in relation to the number and configuration of samples to be tested and / or the number of desired microwell sizes). However, the set of sample processing regions can alternatively be arranged relative to another suitable axis. Furthermore, in other aspects, each of the sets of sample processing regions may not be configured as a lane with a longitudinally defined microwell subarray. For example, in another aspect shown in FIG. 2, each of the sets of sample processing regions can be defined as a zone (e.g., a circular zone, an ellipsoidal zone, a polygonal zone, an amorphous zone, etc.), and the microwell subarray can be arranged along another suitable axis (e.g., a radial axis, a circumferential axis, etc.) or within another suitable coordinate system.

[0033] In embodiments, the number of sample processing regions included in the microwell assembly 100 can be adjusted by the dimensions of the base substrate 110 (with examples given above) in relation to the characteristic microwell dimensions and number of individual microwells desired for each microwell subarray, and the microwell dimensions and number of implemented microwells can be optimized for serial dilution test operations in relation to the sample volume accepted per region for MPN determination. In examples (two of which are shown in Figures 3A and 3B), a set of sample processing regions can include between two and seven sample processing regions. However, in other embodiments, a set of sample processing regions can include another suitable number of sample processing regions (e.g., fewer than two sample processing regions, more than seven sample processing regions).

[0034] As briefly described above, each set of sample processing regions can include a set of microwell subarrays 130 arranged at a gradient between the upstream end 10 and downstream end 90 of the sample processing region 120, and a boundary 190 separating the sample processing region 120 from adjacent sample processing regions. The set of microwell subarrays 130 functions to provide, for each sample processed, a set of partitions with known volume distributions for determining MPNs and / or performing other assays for target detection from the sample. Thus, each set of microwell subarrays can have partitions (e.g., microwells) with different characteristic volumes for each partition to provide an appropriate number of dilutions and partitions per dilution to generate minimum and maximum detectable MPN values ​​with appropriate confidence limits. Aspects of MPN determination and confidence limits are further described below.

[0035] In embodiments, the sample processing area of ​​the base substrate 110 can include 2 to 10 microwell subarrays, each set of microwell subarrays having 10 to 100,000 partitions. Each sample processing area can accept 0.01 mL to 10 mL of sample (by providing a total volume) to provide MPN values ​​for the MPN assay in a minimum and maximum range of 5 to 3,000,000, with appropriate confidence limits. However, the sample processing area can include other suitable numbers of microwell subarrays (e.g., fewer than 2 subarrays, more than 10 subarrays), each with other suitable numbers of partitions (e.g., fewer than 10 partitions, more than 100,000 partitions), to accept other sample volume sizes (e.g., less than 0.01 mL, more than 10 mL) and enable determination of MPNs within other suitable ranges.

[0036] In particular, the number / characteristic volume of microwell subarrays and the number of partitions per microwell subarray can be constructed in relation to solving for λ in the following equation [1], where exp(x) is e x where K is the dilution number and g j denotes the number of positive (or growing) tubes at the jth dilution, and m j denotes the amount of original sample placed in each tube at the jth dilution, and t j denotes the number of tubes in the jth dilution. TIFF2025148342000002.tif20170

[0037] In the specific example shown in Figures 3A and 3B, each set of sample processing regions can have three microwell subarrays distributed in a gradient along the longitudinal axis of the sample processing region, with the first microwell subarray 121 having a characteristic volume of 0.03 microliters per partition and 300 microwell partitions. As shown in Figure 3B, each microwell in the first microwell subarray 121 can have a width of 0.35 mm, a height of 0.25 mm, and a 0.2 mm wide rib separating each microwell from adjacent microwells. The sample processing region can also include a second microwell subarray 122 having a characteristic volume of 0.3 microliters per partition and 300 microwell partitions. As shown in Figure 3B, each microwell in the second microwell subarray 122 can have a width of 0.70 mm, a height of 0.61 mm, and a 0.35 mm wide rib separating each microwell from adjacent microwells. The sample processing area can also include a third microwell subarray 123, which has a characteristic volume of 3 microliters per partition and 300 microwell partitions. As shown in FIG. 3B, each microwell in the third microwell subarray 123 can have a width of 1.45 mm, a height of 1.43 mm, and a 0.70 mm wide rib separating each microwell from adjacent microwells. The microwells can have an appropriate pitch to facilitate distribution of sample fluid across the microwell subarray. With this configuration, each sample processing area can process approximately 1 mL of sample to enable determination of the MPN per sample.

[0038] In embodiments, the cross-section of each microwell can be polygonal (e.g., hexagonal, rectangular, etc.) or non-polygonal (e.g., circular, elliptical, amorphous, etc.) (e.g., cross-section along a plane parallel to the broad surface of the base substrate 110). Additionally or alternatively, the cross-section of each microwell can taper toward the base of each microwell along a direction away from the broad surface of the substrate 110. Thus, each well can have an opening in the broad surface of the base substrate 110, thereby allowing a subvolume of sample to enter the microwell from a direction perpendicular to the broad surface of the base substrate 110. However, the opening or openings of a microwell can be configured in another suitable manner. Furthermore, the microwells can be arranged in a packed configuration (e.g., hexagonal close-packed, square close-packed, other close-packed configurations, etc.) or an unpacked configuration. For example, the wells of an initial subarray of a set of microwell subarrays can be arranged in a first packing configuration (e.g., hexagonal close-packed, square close-packed, other close-packed configuration, etc.), and the wells of a terminal subarray of the set of microwell subarrays can be arranged in a second packing configuration (e.g., hexagonal close-packed, square close-packed, other close-packed configuration, etc.).

[0039] In relation to the gradient of microwell subarrays for each sample processing region, a first microwell subarray 130 containing wells having a first characteristic dimension (e.g., smallest characteristic dimension) can be located at the upstream end 10 of the sample processing region, and a terminal microwell subarray 170 containing wells having a second characteristic dimension (e.g., largest characteristic dimension) can be located at the downstream end 90 of the sample processing region 120. Thus, the microwell subarrays can have larger characteristic microwell dimensions in the upstream-to-downstream direction. Alternatively, the microwell subarrays can have smaller characteristic microwell dimensions in the upstream-to-downstream direction (e.g., the first microwell subarray 130 can contain wells having the largest characteristic dimension, and the terminal subarray 170 can contain wells having the smallest characteristic dimension). Further alternatively, the microwell arrays can be organized in another suitable manner (e.g., along another directional axis, associated with another microwell characteristic), gradient or non-gradient. Further alternatively, each sample processing region can be configured in other manners (e.g., with step increments across the microwell gradient). For example, the dimensions of the wells can be arranged to have a lateral gradient (eg, perpendicular to the upstream-to-downstream direction) rather than a gradient in the upstream-to-downstream direction.

[0040] As shown in FIGS. 3A and 3B, the base substrate 110 can include a set of boundaries (including boundary 190 shown in FIG. 1A) that separate each sample processing region from an adjacent sample processing region, thereby functioning to prevent sample crosstalk. Boundaries 190 can be configured as recesses (e.g., as moats, as concave channels forming a perimeter), or as protrusions, or can include alternating recesses and protrusions. Boundaries 190 can also be configured as regions configured to promote evaporation or absorption of overflow sample, such that as the sample enters the region, the sample evaporates and / or is absorbed by the walls of boundary 190. In embodiments in which boundary 190 is defined as a concave perimeter around a sample processing region, boundary 190 can function as a moat into which overflow sample is received during sample processing. Alternatively, boundary 190 can serve another suitable purpose.

[0041] Additionally or alternatively, the boundary 190 may be comprised of an absorbent material configured to receive and absorb material that overflows from the sample processing area.

[0042] Additionally or alternatively, the one or more boundaries may include one or more outlets away from the sample processing area (e.g., an outlet to a waste chamber), thereby directing overflow material away from the sample processing area and preventing it from re-entering the sample processing area.

[0043] Additionally, while the base substrate 110 can be physically contiguous, in embodiments, the base substrate 110 can be configured to allow separation between adjacent sample processing regions (e.g., by perforations, reversible locking components, etc.). However, the base substrate 110 can alternatively be configured to be non-separable.

[0044] Although embodiments, implementations, and examples of microwells in the sample processing area are described above, aspects of the microwells and / or sample processing area may also be found in U.S. application Ser. No. 16 / 048,104, filed July 27, 2018; U.S. application Ser. No. 16 / 049,057, filed July 30, 2018; U.S. application Ser. No. 15 / 720,194, filed September 29, 2017; U.S. application Ser. No. 15 / 430,833, filed February 13, 2017; U.S. application Ser. No. 15 / 821,329, filed November 22, 2017; U.S. application Ser. No. 15 / 821,329, filed October 12, 2017; No. 15 / 782,270, filed July 30, 2018, U.S. Application No. 16 / 049,240, filed November 16, 2017, U.S. Application No. 15 / 815,532, filed November 16, 2017, U.S. Application No. 16 / 115,370, filed August 28, 2018, U.S. Application No. 16 / 564,375, filed September 9, 2019, and U.S. Application No. 16 / 816,817, filed March 12, 2020, each of which is incorporated by reference in its entirety.

[0045] 2.2 Cover Board and Optional Elements 2.2.1 Cover board 1B and 4, in some embodiments, the microwell assembly 100 can include a cover substrate 210 configured to mate with the base substrate 110. The cover substrate 210 functions to protect samples being processed and / or incubated in the base substrate 110 from contamination while allowing gas exchange with the environment (e.g., the environment surrounding the microwell assembly, the local environment between the base substrate 110 and the cover substrate 210, etc.).

[0046] In terms of material composition, the cover substrate 210 can be composed of one or more of a polymer (e.g., polypropylene, polydimethylsiloxane, polystyrene, polyvinyl chloride, polymethyl methacrylate, cyclic olefin copolymer, polycarbonate, silicone, polydimethylsiloxane), a silicon-derived material, glass, a metallic material, a ceramic material, a natural material, an elastomeric material, a porous material, a synthetic material, and / or any other suitable material. In particular, the selection of materials can be based on one or more of manufacturing considerations, surface properties desired for sample processing, optical properties, bulk properties (e.g., with respect to porosity, with respect to density, etc.), surface properties, thermal properties, mechanical properties, and / or any other suitable properties. Furthermore, all portions of the cover substrate 210 can be constructed using the same material(s), different materials (e.g., if each portion of the cover substrate 210 has different design constraints), and / or any combination of materials. Furthermore, the base substrate 110 can be a unitary body or a base substrate 110 having separate portions that are bonded together (e.g., during manufacturing).

[0047] With respect to optical properties, the material or materials of the cover substrate 210 can have any degree of transparency, elasticity, reflectivity, or other optical properties. For example, a material can be transparent to allow for optical analysis, examination, or observation (e.g., from the top surface of the cover substrate 210), but can also be opaque, transparent, translucent, and / or of any suitable opacity. For example, with respect to bulk properties such as porosity, if a high degree of porosity is desired (e.g., to provide gas exchange functionality), the cover substrate 210 need not be transparent.

[0048] With respect to bulk properties, the material(s) of cover substrate 210 can be configured to have a level of porosity that allows gas exchange between the sample and the environment during processing while preventing liquid exchange, thereby providing humidity control and preventing evaporation. Additionally or alternatively, with respect to bulk properties, the material(s) of cover substrate 210 can be configured to have a level of density or other bulk properties appropriate for sample processing and / or incubation purposes. In embodiments, cover substrate 210 can be constructed from or otherwise incorporate a polymer (e.g., polytetrafluoroethylene (PTFE), polyethylene (PE), polyvinyl alcohol (PVA), etc.), ceramic, or another suitable material (e.g., natural material, synthetic material) with an appropriate ingress rating (e.g., according to the IP scale, according to another rating scale) or particle retention properties (e.g., rated retention for particles less than 1 micron, rated retention for particles greater than 1 micron). In one example, the cover substrate 210 can be constructed of a PTFE-based material having an IP rating (e.g., IP65-69), although the cover substrate 210 can alternatively be constructed of a non-porous material that allows gas exchange with other elements of the microwell assembly 100. Additionally or alternatively, the cover substrate 210 can be converted from a porous state to a non-porous state to prevent contamination of the environment at the end of the assay.

[0049] With respect to surface properties, the material(s) of the base substrate 110 can be configured to have desired hydrophilic / hydrophobic properties (e.g., highly hydrophilic), determined, for example, by contact angle and wettability. With respect to other electrical and physical properties, the material(s) of the cover substrate 210 can be configured to have desired charge (e.g., related to the properties of the sample fluid and / or sample processing fluid used), field characteristics, conductivity, resistance, and / or any other suitable surface or physical property. Additionally or alternatively, the material(s) of the cover substrate 210 is preferably configured to be non-reactive with fluids used during sample processing.

[0050] With respect to thermal properties, the material or materials of the cover substrate 210 can be configured to have desired thermal properties with respect to heat transfer and / or heat retention properties. In particular, the cover substrate 210 can be configured to have desired thermal conductivity and / or heat capacity properties (e.g., as appropriate for sample incubation steps). In one aspect, the cover substrate 210 can be configured to have thermal properties that allow for efficient transfer of heat to and from the microwell assembly 100 during sample processing and / or incubation.

[0051] With respect to mechanical properties, the material or materials of the base substrate 110 can be configured to have desired mechanical properties, including one or more of stiffness, strength, elastic behavior, hardness, and other properties. For example, as shown in FIG. 5, an embodiment of the cover substrate 210 can be constructed of an elastomeric material that can elastically deform, and the reversible deformation of the elastomeric cover substrate 210 can enable modes of operation that facilitate sample handling. For example, as shown in FIG. 5, the elastic properties of the cover substrate 210 can provide a deformation mode of operation 211 in which a subvolume (e.g., an upper portion of fluid) is displaced from each micro-well of the sample processing region, and a relaxation mode of operation 212 in which the cover substrate relaxes to a baseline state, forming pockets (e.g., air pockets) between the cover substrate 210 and the micro-wells of the base substrate. Such modes of operation can further enable the micro-well assembly 100 to process samples while providing humidity control, evaporation prevention, and prevention of crosstalk between the micro-wells of the base substrate 110 (e.g., after sample distribution to a set of sample processing regions). However, the cover substrate 210 can have other suitable mechanical properties to accommodate the operating mode.

[0052] In embodiments in which the cover substrate 210 is elastomeric, the cover substrate 210 can be constructed of an elastomer (e.g., a polyether / polyamide material, a polyurethane material, a polyester material, etc.), which may be porous to provide gas exchange with the environment. However, the cover substrate 210 can be constructed of other suitable materials (e.g., microporous polycarbonate, cellulose acetate, nitrocellulose, glass fiber, microporous nylon, polytetrafluoroethylene, regenerated cellulose, polyvinyl fluoride, polypropylene, microporous polyester, polyvinylidene fluoride, reproducibly charged nylon, etc.).

[0053] 6A and 6B, the cover substrate 210 defines a network of venting channels 220 that face the base substrate 110 (e.g., face the base substrate 110 when assembled with the cover substrate 210). The venting channels 220 preferably align with the set of sample processing regions when the base substrate 110 is mated with the cover substrate 210, thereby providing gas exchange between the contents of the base substrate 110 and the environment 50 surrounding the microwell assembly 100.

[0054] As shown in the cross-sectional image of FIG. 6A, the vent channels 220 can extend throughout the cover substrate 210 (e.g., along one or more axes) to allow gas exchange between the microwell subarrays of each sample processing region and the environment (e.g., the incubation environment). In the example shown in FIG. 6B, a first subset of channels 221 can extend through the cover substrate 210 parallel to a first axis, and a second subset of channels 222 can extend through the cover substrate 210 parallel to a second axis, such that the first subset of channels 221 and the second subset of channels 220 intersect each other (e.g., are perpendicular to each other and / or are in fluid communication with each other). However, the individual subsets of channels can be arranged in another suitable manner. While the example shown in FIGS. 6A and 6B shows the network of channels 220 open to the environment at the sides / periphery of the cover substrate 210, in other embodiments, the network of channels 220 can open to the environment at another suitable portion of the cover substrate 210 (e.g., by entering the thickness of the cover substrate 210 and opening at another surface). The vent channels 220 are preferably hydrophobic and have small capillary dimensions (e.g., <200 microns) to prevent fluid intrusion from the sample. Alternatively, the vent channels 220 can have another degree of hydrophobicity and / or capillary dimensions of 200 microns or greater. Additionally or alternatively, the dimensions of the vent channels can be smaller (e.g., less than 50% and / or substantially less than 25% of the cross section of the characteristic microwell dimensions). However, the vent channels 220 can have other suitable dimensions relative to the characteristic microwell dimensions.

[0055] In forming an assembly, the cover substrate 210 can mate with the base substrate 110 by including an interlocking mechanism (e.g., a first locking portion on the cover substrate 210 that is complementary to a second locking portion on the base substrate 110). For example, as shown in FIG. 4 , the cover substrate can include a set of lips or tabs that engage with a surface (e.g., bottom surface, outer periphery) of the base substrate 110, thereby providing a bond between the base substrate 110 and the cover substrate 210. In other embodiments, the bond can be provided by another suitable mechanism (e.g., a press-fit mechanism, a snap-fit ​​mechanism, a magnetic mechanism, an adhesive mechanism, a gravity mechanism, etc.). The bond between the base substrate 110 and the cover substrate 210 can be reversible or permanent. In still other embodiments, the cover substrate 210 may not be configured to bond with the base substrate 110.

[0056] In yet other embodiments, the cover substrate 210 can have the form of one or more films (e.g., adhesive films, porous films, porous adhesive films) covering a set of sample processing areas. For example, in one such embodiment, the cover substrate 210 can include a set of films (e.g., porous adhesive films) corresponding to the set of sample processing areas or the number of samples to be processed, and the set of films can be affixed to the base substrate after sample dispensing. The set of films can then be removed from the base substrate 110 as needed (e.g., by a user, by an embodiment of the described automated platform 300, etc.) according to the various assays being performed and / or the operating modes described in more detail below.

[0057] 2.2.2 Optional Elements The cover substrate 210 can also be separated from the base substrate 110 by one or more functional layers. For example, as shown in FIG. 7A , the cover substrate 210 can be separated from the base substrate 110 by a film layer 225, which can facilitate bonding between the cover substrate 210 and the base substrate 110, provide additional separation between adjacent sample processing regions to prevent sample crosstalk, perform a sealing function for the microwells, and enable gas exchange between the samples in the base substrate 110 and the environment. More specifically, the film layer 225 and / or the cover substrate 220 can function to prevent fluorescent and / or chromogenic substrates or other sample processing materials / reagents from contaminating adjacent microwell partitions, promote growth of targets (e.g., bacteria, yeast, mold, etc.) for detection, enable gas exchange, and prevent evaporation, all while minimizing manufacturing costs.

[0058] The film layer 225 can be entirely located between the cover substrate 210 and the base substrate 110. Alternatively, as shown in FIG. 7B, the cover substrate 210 can be configured to form a boundary around the base substrate 110, and the film layer 225 can bond the base substrate 110 to the cover substrate 210 while interacting with the sample processing regions to provide gas exchange. In an embodiment, the film layer 225 can be composed of a porous polymer (e.g., a polyether / polyamide material, a polyurethane material, a polyester material, a nylon material, etc.) to provide gas exchange with the environment. However, the film layer 225 can be composed of other suitable materials. For example, in some applications, the film layer 225 can be replaced or supplemented by a hydrogel material that binds a sample processing substrate (e.g., a fluorescent substrate, a colorimetric substrate) for detection, where the hydrogel material is delivered to the set of sample processing regions in a fluid state and transitions to a sol state during incubation. The film layer 225 can be continuous or divided into a number of subregions corresponding to the set of sample processing regions or the number of samples to be processed.

[0059] Additionally, the microwell assembly may include multiple membranes depending on the design features and / or intended use of the system 100 .

[0060] Although embodiments of porous base substrate 110 and / or cover substrate 210 materials have been described, one or more of the base substrate 110 and cover substrate 210 may not be porous, as shown in FIG. 8, and may not be composed of a material that promotes gas exchange with the environment.

[0061] Furthermore, embodiments of microwell assembly 100 may additionally or alternatively include or support other suitable elements (e.g., oil layers, other partitioning material layers, buoyant hydrophobic particles, buoyant self-polymerizing materials, biological membranes, cell layers, biological coatings, sample processing substrates contained or bound to the microwell surface, composition media, diluent media, media provided in a lyophilized state, etc.) that facilitate gas exchange with the environment and prevent sample crosstalk while facilitating sample processing and / or sample incubation.

[0062] 3. Platform As shown in FIG. 9 , one embodiment of a platform 300 for automated sample processing (e.g., for processing samples using units of the microwell assembly 100 described above) includes a deck 310 that supports and positions a set of sample processing elements, a gantry 370 that actuates tools for interacting with the set of sample processing elements supported by the deck 310, and a base 380 that supports various processing subsystems and a control subsystem that communicates with the processing subsystems and controls the state of the deck 310, the set of sample processing elements, and the gantry 370 to transition the platform 300 between various operational modes. In an example, the platform 300 may provide high-throughput sample loading capabilities (e.g., loading samples in less than 60 seconds per sample, processing more than 600 samples in 8 hours, etc.) and / or high-throughput sample reading capabilities (e.g., reading samples faster than 10 seconds per sample, reading more than 500 samples per hour, etc.). Embodiments, implementations, and examples of operational modes providing various workflows are described in more detail in Section 4, below.

[0063] 3.1. Deck and Deck Supporting Elements As shown in FIG. 9 , deck 310 functions as a platform for supporting and positioning one or more components (e.g., a top broad plane, top and bottom broad planes, sides, etc.) for automated processing of samples using the units of microwell assembly 100 described above. Additionally, deck 310 can function to position one or more components to align with or otherwise interact with a fluid processing subsystem, an imaging subsystem, a gripping / manipulation subsystem, and / or other subsystems coupled to gantry 370 and / or base 380, as described below. In this regard, deck 310 can be fixed as a reference platform, while other components are actuated into position for interacting with elements of deck 310. Alternatively, deck 310 can be coupled to one or more actuators for positioning elements of deck 310 to interact with other subsystems.

[0064] 9 , deck 310 provides a platform for supporting a set of sample processing elements, which may include disposable and / or reusable components, including containers for housing sample processing materials and / or tools for processing samples (e.g., in connection with fluid processing, in connection with material separation, in connection with heating and cooling, etc.). In an embodiment, deck 310 may support a set of sample processing elements, including one or more units of reagent cartridges 320, units of microwell assembly 100 described above (e.g., microwell assemblies in storage, microwell assemblies disassembled with separate base substrate 110 and cover substrate 210, and microwell assemblies in use for processing samples), sample staging container 330 (for staging samples prior to transfer to microwell assembly 100), tool container 340, and / or other subsystems.

[0065] Additionally or alternatively, deck 310 may include other suitable components associated with an imaging subsystem (e.g., a fluorescence detection subsystem, a brightfield camera subsystem, a confocal microscope subsystem, a spectroscopic detection subsystem, a total internal reflection fluorescence (TIRF) subsystem, a nuclear magnetic resonance (NMR) subsystem, a Raman spectroscopy (RS) subsystem, a cell phone with optical accessories to improve pixel resolution, etc.) Additionally or alternatively, deck 310 or other components of platform 300 may include a barcode reader to support operations related to reading and tracking information with system components (e.g., disposables) and samples for traceability, as described in the following incorporated-by-reference applications:

[0066] The sample processing elements may be supported in the same plane by the deck 310, or alternatively, may be supported in different planes. Preferably, the separate elements supported by the deck do not overlap, although alternative embodiments of the deck 310 may support the sample processing elements in an overlapping manner (e.g., to conserve space, etc., for operational efficiency, etc.).

[0067] 3.1.1 Deck-Supported Elements: Reagent Cartridges As shown in FIG. 9 , deck 310 includes at least one area for supporting a unit of reagent cartridge 320, which functions to contain materials in one or more compartments for microbial cell capture and / or sample processing according to one or more workflows for various applications. To this end, reagent cartridge 320 can define a set of storage volumes distributed across a set of domains, which can be configured to provide an environment appropriate for the material content of each domain. The set of storage volumes can directly contain sample processing materials and / or can alternatively be configured to receive and maintain the position of individual containers (e.g., tubes, etc.) containing sample processing materials. The storage volumes of each domain can be distributed in an array or otherwise arranged. While reagent cartridge 320 is described as being supported by deck 310, embodiments of reagent cartridge 320 can alternatively be configured to operate independently of deck 110.The reagent cartridge 120 may further additionally or alternatively be any of U.S. application Ser. No. 16 / 867,235 filed May 5, 2020, U.S. application Ser. No. 16 / 867,256 filed May 5, 2020, U.S. application Ser. No. 16 / 816,817 filed March 12, 2020, U.S. application Ser. No. 16 / 564,375 filed September 9, 2019, U.S. application Ser. No. 16 / 115,370 filed August 28, 2018, U.S. application Ser. No. 16 / 115,059 filed August 28, 2018, U.S. application Ser. No. 16 / 048,104 filed July 27, 2018, U.S. application Ser. No. 16 / 048,104 filed July 30 ... No. 15 / 720,194, filed September 29, 2017, U.S. Application No. 15 / 430,833, filed February 13, 2017, U.S. Application No. 15 / 821,329, filed November 22, 2017, U.S. Application No. 15 / 782,270, filed October 12, 2017, U.S. Application No. 16 / 049,240, filed July 30, 2018, and U.S. Application No. 15 / 815,532, filed November 16, 2017, all of which applications are incorporated by reference in their entireties.

[0068] 3.1.2 Deck-supported elements: tool containers As shown in FIG. 9 , deck 310 includes at least one area for supporting a unit of tool container 340, which functions to position tool container 340 relative to fluid handling devices of gantry 370, described below. Tool container 340 functions to house one or more units of various tools for fluid aspiration, fluid delivery, fluid dispensing, separation of target from non-target materials in a sample, and / or other tools in one or more compartments according to one or more workflows for various applications. As such, tool container 340 can facilitate the transfer and / or mixing of reagents with a sample, fluidly couple and / or detach elements in various areas of deck 310, facilitate the transfer of a microwell plate / lid from one location to another, or otherwise interact with one or more components of platform 300. While tool container 340 is described as being supported by deck 110, embodiments of tool container 340 can alternatively be configured to operate independently of deck 310.The tool container 340 may additionally or alternatively include any of U.S. application Ser. No. 16 / 867,235 filed May 5, 2020, U.S. application Ser. No. 16 / 867,256 filed May 5, 2020, U.S. application Ser. No. 16 / 816,817 filed March 12, 2020, U.S. application Ser. No. 16 / 564,375 filed September 9, 2019, U.S. application Ser. No. 16 / 115,370 filed August 28, 2018, U.S. application Ser. No. 16 / 115,059 filed August 28, 2018, U.S. application Ser. No. 16 / 048,104 filed July 27, 2018, U.S. application Ser. No. 16 / 048,104 filed July 30 ... No. 15 / 720,194, filed September 29, 2017, U.S. Application No. 15 / 430,833, filed February 13, 2017, U.S. Application No. 15 / 821,329, filed November 22, 2017, U.S. Application No. 15 / 782,270, filed October 12, 2017, U.S. Application No. 16 / 049,240, filed July 30, 2018, and U.S. Application No. 15 / 815,532, filed November 16, 2017, all of which applications are incorporated by reference in their entireties.

[0069] 3.1.4 Heating and / or Cooling Subsystem The deck 310 may additionally or alternatively include or support a heating and cooling subsystem 350 that functions to transfer heat to and / or from desired regions of the substrates (e.g., base substrate 110, cover substrate 210), reagent cartridge 320, tool container 340, and / or other components. The heating and cooling subsystem 350 may additionally or alternatively function to maintain a desired temperature within the interior volume of the platform 300. In embodiments, the heating and cooling subsystem 350 may include one or more units of heating elements (e.g., Peltier heating elements, resistive heating elements, other heating elements), cooling elements (e.g., Peltier cooling elements, chilled aluminum blocks, fluid path systems for circulating coolant, etc.), thermal contacts or non-contacts for transferring heat between the heating and cooling elements and other objects, heat sinks, fans, temperature sensors, and thermal control circuitry (e.g., having electrical coupling with processing elements of the base 180, described below). In embodiments, the one or more cooling elements can maintain the storage volume and / or sample at 2-8° C., more preferably 4° C. Additionally or alternatively, the cooling elements can maintain the one or more storage volumes / samples at any suitable temperature (e.g., below 2° C., above 8° C., etc.).

[0070] One or more portions of heating and cooling subsystem 350 may extend into openings in deck 310 to thermally interact with or otherwise couple to desired portions of other system elements supported by deck 310 to provide heat transfer functionality for various applications. Alternatively, deck 310 may be constructed of thermally conductive material in desired areas for heat transfer applications, and portions of heating and cooling subsystem 350 may be configured to contact areas of the thermally conductive material of deck 310 for heat transfer.

[0071] In embodiments, the heating and cooling subsystem 350 may include a set of thermal bodies (e.g., that may be coupled to heat sink elements) to provide a larger surface area for heat transfer. Additionally, the area between the deck 310 and other volumes of the platform 300 may include one or more fans and / or ducts to provide a thermal mechanism for convective heat transfer away from the set of thermal bodies and / or other system components as needed. Additionally, in such embodiments, one or more portions of the heating and cooling subsystem 350 (e.g., thermal bodies, etc.) may include features that facilitate holding a corresponding cartridge (e.g., reagent cartridge, substrate, etc.) in place.

[0072] In embodiments, the thermal body and / or one or more of the other portions of the heating and cooling subsystem 350 may be coupled to actuators that move the thermal body into or out of thermal communication with elements supported by the deck 310, although embodiments of the system 100 may omit actuators in the heating and cooling subsystem 350.

[0073] The heating and cooling subsystem 350 may additionally or alternatively be any of U.S. application Ser. No. 16 / 867,235 filed May 5, 2020, U.S. application Ser. No. 16 / 867,256 filed May 5, 2020, U.S. application Ser. No. 16 / 816,817 filed March 12, 2020, U.S. application Ser. No. 16 / 564,375 filed September 9, 2019, U.S. application Ser. No. 16 / 115,370 filed August 28, 2018, U.S. application Ser. No. 16 / 115,059 filed August 28, 2018, U.S. application Ser. No. 16 / 048,104 filed July 27, 2018, U.S. application Ser. No. 16 / 048,104 filed July 30 ... No. 16 / 049,057, filed September 29, 2017, U.S. Application No. 15 / 720,194, filed September 29, 2017, U.S. Application No. 15 / 430,833, filed February 13, 2017, U.S. Application No. 15 / 821,329, filed November 22, 2017, U.S. Application No. 15 / 782,270, filed October 12, 2017, U.S. Application No. 16 / 049,240, filed July 30, 2018, and U.S. Application No. 15 / 815,532, filed November 16, 2017, all of which applications are incorporated by reference in their entireties.

[0074] 3.1.4 Gantry 9 , the platform 100 can include a gantry 370 coupled to the deck 110, which functions to support and / or enable actuation of one or more tools for various interactions with elements of the deck 110 along a set of axes. In an embodiment, the gantry 370 includes one or more rails / tracks for moving a tool, such as a pipettor 374 having a pipette interface and / or a gripping tool 375 (e.g., for gripping a microwell assembly portion and / or other tool, etc.), within three-dimensional space (e.g., a three-dimensional volume enclosed by a first side of the deck 310). In an embodiment, a tool actuated using the gantry 370 can move relative to a sample processing consumable, a reagent cartridge 320 unit, a tool container 340, or other element to transfer material across various components supported by the deck 310. Additionally or alternatively, tools supported by gantry 370 can be used to image and / or read barcodes (e.g., associated with identifying proper setup for a run, inventory control, etc.) associated with the various disposables supported by deck 310. For example, as shown in FIG. 9, gantry 370 can support or be coupled to a camera 376 (e.g., a fluorescent imaging camera, a brightfield imaging camera, etc.) for sample reading. Additionally or alternatively, camera 376 can be coupled to another portion of platform 300 (e.g., based on the orientation of the imaging relative to the surface of microwell assembly 100 configured to optically detect a signal).

[0075] Gantry 370 preferably can move one or more tools along one or more axes parallel to the broad surfaces of reagent cartridge 320, sample processing disposables (e.g., microwell assembly units), and tool container 340, and additionally along axes perpendicular to the broad surfaces. Gantry 370 can additionally or alternatively enable movement along a subset of those directions or along any other suitable direction. To enable movement, gantry 370 includes or is otherwise coupled to one or more motors (e.g., a motor for each axis or direction of movement), one or more encoders for position identification in each axis or direction of movement, and / or one or more switches (e.g., optical switches for each axis) for control of gantry 370 (e.g., if the switches are electrically coupled to the control circuitry described in connection with base 180 below).

[0076] 9, the gantry 370 can include and / or be configured to interact with a pipettor 374 that functions to hold, move, and / or otherwise interact with any number of tips or other tools, such as those of the tool container 340 described above. In an embodiment, the pipettor 374 assembly can include one or more of a pump (e.g., a displacement pump) for providing a pressure differential for fluid delivery and aspiration, a pressure sensor for detecting pipetting pressure, a level sensor for detecting fluid level within the pipettor 374, a tip detector (e.g., capable of determining the presence or absence of a tip coupled to the pipettor 374), and a tip ejection motor coupled to a tip ejector for removing a tip from the pipettor 374.

[0077] The gantry 370, pipettor 374, camera / imaging element, and / or gripping element may additionally or alternatively be configured as described in U.S. application Ser. No. 16 / 867,235 filed May 5, 2020, U.S. application Ser. No. 16 / 867,256 filed May 5, 2020, U.S. application Ser. No. 16 / 816,817 filed March 12, 2020, U.S. application Ser. No. 16 / 564,375 filed September 9, 2019, U.S. application Ser. No. 16 / 115,370 filed August 28, 2018, U.S. application Ser. No. 16 / 115,059 filed August 28, 2018, U.S. application Ser. No. 16 / 048,104 filed July 27, 2018, U.S. application Ser. No. 16 / 115,059 filed July 27 ... This invention may include aspects described in U.S. Application No. 16 / 049,057, filed July 30, 2018, U.S. Application No. 15 / 720,194, filed September 29, 2017, U.S. Application No. 15 / 430,833, filed February 13, 2017, U.S. Application No. 15 / 821,329, filed November 22, 2017, U.S. Application No. 15 / 782,270, filed October 12, 2017, U.S. Application No. 16 / 049,240, filed July 30, 2018, and U.S. Application No. 15 / 815,532, filed November 16, 2017, all of which applications are incorporated by reference in their entireties.

[0078] 3.1.5 Other Platform Elements In some embodiments, platform 300 can include or support other sample processing elements (e.g., to provide functionality for other parts of the operation for determining food quality / food safety). In an embodiment, platform 300 can include or support a gas composition modulator with environmental control (e.g., for incubation and culture applications). Such subsystems can include one or more of heating elements, cooling elements, temperature sensors, gas composition sensors, vents, gas inlets, gas outlets, valves, and other suitable elements for environmental control within platform 300 or in a device separate from platform 300. In connection with environmental control, platform 300 can include various enclosures and / or chambers within which the sample environment can be controlled. The platform can regulate O2 and / or CO2 concentrations within microwells. The platform can include elements, such as UV lights, that allow for decontamination of the system between sample runs.

[0079] In an embodiment, the platform 300 (e.g., in the base 380) can support a control and processing architecture for one or more system functions including fluid delivery with respect to the pipettor 374 for sample processing, fluid level sensing (e.g., in the pipettor 374, various storage volumes of the reagent cartridge 320, etc.), actuation of the gripping mode of the gripping tool 375, thermal cycling and / or other heating or cooling functions of the reagent cartridge 320 and / or the microwell assembly 100, functions for control of the gantry 370, functions including receiving sensor signals and returning outputs, functions including receiving sensor signals and performing various operations, functions related to power management of the system, functions related to system status indication elements (e.g., lights, audio output devices, visual output devices, etc.), functions related to system input devices (e.g., buttons, keyboard, keypad, mouse, joystick, switches, touch screen, etc.), functions related to display devices, functions related to system data storage devices, functions related to system transmission devices (e.g., wired transmission devices, wireless transmission devices, etc.), and other suitable functions. In embodiments, platform 300 may support electronic subsystems (e.g., PCBs, power supplies, communication modules, encoders, etc.) associated with a processing architecture (e.g., on-board the system, separate from the system, etc.), or any other suitable components, where the processing architecture may include any or all of a processor (e.g., microprocessor), a controller (e.g., microcontroller), memory, storage, software, firmware, or any other suitable components. Additionally, the processing subsystem may include a machine vision module that functions to read tags, validate protocols, perform error detection (e.g., detect that a reagent does not match an assigned protocol), or perform any other function.

[0080] Embodiments, implementations, and examples of additional elements are described in U.S. Application No. 16 / 048,104, filed July 27, 2018; U.S. Application No. 16 / 049,057, filed July 30, 2018; U.S. Application No. 15 / 720,194, filed September 29, 2017; U.S. Application No. 15 / 430,833, filed February 13, 2017; U.S. Application No. 15 / 821,329, filed November 22, 2017; U.S. Application No. 15 / 782, filed October 12, 2017; ,270, U.S. Application No. 16 / 049,240, filed July 30, 2018, U.S. Application No. 15 / 815,532, filed November 16, 2017, U.S. Application No. 16 / 115,370, filed August 28, 2018, U.S. Application No. 16 / 564,375, filed September 9, 2019, and U.S. Application No. 16 / 816,817, filed March 12, 2020, which applications are incorporated by reference in their entireties.

[0081] 4. Usage and Use As shown in FIG. 10A, one embodiment of a method 400 for target detection and characterization may include step S410 of placing a set of sample processing elements including units of microwell assemblies on a deck of a sample processing system, step S420 of transferring samples and / or other substances between the sets of sample processing elements in an operational sequence, and step S430 of processing the set of samples for target detection in the units of microwell assemblies. Additionally or alternatively, the method 400 may be performed using any of the methods described in U.S. Application Nos. 16 / 048,104, filed July 27, 2018, 16 / 049,057, filed July 30, 2018, 15 / 720,194, filed September 29, 2017, 15 / 430,833, filed February 13, 2017, 15 / 821,329, filed November 22, 2017, 15 / 782,270, filed October 12, 2017, 15 / 782,270, filed May 10 ... This application may include any or all of the processes described in U.S. Application No. 16 / 049,240, filed July 30, 2018, U.S. Application No. 15 / 815,532, filed November 16, 2017, U.S. Application No. 16 / 115,370, filed August 28, 2018, U.S. Application No. 16 / 564,375, filed September 9, 2019, and U.S. Application No. 16 / 816,817, filed March 12, 2020, which applications are incorporated by reference in their entireties.

[0082] The method is preferably performed in an embodiment, implementation, or example of a system described above (e.g., in connection with transferring contents between various elements and / or processing samples), but may additionally or alternatively be performed in any other suitable system. The method 400 is further preferably at least partially automated (e.g., requiring a user to load reagents and select protocols, not requiring user intervention, etc.), but may additionally or alternatively be performed in one or more portions manually (e.g., for quality control steps, all protocols, rare protocols, etc.).

[0083] Specific workflows associated with the method 400 and system elements described above are described in further detail below, where a sample (e.g., a sample derived from a homogenized or non-homogenized consumable) can be processed according to the workflow.

[0084] 4.1 Methods – Example workflow for parallel MPN determination from multiple food samples As shown in FIG. 10B, an embodiment of method 400 configured for processing one or more consumable samples (e.g., individual food sample suspensions) for safety and quality assessment purposes can include step S410′ of placing a set of sample processing elements including a unit of microwell assemblies on a deck of a sample processing system; step S415′ of performing a series of sample preparation operations to process the set of samples; step S420′ of transferring the set of samples to a set of sample processing regions in a base substrate of the microwell assembly (e.g., using predetermined delivery of sample in droplet form across the surface of each microwell associated with each sample by capillary flow from one end of the sample processing region enabled by a spacing between the base substrate and a cover substrate of the microwell assembly); step S425′ of covering the base substrate with a cover substrate of the microwell assembly (e.g., to spread or divide the sample fluid); step S430′ of transferring the microwell assembly for further processing; and step S440′ of detecting one or more targets from the set of samples in the microwell assembly.

[0085] The method 400 functions to implement a system for rapid sample testing, with particular application to most probable number (MPN) determination. In particular, the method 400 can be used to process multiple samples in parallel (e.g., for food safety / food quality applications), in conjunction with enabling rapid operation of serial dilution tests to measure the concentration of target microorganisms in food test portions. During processing, the food test portions can be diluted to prepare a food suspension (e.g., by combining with specific broths / reagents to prepare the sample for analysis).

[0086] In embodiments, the system may position a set of sample processing elements, including a unit of microwell assembly, on the deck of the sample processing system in S410′ in preparation for processing samples in subsequent steps of method 400. The system may configure the set of sample processing elements for downstream processing steps (e.g., as shown in FIG. 9 ) using the gantry described above and / or by manual action by the system operator. In other aspects, step S410′ may configure the sample processing elements in another suitable manner to process multiple samples in parallel. In preparation for downstream sample processing operations, step S410′ may include moving the base substrate of the unit of microwell assembly to a liquid loading position of the platform and reading a plate identifier using a camera on the platform. The system may then match the plate identifier with the identifier of the sample for processing to confirm that the correct sample is being processed and associate the sample processing results with the run associated with the plate identifier. However, step S410′ may include other run preparation steps in other aspects.

[0087] Step S415' recites performing a series of sample preparation operations to process a set of samples. Step S415' functions to process sample material from consumables (e.g., food samples, beverage samples, etc.) so that it is distributed within a set of sample processing regions of the base substrate of the microwell assembly. In an embodiment, step S415' includes homogenizing (e.g., mechanically, chemically) the food test portion with a diluent (e.g., having an appropriate dilution factor); suspending the sample (e.g., with buffered peptone water, with other suspending agents); dissolving the volume of each sample to be processed (e.g., less than 1 mL, more than 1 mL) in a medium (e.g., a medium that suppresses background flora and promotes the growth of specific microorganisms, a medium to provide an appropriate dilution factor such as a 1:1 dilution factor, etc.), revelation chemicals including enzyme substrates (e.g., fluorescent substrates, chromogenic substrates, etc.), or other processing materials in dry or liquid form (e.g., to generate samples with viscosity characteristics and / or total volume suitable for handling and fluid delivery). The process may include one or more of the following: mixing with a soluble solid (e.g., spices, chocolate, food matrices with coloring / autofluorescence, or other consumables) to process samples with high inhibitory properties; filtering the sample (e.g., to remove particulates that may affect sample loading in the microwells, uniform distribution of the sample across microwells with different characteristic dimensions, clogging, detection, etc.) (where the limiting particulate size can be based on the pitch between adjacent microwells (e.g., filter sizes between 20 microns and 280 microns, other filter sizes); as well as any other suitable sample preparation steps. Step S415' may be performed using the fluid handling elements of the platform described in Section 3 above, or using other suitable device components.

[0088] In embodiments, the sample processing steps of block S415' can be used to generate MPN estimates with a lower detection limit of at least 10 cfu / g, an upper detection limit of 1,000,000 cfu / g, or other suitable detection limits (e.g., relative to a volumetric measure such as cfu / mL).

[0089] Step S420′ recites transferring the set of samples to a set of sample processing regions of a base substrate of a microwell assembly (e.g., by using predetermined delivery of samples in droplet form across the surface of each microwell associated with each sample, such as by capillary flow from one end of the sample processing regions enabled by spacing between the base substrate and the cover substrate of the microwell assembly). Step S420′ can be performed using the fluid handling elements of the platform described in Section 3 above, or using other suitable devices. In embodiments, each of the set of samples to be processed may be transferred to be sequentially received by the sample processing regions of the base substrate. Alternatively, the set of samples may be dispensed simultaneously (e.g., using a multi-head fluid dispensing device, using other devices) into the sample processing regions of the base substrate. Notably, transferring the set of samples to the set of sample processing regions of a base substrate of a microwell assembly in step S420′ automatically divides the set of samples for rapid sample testing (e.g., MPN determination) using embodiments, implementations, and examples of a set of gradiently arranged microwell subarrays.

[0090] Step S425' recites covering the base substrate with a cover substrate of the microwell assembly, which functions to protect and facilitate maintaining a suitable environment for the set of samples during downstream processing and incubation steps. In embodiments, step S425' can be performed using elements (e.g., modified gripping elements) coupled to the platform gantry, as described in Section 3 above, or with other suitable devices. In embodiments, as described in Section 2 above, the cover substrate can include ventilation channels and / or be constructed of a porous material, which functions in step S425' to prevent sample cross-contamination, prevent sample evaporation, and allow gas exchange between the samples and the environment during incubation / incubation. Additionally or alternatively, as shown in FIG. 5, the elastomeric properties of the cover substrate can be used in step S425' to provide pockets (e.g., air pockets) over each of the set of samples during processing to further prevent sample crosstalk. However, step S425' can also be performed in another suitable manner.

[0091] Step S430' recites transferring the microwell assembly for further processing and functions to stage the microwell assembly along with the set of samples for incubation, culturing, or other processing steps. Step S430' can be performed using an element (e.g., a gripping element) coupled to the gantry of the platform described in Section 3 above, or using other suitable devices. In embodiments, the system can transport the microwell assembly to a location on the platform where an operator can transfer the microwell assembly to another device for incubation, culturing, or further processing steps. Alternatively, the system can transfer the microwell assembly to a location on the platform where incubation, culturing, or further processing steps can be performed automatically. When transferring the microwell assembly, the system preferably operates (e.g., using a gantry and tool) in a manner that minimizes physical disturbance of the samples in the set of microwells, although step S430' can alternatively be performed in another suitable manner.

[0092] Step S440' recites detecting one or more targets from the set of samples in the microwell assembly. Block S440' can be performed after a suitable incubation period (e.g., 24 hours, less than 24 hours, more than 24 hours). Step S440' can be performed using a camera element (e.g., a fluorescent imaging element, a bright-field imaging element) coupled to the gantry and / or base of the platform described in Section 3 above, or using other suitable equipment. For example, a separate readout subsystem can be implemented for operations performed outside the platform (e.g., in the context of incubation). The output of step S440' can include MPN estimates for each sample, along with confidence limits. More specifically, a computing component in communication with the imaging subsystem can implement an algorithm to process the microwells of each microwell subarray in which a signal (e.g., a fluorescent signal, a colorimetric signal, etc.) is detected and return an analysis indicating the MPN or other quality- and safety-related statistics for each of the set of samples. Detection can be based on fluorescence and / or other optically detected signals (e.g., based on enzyme substrates linked to fluorophores, fluorescent or colorimetric pH indicators), and multiple and / or different fluorophores can be used depending on the intended target for detection.

[0093] In an embodiment of step S440′, the system can process the image of the microwell assembly after sample processing and incubation with one or more transformation algorithms (e.g., Hough transform, filtering operations, fitting operations, microwell recognition operations, registration operations, etc.) for detection of fluorescent / colorimetric signals and related properties (e.g., intensity), and / or perform other appropriate image processing operations. The system can then generate an analysis based on one or more of: aggregate referencing, estimation algorithms (e.g., Thomas's rule), confidence limit determination methods (e.g., Haldane's method, etc.), boundary approximation approaches (e.g., Blodgett's method, etc.), particle counting statistics (e.g., particle / cell counting methods via Poisson statistics applied to microwell arrays), applying error corrections related to partitioning and / or subsampling errors, performing virtual partitioning, or other suitable methods. Additionally or alternatively, once the threshold detection limit is reached, the plate can be sealed to prevent further interaction with and / or contamination of the environment.

[0094] In embodiments of method 400, the medium composition, substrate, incubation conditions, and detection system depend on the target or targets to be detected from a set of samples. For example, to detect total viable count, an exemplary medium base can include plate count agar (e.g., non-selective plate count agar), with a medium composition of tryptone, yeast extract, and glucose, a detection principle based on a substrate targeting general enzyme activity or viability, and an incubation time of less than 72 hours at 30° C. To detect Enterobacteriaceae, an exemplary medium base can include violet red bile glucose (e.g., selective violet red bile glucose), with a medium composition of peptone, yeast extract, glucose, crystal violet, sodium, bile salts / sodium deoxycholate, a detection principle based on pH indication, and an incubation time of less than 24 hours at 30° C. For coliform detection, an exemplary medium base can include Violet Red Bile Lactose (e.g., Selective Violet Red Bile Lactose), with a medium composition of peptone, yeast extract, lactose, crystal violet, sodium, bile salts / sodium deoxycholate, detection based on pH indication, and incubation time of less than 24 hours at 37°C. Furthermore, for detection based on β-galactosidase enzyme substrate, the medium used must be lactose-free to avoid acidification of the medium (and suppression of fluorescence). In such an embodiment, Rapid E. coli medium (e.g., a medium containing peptone, yeast extract, sodium chloride, bile salts / sodium deoxycholate + β-galactosidase enzyme substrate) can be considered. For detecting E. coli, an exemplary medium base can include tryptone bile X-glucuronide (e.g., selective tryptone bile X-glucuronide), the medium composition is peptone / tryptone, yeast extract, bile salts / sodium deoxycholate, the detection principle is based on a substrate that interacts with the beta-glucuronidase enzyme, and the incubation time is less than 24 hours at 44°C.For detecting yeast and mold, an exemplary medium base can include YGC medium, which consists of yeast extract, glucose, and chloramphenicol. The detection principle is based on several nonspecific enzyme activities, and the incubation time is less than 72 hours at 30°C. This system can also perform multi-target detection of E. coli / coliform bacteria. However, other medium compositions, substrates, incubation conditions, and detection systems can be used depending on the intended targets.

[0095] However, one or more embodiments of the system may be configured to implement other workflows, including implementations of the described workflows, and / or other workflows.

[0096] 4.1.1 Method - MPN Counting In connection with the method 400 described above and / or other related methods, the principles and procedures of MPN can be applied as follows.

[0097] principle: Test portions of samples processed according to the above-described embodiments, implementations, and / or examples can be inoculated into media (e.g., liquid media, dry media, rehydrated dry media, etc.) designed to support the growth of a specific microorganism or group of microorganisms and / or inhibit the growth of non-target microorganisms. Various criteria (e.g., visual detection of turbidity, gas evolution, color change, subsequent separation of the microorganism on selective agar media, other mechanisms, etc.) can be used to determine whether growth of the target microorganism has occurred. The composition of the growth media and the criteria for discriminating between positive and negative results are further described below. Using these approaches, only a qualitative value can be attributed to each test portion (i.e., the result will be either positive or negative). To obtain an estimate of the amount of microorganism present, several test portions must be tested and a most probable number (MPN) determined using statistical procedures.

[0098] Inoculation Procedure:When using selective growth media, the addition of the test moiety must not reduce their selective properties, allowing the growth of non-target microorganisms. While most standards provide information on the compatibility of specific matrices with liquid media, caution is advised for some matrices that may contain growth inhibitors (e.g., spices, cocoa, bouillon, etc.). When such matrices are present, sample and matrix processing methods involving neutralizing compounds can be implemented using high dilution factors, centrifugation, buoyancy-based separation (e.g., involving binding of target sample materials to buoyant particles and washing of non-target matrix materials), filtration, immunomagnetic separation to separate target microorganisms from the matrix, and / or other mechanisms to mitigate the effects of the problematic matrix. If incompatibility is due to the biological composition of the matrix (e.g., heavily contaminated environmental samples, fermentation products, products containing probiotic bacteria, etc.), this method can be further implemented using spike-based experiments and / or the generation of appropriate controls.

[0099] In an example, a small-volume (e.g., less than 1 milliliter, 1 milliliter, etc.) test portion can be added to a volume (e.g., 5-10 times the volume) of a single-strength medium. In an example, a medium-volume (e.g., 1 milliliter to 100 milliliters) test portion can be added to a volume (e.g., an equal volume) of a higher-strength (e.g., double-strength) medium. In an example, a large-volume (e.g., more than 100 milliliters) test portion can be combined with a more concentrated medium. For special purposes, as described in the above system and platform embodiments, a sterile dehydrated medium can be dissolved in the sample (e.g., a cold or pre-warmed sample) for analysis. In an example, the time lag between preparing the first dilution of the sample and inoculating the last portion must be less than a threshold time (e.g., 15 minutes, another appropriate time) by implementing aseptic procedures. The inoculated partitions are then incubated for an appropriate incubation period and / or temperature (e.g., depending on the target microorganism involved). For some target microorganisms, multi-step incubation procedures and / or confirmation steps can be implemented. The criteria for distinguishing between positive and negative results may vary for each microorganism or group of microorganisms. Using these criteria, the MPN determination method counts the number of positive results obtained in all test portions derived from a single sample.

[0100] Selection of inoculation system:According to the MPN method, a sample is diluted across multiple partitions to the extent that the inoculum sometimes, but not always, contains viable microorganisms of interest. The "result" (i.e., the number of inocula that result in growth at each dilution) allows for an estimation of the initial concentration of one or more microorganisms in the sample. To obtain estimates of a wide range of possible concentrations, serial dilutions and / or incubations in multiple partitions (e.g., tubes, plate wells, the microwell system described above, emulsion droplets, etc.) can be used. Based on the number of positive and negative partitions observed after incubation at one or more dilutions, the estimated MPN of the microorganisms present in the original sample, and the accuracy of that estimate, can then be calculated using statistical procedures. The MPN inoculum system can be selected based on one or more of the expected number of microorganisms in the sample under investigation, regulatory requirements, the required accuracy, and other practical considerations. Measurement uncertainty depends on the number of observed positive test fractions and increases as a function of the square root of the number of partitions used. To halve the measurement uncertainty, the number of tubes must be quadrupled. When using a system with only a small number of replicate partitions, the measurement uncertainty is low.

[0101] Inoculation System Implementation - Single Dilution System: If the expected microbial concentrations are low or expected to vary only moderately, a suitable inoculation system is a single series of equal test sections. If the expected ratio between the maximum and minimum numbers of microorganisms is less than about 25, 10 parallel test sections is the minimum number expected to work, and with 50 parallel sections, a ratio of 200 is the limit.

[0102] Inoculation System Implementation - Multiple Dilution System:When the concentration of microorganisms in a sample is unknown or large variations are expected, a suitable inoculation system is a multiple dilution system, in which a series of partitions from multiple dilutions is performed. In such a platform, a sufficient number of dilutions are inoculated to ensure a system that produces both positive and negative results. The number of dilutions also depends on the calculation method used to estimate the MPN value (e.g., theoretical model, reference table, etc.).

[0103] Inoculation System Implementation - Symmetric Dilution System: In a symmetric MPN system, 3 or 5 parallel partitions (or another appropriate number of partitions) can be used per dilution. The precision obtained with this system decreases rapidly with fewer tubes per dilution. If greater precision is required, it is recommended to choose 5 or more partitions.

[0104] Inoculation System Implementation - Asymmetric Dilution System: In asymmetric systems, the number of tubes is not the same for different dilution levels. Such a configuration is suitable for estimating the number of microorganisms within a well-defined range (examples of which are given in ISO 8199).

[0105] Determining MPN value: In embodiments, the MPN value may be determined by one or more of the following methods: calculating a formula, referencing an MPN table, and utilizing other algorithms. These three methods are described in detail below.

[0106] Determining MPN value - Formula: The approximate MPN value for any number of dilutions and parallel tubes can be derived by applying the following formula: p is the number of positive partitions, m r is the sample reference mass (e.g., grams), m s is the total mass (e.g., grams) of the sample in all partitions with negative reactions, m t is the total mass (e.g., grams) of the sample in all partitions. TIFF2025148342000003.tif22170

[0107] The MPN value for a single series of partitions is derived from the following formula: where m r is the sample reference mass (e.g., grams), m m is the mass of the sample in each partition of the series (e.g., grams), ln is the natural logarithm, n is the number of partitions in the series, z p is the number of partitions with positive responses. TIFF2025148342000004.tif21170

[0108] The 95% confidence limits for the MPN estimate can be approximately calculated using the following formula: where x is the upper or lower 95% confidence limit, m r is the sample reference mass (e.g., grams), m m is the mass of the sample in each partition of the series (e.g., grams), ln is the natural logarithm, n is the number of partitions in the series, z n is the number of partitions with negative reactions. TIFF2025148342000005.tif35170

[0109] Log of symmetric multiple dilution MPN system 10 The standard uncertainty can be obtained from the following formula, where SE is log 10 where f is the standard error of the MPN, f is the dilution factor between successive dilutions, and n is the number of partitions per dilution. TIFF2025148342000006.tif23170

[0110] Variations of the above formula can be adapted to volumetric parameters that allow for mass extraction from the associated volume, and / or the formula can be adapted to take volume into account.

[0111] Determining MPN Values ​​- Table:The values ​​in the table can be processed (e.g., by multiplying the MPN and 95% limit by a ratio such as [reference mass] / [test portion mass]) to express results per reference mass of sample (or volume, for liquid samples). In a symmetric system, the method can include performing multiple serial dilutions (e.g., three serial dilutions) with an appropriate number of replicates, an appropriate number of embodiments, implementations, and examples described above to support the system components. Using these methods, the number of positive results for each set of partitions can be obtained (e.g., by using the platform 300 described above), and the MPN value of the associated microorganism present in the reference volume of the sample can be obtained from the MPN table for the inoculation system used. Because various combinations of positive partitions can be statistically more likely than others, combinations of positive partition results can be classified into various categories (e.g., high probability results, medium probability results, low probability results, etc.).

[0112] Determination of MPN value - algorithm: In the method embodiments described above, various algorithms can be implemented (eg, using an MPN assay analyzer, or can be performed using another suitable system).

[0113] Additionally or alternatively, the methods may be adapted from those described in Appendix A and / or U.S. Patent Application No. 16 / 072,712, entitled "Digital Microbiology," filed January 25, 2016, which are incorporated by reference in their entirety.

[0114] 5. Conclusion The drawings illustrate the architecture, functionality, and operation of possible implementations of systems, methods, and computer program products according to preferred embodiments, exemplary configurations, and aspects thereof. In this regard, each block in a flowchart or block diagram may represent a module, segment, or portion of code, including one or more executable instructions for implementing one or more specified logical functions. It should also be noted that in some alternative aspects, the functions noted in the blocks may occur out of the order noted in the figures. For example, two blocks shown in succession may, in fact, be executed substantially simultaneously, or may be executed in the reverse order, depending on the functionality involved. It should also be noted that each block of the block diagrams and / or flowcharts, and combinations of blocks in the block diagrams and / or flowcharts, may be implemented by a dedicated hardware-based system that performs the specified functions or operations, or by a combination of dedicated hardware and computer instructions.

[0115] As can be appreciated from the above detailed description, drawings and claims, those skilled in the art can make modifications and variations to the preferred embodiments of the invention without departing from the scope of the invention as defined in the following claims.

Claims

1. 1. A system for target detection, comprising: A base substrate; a set of sample processing regions defined on a broad surface of the substrate, each of the set of sample processing regions comprising: a set of microwell subarrays arranged in a gradient fashion between the upstream and downstream ends of each sample processing region; and a boundary separating each sample processing area from an adjacent sample processing area.

2. 10. The system of claim 1, The set of microwell subarrays comprises a first microwell subarray including wells having a first characteristic dimension located at an upstream end, and a terminal microwell subarray including wells having a second characteristic dimension located at a downstream end.

3. 3. The system of claim 2, the first characteristic dimension is smaller than the second characteristic dimension, the initial microwell subarray occupies a first footprint on the base substrate, and the terminal microwell subarray occupies a second footprint on the base substrate that is larger than the first footprint.

4. 3. The system of claim 2, The system, wherein the wells of the initial subarray are arranged in a first filling configuration and the wells of the terminal subarray are arranged in a second filling configuration.

5. 10. The system of claim 1, A system characterized in that the base substrate is made of a material having a level of porosity that allows gas exchange between the contents of the wells of the set of sample processing areas and the environment of the system while preventing liquid exchange.

6. 10. The system of claim 1, The system, wherein the set of sample processing regions includes dry sample processing materials including at least one of a medium, a fluorescent substrate, and a colorimetric substrate.

7. 10. The system of claim 1, 1. A system, wherein each sample processing area has a total volume of 0.01 to 10 milliliters, and each of said sets of microwell subarrays has 10 to 100,000 partitions, corresponding to a most probable number (MPN) range of 5 to 3,000,000 for an MPN assay.

8. 10. The system of claim 1, The system wherein the boundary includes a recessed channel operable to receive overflow sample.

9. 10. The system of claim 1, The system further comprises a cover substrate configured to mate with the base substrate in a bonding mode, the cover substrate comprising a network of ventilation channels that align with the set of sample processing regions when the base substrate and the cover substrate are mated in the bonding mode, the network of ventilation channels providing gas exchange between the base substrate and an environment surrounding the microwell assembly.

10. 10. The system of claim 9, A system characterized in that the cover substrate is composed of a material having a level of porosity that allows gas exchange between the contents of the system and the environment of the system while preventing liquid exchange.

11. 10. The system of claim 9, The system, wherein the cover substrate is constructed from an elastomeric material and provides a deformation mode of operation in which sub-volumes of fluid are displaced from a sample dispensed into the set of sample processing regions in the base substrate, and a relaxation mode of operation in which the cover substrate relaxes to a baseline state to create pockets between the cover substrate and the microwells of the base substrate.

12. 10. The system of claim 9, the network of ventilation channels includes a first subset of channels extending through the cover substrate parallel to a first axis and a second subset of channels extending through the cover substrate parallel to a second axis, the first subset of channels intersecting the second subset of channels.

13. 10. The system of claim 9, The system, wherein the network of ventilation channels is open to the environment of the system at the periphery of the cover substrate.

14. 10. The system of claim 9, The system, wherein the cover substrate includes a first locking portion complementary to a second locking portion of the base substrate.

15. 10. The system of claim 9, The system, wherein the cover substrate is separated from the base substrate by one or more functional layers.

16. 16. The system of claim 15, The system, characterized in that the one or more functional layers include a porous adhesive film layer disposed between the cover substrate and the base substrate and providing gas exchange from the wells of the set of sample processing areas.

17. 1. A system for target detection, comprising: A base substrate; a set of sample processing regions defined on a broad surface of the substrate, each of the set of sample processing regions comprising: a set of microwell subarrays arranged in a gradient fashion between the upstream and downstream ends of each sample processing region; a set of sample processing areas, each sample processing area having a boundary separating it from an adjacent sample processing area; and a cover substrate configured to mate with the base substrate in a mating mode, the cover substrate including a network of ventilation channels that align with the set of sample processing regions when the base substrate and the cover substrate are mated in the mating mode, the network of ventilation channels providing gas exchange between the base substrate and an environment surrounding the microwell assembly.

18. 18. The system of claim 17, the set of microwell subarrays includes a first microwell subarray including wells having a first characteristic dimension located at an upstream end and a terminal microwell subarray including wells having a second characteristic dimension located at a downstream end, the first characteristic dimension being smaller than the second characteristic dimension.

19. 18. The system of claim 17, A system characterized in that at least one of the base substrate and the cover substrate is made of a material having a level of porosity that allows gas exchange between the contents of the wells of the set of sample processing areas and the environment of the system while preventing liquid exchange.

20. 18. The system of claim 17, The system, wherein the cover substrate is constructed from an elastomeric material and provides a deformation mode of operation in which a subvolume of fluid is displaced from a sample dispensed into the set of sample processing regions of the base substrate, and a relaxation mode of operation in which the cover substrate relaxes to a baseline state to create a pocket between the cover substrate and the microwells of the base substrate.

Images