Systems and methods for target detection applicable to the characterization evaluation of food quality and the improvement of food safety

Abstract

A system, method, and platform for target detection includes 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 including a set of microwell subarrays arranged at a gradient between the upstream and downstream ends of each sample processing region and a boundary separating each sample processing region from an adjacent sample processing region, and a cover substrate configured to mate with the base substrate in a binding mode, the cover substrate including a network of vent channels that align with the set of sample processing regions when the base substrate and cover substrate are mated in the binding mode, the network of vent channels providing gas exchange between the base substrate and the environment surrounding the microwell assembly. The present invention can be used in MPN assays.

Description

Technical Field

[0001] The present invention generally relates to the field of food safety, and more specifically, to novel and useful systems and methods for target detection applied to the characterization of food quality and the improvement of 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, the entire disclosure of which is hereby incorporated by reference herein.

Background Art

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

[0004] The food industry is subject to numerous requirements for monitoring a large number of parameters related to food safety and hygiene, including contaminants, pathogens, and quality indicators, across various stages from the production, procurement, and handling of raw materials to the manufacturing, distribution, and consumption of finished products. One aspect of these requirements is the counting of microbial food quality indicators (QIs). The characterization of such QIs provides indicators related to the quality of the product (e.g., for spoilage, shelf life) and the hygiene of the process and helps predict the occurrence of pathogenic bacteria. There are several methods for counting QIs in food and environmental samples, with the most widely used being colony counting methods (e.g., implementation of agar media, implementation of ready-to-use pads containing dry reagents, etc.), the most probable number (MPN) method. However, such methods are prone to one or more of human error, inherent assay variability, low technical reproducibility, long time to results, lack of automation, inhibition related to detection, limitations in the counting range, low sensitivity, analog as opposed to digital readout, high cost, high waste, complex workflows, low throughput, and incompatibility with the food matrix. Furthermore, this method may require special reagents for the counting and characterization of various target microorganisms.

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

[0006] Therefore, there is a need for novel and useful systems and methods for target detection that can be applied to characterize the quality of food and improve food safety.

Brief Description of the Drawings

[0007]

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Embodiments for Carrying Out the Invention

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

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

[0010] In particular, the present invention can provide the advantage of providing an innovative solution for a target detection assay involving sample dispensing (e.g., for a MPN assay) in a rational, efficient and / or automated manner with respect to minimizing manual steps associated with dispensing. Further, the present invention includes an innovative design of sample processing consumables such that a plurality of samples can be processed in parallel in a throughput-high manner that is not cost-disadvantageous in various industries. Such a design optionally includes structures and functions that function to provide automated sample application and dispensing, controlled liquid diffusion, controlled sample containment, variable volume sample dispensing, humidity control, evaporation prevention, and / or crosstalk 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 form that significantly reduces human error, workflow complexity, inherent assay variability, and the time required for target characterization and counting.

[0012] The present invention also provides the advantage of automating and / or simplifying the processing steps, and in some aspects, can automatically dispense samples (e.g., samples having a volume of 1 mL or less, samples having a volume exceeding 1 mL) for a MPN assay. By efficiently processing small amounts, the present invention can also significantly reduce waste associated with assay operations, optimize the success of execution, and optimize consistency between executions and / or between different users.

[0013] In addition, in the embodiments, the present invention can also provide the advantage of significantly shortening the time required for a small amount of the converted enzyme substrate, such as a chromogenic substrate, to reach the detection threshold, thereby significantly shortening the time to the result and the inspection time. Further, by dividing the sample into small partitions in a rapid manner, the counting range is expanded and the accuracy of MPN estimation is improved.

[0014] The present invention also provides the advantage of alleviating the inhibition problems associated with the generation and / or colorimetric and / or fluorescence (e.g., multi-channel fluorescence) detection of the converted enzyme substrate.

[0015] Also, aspects of the present invention provide a structure and environment for growing microorganisms, and also provide the advantage of providing kits, compositions, methods, and devices for rapidly analyzing the growth and / or number of microorganisms in a cost-effective and time-efficient manner.

[0016] Furthermore, the present invention provides a closed system for the growth and detection of microorganisms, which can prevent laboratory contamination by potentially harmful pathogens.

[0017] Furthermore, through improvements in software and workflows, 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 system and / or method can provide any other suitable advantages.

[0019] 2. Microplate As shown in FIGS. 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 defined on a broad surface of the substrate 110 (including sample processing region 120), each of the set of sample processing regions including a set of microwell subarrays 130 disposed with a gradient between an upstream end 10 and a downstream end 90 of the sample processing region 120, and a boundary 190 separating the sample processing region 120 from an adjacent sample processing region. In relation to the gradient of the microwell subarrays for each sample processing region, a first microwell subarray 130 including wells having a first characteristic dimension (e.g., the smallest characteristic dimension) can be disposed at the upstream end 10, and a terminal microwell subarray 170 including wells having a second characteristic dimension (e.g., the largest characteristic dimension) can be disposed at the downstream end 90 of the sample processing region 120, with other aspects to be described in more detail below.

[0020] In some embodiments, as shown in FIG. 1B, the microwell assembly 100 can include a cover substrate 210 configured to mate with the base substrate 110 in a coupling mode, the cover substrate 210 including a network 220 of ventilation channels facing the base substrate 110 (in the coupling mode), the network of ventilation channels being aligned with the set of sample processing regions when the base substrate 110 and the cover substrate 210 are mated, and providing gas exchange between the base substrate 110 and the environment 50 (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 will be described in more detail below.

[0021] The microwell assembly 100 functions to provide a mechanism for low-cost and rapid sample dispensing and processing for specific applications in most probable number (MPN) determination. In particular, the microwell assembly can receive a sample volume and dispense it across a plurality of sub-arrays of microwells (e.g., with minimal user manual intervention), each sub-array having characteristic dimensions, thereby facilitating the rapid operation and execution of serial dilution tests across multiple samples in parallel (e.g., for food safety / food quality applications, other applications), and enabling the measurement of the concentration of one or more target microorganisms in the sample. 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. Also, the microwell assembly 100 can prevent cross-talk of liquids, pathogens, and / or conversion substrates between different samples being processed. Additional features of the microwell assembly 100 will be described in more detail below with respect to the individual elements of the microwell assembly 110.

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

[0023] In terms of the 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 suitable material. In particular, the selection of the material can be made based on one or more of manufacturing considerations, surface characteristics desirable for sample processing, optical characteristics, bulk characteristics (e.g., with respect to porosity, density, etc.), surface characteristics, thermal characteristics, mechanical characteristics, and / or any other suitable characteristics. Further, all parts of the base substrate 110 can be constructed using one or more of the same material, different materials (e.g., when each part of the base substrate 110 has different design constraints), and / or any combination of materials. Further, the base substrate 110 may be a single body or a base substrate 110 having separate parts that are joined to each other (e.g., during manufacturing).

[0024] In relation to optical characteristics, one or more materials of the base substrate 110 can have any degree of transparency, reflectivity, or other optical characteristics. For example, the material can be transparent (e.g., from the bottom surface of the base substrate 110, from the top surface of the base substrate 110, etc.) to enable optical analysis, investigation, or observation, but can also be opaque, transparent, translucent, and / or any suitable opacity. For example, in relation to bulk characteristics such as porosity (e.g., to provide a gas exchange function), a high degree of porosity may be desired, and the base substrate 110 may not need to be transparent. Further, the material and / or configuration aspects can be configured to facilitate the confinement of detectable signals within the individual microwells of the base substrate 110 (e.g., with respect to the confinement of fluorescence, with respect to the confinement of other conversion substrates). Further, the material aspects of the substrate can be configured and / or processed to prevent the absorption of sample processing materials (e.g., fluorescent substrates, colorimetric substrates, other conversion substrates, samples, etc.).

[0025] In relation to the bulk properties, one or more materials 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, in relation to the bulk properties, one or more materials of the base substrate 110 can be configured to have a level of density or other bulk properties suitable for sample processing and / or incubation purposes necessary to maintain the viability of microorganisms. In an embodiment, the base substrate 110 can be composed of a polymer (e.g., polytetrafluoroethylene (PTFE), polyethylene (PE), polyvinyl alcohol (PVA), etc.), ceramic, or another suitable material (e.g., natural material, synthetic material) having an appropriate ingress rating (e.g., according to an IP scale, according to another rating scale) or particle retention characteristics (e.g., rated retention of particles less than 1 micron, rated retention of particles 1 micron or greater), or can be incorporated in other ways. In one example, the base substrate 110 can be composed of a PTFE-based material having an IP rating (e.g., IP65 - 69), but alternatively, the base substrate 110 can be composed of a non-porous material that allows gas exchange by other elements of the microwell assembly 100 (e.g., vents, microscale channels, nanoscale channels, etc.).

[0026] In relation to surface properties, one or more materials of the base substrate 110 can be configured to have desired hydrophilic / hydrophobic properties (e.g., a high degree of hydrophilicity), which are determined by, for example, the contact angle and wettability. In relation to other electrical and physical properties, one or more materials of the base substrate 110 can be configured to have desired charge (e.g., in relation to the properties of the sample fluid and / or sample processing fluid used), electric field properties, conductivity, resistance, and / or any other suitable surface or physical properties. Additionally or alternatively, one or more materials of the base substrate 110 are preferably configured not to react with the fluids and microorganisms used during sample processing. Additionally or alternatively, one or more materials of the base substrate 110 are configured to absorb inhibitors that prevent the survival rate of microorganisms and / or the conversion of detectable enzyme substrates. Additionally or alternatively, the surface of the base substrate 110 exposed to the receiving fluid can have a desired surface finish.

[0027] In relation to 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 characteristics. In particular, the base substrate 110 can be configured to have desired thermal conductivity and / or heat capacity characteristics (e.g., suitable for a sample incubation step). In one aspect, the base substrate 110 can be configured to have thermal properties such that it can efficiently transfer heat to or from the fluid in contact with the base substrate 110. For example, in an aspect where the base substrate 110 is coupled to a heating or cooling element, the base substrate 110 can be configured to facilitate the transfer of heat to the contents of the microwells during incubation and / or the transfer of heat from the fluid. However, the base substrate 110 can have other suitable thermal properties based on the intended use. For example, the base substrate 110 can be configured to have a low thermal conductivity (e.g., as a heat insulating material) so that the material does not significantly affect the temperature of the fluid with which it comes into contact during operation.

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

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

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

[0031] 2.1.2 Sample Processing Region 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), and each of the set of sample processing regions has a gradient (e.g., volume, size, surface area, footprint, cross-sectional area, etc.) between the upstream end 10 and the downstream end 90 of the sample processing region 120 and includes a set of microwell subarrays 130 arranged with the gradient. The set of sample processing regions functions to receive a set of samples and facilitate distribution of the set of samples across the set of microwell subarrays to enable performance 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 a dried sample processing material (e.g., a medium, a fluorescent substrate, a colorimetric substrate, other dyes, etc.) prior to receiving a sample 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, the set of sample processing regions can be configured to be hydrophilic, hydrated, processed, and / or blocked with a non-specific absorbent.

[0032] In an embodiment, a set of sample processing regions can be arranged as a set of lanes spanning a wide surface of the base substrate 110, each region being configured to receive a separate sample and enabling the samples to be processed in parallel with high throughput. In an embodiment where the wide 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 test samples and / or the number of desired microwell sizes). However, the set of sample processing regions can alternatively be arranged with respect to another suitable axis. Further, in other embodiments, each of the set of sample processing regions may not be configured as a lane having a longitudinally defined microwell subarray. For example, in another embodiment shown in FIG. 2, each of the set 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 an embodiment, the number of sample processing regions included in the microwell assembly 100 can be adjusted according to the dimensions of the base substrate 110 (having the examples given above) in relation to the characteristic microwell dimensions desired for each microwell subarray and the number of individual microwells, and the microwell dimensions and the number of microwells implemented can be optimized for the operation of serial dilution tests in relation to the amount of sample received per region for MPN determination. In examples (two of which are shown in FIGS. 3A and 3B), the set of sample processing regions can include from 2 to 7 sample processing regions. However, in other embodiments, the set of sample processing regions can include another suitable number of sample processing regions (e.g., less than 2 sample processing regions, more than 7 sample processing regions).

[0034] As briefly described above, each set of sample processing regions can include a set of microwell subarrays 130 arranged with a gradient between the upstream end 10 and the downstream end 90 of the sample processing region 120, and a boundary 190 that separates the sample processing region 120 from adjacent sample processing regions. The set of microwell subarrays 130 functions to provide a set of partitions having a known volume distribution for determining the MPN for target detection from the sample and / or performing other assays for each sample being processed. Thus, each of the sets 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. The aspects of MPN determination and confidence limits are further described below.

[0035] In an embodiment, the sample processing region of the base substrate 110 can include 2 to 10 microwell subarrays, and each of the sets of microwell subarrays can have 10 to 100,000 partitions. Each sample processing region can receive a sample of 0.01 mL to 10 mL (by providing the total volume) to provide minimum and maximum ranges of MPN values of 5 to 3,000,000 for the MPN assay with appropriate confidence limits. However, the sample processing region can receive other sizes of sample amounts (e.g., less than 0.01 mL, greater than 10 mL) and can include other appropriate numbers of microwell subarrays (e.g., less than 2 subarrays, more than 10 subarrays) to enable determination of MPN within another appropriate range, with each having other appropriate numbers of partitions (e.g., less than 10 partitions, more than 100,000 partitions).

[0036] In particular, the number / characteristic volume of the microwell subarrays and the number of partitions per microwell subarray can be configured in relation to finding the solution of λ in the following equation [1]. Here, exp(x) is e x where K indicates the dilution factor, and g j indicates the number of positive (or growing) tubes in the j-th dilution, and m j indicates the amount of the original sample placed in each tube in the j-th dilution, and t j indicates the number of tubes in the j-th dilution. TIFF0007697020000001.tif20170

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

[0038] In an embodiment, the cross-section of each microwell can be a cross-section of a polygon (e.g., hexagon, rectangle, etc.) or a non-polygon (e.g., circle, ellipse, amorphous, etc.) (e.g., a 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 towards 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, whereby a sub-volume of the sample can enter the microwell from a direction perpendicular to the broad surface of the base substrate 110. However, one or more openings of the microwell can be configured in another suitable way. Further, the microwells can be arranged in a filled configuration (e.g., hexagonal closest packing, square closest packing, other closest packing configurations, etc.) or an unfilled configuration. For example, the wells of the first subarray of a set of microwell subarrays can be arranged in a first filled configuration (e.g., hexagonal closest packing, square closest packing, other closest packing configurations, etc.), and the wells of the terminal subarray of the set of microwell subarrays can be arranged in a second filled configuration (e.g., hexagonal closest packing, square closest packing, other closest packing configurations, etc.).

[0039] In connection with the gradient of the microwell subarray for each sample processing region, the first microwell subarray 130 including wells having a first characteristic dimension (e.g., the smallest characteristic dimension) can be disposed at the upstream end 10 of the sample processing region, and the terminal microwell subarray 170 including wells having a second characteristic dimension (e.g., the largest characteristic dimension) can be disposed at the downstream end 90 of the sample processing region 120. Thus, the microwell subarray can have larger characteristic microwell dimensions in the upstream-to-downstream direction. Alternatively, the microwell subarray can have smaller characteristic microwell dimensions in the upstream-to-downstream direction (e.g., the first microwell subarray 130 can include wells having the largest characteristic dimension and the terminal subarray 170 can include wells having the smallest characteristic dimension). Further alternatively, the microwell array can be arranged in a gradient or non-gradient manner in another suitable way (e.g., in relation to another microwell characteristic, along another direction axis). Further alternatively, each sample processing region can be configured in other ways (e.g., in stepwise increments across the gradient of the microwells). For example, the dimensions of the wells can be arranged to have a gradient in the lateral direction (e.g., a direction orthogonal 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 adjacent sample processing regions, thereby functioning to prevent crosstalk between samples. The boundary 190 can be configured as a recess (e.g., as a trench, such as a concave channel forming a perimeter), or as a protrusion, or can include alternating recesses and protrusions. Also, the boundary 190 can be configured as a region configured to promote evaporation or absorption of an overflowed sample, such that when a sample enters the region, the sample evaporates and / or is absorbed by the walls of the boundary 190. In an aspect where the boundary 190 is defined as a concave outer perimeter around the sample processing region, the boundary 190 can function as a trench into which an overflowed sample during sample processing can be received. Alternatively, the boundary 190 can serve another suitable purpose.

[0041] Additionally or alternatively, the boundary 190 can be composed of an absorbent material configured to receive and absorb a substance that has overflowed from the sample processing region.

[0042] Additionally or alternatively, one or more boundaries can include one or more outlets (e.g., an outlet to a waste chamber) away from the sample processing region, thereby delivering an overflowed substance away from the sample processing region and preventing it from re-entering the sample processing region.

[0043] Furthermore, the base substrate 110 can be physically continuous, but in an embodiment, the base substrate 110 can be configured to be separable (e.g., by a perforation, a reversibly locking component, etc.) between adjacent sample processing regions. However, the base substrate 110 can alternatively be configured to be inseparable.

[0044] Embodiments, aspects, and examples of the microwells in the sample processing region have been described above. However, aspects of the microwells and / or the sample processing region can be adapted from one or more of U.S. Application No. 16 / 048,104, filed Jul. 27, 2018; U.S. Application No. 16 / 049,057, filed Jul. 30, 2018; U.S. Application No. 15 / 720,194, filed Sep. 29, 2017; U.S. Application No. 15 / 430,833, filed Feb. 13, 2017; U.S. Application No. 15 / 821,329, filed Nov. 22, 2017; U.S. Application No. 15 / 782,270, filed Oct. 12, 2017; U.S. Application No. 16 / 049,240, filed Jul. 30, 2018; U.S. Application No. 15 / 815,532, filed Nov. 16, 2017; U.S. Application No. 16 / 115,370, filed Aug. 28, 2018; U.S. Application No. 16 / 564,375, filed Sep. 9, 2019; and U.S. Application No. 16 / 816,817, filed Mar. 12, 2020, which are hereby incorporated by reference in their entirety for each.

[0045] 2.2 Cover Substrate and Optional Elements 2.2.1 Cover Substrate As shown in FIGS. 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 the sample being processed and / or incubated on 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] Regarding the 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 suitable material. In particular, the material selection can be made based on one or more of manufacturing considerations, surface characteristics desirable for sample processing, optical characteristics, bulk characteristics (e.g., regarding porosity, regarding density, etc.), surface characteristics, thermal characteristics, mechanical characteristics, and / or any other suitable characteristics. Further, all parts of the cover substrate 210 can be constructed using the same one or more materials, different materials (e.g., if each part of the cover substrate 210 has different design constraints), and / or any combination of materials. Further, the base substrate 110 can be a single unit or a base substrate 110 having separate parts that are joined together (e.g., during manufacturing).

[0047] Regarding optical characteristics, one or more materials of the cover substrate 210 can have any degree of transparency, elasticity, reflectivity, or other optical characteristics. For example, the material can be transparent (e.g., to enable optical analysis, investigation, or observation from, e.g., the upper surface of the cover substrate 210), but can also be opaque, transparent, translucent, and / or any suitable opacity. For example, if a high degree of porosity is desired in relation to bulk characteristics such as porosity (e.g., to provide a gas exchange function), the cover substrate 210 need not be transparent.

[0048] In relation to the bulk properties, one or more materials of the cover substrate 210 can be configured to have a level of porosity that allows gas exchange between the sample being processed and the environment while preventing liquid exchange, thereby providing humidity control and preventing evaporation. Additionally or alternatively, in relation to the bulk properties, one or more materials of the cover substrate 210 can be configured to have a level of density or other bulk properties suitable for sample processing and / or incubation purposes. In an embodiment, the cover substrate 210 can be composed of a polymer (e.g., polytetrafluoroethylene (PTFE), polyethylene (PE), polyvinyl alcohol (PVA), etc.), ceramic, or another suitable material (e.g., natural material, synthetic material) having an appropriate ingress rating (e.g., according to the IP scale, according to another evaluation scale) or particle retention characteristics (e.g., rated retention of particles less than 1 micron, rated retention of particles 1 micron or greater), or can be incorporated in other ways. In one example, the cover substrate 210 can be composed of a PTFE-based material having an IP rating (e.g., IP65 - 69), but alternatively, the cover substrate 210 can be composed of a non-porous material that allows gas exchange by 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 environmental contamination at the end of the assay.

[0049] In relation to the surface properties, one or more materials of the base substrate 110 can be configured to have desired hydrophilic / hydrophobic properties (e.g., a high degree of hydrophilicity) determined, for example, by the contact angle and wettability. In relation to other electrical and physical properties, one or more materials of the cover substrate 210 can be configured to have a desired charge (e.g., in relation to the properties of the sample fluid and / or sample processing fluid being used), electric field properties, conductivity, resistance, and / or any other suitable surface or physical properties. Additionally or alternatively, one or more materials of the cover substrate 210 are preferably configured not to react with the fluids used during sample processing.

[0050] In relation to thermal properties, one or more materials of the cover substrate 210 can be configured to have desired thermal properties with respect to heat transfer and / or heat retention characteristics. In particular, the cover substrate 210 can be configured to have desired thermal conductivity and / or heat capacity characteristics (e.g., as appropriate for a sample incubation step). In one aspect, the cover substrate 210 can be configured to have thermal properties such that heat can be efficiently transferred to or from the microwell assembly 100 during sample processing and / or incubation.

[0051] In relation to mechanical properties, one or more materials of the base substrate 110 can be configured to have desired mechanical properties including one or more of rigidity, strength, elastic behavior, hardness, and other properties. For example, as shown in FIG. 5, an embodiment of the cover substrate 210 can be composed of an elastomeric material that can be elastically deformed, and the reversible deformation of the elastomeric cover substrate 210 can enable an operating mode that facilitates sample handling. For example, as shown in FIG. 5, the elastic properties of the cover substrate 210 can provide a deformation operating mode 211 in which a sub-volume (e.g., the upper part of a fluid) moves from each microwell in the sample processing region, and a relaxation operating mode 212 in which the cover substrate relaxes to a baseline state to form a pocket (e.g., an air pocket) between the cover substrate 210 and the microwells of the base substrate. Such operating modes can enable the microwell assembly 100 to further process samples while providing humidity control, preventing evaporation, and preventing crosstalk between the microwells of the base substrate 110 (e.g., after sample dispensing into a set of sample processing regions). However, the cover substrate 210 can have other suitable mechanical properties to provide the operating modes.

[0052] In an embodiment where the cover substrate 210 is an elastomer, the cover substrate 210 can be composed of an elastomer (e.g., a polyether / polyamide material, a polyurethane material, a polyester material, etc.), and the elastomer may be porous to provide gas exchange with the environment. However, the cover substrate 210 can be composed 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, reproving charged nylon, etc.).

[0053] As shown in FIGS. 6A and 6B, the cover substrate 210 defines a network of ventilation channels 220 that face the base substrate 110 (e.g., that are opposite the base substrate 110 when assembled with the cover substrate 210). The ventilation 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 ventilation channel 220 can extend across the cover substrate 210 (e.g., along one or more axes) to enable gas exchange between the microwell subarray of each sample processing region and the environment (e.g., the incubation environment). In the embodiment shown in FIG. 6B, a first subset 221 of the channels extends in the cover substrate 210 parallel to a first axis, and a second subset 222 of the channels can extend in the cover substrate 210 parallel to a second axis, such that the first subset 221 of the channels and the second subset 220 of the channels intersect each other (e.g., are orthogonal to each other and / or in fluid communication with each other). However, the individual subsets of the channels can be arranged in another suitable manner. In the embodiments shown in FIGS. 6A and 6B, the network of channels 220 is open to the environment at the lateral / peripheral edge of the cover substrate 210, but in other aspects, the network of channels 220 can be open to the environment at another suitable portion of the cover substrate 210 (e.g., by entering the thickness portion of the cover substrate 210 and opening at another surface). The ventilation channel 220 is preferably hydrophobic and has small capillary dimensions (e.g., <200 microns) to prevent the intrusion of fluid from the sample. Alternatively, the ventilation channel 220 can have another degree of hydrophobicity and / or can have capillary dimensions of 200 microns or more. Additionally or alternatively, the dimensions of the ventilation channels can be made smaller (e.g., less than 50% of the cross-section of the characteristic microwell dimensions and / or substantially less than 25%). However, the ventilation channel 220 can have other suitable dimensions relative to the characteristic microwell dimensions.

[0055] When forming the assembly, the cover substrate 210 can be fitted with the base substrate 110 by including an interlock mechanism (e.g., a first lock portion on the cover substrate 210 that is complementary to a second lock portion on the base substrate 110). For example, as shown in FIG. 4, the cover substrate includes a set of lips or tabs that engage the surface (e.g., the bottom surface, the outer peripheral surface) 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 yet other embodiments, the cover substrate 210 may not be configured to couple with the base substrate 110.

[0056] In yet other embodiments, the cover substrate 210 can be in the form of one or more films (e.g., an adhesive film, a porous film, a porous adhesive film) that cover a set of sample processing regions. 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 regions 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 further be removed from the base substrate 110 as needed (e.g., by a user, etc. according to the described embodiments of the automated platform 300) 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, thereby facilitating the bonding between the cover substrate 210 and the base substrate 110, providing further separation between adjacent sample processing regions to prevent sample crosstalk, performing the sealing function of the microwells, and enabling gas exchange between the samples on the base substrate 110 and the environment. More specifically, the film layer 225 and / or the cover substrate 220 can prevent the fluorescent substrate and / or the chromogenic substrate or other sample processing materials / reagents from contaminating the adjacent microwell partitions while not incurring manufacturing costs, promote the growth of targets (e.g., bacteria, yeast, mold, etc.) for detection, enable gas exchange, and perform the function of preventing evaporation.

[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 being able to interact 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, and the hydrogel material is delivered to a 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 sub-regions corresponding to the set of sample processing regions or the number of samples being processed.

[0059] Furthermore, the microwell assembly can include multiple membranes in relation to the design features and / or intended uses of the system 100.

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

[0061] Furthermore, embodiments of the microwell assembly 100 can 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, biocoatings, sample processing substrates incorporated or bound to the microwell surface, constituent media, diluent media, media provided in a lyophilized state, etc.) that promote gas exchange with the environment and prevent sample cross-talk while promoting 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 the 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 a control subsystem that communicates with various processing subsystems and the processing subsystems. The control subsystem controls the states of the deck 310, the set of sample processing elements, and the gantry 370 to transition the platform 300 between various operating modes. In an example, the platform 300 can provide a function of loading samples at high throughput (e.g., loading samples in less than 60 seconds / sample, processing more than 600 samples in 8 hours, etc.) and / or a function of reading samples at high throughput (e.g., reading samples faster than 10 seconds / sample, reading more than 500 samples per hour, etc.). Embodiments, implementations, and examples of operating modes that provide various workflows are described in more detail in Section 4 below.

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

[0064] In the embodiment shown in FIG. 9, the deck 310 provides a platform for supporting a set of sample processing elements, which can include disposable and / or reusable components, and these components include containers for containing sample processing materials and / or tools for processing samples (e.g., related to fluid processing, related to material separation, related to heating and cooling, etc.). In an embodiment, the deck 310 can support a set of sample processing elements, and the set of sample processing elements includes a reagent cartridge 320, units of the above-described microwell assembly 100 (e.g., a microwell assembly during storage, a microwell assembly disassembled into a separate base substrate 110 and a cover substrate 210, and a microwell assembly in an in-use position for processing samples), a sample staging container 330 (for staging samples prior to transfer to the microwell assembly 100), a tool container 340, and / or one or more units of other subsystems.

[0065] Additionally or alternatively, deck 310 can 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 having an optical accessory for improving pixel resolution, etc.). Additionally or alternatively, deck 310 or other components of platform 300 can include a barcode reader to support system components (e.g., disposables) for traceability and operations related to reading and tracking information with samples, as described in the application incorporated by reference below.

[0066] The sample processing elements can be supported on the same plane by deck 310 or, alternatively, on different planes. Preferably, the separate elements supported by the deck do not overlap, but alternative embodiments of deck 310 can support the sample processing elements to overlap (e.g., for space savings, for operational efficiency, etc.).

[0067] 3.1.1 Elements Supported by the Deck: Reagent Cartridge As shown in FIG. 9, the deck 310 includes at least one region for supporting units of the reagent cartridge 320, and this region functions to include materials for capturing microbial cells and / or processing samples in one or more compartments according to one or more workflows for various applications. For this purpose, the reagent cartridge 320 can define a set of storage volumes dispersed across a set of domains, and the set of domains can be configured to provide an environment suitable for the material content of each domain. The set of storage volumes can directly include 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 volume of each domain can be distributed in an array or arranged in other ways. Although the reagent cartridge 320 is described as being supported by the deck 310, aspects of the reagent cartridge 320 can alternatively be configured to operate independently of the deck 110.The reagent cartridge 120 can further additionally or alternatively include aspects described in U.S. Application No. 16 / 867,235, filed May 5, 2020; U.S. Application No. 16 / 867,256, filed May 5, 2020; U.S. Application No. 16 / 816,817, filed Mar. 12, 2020; U.S. Application No. 16 / 564,375, filed Sep. 9, 2019; U.S. Application No. 16 / 115,370, filed Aug. 28, 2018; U.S. Application No. 16 / 115,059, filed Aug. 28, 2018; U.S. Application No. 16 / 048,104, filed Jul. 27, 2018; U.S. Application No. 16 / 049,057, filed Jul. 30, 2018; U.S. Application No. 15 / 720,194, filed Sep. 29, 2017; U.S. Application No. 15 / 430,833, filed Feb. 13, 2017; U.S. Application No. 15 / 821,329, filed Nov. 22, 2017; U.S. Application No. 15 / 782,270, filed Oct. 12, 2017; U.S. Application No. 16 / 049,240, filed Jul. 30, 2018; and U.S. Application No. 15 / 815,532, filed Nov. 16, 2017, the disclosures of which are hereby incorporated by reference in their entireties.

[0068] 3.1.2 Elements Supported on the Deck: Tool Container As shown in FIG. 9, the deck 310 includes at least one area for supporting units of the tool container 340, and that area functions to position the tool container 340 relative to the fluid processing apparatus of the gantry 370 described below. The tool container 340 functions to accommodate one or more units of various tools for fluid suction, fluid delivery, fluid spraying, separation of target substances from non-target substances in samples, and / or other tools, according to one or more workflows for various applications, in one or more compartments. For this purpose, the tool container 340 facilitates the transfer and / or mixing of reagents with samples, fluidly couples and / or disconnects elements in various areas of the deck 310, facilitates the transfer of microplate / lid from one position to another position, or can interact with one or more components of the platform 300 in other ways. Although the tool container 340 is described as being supported by the deck 110, aspects of the tool container 340 can alternatively be configured to operate independently of the deck 310.The tool container 340 can further additionally or alternatively include the aspects described in U.S. Application No. 16 / 867,235, filed May 5, 2020; U.S. Application No. 16 / 867,256, filed May 5, 2020; U.S. Application No. 16 / 816,817, filed Mar. 12, 2020; U.S. Application No. 16 / 564,375, filed Sep. 9, 2019; U.S. Application No. 16 / 115,370, filed Aug. 28, 2018; U.S. Application No. 16 / 115,059, filed Aug. 28, 2018; U.S. Application No. 16 / 048,104, filed Jul. 27, 2018; U.S. Application No. 16 / 049,057, filed Jul. 30, 2018; U.S. Application No. 15 / 720,194, filed Sep. 29, 2017; U.S. Application No. 15 / 430,833, filed Feb. 13, 2017; U.S. Application No. 15 / 821,329, filed Nov. 22, 2017; U.S. Application No. 15 / 782,270, filed Oct. 12, 2017; U.S. Application No. 16 / 049,240, filed Jul. 30, 2018; and U.S. Application No. 15 / 815,532, filed Nov. 16, 2017, which applications are hereby incorporated by reference in their entireties.

[0069] 3.1.4 Heating and / or Cooling Subsystem The deck 310 can include or support a heating and cooling subsystem 350 that functions to transfer heat to and / or from desired regions of a substrate (e.g., base substrate 110, cover substrate 210), reagent cartridge 320, tool container 340, and / or other components. The heating and cooling subsystem 350 can alternatively or additionally function to maintain a desired temperature within the internal volume of the platform 300. In an embodiment, the heating and cooling subsystem 350 can include one or more units of a heating element (e.g., Peltier heating element, resistive heating element, other heating element), a cooling element (e.g., Peltier cooling element, chilled aluminum block, fluid path system for circulating coolant, etc.), a thermal contact or non-contact body for transferring heat between the heating and cooling elements and other objects, a heat sink, a fan, a temperature sensor, and a thermal control circuit (e.g., having an electrical coupling with a processing element of base 180 described later). In an embodiment, one or more cooling elements can maintain a storage volume and / or sample at 2 to 8 °C, more preferably 4 °C. Alternatively or additionally, the cooling element can maintain one or more storage volumes / samples at any suitable temperature (e.g., less than 2 °C, greater than 8 °C, etc.).

[0070] One or more portions of the heating and cooling subsystem 350 can enter into an opening of the deck 310 and thermally interact with or otherwise couple to desired portions of other system elements supported by the deck 310 to provide heat transfer functions for various applications. Alternatively, the deck 310 can be constructed of a thermally conductive material in desired regions for heat transfer applications, and a portion of the heating and cooling subsystem 350 can be configured to contact the thermally conductive material region of the deck 310 for heat transfer.

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

[0072] In an embodiment, one or more of the heat bodies and / or other portions of the heating and cooling subsystem 350 can be coupled to an actuator that moves the heat body in and out of heat transfer with an element supported by the deck 310, although aspects of the system 100 can omit the actuator of the heating and cooling subsystem 350.

[0073] The heating and cooling subsystem 350 can additionally or alternatively include aspects described in U.S. Application No. 16 / 867,235, filed May 5, 2020; U.S. Application No. 16 / 867,256, filed May 5, 2020; U.S. Application No. 16 / 816,817, filed Mar. 12, 2020; U.S. Application No. 16 / 564,375, filed Sep. 9, 2019; U.S. Application No. 16 / 115,370, filed Aug. 28, 2018; U.S. Application No. 16 / 115,059, filed Aug. 28, 2018; U.S. Application No. 16 / 048,104, filed Jul. 27, 2018; U.S. Application No. 16 / 049,057, filed Jul. 30, 2018; U.S. Application No. 15 / 720,194, filed Sep. 29, 2017; U.S. Application No. 15 / 430,833, filed Feb. 13, 2017; U.S. Application No. 15 / 821,329, filed Nov. 22, 2017; U.S. Application No. 15 / 782,270, filed Oct. 12, 2017; U.S. Application No. 16 / 049,240, filed Jul. 30, 2018; and U.S. Application No. 15 / 815,532, filed Nov. 16, 2017, which are hereby incorporated by reference in their entirety.

[0074] 3.1.4 Gantry As shown in FIG. 9, the platform 100 can include a gantry 370 coupled to the deck 110, which functions to support and / or enable the operation 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 rails / tracks for moving tools, such as a pipetter 374 having a pipette interface and / or a gripping tool 375 (e.g., for gripping a microwell assembly portion and / or other tools), within a three-dimensional space (e.g., a three-dimensional volume bounded by a first side of the deck 310). In an embodiment, tools operating using the gantry 370 can be moved relative to sample processing consumables, reagent cartridge 320 units, tool containers 340, or other elements to transfer substances across various components supported by the deck 310. Additionally or alternatively, tools supported by the gantry 370 can be used for imaging and / or reading barcodes associated with various disposable items supported by the deck 310 (e.g., related to identification of a proper set-up for execution, inventory management, etc.). For example, as shown in FIG. 9, the gantry 370 can support or be coupled to a camera 376 (e.g., a fluorescence imaging camera, a brightfield imaging camera, etc.) for sample reading. Additionally or alternatively, the camera 376 can be coupled to another part of the platform 300 (e.g., based on the imaging direction with respect to the surface of the microwell assembly 100 configured to optically detect signals).

[0075] The gantry 370 can preferably move one or more tools along one or more axes parallel to the broad surfaces of the reagent cartridge 320, the sample processing disposable items (e.g., the microwell assembly unit), and the tool container 340, and additionally along an axis perpendicular to the broad surfaces. The gantry 370 can additionally or alternatively enable movement along a subset of those directions or along any other suitable direction. To enable movement, the gantry 370 includes one or more motors (e.g., a motor for each axis or a motor for the direction of movement), one or more encoders for position identification in each axis or direction of movement, and / or one or more switches for controlling the gantry 370 (e.g., an optical switch for each axis) (e.g., when electrically coupled to a control circuit described in relation to the base 180 where the switch is described later), or is coupled in some other way.

[0076] As shown in FIG. 9, the gantry 370 can include 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, and / or can be configured to interact with the pipettor. 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 sensing pipetting pressure, a level sensor for sensing the 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 the tip from the pipettor 374.

[0077] The gantry 370, pipettor 374, camera / imaging element, and / or gripping element can additionally or alternatively include the aspects described in U.S. Application No. 16 / 867,235, filed May 5, 2020; U.S. Application No. 16 / 867,256, filed May 5, 2020; U.S. Application No. 16 / 816,817, filed Mar. 12, 2020; U.S. Application No. 16 / 564,375, filed Sep. 9, 2019; U.S. Application No. 16 / 115,370, filed Aug. 28, 2018; U.S. Application No. 16 / 115,059, filed Aug. 28, 2018; U.S. Application No. 16 / 048,104, filed Jul. 27, 2018; U.S. Application No. 16 / 049,057, filed Jul. 30, 2018; U.S. Application No. 15 / 720,194, filed Sep. 29, 2017; U.S. Application No. 15 / 430,833, filed Feb. 13, 2017; U.S. Application No. 15 / 821,329, filed Nov. 22, 2017; U.S. Application No. 15 / 782,270, filed Oct. 12, 2017; U.S. Application No. 16 / 049,240, filed Jul. 30, 2018; and U.S. Application No. 15 / 815,532, filed Nov. 16, 2017, the disclosures of which are hereby 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., for functions other than the operations 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 a subsystem can include one or more of a heating element, a cooling element, a temperature sensor, a gas composition sensor, a vent, a gas inlet, a gas outlet, a valve, and other suitable elements for environmental control within or external to platform 300. In connection with environmental control, platform 300 can include various enclosures and / or chambers that can control the sample environment internally. The platform can regulate the O2 and / or CO2 concentration within the microwells. The platform can include elements, such as UV light, that enable system decontamination during sample runs.

[0079] In an embodiment, the platform 300 (e.g., at the base 380) supports control and processing architectures for one or more system functions including fluid delivery for a pipettor 374 for sample processing, fluid level sensing (e.g., in the pipettor 374, various storage volumes of the reagent cartridge 320, etc.), operation 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 controlling the gantry 370, functions including receiving sensor signals and returning an output, functions including receiving sensor signals and performing various operations, functions related to power management of the system, functions related to system status display elements (e.g., lights, audio output devices, visual output devices, etc.), functions related to system input devices (e.g., buttons, keyboards, keypads, mice, joysticks, switches, touchscreens, 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 appropriate functions. In an embodiment, the platform 300 can support an electronic subsystem (e.g., a PCB, a power supply, a communication module, an encoder, etc.) related to a processing architecture (e.g., mounted on the system, separated from the system, etc.) or any other appropriate component, and the processing architecture can include any or all of a processor (e.g., a microprocessor), a controller (e.g., a microcontroller), memory, storage, software, firmware, or any other appropriate component. Further, the processing subsystem can include a machine vision module that functions to perform tag reading, protocol verification, error detection (e.g., detecting that a reagent does not match the assigned protocol), or any other function.

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

[0081] 4. Methods of Use and Applications As shown in FIG. 10A, one embodiment of a method 400 for target detection and characterization can include step S410 of arranging a set of sample processing elements including units of a microwell assembly on a deck of a sample processing system, step S420 of transferring a sample and / or other substances in an operating sequence among the set of sample processing elements, and step S430 of processing a set of samples for target detection in the units of the microwell assembly. Additionally or alternatively, the method 400 can include any or all of the processes described in U.S. Application No. 16 / 048,104, filed Jul. 27, 2018; U.S. Application No. 16 / 049,057, filed Jul. 30, 2018; U.S. Application No. 15 / 720,194, filed Sep. 29, 2017; U.S. Application No. 15 / 430,833, filed Feb. 13, 2017; U.S. Application No. 15 / 821,329, filed Nov. 22, 2017; U.S. Application No. 15 / 782,270, filed Oct. 12, 2017; U.S. Application No. 16 / 049,240, filed Jul. 30, 2018; U.S. Application No. 15 / 815,532, filed Nov. 16, 2017; U.S. Application No. 16 / 115,370, filed Aug. 28, 2018; U.S. Application No. 16 / 564,375, filed Sep. 9, 2019; and U.S. Application No. 16 / 816,817, filed Mar. 12, 2020, the disclosures of which are hereby incorporated by reference in their entireties.

[0082] The method is preferably implemented in an embodiment, implementation, or example of the system described above (e.g., in connection with the transfer of contents between various elements and / or sample processing), but additionally or alternatively, it can be implemented in any other suitable system. The method 400 is more preferably at least partially automated (e.g., when a user is required to load reagents and select a protocol, when no user intervention is required, etc.), but additionally or alternatively, one or more parts can be performed manually (e.g., for quality control steps, for all protocols, for rare protocols, etc.).

[0083] The specific workflows related to the above-described method 400 and system elements are further described in more detail below. Here, samples (e.g., samples derived from homogenized or non-homogenized consumables) can be processed according to the workflow.

[0084] 4.1 Method - Example of a Workflow for Determining MPN in Parallel from Multiple Food Samples As shown in FIG. 10B, an embodiment of method 400 configured to process one or more consumable samples (e.g., individual food sample suspensions) for the purpose of evaluating safety and quality includes step S410' of placing a set of sample processing elements including a unit of a microwell assembly on the deck of a sample processing system, step S415' of performing a series of sample preparation operations for processing the set of samples, step S420' of transferring the set of samples to a set of sample processing regions of the base substrate of the microwell assembly (using, for example, a predetermined delivery of the 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 the spacing between the base substrate and the cover substrate of the microwell assembly), step S425' of covering the base substrate with the cover substrate of the microwell assembly (e.g., for spreading the sample fluid, for dividing 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] Method 400 functions to implement a system for rapid sample testing using an application specific to most probable number (MPN) determination. In particular, method 400 is related to enabling rapid operation of serial dilution tests for measuring the concentration of target microorganisms in a food test portion (e.g., for food safety / food quality applications) and can be used to process multiple samples in parallel. During processing, the food test portion can be diluted (e.g., by combination with a specific broth / reagent for preparing a sample for analysis) to prepare a food suspension.

[0086] In an embodiment, the system can place a set of sample processing elements including units of a microwell assembly on the deck of the sample processing system in S410’ as preparation for processing the sample in subsequent steps of method 400. The system can configure a 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 operation by a system operator. In other aspects, step S410’ can configure the sample processing elements in another suitable way for processing multiple samples in parallel. In preparation for downstream sample processing operations, step S410’ can include moving the base substrate of the unit of the microwell assembly to the liquid loading position of the platform and using the camera of the platform to read a plate identifier. Thereafter, the system can compare the plate identifier with the identifier of the sample to be processed and associate the sample processing result with the execution associated with the plate identifier to confirm that the correct sample is being processed. However, step S410’ can include other execution preparation steps in other aspects.

[0087] Step S415’ describes performing a series of sample preparation operations to process a set of samples. Step S415’ functions to process the sample material such that the sample material derived from consumables (e.g., food samples, beverage samples, etc.) is dispensed into the set of sample processing regions of the base substrate of the microwell assembly. In an embodiment, Step S415’ includes homogenizing the food test portion with a diluent (e.g., having an appropriate dilution ratio) (e.g., mechanically, chemically); suspending the sample (e.g., in buffered peptone water, with other suspending agents); mixing the volume of each sample to be processed (e.g., less than 1 mL, 1 mL or more) with a medium (e.g., a medium that suppresses background flora and promotes the growth of specific microorganisms, a medium that provides an appropriate dilution ratio such as a 1:1 dilution ratio, etc.), a revelation chemical containing an enzyme substrate (e.g., a fluorescent substrate, a chromogenic substrate, etc.), or other processing materials in dry or liquid form (e.g., spices, chocolate, food matrices with coloring / auto-fluorescence, or other consumables, etc., to process samples with high inhibitory properties to produce a sample having viscosity characteristics and / or a total amount suitable for handling and fluid delivery); filtering the sample (e.g., to remove fine particles that may affect sample loading in microwells, uniform distribution of the sample across microwells with different characteristic dimensions, clogging, detection, etc.), where the limiting fine particle size can cover based on the pitch between adjacent microwells (e.g., a filter size of 20 microns to 280 microns, having other filter sizes); and can include one or more of any other suitable sample preparation steps. Step S415’ can be performed using the fluid processing elements of the platform described in Section 3 above or using other suitable device components.

[0088] In an embodiment, the sample processing step of block S415’ can be used to generate an MPN estimate having 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., related to volume measurements such as cfu / mL).

[0089] Step S420’ describes transferring a set of samples to a set of sample processing regions of a base substrate of a microwell assembly (e.g., by capillary flow from one end of a sample processing region enabled by a spacing between a base substrate and a cover substrate of the microwell assembly, using a predetermined delivery of the sample in droplet form across each microwell surface associated with each sample). Step S420’ can be performed using the fluid processing elements of the platform described in Section 3 above or using other suitable devices. In an embodiment, each of the set of samples to be processed can be transferred so as to be received in turn in the sample processing regions of the base substrate. Alternatively, the set of samples can be dispensed simultaneously (e.g., using a multi-head fluid dispenser, using other devices) into the sample processing regions of the base substrate. In particular, transferring the set of samples to the set of sample processing regions of the base substrate of the microwell assembly in step S420’ will result in an automatic splitting of the set of samples using embodiments, implementations, and examples of sets of microwell subarrays arranged in a gradient for rapid sample testing (e.g., MPN determination).

[0090] Step S425’ describes covering the base substrate with the cover substrate of the microwell assembly and functions to protect and facilitate the maintenance of an appropriate environment for the set of samples during downstream processing and incubation steps. In an embodiment, step S425’ can be performed using an element (e.g., a modified gripping element) coupled to the gantry of the platform described in section 3 above or using other suitable apparatus. In an embodiment, as described in section 2 above, the cover substrate can include ventilation channels and / or can be composed of a porous material, thereby functioning in step S425’ to prevent cross-contamination of the samples, prevent evaporation of the samples, and allow gas exchange between the samples and the environment during incubation / culture. 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 cross-talk of the samples. However, step S425’ can also be performed in another suitable manner.

[0091] Step S430’ describes transferring the microwell assembly for further processing and functions to stage the microwell assembly with a 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 apparatus. In an embodiment, the system can transport the microwell assembly to a location on the platform where an operator can transfer the microwell assembly to another apparatus for incubation, culturing, or further processing steps. Alternatively, the system can transport the microwell assembly to a location on the platform that can automatically perform incubation, culturing, or further processing steps. When transferring the microwell assembly, the system preferably operates in a manner that minimizes physical disturbance of the samples in the set of microwells (e.g., using the gantry and tools), but Step S430’ can alternatively be performed in another suitable manner.

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

[0093] In the embodiment of step S440', the system can process the image of the microwell assembly after sample processing and incubation with one or more conversion algorithms (e.g., Hough transform, filtering operations, fitting operations, microwell recognition operations, registration operations, etc.) for the detection of fluorescence / colorimetric signals and related characteristics (e.g., intensity), and / or perform other appropriate image processing operations. Thereafter, the system can generate an analysis based on one or more of aggregation reference, estimation algorithms (e.g., Thomas' law), confidence limit determination methods (e.g., Holden's method, etc.), boundary approximation approaches (e.g., Blodgett's method, etc.), particle counting statistics methods (e.g., particle / cell counting method via Poisson statistics applied to microwell arrays), application of error correction related to partitioning error and / or subsampling error, execution of virtual partitioning, or other appropriate methods. Additionally or alternatively, when the threshold detection limit is reached, the plate can be sealed to prevent further interaction with the environment and / or contamination of the environment.

[0094] In an embodiment of method 400, the media composition, substrate, incubation conditions, and detection system depend on one or more targets aimed at detecting from a set of samples. For example, to detect the total viable count, an exemplary media base can include plate count agar (e.g., non-selective plate count agar), the media composition can be tryptone, yeast extract, and glucose, the detection principle is based on a substrate targeting general enzyme activity or viability, and the incubation time is less than 72 hours at 30°C. To detect Enterobacteriaceae, an exemplary media base can include violet red bile glucose (e.g., selective violet red bile glucose), the media composition can be peptone, yeast extract, glucose, crystal violet, sodium, bile salts / sodium deoxycholate, the detection principle is based on pH indication, and the incubation time is less than 24 hours at 30°C. To detect coliforms, an exemplary media base can include violet red bile lactose (e.g., selective violet red bile lactose), the media composition can be peptone, yeast extract, lactose, crystal violet, sodium, bile salts / sodium deoxycholate, the detection principle is based on pH indication, and the incubation time is less than 24 hours at 37°C. Also, in the case of detection based on a β-galactosidase enzyme substrate, lactose needs to be excluded from the media used to avoid acidification of the media (and suppression of fluorescence). In such embodiments, Rapid E. coli media (e.g., media containing peptone, yeast extract, sodium chloride, bile salts / sodium deoxycholate + β-galactosidase enzyme substrate) can be considered. When detecting E. coli, an exemplary media base can include tryptone bile X-glucuronide (e.g., selective tryptone bile X-glucuronide), the media composition can be 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.To detect yeast and mold, an exemplary medium base can include YGC medium, the medium composition can be yeast extract, glucose, and chloramphenicol, the detection principle is based on some non-specific enzyme activities, and the incubation time is less than 72 hours at 30°C. This system can also perform multi-target detection of Escherichia coli / coliforms. However, depending on the intended target, other medium compositions, substrates, incubation conditions, and detection systems can be used.

[0095] However, one or more embodiments of the system can include other workflows, including the described implementation modes of the workflow, and / or be configured to implement other workflows.

[0096] 4.1.1 Method - MPN Count In relation to the above-described method 400 and / or other related methods, the principle and procedure of MPN can be applied as follows.

[0097] Principle: The test portion of the sample processed according to the above-described embodiments, implementation modes, and / or examples can be inoculated into a medium (e.g., liquid medium, dry medium, rehydrated dry medium, etc.) designed to support the growth of specific microorganisms or microbial groups and / or inhibit the growth of non-target microorganisms. To determine whether the growth of the target microorganism has occurred, various criteria (e.g., visual detection of turbidity, gas generation, color change, subsequent isolation of microorganisms on selective agar medium, other mechanisms, etc.) can be used. The composition of the growth medium and the criteria for distinguishing positive and negative results will be described further below. By using these approaches, only qualitative values can be attributed to each test portion (i.e., the result can be either positive or negative). To obtain an estimated value of the amount of microorganisms present, it is necessary to examine several test portions and use statistical procedures to determine the most probable number (MPN).

[0098] Inoculation procedure:When using a selective growth medium, it is necessary to prevent the addition of test portions from reducing its selective properties and enabling the growth of non-target microorganisms. In most specifications, information on the compatibility of a particular matrix with a liquid medium is provided, but caution is required for some matrices that may contain growth inhibitors (e.g., spices, cocoa, broth, etc.). When such a matrix is included, sample and matrix treatment methods involving neutralizing compounds can be implemented using high dilution factors, centrifugation, separation based on buoyancy (e.g., involving binding of the target sample material to buoyant particles and washing of non-target matrix material), filtration, immunomagnetic separation to separate the target microorganism 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., highly contaminated environmental samples, fermented products, products with probiotic bacteria, etc.), the method can further perform spike-based experiments and / or generate appropriate controls.

[0099] In an embodiment, a test portion of a small volume (e.g., less than 1 milliliter, 1 milliliter, etc.) can be added to a volume of a single-strength medium (e.g., 5 to 10 times the volume). In an embodiment, a test portion of an intermediate volume (e.g., 1 milliliter to 100 milliliters) can be added to a volume of a higher-strength (e.g., double-strength) medium (e.g., an equal volume). In an embodiment, a test portion of a large volume (e.g., greater than 100 milliliters) can be combined with a more concentrated medium. For special purposes, as described in embodiments of the above systems and platforms, a sterile dehydrated medium can be dissolved in a sample (e.g., a cold sample or a pre-warmed sample) for analysis. In an embodiment, the time difference from preparing the first dilution of the sample to inoculating the last portion needs to be less than a threshold time (e.g., 15 minutes, another appropriate time) by performing aseptic procedures. Thereafter, the inoculated partitions are incubated at 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 performed. The criteria for distinguishing positive and negative results can vary depending on each microorganism or group of microorganisms. Using those criteria, in the MPN determination method, the number of positive results obtained in all test portions derived from one sample is counted.

[0100] Selection of inoculation system:According to the MPN method, the sample is diluted over a plurality of partitions to an extent where the inoculum may or may not contain the target viable microorganisms. From the "results" (i.e., the number of inocula that result in growth at each dilution), the initial concentration of one or more microorganisms in the sample can be estimated. To obtain an estimate of a wide range of possible concentrations, serial dilution and / or incubation in multiple partitions (e.g., tubes, wells of plates, the micro-well systems described above, droplets of emulsions, etc.) can be used. Subsequently, based on the number of positive and negative partitions at one or more dilutions observed after incubation, the estimated MPN of the microorganisms present in the original sample, and its estimated accuracy, can be calculated by statistical procedures. The MPN inoculation system can be selected based on one or more of the expected number of microorganisms in the sample under investigation, regulatory requirements, required accuracy, and other practical considerations. The uncertainty of the measurement depends on the number of positive test portions observed and increases as a function of the square root of the number of partitions used. To halve the measurement uncertainty, the number of tubes needs to be quadrupled. When using a system with only a small number of replicate partitions, the measurement uncertainty is low.

[0101] Embodiment of inoculation system - single dilution system: If the expected concentration of microorganisms is low or is expected to vary only moderately, a suitable inoculation system is a single series of equal test portions. When the expected ratio between the maximum and minimum number of microorganisms is less than about 25, 10 parallel test portions are the minimum number expected to function, and for 50 parallel partitions, a ratio of 200 is the limit.

[0102] Embodiment of inoculation system - multiple dilution system:When the concentration of microorganisms in the 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 are performed. In such a platform, a sufficient number of dilutions are inoculated to ensure a system that gives both positive and negative results. Also, the number of dilutions depends on the calculation method used to estimate the MPN value (e.g., depends on theoretical models, reference tables, etc.).

[0103] Embodiment of inoculation system - symmetric dilution system: In a symmetrical MPN system, for each dilution, 3 or 5 parallel partitions (or another suitable number of partitions) can be used. The accuracy obtained in this system drops rapidly as the number of tubes per dilution decreases. If higher accuracy is required, it is recommended to select 5 or more partitions.

[0104] Embodiment of inoculation system - asymmetric dilution system: In an asymmetrical system, when the dilution levels are different, the number of tubes is not the same. Such a configuration is suitable for estimating the number of microorganisms within a clearly defined range (an example of which is described in ISO8199).

[0105] Determination of MPN value: In an embodiment, the MPN value can be determined by one or more of mathematical calculation, reference to an MPN table, and utilization of other algorithms. These three methods are detailed below.

[0106] Determination of MPN value - formula: The approximate MPN value for any number of dilutions and parallel tubes can be derived by applying the following formula. Here, Z p is the number of positive partitions, m r is the sample reference mass (e.g., grams), m s is the total mass of the sample in all partitions having a negative reaction (e.g., grams), m t is the total mass of the sample in all partitions (e.g., grams). TIFF0007697020000002.tif22170

[0107] The MPN value for a single series of partitions is derived from the following formula. Here, 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 having a positive reaction. TIFF0007697020000003.tif21170

[0108] The 95% confidence limits of the MPN estimate can be approximately calculated using the following formula. Here, x is the upper or lower limit of the 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 a negative reaction. TIFF0007697020000004.tif35170

[0109] The log 10 standard uncertainty of a symmetric multiple dilution MPN system can be obtained from the following formula. Here, SE is the standard error of log 10 MPN, f is the dilution factor between successive dilutions, and n is the number of partitions per dilution. TIFF0007697020000005.tif23170

[0110] Variants of the above formula can be adapted to volume parameters that can extract mass from the relevant volume and / or the formula can be adapted to account for volume.

[0111] Determination of MPN value - table:To represent results per sample reference mass (or volume in the case of a liquid sample), the values in the table can be processed (e.g., by multiplying the ratios such as [reference mass] / [test portion mass] for MPN and 95% limits). In a symmetric system, the method can include the implementation of a suitable number of successive dilutions (e.g., 3 successive dilutions) according to a suitable number of replications, embodiments, modes, and examples as described above to support the system components. Using those methods, the number of positive results for each set of partitions can be obtained (e.g., by using the platform 300 described above), and from the MPN table for the inoculation system used, the MPN value of the relevant microorganisms present in the reference volume of the sample can be obtained. Since various combinations of positive partitions can be more likely statistically than others, the combinations of positive partition results can be classified into various categories (e.g., highly probable results, moderately probable results, low-probability results, etc.).

[0112] Determination of MPN value - algorithm: In aspects of the methods described above, various algorithms can be implemented (e.g., using an MPN assay analyzer and can be run using another suitable system).

[0113] Additionally or alternatively, the method can be adapted from those described in Appendix A and / or U.S. Patent Application No. 16 / 072,712, titled "Digital Microbiology," filed on January 25, 2016, which are hereby 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 their aspects. In this regard, each block of the flowchart or block diagram can represent a part of a module, segment, or code that includes one or more executable instructions for implementing the specified one or more logical functions. It should also be noted that in some alternative aspects, the functions noted in the blocks may occur in an order different from that noted in the drawings. 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 functions involved. It should also be noted that each block of the block diagram and / or flowchart, and combinations of blocks in the block diagram and / or flowchart, can 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] Those skilled in the art can make modifications and changes to the preferred embodiments of the present invention without departing from the scope of the present invention as specified in the following claims, as can be recognized from the detailed description, the drawings, and the claims described above.

Claims

1. A system for target detection, comprising a base substrate, and a set of sample processing regions defined on a wide surface of the base substrate, each of the set of sample processing regions comprising a set of microwell subarrays arranged with a gradient between an upstream end and a downstream end of each sample processing region, and a boundary separating each sample processing region from an adjacent sample processing region, Further comprising a cover substrate configured to fit with the base substrate in a coupling 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 fitted together in the coupling mode, the network of ventilation channels providing gas exchange between the base substrate and the environment surrounding the set of sample processing regions, and the cover substrate being 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.

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

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

4. The system according to claim 2, A system, wherein the wells of the first subarray are arranged in a first filling configuration and the wells of the terminal subarray are arranged in a second filling configuration. **Claim 5** In the system according to claim 1, the base substrate is composed of a material having a level of porosity that allows gas exchange between the contents of the wells of the set of sample processing regions and the environment of the system while preventing liquid exchange. **Claim 6** In the system according to claim 1, the set of sample processing regions includes a dry sample processing substance containing at least one of a medium, a fluorescent substrate, and a colorimetric substrate. **Claim 7** In the system according to claim 1, each sample processing region has a total volume of 0.01 to 10 milliliters, and each of the sets of the microwell subarrays has 10 to 100,000 partitions corresponding to a most probable number (MPN) range of 5 to 3,000,000 for the MPN assay. **Claim 8** In the system according to claim 1, the boundary includes a concave channel operable to receive an overflowed sample. **Claim 9** In the system according to claim 1, the cover substrate is composed of an elastomeric material, and provides a deformation operation mode in which a fluid subvolume moves from the sample dispensed to the set of sample processing regions in the base substrate, and a relaxation operation mode 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. **Claim 10** In the system according to claim 1, The network of the ventilation channels includes a first subset of channels extending in the cover substrate parallel to a first axis and a second subset of channels extending in the cover substrate parallel to a second axis, and the first subset of channels intersects the second subset of channels. A system characterized by this.

11. In the system according to claim 1, The network of the ventilation channels is open to the environment of the system at the peripheral edge of the cover substrate. A system characterized by this.

12. In the system according to claim 1, The cover substrate includes a first locking portion complementary to a second locking portion of the base substrate. A system characterized by this.

13. In the system according to claim 1, The cover substrate is separated from the base substrate by one or more functional layers. A system characterized by this.

14. In the system according to claim 13, The one or more functional layers are disposed between the cover substrate and the base substrate and include a porous adhesive film layer that provides gas exchange from the wells of the set of sample processing regions. A system characterized by this.

15. A system for target detection, A base substrate, A set of sample processing regions defined on a wide surface of the base substrate, each of the set of sample processing regions having, A set of micro-well sub-arrays arranged with a gradient between the upstream end and the downstream end of each sample processing region, and A boundary separating each sample processing region from an adjacent sample processing region. A set of sample processing regions, A system comprising a cover substrate configured to fit with the base substrate in a coupling 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 fitted in the coupling mode, the network of ventilation channels providing gas exchange between the environment around the base substrate and the set of micro-well sub-arrays, the cover substrate being composed of a material having a level of porosity that allows gas exchange between the contents of the wells of the set of sample processing regions and the environment of the system while preventing liquid exchange, the cover substrate being composed of an elastomeric material, and the cover substrate providing a deformation operation mode in which a sub-volume of fluid moves from a sample dispensed into the set of sample processing regions of the base substrate, and a relaxation operation mode in which the cover substrate relaxes to a baseline state to create a pocket between the cover substrate and the micro-wells of the base substrate.

16. In the system according to claim 15, the set of micro-well sub-arrays includes an initial micro-well sub-array including wells having a first characteristic dimension disposed at an upstream end and a terminal micro-well sub-array including wells having a second characteristic dimension disposed at a downstream end, and the first characteristic dimension is smaller than the second characteristic dimension.

17. In the system according to claim 15, both the base substrate and the cover substrate are composed of a material having a level of porosity that allows gas exchange between the contents of the wells of the set of sample processing regions and the environment of the system while preventing liquid exchange.

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