Systems and methods of analyte detection

Abstract

The disclosed systems and methods involve the semi-quantitative detection of disease-specific protein signatures in biological samples. The system may include a housing with a motor driven substrate holder on rails. The substrate holder is configured to be positioned at multiple locations along the guide rails. Imaging sensors capture images of portions of the substrate at these locations. The system detects markers on the substrate and determines the location of wells based on these markers. Based on the determined location of the wells, color values for each pixel in the wells can be measured. These values may be analyzed to detect disease-specific protein signatures in a variety of sample types.

Description

[0001] Attorney Docket No. 128036-10402

[0002] 1

[0003] SYSTEMS AND METHODS OF ANALYTE DETECTION

[0004] CROSS REFERENCE TO RELATED APPLICATION

[0005] This application claims priority to U.S. Provisional Patent Application No. 63 / 685,081 entitled “SYSTEMS AND METHODS OF ANALYTE DETECTION” filed August 20, 2024 (Attorney Docket No. 128036-10401), which is hereby incorporated by reference in its entirety.

[0006] FILED OF THE INVENTION

[0007] The present disclosure relates generally to multiplex immunoassays, and more specifically, to systems and methods for the semi-quantitative detection of disease-specific protein signatures in biological samples.

[0008] BACKGROUND

[0009] Multiplex immunoassays are a type of laboratory technique used in the detection and quantification of multiple analytes, such as proteins, in a single sample. This technique is based on the principle of immunoassays, which utilize the specific binding between an antigen and its corresponding antibody to detect the presence of a specific protein in a sample.

[0010] In a typical multiplex immunoassay, a panel of antibodies, each specific to a different target analyte, is immobilized on a solid support, such as a microtiter plate. The sample is then added to the plate, and any target analytes present in the sample bind to their corresponding antibodies. The bound analytes are then detected and quantified using a detection system, which often involves a secondary antibody that is conjugated to a detectable label, such as an enzyme or a fluorescent dye.

[0011] One common type of multiplex immunoassay is the enzyme-linked immunosorbent assay (ELISA), which uses a reaction product as the detectable label. In an ELISA, the enzyme catalyzes a reaction that produces a color change, and the intensity of the color is proportional to the amount of the target analyte in the sample.

[0012] The use of multiplex immunoassays has several advantages over traditional single-analyte assays. For example, they allow for the simultaneous detection of multiple analytes in a single sample, which can save time and resources, and they can provide a more comprehensive profile of the analytes in the sample. However, the performance of a multiplex immunoassay can be influenced by various factors, such as the quality of the antibodies used, the efficiency of the binding between the antibodies and the analytes, and the sensitivity and specificity of the detection system.

[0013] The demand for affordable, efficient and accurate analyte detection systems is driven by the growing complexity of diagnostic requirements in the medical field. Traditional single-analyte assays are limited in their throughput and scope, often requiring separate tests for each analyte of interest. This not only consumes more time and resources but also increases the volume of biological samples necessitated for comprehensive analysis. Thus, an analyte detection system that enables the simultaneous detection of multiple analytes in a single sample is needed. This multiplexing capability streamlines the diagnostic process, reduces sample volume requirements, and provides a more detailed profile of the biological sample, which is particularly beneficial in the rapid identification of disease-specific protein signatures. The integration of automated systems, precise imaging, and advanced data processing within the system further enhances the reliability and quantification of the assay results, making it a valuable tool in clinical diagnostics, research, and therapeutic monitoring.

[0014] SUMMARY

[0015] In some embodiments, a system includes a housing including an opening; a stepper motor; a plate holder configured to receive a well plate including a plurality of wells; one or more guide rails, wherein at least one of the one or more guide rails is mechanically coupled to the stepper motor, wherein the stepper motor is configured to drive the plate holder along the guide rails to a plurality of positions including at least one position inside the housing and at least one position outside the opening of the housing; and one or more imaging sensors affixed inside the housing, wherein the imaging sensor is configured to capture at least one image of at least a portion of the well plate in the plate holder in the at least one position inside the housing, and wherein the portion of the well plate is based on an imaging area of the one or more imaging sensors.

[0016] In some embodiments, the system further includes a processor; and a non-transitory, processor-readable storage medium, wherein the non-transitory, processor-readable storage medium includes one or more programming instructions that, when executed, cause the processor to: actuate the stepper motor to eject the plate holder from the opening; actuate the stepper motor to receive the plate holder from the opening and place the plate holder in a first position in the housing; capture, using the one or more imaging sensors, images of the well plate at the first position, wherein each of the images captures a first marker and a first portion of the plurality of wells based on the imaging area of the one or more imaging sensors with regard to the first position, wherein each well includes a sample including at least one protein; detect a location the first marker; determine a location of the first portion of the plurality of wells based on the location of the first marker; determine color values for each pixel in each of the first portion of the plurality of wells; determine color intensity values based on the color values; and analyze the color intensity values to detect disease-specific protein signatures.

[0017] In some embodiments, the system further includes one or more interface elements attached to the housing and electronically interfaced to the processor.

[0018] In some embodiments, the one or more programming instructions further cause the processor to: actuate the stepper motor to receive the plate holder from the opening and place the plate holder in one or more additional positions in the housing; capture, using the one or more imaging sensors, images of the well plate at each of the one or more additional positions, wherein each of the images captures a unique marker and a unique portion of the plurality of wells based on an imaging area of the one or more imaging sensors with regard to the unique position; detect, in the images of the well plate, a location the unique marker; and determine a location of the unique portion of the plurality of wells based on the location of the unique marker.

[0019] In some embodiments, the one or more imaging sensors include a first imaging sensor and a second imaging sensor.

[0020] In some embodiments, the imaging area of the first image sensor is distinct from the imaging area of the second imaging sensor.

[0021] In some embodiments, the one or more image sensors are at least one of a complimentary metal-oxide semiconductor RGB sensor and charge-coupled device RGB sensor.

[0022] In some embodiments, the disease-specific protein signature includes detection of a biotinstreptavidin complex with at least one antibody and one protein in a well of the well plate.

[0023] In some embodiments, the well plate includes a plurality of alignment spots configured to display intense color intensity when imaged.

[0024] In some embodiments, the plurality of alignment spots is asymmetrically positioned.

[0025] In some embodiments, the positioning of the plurality of alignment spots encodes well plate information including at least one of a make of the well plate, a test type, or an orientation of the well plate in the plate holder.

[0026] In some embodiments, a system, at least one of the plurality of alignment spots includes a reagent control configured to indicate an addition of a detection antibody.

[0027] In some embodiments, at least one of plurality of alignment spots include a sample control configured to indicate an addition of samples to the well plate.

[0028] In some embodiments, the one or more programming instructions further cause the processor to: detect, in the images of the well plate, the plurality of alignment spots; and in response to failing to detect the plurality of alignment spots, return an error.

[0029] In some embodiments, the one or more programming instructions further cause the processor to perform circle detection at an expected target spot.

[0030] In some embodiments, the system includes a laser distance sensor configured to measure a location of the first position.

[0031] In some embodiments, the one or more programming instructions further cause the processor to calibrate the stepper motor based on the location of the first position.

[0032] In some embodiments, the one or more programming instructions further cause the processor to calibrate the one or more imaging sensors based on the location of the first position.

[0033] In some embodiments, the one or more programming instructions further cause the processor to determine a sharpness value metric of each of the one or more imaging sensors; and tune a focal length of each of the one or more imaging sensors to optimize the sharpness value metric. In some embodiments, the system includes one or more background lights within the housing; wherein the one or more programming instructions further cause the processor to: analyze color values and intensity values for the one or more background lights; and return an operational status of the one or more background lights.

[0034] In some embodiments, the well plate includes a sample including at least one of milk, bulk tank milk, saliva, serum, bronchoalveolar lavage, bronchial wash, sputum, swab in transport media, feces, urine, and cerebrospinal fluid.

[0035] In some embodiments, the one or more programming instructions further cause the processor to detect the avidity of the sample.

[0036] In some embodiments, at least a portion of the plurality of alignment spots include antihorseradish peroxidase goat IgG.

[0037] In some embodiments, a method includes actuating, by a processor, a stepper motor to receive a plate holder and a well plate including a plurality of wells, along one or more guide rails, from an opening in a housing and placing the plate holder in a first position in the housing; capturing, using one or more imaging sensors, images of the well plate at the first position, wherein each of the images captures a first marker and a first portion of the plurality of wells based on an imaging area of the one or more imaging sensors with regard to the first position, wherein each well includes a sample including at least one protein; detecting, by the processor, a location of the first marker; determining, by the processor, a location of the first portion of the plurality of wells based on the location of the first marker; determining, by the processor, color values for each pixel in each of the first portion of the plurality of wells; determining, by the processor, color intensity values based on the color values; and analyzing, by the processor, the color intensity values to detect disease-specific protein signatures.

[0038] In some embodiments, the method includes actuating, by the processor, the stepper motor to receive the plate holder from the opening and place the plate holder in one or more additional positions in the housing; capturing, using the one or more imaging sensors, images of the well plate at each of the one or more additional positions, wherein each of the images captures a unique marker and a unique portion of the plurality of wells based on an imaging area of the one or more imaging sensors with regard to the unique position; detecting, by the processor, in the images of the well plate, a location the unique marker; and determining, by the processor, a location of the unique portion of the plurality of wells based on the location of the unique marker.

[0039] In some embodiments, the one or more imaging sensors includes a first imaging sensor and a second imaging sensor.

[0040] In some embodiments, the imaging area of the first imaging sensor is distinct from the imaging area of the second imaging sensor.

[0041] In some embodiments, the one or more imaging sensors are at least one of a complimentary metal-oxide semiconductor RGB sensor and charge-coupled device RGB sensor. In some embodiments, the disease-specific protein signature includes detection of a biotinstreptavidin complex with at least one antibody and one protein in a well of the well plate.

[0042] In some embodiments, the well plate includes a plurality of alignment spots configured to display intense color intensity when imaged.

[0043] In some embodiments, the plurality of alignment spots is asymmetrically positioned.

[0044] In some embodiments, the method includes determining well plate information including at least one of a make of the well plate, a test type, or an orientation of the well plate in the plate holder based on a location of the plurality of alignment spots.

[0045] In some embodiments, at least one of the plurality of alignment spots includes a reagent control configured to indicate an addition of a detection antibody.

[0046] In some embodiments, at least one of plurality of alignment spots include a sample control configured to indicate an addition of samples to the well plate.

[0047] In some embodiments, the method includes detecting, by the processor, in the images of the well plate, the plurality of alignment spots; and in response to failing to detect the plurality of alignment spots, returning an error.

[0048] In some embodiments, the method includes performing, by the processor, circle detection at an expected target spot.

[0049] In some embodiments, the method includes measuring, by a laser distance sensor, a location of the first position.

[0050] In some embodiments, the method includes calibrating, by the processor, the stepper motor based on the location of the first position.

[0051] In some embodiments, the method includes calibrating, by the processor, the one or more imaging sensors based on the location of the first position.

[0052] In some embodiments, the method includes determining, by the processor, a sharpness value metric of each of the one or more imaging sensors; and tuning, by the processor, a focal length of each of the one or more imaging sensors to optimize the sharpness value metric.

[0053] In some embodiments, the method includes analyzing, by the processor, color values and intensity values for one or more background lights in the housing; and returning an operational status of the one or more background lights.

[0054] In some embodiments, the well plate includes a sample including at least one of milk, bulk tank milk, saliva, serum, bronchoalveolar lavage, bronchial wash, sputum, swab in transport media, feces, urine and cerebrospinal fluid.

[0055] In some embodiments, the one or more programming instructions further cause the processor to detect an avidity of the sample. In some embodiments, at least a portion of the plurality of alignment spots include antihorseradish peroxidase goat IgG.

[0056] In some embodiments, a method includes adding at least one sample to a well plate; providing the well plate to the system; and processing the well plate to detect disease-specific protein signatures in the at least one sample.

[0057] In some embodiments, processing the well plate further includes actuating the stepper motor to receive a plate holder and the well plate including a plurality of wells, along the one or more guide rails, from the opening in the housing and placing the plate holder in a first position in the housing; capturing, using the one or more imaging sensors, images of the well plate at the first position, wherein each of the images captures a first marker and a first portion of the plurality of wells based on an imaging area of the one or more imaging sensors with regard to the first position, wherein each well includes a sample including at least one protein; detecting a location of the first marker; determining a location of the first portion of the plurality of wells based on the location of the first marker; determining color values for each pixel in each of the first portion of the plurality of wells; determining color intensity values based on the color values; and analyzing the color intensity values to detect disease-specific protein signatures.

[0058] In some embodiments, the method includes actuating the stepper motor to receive the plate holder from the opening and place the plate holder in one or more additional positions in the housing; capturing, using the one or more imaging sensors, images of the well plate at each of the one or more additional positions, wherein each of the images captures a unique marker and a unique portion of the plurality of wells based on an imaging area of the one or more imaging sensors with regard to the unique position; detecting in the images of the well plate, a location the unique marker; and determining a location of the unique portion of the plurality of wells based on the location of the unique marker.

[0059] In some embodiments, the one or more imaging sensors includes a first imaging sensor and a second imaging sensor.

[0060] In some embodiments, the imaging area of the first imaging sensor is distinct from the imaging area of the second imaging sensor.

[0061] In some embodiments, the one or more imaging sensors are at least one of a complimentary metal-oxide semiconductor RGB sensor and charge-coupled device RGB sensor.

[0062] In some embodiments, the disease-specific protein signature includes detection of a biotinstreptavidin complex with at least one antibody and one protein in a well of the well plate.

[0063] In some embodiments, the well plate includes a plurality of alignment spots configured to display intense color intensity when imaged.

[0064] In some embodiments, the plurality of alignment spots are asymmetrically positioned. In some embodiments, the method includes determining well plate information including at least one of a make of the well plate, a test type, or an orientation of the well plate in the plate holder based on a location of the plurality of alignment spots.

[0065] In some embodiments, at least one of the plurality of alignment spots includes a reagent control configured to indicate an addition of a detection antibody.

[0066] In some embodiments, at least one of plurality of alignment spots include a sample control configured to indicate an addition of samples to the well plate.

[0067] In some embodiments, the method includes detecting in images of the well plate, the plurality of alignment spots; and in response to failing to detect the plurality of alignment spots, returning an error.

[0068] In some embodiments, the method includes performing circle detection at an expected target spot.

[0069] In some embodiments, the method includes measuring, by a laser distance sensor, a location of the first position.

[0070] In some embodiments, the method includes calibrating the stepper motor based on the location of the first position.

[0071] In some embodiments, the method includes calibrating the one or more imaging sensors based on the location of the first position.

[0072] In some embodiments, the method includes determining a sharpness value metric of each of the one or more imaging sensors; and tuning a focal length of each of the one or more imaging sensors to optimize the sharpness value metric.

[0073] In some embodiments, the method includes analyzing color values and intensity values for one or more background lights in the housing; and returning an operational status of the one or more background lights.

[0074] In some embodiments, the well plate includes a sample including at least one of milk, bulk tank milk, saliva, serum, bronchoalveolar lavage, bronchial wash, sputum, swab in transport media, feces, urine, and cerebrospinal fluid.

[0075] In some embodiments, the one or more programming instructions further cause the processor to detect an avidity of the sample.

[0076] In some embodiments, at least a portion of the plurality of alignment spots include antihorseradish peroxidase goat IgG.

[0077] In some embodiments, providing the well plate further includes providing antibody targets ununiformly in at least a portion of the plurality of wells; providing a sample to the portion of the plurality of wells; and incubating the well plate.

[0078] In some embodiments, incubating the well plate is performed for under 1 hour. BRIEF DESCRIPTION OF THE DRAWINGS

[0079] The accompanying drawings, which are incorporated in and form a part of the specification, illustrate the embodiments of the invention and together with the written description serve to explain the principles, characteristics, and features of the invention. In the drawings:

[0080] FIG. 1 depicts an analyte detection device in accordance with an embodiment.

[0081] FIG. 2 depicts an internal view of an analyte detection device in accordance with an embodiment.

[0082] FIG. 3 depicts a well plate in accordance with an embodiment.

[0083] FIG. 4 depicts a motorized plate holder in accordance with an embodiment.

[0084] FIGS. 5A-5C depict components of a motorized plate holder in accordance with an embodiment.

[0085] FIG. 6 depicts an imaging component in accordance with an embodiment.

[0086] FIGS. 7A-7B depict illumination systems in accordance with an embodiment.

[0087] FIG. 8 depicts a user input / output circuit in accordance with an embodiment.

[0088] FIG. 9 illustrates a flow diagram depicting a method of analyte detection in accordance with an embodiment.

[0089] FIGS. 10A-10B illustrate a method for HRP-conjugated ab / biotin-Streptavidin detection in accordance with an embodiment.

[0090] FIG. 11 illustrates an example workflow for HRP-conjugated ab / biotin-Streptavidin detection in accordance with an embodiment.

[0091] FIG. 12 illustrates a graph depicts the differentiation between recent and pass infections in a multiplex assay in accordance with an embodiment.

[0092] FIG. 13 illustrates a block diagram of an exemplary data processing system in which embodiments are implemented.

[0093] DETAILED DESCRIPTION

[0094] This disclosure is not limited to the particular systems, devices and methods described, as these may vary. The terminology used in the description is for the purpose of describing the particular versions or embodiments only and is not intended to limit the scope.

[0095] As used in this document, the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art. Nothing in this disclosure is to be construed as an admission that the embodiments described in this disclosure are not entitled to antedate such disclosure by virtue of prior invention. As used in this document, the term “comprising” means “including, but not limited to.”

[0096] The present invention provides systems and methods for analyte detection using automated multiplex immunoassay (MIA).

[0097] As used herein, the term “substrate” is any surface that supports an immunoassay. The substrate of the invention may be a solid substrate or a porous substrate, for example.

[0098] FIG. 1 depicts an analyte detection device 100 is accordance with an embodiment. A device housing 102 may encase the analyte detection device 100. An opening 104 may allow access to a substrate holder configured for receiving a substrate. In some embodiments, the opening includes a door. The door may include an actuator such that the door may automatically open / close. Alternatively, the door may be manually opened / closed. In certain embodiments, the analyte detection device 100 includes one or more indicators 108 (e.g., light emitting diodes), that may provide status indication. Example status indication includes a mode and / or phase of operation or an error status. In some embodiments the status indicators may be interactable for a user. In certain embodiments, the analyte detection device 100 may include one or more user inputs 106 configured to allow the user to interact with the device. Example interactions include activating the device or configuring the device. The analyte detection device 100 may include a user input (e.g., a button, knob, switch, or combination thereof) allowing for user input and control of the device's 100 functions. In certain embodiments, the analyte detection device includes communication ports 110 (e.g., the communication ports 1313 of FIG. 13).

[0099] The communication ports 110 may be configured to facilitate communication with an external device (e.g., a computing device or storage device). For example, the analyte detection device 100 may provide data to the external device for display, storage, and / or further processing. In some embodiments, communication by the analyte detection device 100 may be performed using a wireless communication interface via one or more wireless protocols (e.g., Wi-Fi, Bluetooth, Zigbee, etc.).

[0100] In certain embodiments, the communication ports 110 may include a power port configured to receive power for an external source. Additionally, or alternatively, the power port may be coupled to a battery within the analyte detection device 100.

[0101] FIG. 2 depicts an internal view of an analyte detection device 100 in accordance with an embodiment. Through the opening 104, the analyte detection device 100 may be configured to receive a substrate on a substrate holder movable along a series of rails 204.

[0102] In certain aspects, the substrate is a solid substrate. Examples of solid substrates include, but are not limited to, 96 well microtiter plates, glass, microbeads, nano / micro- particles and magnetic beads. In one aspect, the bottom of a 96 well microtiter plate is made up of polystyrene, polydimethylsiloxane (PDMS), poly (methyl methacrylate) (PMMA), polycarbonate, cyclic polyolefins, Zeonor, Zeonex, or cellulose acetate. In various aspects, the solid substrate maybe glass beads, nano- / microparticles, magnetic beads or paramagnetic beads. FIG. 3 depicts a substrate 300 (e.g., a 96 well microtiter plate) in accordance with an embodiment. The substrate 300 may include a plurality of wells 302 configured to house assay elements.

[0103] In some embodiments, the substrate 300 may feature a series of alignment spots, each engineered to exhibit intense color intensity upon imaging. These spots may be strategically positioned to encode valuable information about the substrate 300, such as its make, test type, or orientation.

[0104] The assay elements (e.g., control and capture elements) are placed on the substrate surface, with or without an adapter molecule between the substrate and the element. Preferably, the assay elements bind to the substrate by covalent or non-covalent interaction. One of skill in the art will recognize the methods of placing assay elements on the substrate include printing, spotting or other techniques known in the art. For purposes of the present application, the term “printing” can be used to include any of the methods for placing the assay elements on a membrane.

[0105] The terms “array” or “microarray” as used herein refer to a collection of multiple assay elements on a substrate. Specifically, an array is a collection of capture elements and / or control elements on a substrate.

[0106] In various aspects, the elements on the array are placed on the substrate in discrete areas of between 100 pm to 500 pm in diameter. More preferably, the discrete areas are between 350pm to 400 pm in diameter. In certain aspects, the discrete areas of the array are placed in a 5x5 grid. In one aspect, the array comprises up to nine control elements and two replicates of each of eight different capture elements. In one aspect, the capture elements are printed in two or more replicates of four different capture elements and multiples thereof.

[0107] As used herein, the term “assay element” refers to any of a number of different elements for use in an array of the invention. Exemplary assay elements include, but are not limited to, capture elements and control elements.

[0108] The term “capture element” refers to a molecule that is able to bind to a target analyte. Examples of useful capture elements include proteins, protein fragments, polypeptides, polypeptide fragments, binding proteins, binding protein fragments, antibodies (polyclonal, monoclonal, or chimeric), antibody fragments, antibody heavy chains, antibody light chains, single chain antibodies, single-domain antibodies (e.g., a VHH), Fab antibody fragments, Fc antibody fragments, Fv antibody fragments, F(ab')2 antibody fragments, Fab' antibody fragments, single-chain Fv (scFv) antibody fragments, antibody binding domains, antigens, antigenic determinants, epitopes, haptens, immunogens, immunogen fragments, and binding domains. Useful capture elements will correspond to and are able to bind a specific target analyte, such as a molecule or class of molecules that are present in a sample to be tested.

[0109] In one embodiment, the capture element is a protein, a protein fragment, a binding protein, a binding protein fragment, an antibody, an antibody fragment, an antibody heavy chain, an antibody light chain, a single chain antibody, a single-domain antibody (e.g., a VHH), a Fab antibody fragment, an Fc antibody fragment, an Fv antibody fragment, a F(ab')2 antibody fragment, a Fab' antibody fragment, a single-chain Fv (scFv) antibody fragment, an antibody binding domain, an antigen, an antigenic determinant, an epitope, a hapten, an immunogen, an immunogen fragment, and a binding domain.

[0110] In another aspect, the capture elements may comprise antibodies or fragments thereof that are immobilized on the substrate surface and are specific for different antigens or ligands that may be present in a sample. In certain aspects, the capture elements may comprise antigens or ligands and the assay involves the detection of specific antibodies that may be present in a sample. In various aspects, the capture elements may comprise of a receptor or a subunit of a receptor that binds a specific ligand.

[0111] The substrate may include at least one fiduciary marker that will always be detectable on the substrate, preferably detectable irrespective of the performance of the assay or processing of the substrate.

[0112] The term “fiduciary marker” refers to a colored marker or label that will always be detectable on the substrate, preferably irrespective of the performance of the assay or processing of the substrate. The use of at least one fiduciary marker will obviate the necessity of this element being detected based on successful array processing, in comparison to the positive colorimetric controls. The fiduciary marker is therefore a “true” positive control that would always be detectable regardless of array processing and can be used to orient and help to grid the array. In preferred aspects, the fiduciary marker is a dye, dye-conjugated protein or a chromogenic protein such as hemoglobin. US Patent 10,948,486, issued in 2021, is hereby incorporated herein by reference in its entirety.

[0113] The term “negative control” refers to an element comprising print buffer or an unrelated protein to which no complementary binding partner is intended to be present in the assay. Any detectable signal from the negative control may be used to determine the background threshold of the assay and the accuracy of any positive results. In one aspect, the negative control to monitor background signal is print buffer. The print buffer is a solution used to carry and print the capture elements and control elements onto the substrate and may comprise buffered saline, glycerol and a surfactant, preferably a polysorbate surfactant such as Tween 20. The blocking solution is used to reduce non-specific protein binding to the substrate surface and preferably comprises skim milk, casein, bovine serum albumin, gelatins from fish, pigs or other species, dextran or any mixture of any two or more thereof, preferably in a solution of phosphate buffered saline and a surfactant such as Tween 20.

[0114] The term “control capture element” refers to a capture element that functions as a control, either a negative control that should not bind any analyte or a positive control that will bind a non-target analyte. The substrate may include at least one control to monitor assay performance. The control is intended to provide information of the efficiency of the complementary binding interactions or the quality or performance of the reagents used.

[0115] The term “control to monitor assay performance” refers to an element that forms one part of a complementary binding interaction during an assay and is intended to provide information on the accuracy of the assay result. In one embodiment, the positive control to monitor assay performance comprises one binding partner of a complementary binding pair, where the other binding partner is a sample component or an assay reagent. The assay performance control is preferably a target analyte, a binding partner corresponding to and able to bind a non-target analyte that will be present in the sample, a binding partner corresponding to and able to bind an assay reagent, and a colorimetric enzyme label, or any combination of any two or more thereof. An example of a binding partner corresponding to and able to bind a non-target analyte that will be present in the sample is an anti-Ig antibody that will bind an immunoglobulin present in a serum sample, therefore confirming a sample has been added. An example of a binding partner corresponding to and able to bind an assay reagent is an anti-Ig antibody that may bind a secondary immunoglobulin that is used to process the assay, such as biotinylated anti-target analyte antibody. Another example of a binding partner corresponding to and able to bind an assay reagent is a biotinylated antibody that may bind a streptavidin-peroxidase conjugate that is used to process the assay.

[0116] In one aspect, the assay performance control includes one binding partner of a complementary binding pair, wherein the other binding partner is an assay reagent. The assay performance control is preferably the target analyte, a non-specific binding partner or a colorimetric enzyme label.

[0117] In another aspect, the complementary binding partners include antibody-antigen interactions or antibody-ligand interactions.

[0118] The substrate may include at least one control to monitor assay specificity. The control is intended to provide information of the specificity of binding between the capture element and the target analyte, or between the binding partners of the assay detection steps.

[0119] The term “control to monitor assay specificity” refers to an element that is closely related to at least one binding partner of a complementary binding pair present in the assay and is intended to provide information of the specificity of the complementary binding. This control is a negative control that is not expected to generate a detectable result during normal assay processing. For example, in an antibody array for antigen detection, the assay specificity control would comprise an antibody that should not bind any antigen in the sample. Alternatively, in an antigen array for antibody detection, the assay specificity control would comprise an antigen that should not bind any antibody in the sample.

[0120] In one aspect, the assay specificity control is Mycobacterium phlei protein extract. In another aspect, the assay specificity control is Mycobacterium tuberculosis antigen. The term “positive colorimetric control” as used herein refers to an enzyme or enzyme conjugate that provides a detectable signal upon the enzyme substrate’s addition.

[0121] In one embodiment, the positive colorimetric control is an enzyme label conjugate capable of reacting with a colorimetric substrate, comprising an enzyme comprising horseradish peroxidase, alkaline phosphatases, P-D-galactosidase or glucose oxidase.

[0122] The identity of the assay controls may be dependent on the type of array, the identity of the target analyte, and the type of sample to be analyzed.

[0123] For example, either anti -bovine IgG-HRP or specific monoclonal IgG-HRP may be used in arrays printed with antigens and antibodies, respectively. The final detection antibody in antigen arrays may be anti-bovine IgG-HRP, while for antibody arrays it may be a HRP conjugated IgG antibody specific for the targeted analyte or a bovine IgG specific for the target analyte that is detected later on the next step with anti-bovine IgG-HRP. These controls can provide a positive control in addition to providing information on the performance or quality of the HRP substrate.

[0124] The target protein or antigen and mouse IgG, bovine IgG and anti-bovine IgG present on antigen or antibody arrays may act either as positive or negative controls depending on the array format, in addition to providing information of assay specificity. For example, antigen or mouse IgG spots may provide the positive signal in antibody arrays, while the latter two should provide a positive signal in antigen arrays. These controls may also serve as controls for overall assay performance.

[0125] In a MIA, antibodies are determined via an indirect immunoassay (IA). A membrane-free MIA procedure will be used for the printing of microarray spots (e.g., PictArray™, U.S. Patent No. 9,625,453) directly on the solid surface of a 96-well microtiter plate (MTP). The biorecognition elements responsible for binding the antibodies may be printed onto the surface of the MTP well 302 using a leach-proof biomolecular immobilization procedure.

[0126] The terms “sample” and “specimen” as used herein are used in their broadest sense to include any composition that is obtained and / or derived from biological or environmental source, as well as sampling devices (e.g., swabs) that are brought into contact with biological or environmental samples. “Biological samples” include body fluids such as milk, urine, blood, plasma, fecal matter, cerebrospinal fluid (CSF), semen, respiratory tract mucus or washing and saliva. In one embodiment, the biological sample is fluid obtained from a mammal, including milk, blood, plasma, serum, and stool. These examples are illustrative and are not to be construed as limiting the sample types applicable to the present invention.

[0127] In various aspects of the present invention, the sample is a milk, bulk tank milk, saliva, blood sample (e.g., a plasma or serum sample), bronchoalveolar lavage, bronchial wash, sputum, swab in transport media, feces, urine, or cerebrospinal fluid.

[0128] The analyte detection device 100 may be configured to receive substrate 300. FIG. 4 depicts a motorized substrate holder 400 in accordance with an embodiment. FIGS. 5A-5C further depict components of the substrate holder 400 in accordance with an embodiment. The substrate holder 400 may be configured move the substrate 300 in and out of the opening 104. In some embodiments, the substrate holder 400 may be configured to precisely position the substrate 300 at specific locations within the housing 102.

[0129] In some embodiments, the analyte detection device 100 includes a shelf 402 or grasping elements configured to receive and / or support the substrate 300. The shelf 402 or grasping element may be affixed to one or more linear guides 410 configured to translate along the rails 204. The linear guides 410 may include one or more bearings to facilitate smooth motion along the rails 240.

[0130] The substrate holder 400 may include a motor 404 configured to actuate the shelf 402 or grasping element along the rails 204. In certain embodiments, the motor 404 drives a pinion gear 406 to position in the substrate 300 along a mechanical rack 408. Actuation of the pinion 406 may translate the substrate along the rails 204.

[0131] Alternative means of actuating the substrate holder 400 are also considered. For example, the shelf 402 may be actuated along a screw, track, or a telescoping member. Any known method of precisely positioning the substrate 300 may be used.

[0132] FIG. 6 depicts an imaging component 600 in accordance with an embodiment. The imaging component 600 may include one or more imaging sensors 602. The imaging sensors 602 may be a charge coupled device (CCD) or complementary metal oxide semiconductor (CMOS). In some embodiments, the imaging sensors 602 are configured to accurately detect a color range of a specimen on the substrate, as described herein.

[0133] The imaging component 600 may be mounted in the housing 102 such that the field of view of the imaging sensors 602 captures a portion of the substrate 300 (e.g., a predetermined set of wells) based on a positioning of substrate holder 400. In some embodiments, the imaging component 600 is mounted above the substrate holder 400.

[0134] In some embodiments, the one or more imaging sensors 602 may include two or more imaging sensors 602 arranged in array such that each imaging sensor 602. Each imaging sensor 602, of the array of imaging sensors 602, may have a distinct field of view.

[0135] In alternative embodiments, the imaging component 600 may include an actuation system as disclosed above in reference to the substrate holder 400. The actuation system may facilitate the precise positioning of the imaging component 600 relative to the substrate 300.

[0136] The controlled environment of the housing 102 may facilitate the capture, by the imaging sensors 602, of precisely lighted images of the substrate 300. FIG. 7A depicts an overhead illumination system 700 in accordance with an embodiment. In some embodiments, the overhead illumination system 700 may in include an array of light sources 702 (e.g., light emitting diodes). The overhead illumination system 700 may be configured to directly illuminate the substrate 300. FIG. 7B depicts a backlight illumination system 710 in accordance with an embodiment. In some embodiments, the backlight illumination system 710 may include an array of light sources 712 (e.g., light emitting diodes). The backlight illumination system 710 may be configured to backlight the substrate 300. In some embodiments, the backlight illumination system 710 is integrated into the substrate holder 400, and thus moveable along with the substrate 300. In alternative embodiments, the backlight illumination system 710 is rigidly affixed to an inner floor of housing 102 in a position which intersects the field of view of the imaging sensors 602.

[0137] Although a plurality of illumination systems (e.g. overhead illumination system 700 and backlight illumination system 710) are described herein. In some embodiments, a single illumination system may be used. For example, blacklight illumination may be sufficient for properly illuminating substrate 300.

[0138] In some embodiments, the illumination systems 700 / 710 may be configured to emit light which covers the full visible wavelength spectrum (e.g., from approximately 420 nm to 750 nm). The light sources may operate at a color temperature of approximately 5000 K. In some embodiments, the emitted light has notable intensity peaks around 460-480 nm and / or 530-600 nm bands.

[0139] FIG. 8 depicts a user input / output circuit in accordance with an embodiment. The input / output circuit may be interfaced to control circuit, to allow a user to receive notifications from the system. Further, the input / output circuit may allow a user to provide input to the system.

[0140] In some embodiments, the analyte detection device 100 may include a control circuit (e.g., the processing unit 1303 of FIG. 13). The control circuit may be responsible for coordinating the actions of the various mechanical and electronic components within the device, such as the stepper motor 402, the imaging sensors 602, and the illumination systems 700, 710.

[0141] The control circuit may manage the precise actuation of the stepper motor 402, ensuring that the substrate holder 400 is positioned accurately within the housing 102 for image capture. The control circuit may adjust the parameters of imaging sensors 602 and based on the requirements of the assay being performed, such as exposure time and sensitivity.

[0142] Additionally, the control circuit may control the overhead and backlight illumination systems 700, 710, modulating the intensity of the light sources 702, 712 as appropriate for the assay. The control circuit may ensure that the substrate 300 is illuminated uniformly, providing the ideal conditions for the imaging sensors 602 to capture high-quality images of the wells.

[0143] In certain embodiments, the control circuit may further perform some portion of the processing described herein. For example, the control circuit may process image data captured by the imaging sensors 602 to detect disease-specific protein signatures in biological samples. Alternatively, the control circuit may transmit and / or store collected data for processing on an external computing system. In some embodiments, the analyte detection device 100 may be configured for one or more automatic or manual calibration processes. Calibration may ensure the accuracy and reliability of the assay results. The calibration may optimize the performance of the device's mechanical (e.g., the substrate holder) and optical (e.g., the imaging sensors 602) components, as well as to verify the precision of the system's analytical algorithms.

[0144] The stepper motor 404 is responsible for the precise movement of the substrate holder 400 along the guide rails 204 to predetermined positions within the housing 102 for imaging. Calibration may include moving the substrate holder 400 to the predetermined positions within the housing 102 and measuring the actual positions achieved using an integrated laser distance sensor. Any discrepancies between the expected and actual positions, as determined by the laser distance sensor, may be used to adjust the control parameters of the stepper motor 404, ensuring accurate positioning of the substrate 300 for subsequent imaging.

[0145] The imaging sensors 602 may be calibrated to ensure that they capture clear and accurate images of the substrate 300 and an associated specimen. The calibration process may include adjusting the focal length of the sensors to achieve the sharpest possible image, a process that may be automated by the control circuit. The control circuit may determine a sharpness value metric for each sensor and adjust the focal length accordingly. Additionally, the uniformity and intensity of the illumination provided by the background lights within the housing may be calibrated, based on captured images from the image sensors 602, to ensure consistent lighting conditions.

[0146] Furthermore, the analyte detection device 100 may include calibration routines based on the detection of the alignment spots on the substrate 300. These spots may be used to detect the orientation of the substrate within the housing 102 and to encode information such as the make of the substrate, the test type, or the orientation of the substrate 300 in the substrate holder 400. The system may use the images captured by the imaging sensors to detect the alignment spots and confirm their expected positions. If the alignment spots are not detected or are misaligned, the system may prompt the user to reposition the substrate and / or automatically adjust the substrate holder 400 position.

[0147] The calibration process may also include the verification of the imaging sensors’ 602 color accuracy. This may be achieved by capturing images of a color calibration chart that includes a range of known color values. The control circuit may analyze the captured images and compares the detected color values against the known values of the chart. Any deviations are used to adjust the color detection algorithms of the processor, ensuring that the device accurately determines the color values and intensity levels in the wells, which are indicative of the presence and concentration of analytes. In some embodiments, a color calibration chart may be provided in a similar form as the substrate 300.

[0148] Regular calibration of the analyte detection device 100 may maintain its performance over time. The analyte detection device 100 may include a calibration schedule and prompts to remind the user when calibration is due. Alternatively, automatic calibration may be performed based on the calibration schedule. A calibration schedule may be based on a time period, an amount of usage, or a combination thereof. By adhering to a strict calibration protocol, the analyte detection device 100 may ensure that it provides consistent, reliable, and accurate results for the detection of disease-specific protein signatures in biological samples.

[0149] FIG. 9 illustrates a flow diagram depicting a method of analyte detection in accordance with an embodiment.

[0150] In some embodiments, preprocessing a substrate may include providing the substrate, adding at least one sample to the substrate, and processing the substrate.

[0151] In one aspect, the step of processing the substrate or microarray comprises a blocking step during which available protein-binding sites on the substrate or microarray are blocked with a blocker (i.e., blocking agent), an optional wash step, contacting the substrate or microarray with the sample containing the one or more analytes to be measured, a wash step to remove non-bound material from the substrate or microarray, contacting the substrate or microarray with one or more secondary antibodies that correspond to and will bind one or more target analytes and non-target analyte that is bound to an assay performance control, a wash step, and contacting the substrate or microarray with one or both of an enzyme conjugate or an enzyme substrate to generate a detectable result. In certain embodiments, the wash includes the application of urea. The preprocessed substrate 300 may then be analyzed using the analyte detection device 100.

[0152] In some embodiments, a user may initiate an analysis by pressing an input or pushing on the substrate holder 400. The method 900 may include actuating 902 the stepper motor to maneuver substrate holder 400 along the guide rails 204 from the opening 104 to a designated first position within the housing 102.

[0153] The imaging sensors 602 may capture 904 images of a portion of the substrate 300. The designated first position may be selected such that field of view of the imaging sensors 602 captures a particular set of specimens (e.g., particular wells) on the substrate 300. These images may include one or more markers on the substrate 300. The method may include detecting 906 the marker. In some embodiments, a marker is at a specific location on the substrate 300. The marker may include a specifically colored, and thus unambiguously identifiable, well on the substrate 300. Alternatively, any detectable feature may be used as a marker.

[0154] The detected marker location may be used as a fiduciary reference for determining 908 the subsequent locations of local samples. For example, by leveraging the location of the marker, the system may identify the position of the wells on the substrate, correlating the marker's position with a predefined layout of wells. Detecting 906 the marker and / or determining 908 locations of local samples may be performed using circle detection. The method 900 may include evaluating 910 the captured images to ascertain the color values for each pixel within each of the located wells. These color values may be reflective of the analytes' presence and their concentrations within the wells.

[0155] Utilizing the determined color values, the method may include determining 912 color intensity values for each well. The analysis of these intensity values facilitates the detection of diseasespecific protein signatures, which may suggest the presence of a particular disease or condition in the sample. The method 900 may include the detection of a biotin-streptavidin complex.

[0156] The method 900 may include directing the stepper motor to adjust the substrate holder's 400 position to enable the imaging sensors 602 to capture additional substrate portions, each associated with a marker and local samples. This comprehensive imaging ensures that all samples may be analyzed.

[0157] The method 900 may include confirming the presence of controls, such as reagent controls indicating the addition of detection antibodies and / or sample controls verifying the addition of samples to the substrate. An error message may be generated in response to control not being detected.

[0158] The assay techniques used in conjunction with the substrates of the present invention include any of a number of well-known colorimetric enzyme-linked assays. Examples of such systems are well known in the art. The assay techniques are based upon the formation of a complex between a complementary binding pair, followed by detection with a colorimetric detection system comprising an enzyme-conjugate label and a colorimetric substrate. The detection system will be described with reference to enzyme-linked immunosorbent assays (ELISA). However, a skilled person would appreciate that such techniques are not restricted to the use of antibodies but are equally applicable to any colorimetric assay.

[0159] In one embodiment, the ELISA is in the “sandwich” assay format. In this format, the target analyte to be measured is bound between two antibodies - the capture antibody and the detection antibody. In another embodiment, the ELISA is a non-competitive assay in which an antibody binds to the capture antigen and the amount of bound antibody is determined by a secondary detection antibody.

[0160] Either monoclonal or polyclonal antibodies may be used as the capture and detection antibodies in sandwich ELISA systems. Monoclonal antibodies have an inherent monospecificity toward a single epitope that allows fine detection and quantitation of small differences in antigen. A polyclonal antibody can also be used as the capture antibody to bind as much of the antigen as possible, followed by the use of a monoclonal antibody as the detecting antibody in the sandwich assay to provide improved specificity. A monoclonal antibody can also be used as the capture antibody to provide specific analyte capture, followed by the use of a polyclonal antibody as the detection antibody in the sandwich assay. Additionally, both the capture and the detection antibodies could be monoclonal. The term “antibody” as used herein includes naturally occurring antibodies as well as non- naturally occurring antibodies, including, for example, single chain antibodies, chimeric, bifunctional and humanized antibodies, as well as antigen-binding fragments thereof, including, but not limited to F(ab')2, Fab, Fab' and Fv which are antigen-binding fragments that can be generated from the variable region of IgG and IgM. Such non- naturally occurring antibodies can be constructed using solid phase peptide synthesis, can be produced recombinantly or can be obtained, for example, by screening combinatorial libraries consisting of variable heavy chains and variable light chains (see Huse et al, Science 246: 1275- 1281, 1989, which is incorporated herein by reference). These and other methods of making, for example, chimeric, humanized, CDR-grafted, single chain, and bifunctional antibodies are well known (Winter and Harris, Immunol. Today 14:243-246, 1993; Ward et al., Nature 341 :544-546, 1989; Harlow and Lane, Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1999); Hilyard et al., Protein Engineering: A Practical Approach (IRL Press 1992); Borrabeck, Antibody Engineering, 2d ed. (Oxford University Press 1995); each of which is incorporated herein by reference). In addition, modified or derivatized antibodies, or antigen binding fragments of antibodies, such as pegylated (polyethylene glycol modified) antibodies, can be useful for the present methods. As such, Lab, L(ab')2, Ld and Lv fragments of an antibody that retain specific binding activity are included within the definition of an antibody.

[0161] The term “secondary antibody” refers to an antibody that will bind a target analyte and that is conjugated with either an adaptor molecule such as biotin or an enzyme label such as horseradish peroxidase (HRP). Antibody-adaptor conjugates are processed to give a detectable result by contacting the antibody-adaptor conjugate with an adaptor-enzyme conjugate and then the enzyme substrate; for example, antibody -biotin conjugates will bind streptavidin-HRP conjugates. Antibody-enzyme label conjugates include antibody-HRP conjugates. Use of secondary antibodies is discussed and exemplified below.

[0162] The term “binds specifically” or “specific binding activity” or the like, means that two molecules form a complex that is relatively stable under physiologic conditions. The term is also applicable where an antigen-binding domain is specific for a particular epitope, which is carried by a number of antigens, in which case the antibody carrying the antigen-binding domain will be able to bind to the various antigens carrying the epitope. Specific binding is characterized by a high affinity and a low to moderate capacity. Typically, the binding is considered specific when the affinity constant is about 1 x 10'6M, generally at least about 1 x 10'7M, usually at least about 1 x io-8M, and particularly at least about 1 x 10'9M or less.

[0163] After array manufacture and prior to sample addition, all available protein-binding sites on the substrate surface are blocked by addition and incubation with one or a combination of reagents. These reagents are called “Blockers” and serve to decrease or at best eliminate nonspecific protein binding from the sample on the substrate surface, thereby decreasing overall background signal. This increases the ratio of signal to noise, thereby increasing the overall sensitivity of the assay. Blockers play no active part in the subsequent reactions between the sample and other assay reagents and the immobilized proteins on the substrate. Exemplary blockers include, but are not limited to, bovine serum albumin, casein, non-fat dry milk, gelatin derived from fish, pigs and other sources, dextran, serum derived from sources other than the sample being analyzed such as from steelhead salmon, guinea pigs, hamsters, rabbit and other sources, polyethylene glycol, polyvinyl pyrrollidone, and commercial preparations including HeteroBlock® (Omega Biologicals, Bozeman, Mont.), SuperBlock™, StartingBlock™, SEA BLOCK (Pierce, Rockford, Ill.). Typically, blockers are made up in buffer solutions such as, for example, phosphate buffer, phosphate buffered saline, Tris buffer, acetate buffer and others. The blockers may also be supplemented with detergents such as, for example, Tween 20, Tween 80, Nonidet P40, sodium dodecyl sulfate and others.

[0164] An important consideration in designing an array is that the capture and detection antibodies of each binding pair must recognize two non-overlapping epitopes so that when the antigen binds to the capture antibody, the epitope recognized by the detection antibody must not be obscured or altered. A large number of complementary binding pairs have already been developed for ELISA and can be used in the present invention.

[0165] For multiplexed assays, it is also important that there is no overlap between each of the binding pairs to eliminate cross-reactivity. A number of multiplexed ELISAs have been developed and it is anticipated other combinations of binding pairs could be configured through testing.

[0166] In one aspect, the enzyme-conjugate label comprises an enzyme including but not limited to horseradish peroxidase, alkaline phosphatase, P-D-galactosidase or glucose oxidase.

[0167] In an additional aspect, the enzyme label may be conjugated directly to a primary antibody or introduced through a secondary antibody that recognizes the primary antibody. It may also be conjugated to a protein such as streptavidin if the primary antibody is biotin labelled.

[0168] In a further aspect, the analyte detection system comprises a colorimetric detection substrate comprising 3,3', 5,5'-tetramethylbenzidine, diaminobenzidine, metal-enhanced diaminobenzidine, 4-chloro-l -naphthol, colloidal gold, nitro-blue tetrazolium chloride, 5-bromo- 4-chl oro-3 '-indolylphosphate p-toluidine salt and naphthol AS-MX phosphate+Fast Red TR Salt.

[0169] In certain aspects, the colorimetric reaction can be detected and optionally quantified and analyzed using an image capture device such as a digital camera or a desktop scanner attached to a computer. Known methods for image analysis may be used. For example, the concentration values of known standard elements can be used to generate standard curves. Concentration values for unknown analytes can be analyzed using the standard curve for each analyte to calculate actual concentrations. Values for each analyte can be identified based on the spotting position of each capture element within the array. As used herein, the terms “biomarker” refers to any substance used as an indicator of a biological state. Thus, a biomarker can be any substance whose detection indicates a particular disease state (for example, the presence of an antibody may indicate an infection). Furthermore, a biomarker can be indicative of a change in expression or state of a protein that correlates with the risk or progression of a disease, or with the susceptibility of the disease to a given treatment. Once a proposed biomarker has been validated, it can be used to diagnose disease risk, presence of disease in an individual, or to tailor treatments for the disease in an individual (e.g., choices of drug treatment or administration regimes). In evaluating potential drug therapies, a biomarker may be used as a surrogate for a natural endpoint such as survival or irreversible morbidity. If a treatment alters the biomarker, which has a direct connection to improved health, the biomarker serves as a “surrogate endpoint” for evaluating clinical benefit. In one aspect, the target analyte is a biomarker.

[0170] In one embodiment, the target analyte is a protein, a protein fragment, a peptide, a polypeptide, a polypeptide fragment, an antibody, an antibody fragment, an antibody binding domain, an antigen, an antigen fragment, an antigenic determinant, an epitope, a hapten, an immunogen, an immunogen fragment, or any combination of any two or more thereof.

[0171] In one aspect, the target analyte is an antibody to SARS-CoV-2 protein.

[0172] Capture elements specific for a target analyte are used to detect the presence or absence of the analyte in a sample. A wide range of complementary binding or coupling partners are known, with the choice of capture elements determined by the analytes to be detected, the requirement for adapter molecules and the level of specificity required for the assay. In various aspects, the capture elements are specific for binding / detecting the target analyte.

[0173] The term “control element” refers to an element that is used to provide information on the function of the assay, for example binding specificity, the level of non-specific background binding, the degree of binding cross-reactivity, and the performance of assay reagents and the detection system. Preferred controls useful herein include at least one negative control to monitor background signal, at least one negative control to monitor assay specificity, at least one positive colorimetric control, and at least one positive control to monitor assay performance.

[0174] FIGS. 10A-10B illustrate methods for HRP-conjugated ab / biotin-Streptavidin detection in accordance with an embodiment.

[0175] FIG. 10A depicts an example method utilizing HRP-conjugated antibodies for the detection of analytes. In the example, the well plate contains immobilized antigens that capture target analytes from a sample. A secondary antibody, conjugated with horseradish peroxidase (HRP), is then introduced to bind to the captured analytes. Upon addition of a chromogenic substrate, such as TMB, a colorimetric reaction occurs, indicating the presence of the target analytes.

[0176] FIG. 10B presents an alternative method employing a biotin-streptavidin system for enhanced detection sensitivity. Similar to FIG. 10A, target analytes are captured by immobilized antigens on the well plate. A biotinylated secondary antibody is applied to bind the analytes, followed by the addition of streptavidin conjugated with HRP. The subsequent addition of a chromogenic substrate leads to a colorimetric reaction, with the biotin-streptavidin interaction amplifying the signal for improved detection of the analytes.

[0177] In either example, the colorimetric response may be detected by the systems and methods described herein.

[0178] FIG. 11 illustrates an example workflow for HRP-conjugated ab / biotin-Streptavidin detection in accordance with an embodiment. The workflow outlines the sequential steps that may be involved in conducting a multiplex immunoassay using HRP-conjugated antibodies or a biotinstreptavidin system. The process may include the collection 1102 of samples including target antigens. In some embodiments, a control or diluted sample may be added samples. The samples may be mixed, covered and incubated 1104 for a given time (e.g., 30 minutes) and at a given temperature (e.g., 37oC). The process may include washing 1106 (e.g., 5 times) to remove unbound components, addition of detection antibodies, and further incubation. In certain embodiments, a chromogenic substrate may be applied 1108 to visualize the binding events. After further incubation, (e.g. 20 minutes) the intensity of a color change may correlate to the quantity of target analytes present in the samples.

[0179] The term “avidity” refers to the strength of a multivalent interaction in a sample.

[0180] FIG. 12 illustrates a graph depicting the differentiation between recent and past infections in a multiplex assay in accordance with an embodiment. The graph provides a visual representation of the antibody response over time following an infection. It displays the kinetics of various antibody isotypes, such as IgM, IgA, and IgG, as well as the antigen levels. The illustrated relationships may facilitate distinguishing between recent and past infections by comparing the temporal patterns of antibody titers, with specific emphasis on the maturation of the IgG response and its avidity, which increases over time and is indicative of a chronic infection.

[0181] Data Processing Systems for Implementing Embodiments Herein

[0182] FIG. 13 illustrates a block diagram of an exemplary data processing system 1300 in which embodiments are implemented. The data processing system 1300 is an example of a computer, such as a server or client, in which computer usable code or instructions implementing the process for illustrative embodiments of the present invention are located. In some embodiments, the data processing system 1300 may be a server computing device. For example, the data processing system 1300 may be implemented in a server or another similar computing device operably connected to a surgical system 100 as described above. The data processing system 1300 may be configured to, for example, transmit and receive information related to a patient and / or a related surgical plan with the surgical system 100.

[0183] In the depicted example, the data processing system 1300 may employ a hub architecture including a north bridge and memory controller hub (NB / MCH) 1301 and south bridge and input / output (I / O) controller hub (SB / ICH) 1302. A processing unit 1303, a main memory 1304, and a graphics processor 1305 may be connected to the NB / MCH 1301. The graphics processor 1305 may be connected to the NB / MCH 1301 through, for example, an accelerated graphics port (AGP).

[0184] In the depicted example, a network adapter 1306 connects to the SB / ICH 1302. An audio adapter 1307, a keyboard and mouse adapter 1308, a modem 1309, a read only memory (ROM) 1310, a hard disk drive (HDD) 1311, an optical drive (e.g., CD or DVD) 1312, a universal serial bus (USB) ports and other communication ports 1313, and PCI / PCIe devices 1314 may connect to the SB / ICH 1302 through a bus system 1316. The PCI / PCIe devices 1314 may include Ethernet adapters, add-in cards, and / or PC cards for notebook computers. The ROM 1310 may be, for example, a flash basic input / output system (BIOS). The HDD 1311 and the optical drive 1312 may use an integrated drive electronics (IDE) or serial advanced technology attachment (SATA) interface. A super I / O (SIO) device 1315 may be connected to the SB / ICH 1302.

[0185] An operating system may run on the processing unit 1303. The operating system may coordinate and provide control of various components within the data processing system 1300. As a client, the operating system may be a commercially available operating system. An object- oriented programming system, such as the JavaTM programming system, may run in conjunction with the operating system and provide calls to the operating system from the object-oriented programs or applications executing on the data processing system 1300. As a server, the data processing system 1300 may be an IBM® eServer TM System® running the Advanced Interactive Executive operating system or the Linux operating system. The data processing system 1300 may be a symmetric multiprocessor (SMP) system that includes a plurality of processors in the processing unit 1303. Alternatively, a single processor system may be employed.

[0186] Instructions for the operating system, the object-oriented programming system, and applications or programs are located on storage devices, such as the HDD 1311, and are loaded into the main memory 1304 for execution by the processing unit 1303. The processes for embodiments described herein may be performed by the processing unit 1303 using computer usable program code, which can be located in a memory such as, for example, main memory 1304, ROM 1310, or in one or more peripheral devices.

[0187] A bus system 1316 may comprise one or more busses. The bus system 1316 may be implemented using any type of communication fabric or architecture that provides for a transfer of data between different components or devices attached to the fabric or architecture. A communication unit such as the modem 1309 or the network adapter 1306 may include one or more devices that can be used to transmit and receive data.

[0188] Those of ordinary skill in the art will appreciate that the hardware depicted in FIG. 13 may vary depending on the implementation. Other internal hardware or peripheral devices, such as flash memory, equivalent non-volatile memory, or optical disk drives may be used in addition to or in place of the hardware depicted. Moreover, the data processing system 1300 can take the form of any of a number of different data processing systems, including but not limited to, client computing devices, server computing devices, tablet computers, laptop computers, telephone or other communication devices, personal digital assistants, and the like. Essentially, data processing system 1300 can be any known or later developed data processing system without architectural limitation.

[0189] ADDITIONAL EMBODIMENTS

[0190] Embodiment 1- A system comprising: a housing comprising an opening; a stepper motor; a plate holder configured to receive a well plate comprising a plurality of wells; one or more guide rails, wherein at least one of the one or more guide rails is mechanically coupled to the stepper motor, wherein the stepper motor is configured to drive the plate holder along the guide rails to a plurality of positions comprising at least one position inside the housing and at least one position outside the opening of the housing; and one or more imaging sensors affixed inside the housing, wherein the imaging sensor is configured to capture at least one image of at least a portion of the well plate in the plate holder in the at least one position inside the housing, and wherein the portion of the well plate is based on an imaging area of the one or more imaging sensors.

[0191] Embodiment 2. The system of Embodiment 1, further comprising: a processor; and a non- transitory, processor-readable storage medium, wherein the non-transitory, processor-readable storage medium comprises one or more programming instructions that, when executed, cause the processor to: actuate the stepper motor to eject the plate holder from the opening; actuate the stepper motor to receive the plate holder from the opening and place the plate holder in a first position in the housing; capture, using the one or more imaging sensors, images of the well plate at the first position, wherein each of the images captures a first marker and a first portion of the plurality of wells based on the imaging area of the one or more imaging sensors with regard to the first position, wherein each well comprises a sample comprising at least one protein; detect a location the first marker; determine a location of the first portion of the plurality of wells based on the location of the first marker; determine color values for each pixel in each of the first portion of the plurality of wells; determine color intensity values based on the color values; and analyze the color intensity values to detect disease-specific protein signatures.

[0192] Embodiment 3. The system of any one of Embodiments 1-2, furthering comprising one or more interface elements attached to the housing and electronically interfaced to the processor.

[0193] Embodiment 4. The system of any one of Embodiments 1-3, wherein the one or more programming instructions further cause the processor to: actuate the stepper motor to receive the plate holder from the opening and place the plate holder in one or more additional positions in the housing; capture, using the one or more imaging sensors, images of the well plate at each of the one or more additional positions, wherein each of the images captures a unique marker and a unique portion of the plurality of wells based on an imaging area of the one or more imaging sensors with regard to the unique position; detect, in the images of the well plate, a location the unique marker; and determine a location of the unique portion of the plurality of wells based on the location of the unique marker.

[0194] Embodiment 5. The system of any one of Embodiments 1-4, wherein the one or more imaging sensors comprises a first imaging sensor and a second imaging sensor.

[0195] Embodiment 6. The system of any one of Embodiments 1-5, wherein the imaging area of the first image sensor is distinct from the imaging area of the second imaging sensor.

[0196] Embodiment 7. The system of any one of Embodiments 1-6, wherein the one or more image sensors are at least one of a complimentary metal-oxide semiconductor RGB sensor and charge- coupled device RGB sensor.

[0197] Embodiment 8. The system of any one of Embodiments 1-7, wherein the disease-specific protein signature comprises detection of a biotin-streptavidin complex with at least one antibody and one protein in a well of the well plate.

[0198] Embodiment 9. The system of any one of Embodiments 1-8, wherein the well plate comprises a plurality of alignment spots configured to display intense color intensity when imaged.

[0199] Embodiment 10. The system of any one of Embodiments 1-9, wherein the plurality of alignment spots are asymmetrically positioned.

[0200] Embodiment 11. The system of any one of Embodiments 1-10, wherein the positioning of the plurality of alignment spots encodes well plate information comprising at least one of a make of the well plate, a test type, or an orientation of the well plate in the plate holder.

[0201] Embodiment 12. The system of any one of Embodiments 1-11, wherein at least one of the plurality of alignment spots comprises a reagent control configured to indicate an addition of a detection antibody.

[0202] Embodiment 13. The system of any one of Embodiments 1-12, wherein at least one of plurality of alignment spots comprises a sample control configured to indicate an addition of samples to the well plate.

[0203] Embodiment 14. The system of any one of Embodiments 1-13, wherein the one or more programming instructions further cause the processor to: detect, in the images of the well plate, the plurality of alignment spots; and in response to failing to detect the plurality of alignment spots, return an error.

[0204] Embodiment 15. The system of any one of Embodiments 1-14, wherein the one or more programming instructions further cause the processor to: perform circle detection at an expected target spot.

[0205] Embodiment 16. The system of any one of Embodiments 1-15, further comprising a laser distance sensor configured to measure a location of the first position. Embodiment 17. The system of any one of Embodiments 1-16, wherein the one or more programming instructions further cause the processor to: calibrate the stepper motor based on the location of the first position.

[0206] Embodiment 18. The system of any one of Embodiments 1-17, wherein the one or more programming instructions further cause the processor to: calibrate the one or more imaging sensors based on the location of the first position.

[0207] Embodiment 19. The system of any one of Embodiments 1-18, wherein the one or more programming instructions further cause the processor to: determine a sharpness value metric of each of the one or more imaging sensors; and tune a focal length of each of the one or more imaging sensors to optimize the sharpness value metric.

[0208] Embodiment 20. The system of any one of Embodiments 1-19, further comprising one or more background lights within the housing; wherein the one or more programming instructions further cause the processor to: analyze color values and intensity values for the one or more background lights; and return an operational status of the one or more background lights.

[0209] Embodiment 21. The system of any one of Embodiments 1-20, wherein the well plate comprises a sample comprising at least one of milk, bulk tank milk, saliva, serum, bronchoalveolar lavage, bronchial wash, sputum, swab in transport media, feces, urine, and cerebrospinal fluid.

[0210] Embodiment 22. The system of any one of Embodiments 1-21, wherein the one or more programming instructions further cause the processor to detect an avidity of the sample.

[0211] Embodiment 23. The system of any one of Embodiments 1-22, wherein at least a portion of the plurality of alignment spots comprise anti-horseradish peroxidase goat IgG.

[0212] Embodiment 24. A method comprising: actuating, by a processor, a stepper motor to receive a plate holder and a well plate comprising a plurality of wells, along one or more guide rails, from an opening in a housing and placing the plate holder in a first position in the housing; capturing, using one or more imaging sensors, images of the well plate at the first position, wherein each of the images captures a first marker and a first portion of the plurality of wells based on an imaging area of the one or more imaging sensors with regard to the first position, wherein each well comprises a sample comprising at least one protein; detecting, by the processor, a location of the first marker; determining, by the processor, a location of the first portion of the plurality of wells based on the location of the first marker; determining, by the processor, color values for each pixel in each of the first portion of the plurality of wells; determining, by the processor, color intensity values based on the color values; and analyzing, by the processor, the color intensity values to detect disease-specific protein signatures.

[0213] Embodiment 25. The method of embodiment 24, further comprising: actuating, by the processor, the stepper motor to receive the plate holder from the opening and place the plate holder in one or more additional positions in the housing; capturing, using the one or more imaging sensors, images of the well plate at each of the one or more additional positions, wherein each of the images captures a unique marker and a unique portion of the plurality of wells based on an imaging area of the one or more imaging sensors with regard to the unique position; detecting, by the processor, in the images of the well plate, a location the unique marker; and determining, by the processor, a location of the unique portion of the plurality of wells based on the location of the unique marker.

[0214] Embodiment 26. The method of any one of Embodiments 24-25, wherein the one or more imaging sensors comprises a first imaging sensor and a second imaging sensor.

[0215] Embodiment 27. The method of any one of Embodiments 24-26, wherein the imaging area of the first imaging sensor is distinct from the imaging area of the second imaging sensor.

[0216] Embodiment 28. The method of any one of Embodiments 24-27, wherein the one or more imaging sensors are at least one of a complementary metal-oxide semiconductor RGB sensor and charge-coupled device RGB sensor.

[0217] Embodiment 29. The method of any one of Embodiments 24-28, wherein the disease-specific protein signature comprises detection of a biotin-streptavidin complex with at least one antibody and one protein in a well of the well plate.

[0218] Embodiment 30. The method of any one of Embodiments 24-29, wherein the well plate comprises a plurality of alignment spots configured to display intense color intensity when imaged.

[0219] Embodiment 31. The method of any one of Embodiments 24-30, wherein the plurality of alignment spots are asymmetrically positioned.

[0220] Embodiment 32. The method of any one of Embodiments 24-31, further comprising determining well plate information comprising at least one of a make of the well plate, a test type, or an orientation of the well plate in the plate holder based on a location of the plurality of alignment spots.

[0221] Embodiment 33. The method of any one of Embodiments 24-32, wherein at least one of the plurality of alignment spots comprises a reagent control configured to indicate an addition of a detection antibody.

[0222] Embodiment 34. The method of any one of Embodiments 24-33, wherein at least one of plurality of alignment spots comprises a sample control configured to indicate an addition of samples to the well plate.

[0223] Embodiment 35. The method of any one of Embodiments 24-34, further comprising: detecting, by the processor, in the images of the well plate, the plurality of alignment spots; and in response to failing to detect the plurality of alignment spots, returning an error.

[0224] Embodiment 36. The method of any one of Embodiments 24-35, further comprising performing, by the processor, circle detection at an expected target spot. Embodiment 37. The method of any one of Embodiments 24-36, further comprising measuring, by a laser distance sensor, a location of the first position.

[0225] Embodiment 38. The method of any one of Embodiments 24-37, further comprising calibrating, by the processor, the stepper motor based on the location of the first position.

[0226] Embodiment 39. The method of any one of Embodiments 24-38, further comprising calibrating, by the processor, the one or more imaging sensors based on the location of the first position.

[0227] Embodiment 40. The method of any one of Embodiments 24-39, further comprising: determining, by the processor, a sharpness value metric of each of the one or more imaging sensors; and tuning, by the processor, a focal length of each of the one or more imaging sensors to optimize the sharpness value metric.

[0228] Embodiment 41. The method of any one of Embodiments 24-40 , further comprising: analyzing, by the processor, color values and intensity values for one or more background lights in the housing; and returning an operational status of the one or more background lights.

[0229] Embodiment 42. The method of any one of Embodiments 24-41, wherein the well plate comprises a sample comprising at least one of milk, bulk tank milk, saliva, serum, bronchoalveolar lavage, bronchial wash, sputum, swab in transport media, feces, urine and cerebrospinal fluid.

[0230] Embodiment 43. The method of any one of Embodiments 24-42, wherein the one or more programming instructions further cause the processor to detect an avidity of the sample.

[0231] Embodiment 44. The method of any one of Embodiments 24-43, wherein at least a portion of the plurality of alignment spots comprise anti-horseradish peroxidase goat IgG.

[0232] Embodiment 45. A method comprising: adding at least one sample to a well plate; providing the well plate to the system of embodiment 1; and processing the well plate to detect diseasespecific protein signatures in the at least one sample.

[0233] Embodiment 46. The method of embodiment 45, wherein processing the well plate further comprises: actuating the stepper motor to receive a plate holder and the well plate comprising a plurality of wells, along the one or more guide rails, from the opening in the housing and placing the plate holder in a first position in the housing; capturing, using the one or more imaging sensors, images of the well plate at the first position, wherein each of the images captures a first marker and a first portion of the plurality of wells based on an imaging area of the one or more imaging sensors with regard to the first position, wherein each well comprises a sample comprising at least one protein; detecting a location of the first marker; determining a location of the first portion of the plurality of wells based on the location of the first marker; determining color values for each pixel in each of the first portion of the plurality of wells; determining color intensity values based on the color values; and analyzing the color intensity values to detect disease-specific protein signatures.

[0234] Embodiment 47. The method of any one of Embodiments 45-46, further comprising: actuating the stepper motor to receive the plate holder from the opening and place the plate holder in one or more additional positions in the housing; capturing, using the one or more imaging sensors, images of the well plate at each of the one or more additional positions, wherein each of the images captures a unique marker and a unique portion of the plurality of wells based on an imaging area of the one or more imaging sensors with regard to the unique position; detecting in the images of the well plate, a location the unique marker; and determining a location of the unique portion of the plurality of wells based on the location of the unique marker.

[0235] Embodiment 48. The method of any one of Embodiments 45-47, wherein the one or more imaging sensors comprise a first imaging sensor and a second imaging sensor.

[0236] Embodiment 49. The method of any one of Embodiments 45-48, wherein the imaging area of the first imaging sensor is distinct from the imaging area of the second imaging sensor.

[0237] Embodiment 50. The method of any one of Embodiments 45-49, wherein the one or more imaging sensors are at least one of a complimentary metal-oxide semiconductor RGB sensor and charge-coupled device RGB sensor.

[0238] Embodiment 51. The method of any one of Embodiments 45-50, wherein the disease-specific protein signature comprises detection of a biotin-streptavidin complex with at least one antibody and one protein in a well of the well plate.

[0239] Embodiment 52. The method of any one of Embodiments 45-51, wherein the well plate comprises a plurality of alignment spots configured to display intense color intensity when imaged.

[0240] Embodiment 53. The method of any one of Embodiments 45-52, wherein the plurality of alignment spots is asymmetrically positioned.

[0241] Embodiment 54. The method of any one of Embodiments 45-53, further comprising determining well plate information comprising at least one of a make of the well plate, a test type, or an orientation of the well plate in the plate holder based on a location of the plurality of alignment spots.

[0242] Embodiment 55. The method of any one of Embodiments 45-54, wherein at least one of the plurality of alignment spots comprises a reagent control configured to indicate an addition of a detection antibody.

[0243] Embodiment 56. The method of any one of Embodiments 45-55, wherein at least one of plurality of alignment spots comprise a sample control configured to indicate an addition of samples to the well plate. Embodiment 57. The method of any one of Embodiments 45-56, further comprising: detecting in images of the well plate, the plurality of alignment spots; and in response to failing to detect the plurality of alignment spots, returning an error.

[0244] Embodiment 58. The method of any one of Embodiments 45-57, further comprising performing circle detection at an expected target spot.

[0245] Embodiment 59. The method of any one of Embodiments 45-58, further comprising measuring, by a laser distance sensor, a location of the first position.

[0246] Embodiment 60. The method of any one of Embodiments 45-59, further comprising calibrating the stepper motor based on the location of the first position.

[0247] Embodiment 61. The method of any one of Embodiments 45-60, further comprising calibrating the one or more imaging sensors based on the location of the first position.

[0248] Embodiment 62. The method of any one of Embodiments 45-61, further comprising: determining a sharpness value metric of each of the one or more imaging sensors; and tuning a focal length of each of the one or more imaging sensors to optimize the sharpness value metric.

[0249] Embodiment 63. The method of any one of Embodiments 45-62, further comprising: analyzing color values and intensity values for one or more background lights in the housing; and returning an operational status of the one or more background lights.

[0250] Embodiment 64. The method of any one of Embodiments 45-63, wherein the well plate comprises a sample comprising at least one of milk, bulk tank milk, saliva, serum, bronchoalveolar lavage, bronchial wash, sputum, swab in transport media, feces, urine and cerebrospinal fluid.

[0251] Embodiment 65. The method of any one of Embodiments 45-64, wherein the one or more programming instructions further cause the processor to detect an avidity of the sample.

[0252] Embodiment 66. The method of any one of Embodiments 45-65, wherein at least a portion of the plurality of alignment spots comprise anti-horseradish peroxidase goat IgG.

[0253] Embodiment 67. The method of any one of Embodiments 45-66, wherein providing the well plate further comprises: providing antibody targets ununiformly in at least a portion of the plurality of wells; providing a sample to the portion of the plurality of wells; and incubating the well plate.

[0254] Embodiment 68. The method of any one of Embodiments 45-67, wherein incubating the well plate is performed for under 1 hour.

[0255] While various illustrative embodiments incorporating the principles of the present teachings have been disclosed, the present teachings are not limited to the disclosed embodiments. Instead, this application is intended to cover any variations, uses, or adaptations of the present teachings and use its general principles. Further, this application is intended to cover such departures from the present disclosure as come within known or customary practice in the art to which these teachings pertain.

[0256] In the above detailed description, reference is made to the accompanying drawings, which form a part hereof. In the drawings, similar symbols typically identify similar components, unless context dictates otherwise. The illustrative embodiments described in the present disclosure are not meant to be limiting. Other embodiments may be used, and other changes may be made, without departing from the spirit or scope of the subject matter presented herein. It will be readily understood that various features of the present disclosure, as generally described herein, and illustrated in the Figures, can be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which are explicitly contemplated herein.

[0257] The present disclosure is not to be limited in terms of the particular embodiments described in this application, which are intended as illustrations of various features. Many modifications and variations can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. Functionally equivalent methods and apparatuses within the scope of the disclosure, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing descriptions. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0258] With respect to the use of substantially any plural and / or singular terms herein, those having skill in the art can translate from the plural to the singular and / or from the singular to the plural as is appropriate to the context and / or application. The various singular / plural permutations may be expressly set forth herein for sake of clarity.

[0259] It will be understood by those within the art that, in general, terms used herein are generally intended as “open” terms (for example, the term “including” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “having at least,” the term “includes” should be interpreted as “includes but is not limited to,” et cetera). While various compositions, methods, and devices are described in terms of “comprising” various components or steps (interpreted as meaning “including, but not limited to”), the compositions, methods, and devices also can “consist essentially of’ or “consist of’ the various components and steps, and such terminology should be interpreted as defining essentially closed-member groups.

[0260] In addition, even if a specific number is explicitly recited, those skilled in the art will recognize that such recitation should be interpreted to mean at least the recited number (for example, the bare recitation of "two recitations," without other modifiers, means at least two recitations, or two or more recitations). Furthermore, in those instances where a convention analogous to “at least one of A, B, and C, et cetera” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (for example, “a system having at least one of A, B, and C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, et cetera). In those instances where a convention analogous to “at least one of A, B, or C, et cetera” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (for example, “a system having at least one of A,

[0261] B, or C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, et cetera). It will be further understood by those within the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description, sample embodiments, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or “B” or “A and B.”

[0262] In addition, where features of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0263] As will be understood by one skilled in the art, for any and all purposes, such as in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, et cetera. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, et cetera. As will also be understood by one skilled in the art all language such as “up to,” “at least,” and the like include the number recited and refer to ranges that can be subsequently broken down into subranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 cells refers to groups having 1, 2, or 3 cells. Similarly, a group having 1-5 cells refers to groups having 1, 2, 3, 4, or 5 cells, and so forth.

[0264] The term “about,” as used herein, refers to variations in a numerical quantity that can occur, for example, through measuring or handling procedures in the real world; through inadvertent error in these procedures; through differences in the manufacture, source, or purity of compositions or reagents; and the like. Typically, the term “about” as used herein means greater or lesser than the value or range of values stated by 1 / 10 of the stated values, e.g., ±10%. The term “about” also refers to variations that would be recognized by one skilled in the art as being equivalent so long as such variations do not encompass known values practiced by the prior art. Each value or range of values preceded by the term “about” is also intended to encompass the embodiment of the stated absolute value or range of values. Whether or not modified by the term “about,” quantitative values recited in the present disclosure include equivalents to the recited values, e.g., variations in the numerical quantity of such values that can occur, but would be recognized to be equivalents by a person skilled in the art.

[0265] Various of the above-disclosed and other features and functions, or alternatives thereof, may be combined into many other different systems or applications. Various presently unforeseen or unanticipated alternatives, modifications, variations or improvements therein may be subsequently made by those skilled in the art, each of which is also intended to be encompassed by the disclosed embodiments.

[0266] Although the invention has been described with reference to the presently preferred embodiment, it should be understood that various modifications can be made without departing from the spirit of the invention. Accordingly, the invention is limited only by the following claims.

Claims

What is claimed is:

1. A system comprising: a housing comprising an opening; a stepper motor; a plate holder configured to receive a well plate comprising a plurality of wells; one or more guide rails, wherein at least one of the one or more guide rails is mechanically coupled to the stepper motor, wherein the stepper motor is configured to drive the plate holder along the guide rails to a plurality of positions comprising at least one position inside the housing and at least one position outside the opening of the housing; and one or more imaging sensors affixed inside the housing, wherein the imaging sensor is configured to capture at least one image of at least a portion of the well plate in the plate holder in the at least one position inside the housing, and wherein the portion of the well plate is based on an imaging area of the one or more imaging sensors.

2. The system of claim 1, further comprising: a processor; and a non-transitory, processor-readable storage medium, wherein the non-transitory, processor- readable storage medium comprises one or more programming instructions that, when executed, cause the processor to: actuate the stepper motor to eject the plate holder from the opening; actuate the stepper motor to receive the plate holder from the opening and place the plate holder in a first position in the housing; capture, using the one or more imaging sensors, images of the well plate at the first position, wherein each of the images captures a first marker and a first portion of the plurality of wells based on the imaging area of the one or more imaging sensors with regard to the first position, wherein each well comprises a sample comprising at least one protein; detect a location the first marker; determine a location of the first portion of the plurality of wells based on the location of the first marker; determine color values for each pixel in each of the first portion of the plurality of wells; determine color intensity values based on the color values; and analyze the color intensity values to detect disease-specific protein signatures.

3. The system of claim 2, furthering comprising one or more interface elements attached to the housing and electronically interfaced to the processor.

4. The system of claim 2, wherein the one or more programming instructions further cause the processor to:actuate the stepper motor to receive the plate holder from the opening and place the plate holder in one or more additional positions in the housing; capture, using the one or more imaging sensors, images of the well plate at each of the one or more additional positions, wherein each of the images captures a unique marker and a unique portion of the plurality of wells based on an imaging area of the one or more imaging sensors with regard to the unique position; detect, in the images of the well plate, a location the unique marker; and determine a location of the unique portion of the plurality of wells based on the location of the unique marker.

5. The system of claim 1, wherein the one or more imaging sensors comprises a first imaging sensor and a second imaging sensor.

6. The system of claim 5, wherein the imaging area of the first image sensor is distinct from the imaging area of the second imaging sensor.

7. The system of claim 1, wherein the one or more image sensors are at least one of a complimentary metal-oxide semiconductor RGB sensor and charge-coupled device RGB sensor.

8. The system of claim 2, wherein the disease-specific protein signature comprises detection of a biotin-streptavidin complex with at least one antibody and one protein in a well of the well plate.

9. The system of claim 2, wherein the well plate comprises a plurality of alignment spots configured to display intense color intensity when imaged.

10. The system of claim 9, wherein the plurality of alignment spots are asymmetrically positioned.

11. The system of claim 9, wherein the positioning of the plurality of alignment spots encodes well plate information comprising at least one of a make of the well plate, a test type, or an orientation of the well plate in the plate holder.

12. The system of claim 9, wherein at least one of the plurality of alignment spots comprise a reagent control configured to indicate an addition of a detection antibody.

13. The system of claim 9, wherein at least one of plurality of alignment spots comprise a sample control configured to indicate an addition of samples to the well plate.

14. The system of claim 9, wherein the one or more programming instructions further cause the processor to: detect, in the images of the well plate, the plurality of alignment spots; and in response to failing to detect the plurality of alignment spots, return an error.

15. The system of claim 9, wherein the one or more programming instructions further cause the processor to: perform circle detection at an expected target spot.

16. The system of claim 2, further comprising a laser distance sensor configured to measure a location of the first position.

17. The system of claim 16, wherein the one or more programming instructions further cause the processor to: calibrate the stepper motor based on the location of the first position.

18. The system of claim 16, wherein the one or more programming instructions further cause the processor to: calibrate the one or more imaging sensors based on the location of the first position.

19. The system of claim 16, wherein the one or more programming instructions further cause the processor to: determine a sharpness value metric of each of the one or more imaging sensors; and tune a focal length of each of the one or more imaging sensors to optimize the sharpness value metric.

20. The system of claim 2, further comprising one or more background lights within the housing; wherein the one or more programming instructions further cause the processor to: analyze color values and intensity values for the one or more background lights; and return an operational status of the one or more background lights.

21. The system of claim 1, wherein the well plate comprises a sample comprising at least one of milk, bulk tank milk, saliva, serum, bronchoalveolar lavage, bronchial wash, sputum, swab in transport media, feces, urine, and cerebrospinal fluid.

22. The system of claim 21, wherein the one or more programming instructions further cause the processor to detect an avidity of the sample.

23. The system of claim 9, wherein at least a portion of the plurality of alignment spots comprise anti-horseradish peroxidase goat IgG.

24. A method comprising: actuating, by a processor, a stepper motor to receive a plate holder and a well plate comprising a plurality of wells, along one or more guide rails, from an opening in a housing and placing the plate holder in a first position in the housing; capturing, using one or more imaging sensors, images of the well plate at the first position, wherein each of the images captures a first marker and a first portion of the plurality of wells based on an imaging area of the one or more imaging sensors with regard to the first position, wherein each well comprises a sample comprising at least one protein; detecting, by the processor, a location of the first marker; determining, by the processor, a location of the first portion of the plurality of wells based on the location of the first marker; determining, by the processor, color values for each pixel in each of the first portion of the plurality of wells; determining, by the processor, color intensity values based on the color values; and analyzing, by the processor, the color intensity values to detect disease-specific protein signatures.

25. The method of claim 24, further comprising: actuating, by the processor, the stepper motor to receive the plate holder from the opening and place the plate holder in one or more additional positions in the housing; capturing, using the one or more imaging sensors, images of the well plate at each of the one or more additional positions, wherein each of the images captures a unique marker and a unique portion of the plurality of wells based on an imaging area of the one or more imaging sensors with regard to the unique position; detecting, by the processor, in the images of the well plate, a location the unique marker; and determining, by the processor, a location of the unique portion of the plurality of wells based on the location of the unique marker.

26. The method of claim 24, wherein the one or more imaging sensors comprises a first imaging sensor and a second imaging sensor.

27. The method of claim 26, wherein the imaging area of the first imaging sensor is distinct from the imaging area of the second imaging sensor.

28. The method of claim 24, wherein the one or more imaging sensors are at least one of a complimentary metal-oxide semiconductor RGB sensor and charge-coupled device RGB sensor.

29. The method of claim 24, wherein the disease-specific protein signature comprises detection of a biotin-streptavidin complex with at least one antibody and one protein in a well of the well plate.

30. The method of claim 24, wherein the well plate comprises a plurality of alignment spots configured to display intense color intensity when imaged.

31. The method of claim 30, wherein the plurality of alignment spots are asymmetrically positioned.

32. The method of claim 30, further comprising determining well plate information comprising at least one of a make of the well plate, a test type, or an orientation of the well plate in the plate holder based on a location of the plurality of alignment spots.

33. The method of claim 30, wherein at least one of the plurality of alignment spots comprise a reagent control configured to indicate an addition of a detection antibody.

34. The method of claim 30, wherein at least one of plurality of alignment spots comprise a sample control configured to indicate an addition of samples to the well plate.

35. The method of claim 30, further comprising: detecting, by the processor, in the images of the well plate, the plurality of alignment spots; and in response to failing to detect the plurality of alignment spots, returning an error.

36. The method of claim 30, further comprising performing, by the processor, circle detection at an expected target spot.

37. The method of claim 24, further comprising measuring, by a laser distance sensor, a location of the first position.

38. The method of claim 37, further comprising calibrating, by the processor, the stepper motor based on the location of the first position.

39. The method of claim 37, further comprising calibrating, by the processor, the one or more imaging sensors based on the location of the first position.

40. The method of claim 37, further comprising: determining, by the processor, a sharpness value metric of each of the one or more imaging sensors; and tuning, by the processor, a focal length of each of the one or more imaging sensors to optimize the sharpness value metric.

41. The method of claim 24, further comprising: analyzing, by the processor, color values and intensity values for one or more background lights in the housing; and returning an operational status of the one or more background lights.

42. The method of claim 24, wherein the well plate comprises a sample comprising at least one of milk, bulk tank milk, saliva, serum, bronchoalveolar lavage, bronchial wash, sputum, swab in transport media, feces, urine and cerebrospinal fluid.

43. The method of claim 42, wherein the one or more programming instructions further cause the processor to detect an avidity of the sample.

44. The method of claim 30, wherein at least a portion of the plurality of alignment spots comprise anti-horseradish peroxidase goat IgG.

45. A method comprising: adding at least one sample to a well plate; providing the well plate to the system of claim 1; and processing the well plate to detect disease-specific protein signatures in the at least one sample.

46. The method of claim 45, wherein processing the well plate further comprises: actuating the stepper motor to receive a plate holder and the well plate comprising a plurality of wells, along the one or more guide rails, from the opening in the housing and placing the plate holder in a first position in the housing; capturing, using the one or more imaging sensors, images of the well plate at the first position, wherein each of the images captures a first marker and a first portion of the plurality of wells based on an imaging area of the one or more imaging sensors with regard to the first position, wherein each well comprises a sample comprising at least one protein; detecting a location of the first marker; determining a location of the first portion of the plurality of wells based on the location of the first marker;determining color values for each pixel in each of the first portion of the plurality of wells; determining color intensity values based on the color values; and analyzing the color intensity values to detect disease-specific protein signatures.

47. The method of claim 46, further comprising: actuating the stepper motor to receive the plate holder from the opening and place the plate holder in one or more additional positions in the housing; capturing, using the one or more imaging sensors, images of the well plate at each of the one or more additional positions, wherein each of the images captures a unique marker and a unique portion of the plurality of wells based on an imaging area of the one or more imaging sensors with regard to the unique position; detecting in the images of the well plate, a location the unique marker; and determining a location of the unique portion of the plurality of wells based on the location of the unique marker.

48. The method of claim 45, wherein the one or more imaging sensors comprises a first imaging sensor and a second imaging sensor.

49. The method of claim 48, wherein the imaging area of the first imaging sensor is distinct from the imaging area of the second imaging sensor.

50. The method of claim 45, wherein the one or more imaging sensors are at least one of a complimentary metal-oxide semiconductor RGB sensor and charge-coupled device RGB sensor.

51. The method of claim 45, wherein the disease-specific protein signature comprises detection of a biotin-streptavidin complex with at least one antibody and one protein in a well of the well plate.

52. The method of claim 45, wherein the well plate comprises a plurality of alignment spots configured to display intense color intensity when imaged.

53. The method of claim 52, wherein the plurality of alignment spots are asymmetrically positioned.

54. The method of claim 52, further comprising determining well plate information comprising at least one of a make of the well plate, a test type, or an orientation of the well plate in the plate holder based on a location of the plurality of alignment spots.

55. The method of claim 52, wherein at least one of the plurality of alignment spots comprise a reagent control configured to indicate an addition of a detection antibody.

56. The method of claim 52, wherein at least one of plurality of alignment spots comprise a sample control configured to indicate an addition of samples to the well plate.

57. The method of claim 52, further comprising: detecting in images of the well plate, the plurality of alignment spots; and in response to failing to detect the plurality of alignment spots, returning an error.

58. The method of claim 52, further comprising performing circle detection at an expected target spot.

59. The method of claim 46, further comprising measuring, by a laser distance sensor, a location of the first position.

60. The method of claim 59, further comprising calibrating the stepper motor based on the location of the first position.

61. The method of claim 59, further comprising calibrating the one or more imaging sensors based on the location of the first position.

62. The method of claim 45, further comprising: determining a sharpness value metric of each of the one or more imaging sensors; and tuning a focal length of each of the one or more imaging sensors to optimize the sharpness value metric.

63. The method of claim 45, further comprising: analyzing color values and intensity values for one or more background lights in the housing; and returning an operational status of the one or more background lights.

64. The method of claim 45, wherein the well plate comprises a sample comprising at least one of milk, bulk tank milk, saliva, serum, bronchoalveolar lavage, bronchial wash, sputum, swab in transport media, feces, urine and cerebrospinal fluid.

65. The method of claim 45, wherein the one or more programming instructions further cause the processor to detect an avidity of the sample.

66. The method of claim 52, wherein at least a portion of the plurality of alignment spots comprise anti-horseradish peroxidase goat IgG.

67. The method of claim 45, wherein providing the well plate further comprises: providing antibody targets ununiformly in at least a portion of the plurality of wells; providing a sample to the portion of the plurality of wells; and incubating the well plate.

68. The method of claim 67, wherein incubating the well plate is performed for under 1 hour.

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