Parallel determination of cellular and non-cellular analytes in samples

Abstract

Among other things, the present invention relates to biological / chemical sampling, sensing, assays and applications.

Description

[0001] Cross-references

[0002] This application is a national phase application (§371) of International Application PCT / US19 / 15309, filed on January 25, 2019, which claims the benefit of U.S. Provisional Patent Application No. 62 / 621,761, filed on January 25, 2018, and International Application PCT / US2019 / 013388, filed on January 11, 2019, the contents of which are relied upon and incorporated herein by reference in their entirety.

[0003] The entire disclosure of any publication or patent document mentioned herein is incorporated by reference in its entirety.

[0004] field

[0005] The present disclosure relates to the fields of biological / chemical sampling, sensing, assays and applications.

[0006] background

[0007] A large proportion of antibiotics are used to treat diseases that are rarely resistant to antibiotics. Excessive and inappropriate use of antibiotics has led to the emergence of increasingly resistant bacteria, which has become a serious problem worldwide. Individual healthcare practitioners are often faced with the difficult problem of how to identify patients who need (and especially those who do not need) antibiotic treatment.

[0008] Brief description

[0009] In certain embodiments, the present disclosure provides a device for determining target cells and non-cellular analytes in a liquid sample, comprising a first plate, a second plate, a plurality of spacers, a plurality of particles and a capture agent, wherein: the first plate and the second plate can be moved relative to each other into different configurations, including an open configuration and a closed configuration; each plate has a sample contact area on its respective surface for contacting a sample containing or suspected of containing one or more target cells and non-cellular analytes. The spacer comprises periodically arranged pillars of uniform height and is fixed on the first plate. The particles have a size of 0.2 μm to 100 μm and are randomly distributed in the sample contact area of ​​the first plate in an open configuration. The capture agent specifically binds to the non-cellular analyte, which is capable of binding to form an optical marker of the labeled non-cellular analyte. wherein an open configuration is a configuration in which: two plates are separated and a sample is deposited on one or both plates; wherein a closed configuration is a configuration configured in the open configuration after sample deposition; wherein in the closed configuration, a relevant sample volume is compressed by the two plates into a layer of substantially uniform thickness and substantially stagnant relative to the plates, and the uniform thickness of the layer is limited by the plates and regulated by the plates and spacers; wherein the relevant sample volume of the sample is analyzed for non-cellular analytes and target cells; and wherein the uniform thickness and geometry of the particles are configured such that in the closed configuration there is no significant overlap between one or more target cells, between particles, and between target cells and particles.

[0010] In certain embodiments, the present disclosure provides a device for determining target cells and non-cellular analytes in a liquid sample, the device comprising: a first plate, a second plate, a plurality of spacers, a plurality of particles and a capture agent, wherein: the first plate and the second plate can be moved relative to each other into different configurations, including an open configuration and a closed configuration; each plate has a sample contact area on its respective surface for contacting a sample containing or suspected of containing one or more target cells and non-cellular analytes; the spacer comprises an array of pillars having a uniform height of less than 150 μm fixed to the first plate. The particles have a size of 0.2 μm to 100 μm and are randomly distributed in the sample contact area of ​​the first plate in an open configuration. The capture agent specifically binds to the non-cellular analyte, which is capable of binding to form an optical marker of the labeled non-cellular analyte. wherein the uniform thickness and geometry of the particles are configured such that in a closed configuration there is no significant overlap between one or more target cells, between particles, and between target cells and particles, and the number of layers of uniform thickness is limited by the plate and regulated by the plate and spacers; and wherein the relevant sample volume is a portion or all of the volume of the sample, wherein the relevant sample volume is analyzed for non-cellular analytes and target cells, and wherein the number of particles in the relevant sample volume is configured, in the closed configuration, to be at least 10.

[0011] In certain embodiments, the present disclosure provides a device for determining target cells and non-cellular analytes in a liquid sample, comprising: a first plate, a second plate, a plurality of spacers, a plurality of particles, a capture agent, an imager, and a processor, wherein: the first plate and the second plate can be moved relative to each other into different configurations, including an open configuration and a closed configuration; each plate has a sample contact area on its respective surface for contacting a sample containing one or more target cells and non-cellular analytes. The particles are 0.2 μm to 100 μm in size and are randomly distributed in the sample in a closed configuration; the spacer comprises an array of pillars having a uniform height of 150 μm fixed to the first plate. The capture agent is attached to the particle, wherein the capture agent specifically binds to the non-cellular analyte, wherein the non-cellular analyte is capable of binding to form an optical marker of the labeled non-cellular analyte. an imager captures at least one image of a sample volume; the processor is configured to process the at least one image to (i) detect the non-cellular analyte by analyzing images of at least 10 particles, and (ii) detect the target cell analyte; wherein the open configuration is a configuration in which: two plates are separated and the sample is deposited on one or both plates; wherein the closed configuration is a configuration in which the sample is deposited on the open configuration; in the closed configuration, the sample volume of interest is compressed by the two plates into a layer of substantially uniform thickness and substantially stagnant relative to the plates, and the uniform thickness of the layer is limited by the plates and regulated by the plates and spacers; wherein the uniform thickness and geometry of the particles are configured such that in the closed configuration there is no significant overlap between one or more target cells, between particles, and between target cells and particles; and wherein the sample volume of interest is a portion or all of the volume of the sample, and wherein the number of particles in the volume of interest is configured to be at least 10 in the closed configuration; wherein the processor is configured to process the at least one image to (i) detect the non-cellular analyte by analyzing images of at least 10 particles, and (ii) detect the target cell analyte.

[0012] In certain embodiments, the present disclosure provides a device comprising: a first plate; and a second plate. The second plate has a sample contact area on its surface; wherein the first plate and the second plate are movable relative to each other into: an open configuration, wherein the first plate and the second plate are at least partially separated so that a sample containing one or more target cells and non-cellular analytes can be placed between them, and a closed configuration in which the first plate is placed on top of the second plate to compress at least a portion of the sample between the first plate and the second plate into a layer having a uniform thickness; one or more spacers, the one or more spacers comprising a periodic array of pillars; and wherein the uniform thickness of the layer is adjusted by one or more spacers, a plurality of particles having one or more capture agents disposed on their surfaces, wherein, in the closed configuration, the first plate and the second plate compress at least a portion of the sample so that there is substantially no overlap between (i) one or more target cells, (ii) a plurality of particles, and (iii) one or more target cells and a plurality of particles.

[0013] In certain embodiments, one or more spacers are fixed on the surface of the second plate. In certain embodiments, the diameter of the plurality of particles is 0.2 μm to 100 μm. In certain embodiments, when the first plate and the second plate are in an open configuration, the plurality of particles are randomly distributed in the sample contact area of ​​the second plate. In certain embodiments, the capture agent specifically binds to a labeled targeted analyte, which includes a non-cellular analyte attached to an optical label. In certain embodiments, one or more spacers have a uniform height. In certain embodiments, the uniform height of one or more spacers is at least about 0.8 times the particle size. In certain embodiments, the uniform height of one or more spacers is less than about 200 μm. In certain embodiments, the uniform height of one or more spacers is between at least about 0.8 times and about 200 μm of the particle size. In certain embodiments, the device further includes an imager for obtaining an image of a relevant volume of the sample. In certain embodiments, the relevant volume is a portion or the entire volume of at least a portion of the sample, and wherein the number of particles in the relevant volume is configured to be at least 10 under a closed configuration. The device also includes a processor for processing images. In certain embodiments, the processing comprises at least one of: (i) detecting a non-cellular analyte by analyzing one or more images of at least 10 particles, and (ii) detecting a target cellular analyte.

[0014] In certain embodiments, the present disclosure provides a method for determining cellular and non-cellular analytes in a liquid sample, comprising: having a device of the present disclosure; having a sample containing or suspected of containing one or more target cells and non-cellular analytes; having an optical tag of the non-cellular analyte capable of binding to the non-cellular analyte to form a label; depositing the sample in the sample contact area of ​​the device in an open configuration, and after step (d), making the device into a closed configuration, thereby compressing at least a portion of the sample into a layer with uniform thickness; using an imager to obtain at least one image of a sample volume associated with the sample, wherein the imager is capable of imaging the optical tag and the target cells. Analyzing at least one image to detect the presence or amount of (i) target cells and / or (ii) non-cellular analytes.

[0015] In certain embodiments, the present disclosure provides a method for determining cellular and non-cellular analytes in a liquid sample, comprising: having a device of the present disclosure; having a sample containing or suspected of containing one or more target cells and non-cellular analytes; having an optical tag of a non-cellular analyte capable of binding to a non-cellular analyte to form a label; after step (d), depositing the sample in the sample contact area of ​​the device in an open state, placing the device in a closed state, thereby compressing at least a portion of the sample into a layer of uniform thickness; using an imager to obtain at least one image of a sample volume associated with the sample, wherein the imager is capable of imaging the optical tag and the target cells. Using a processor to analyze at least one image to detect the presence or amount of (i) target cells and / or (ii) non-cellular analytes; wherein the processor is configured to process the at least one image to (i) detect the non-cellular analyte by analyzing images of at least 10 particles, and (ii) detect the target cell analyte.

[0016] In certain embodiments, when detecting a non-cellular analyte, an image of at least 10 particles is analyzed by digital counting, wherein in the digital counting, the presence or amount of the non-cellular analyte in the sample is determined from the sample. The number or percentage of beads with an optical signal above a threshold, wherein the percentage is the ratio of the number of beads above the threshold to the number below the threshold. In certain embodiments, when detecting a non-cellular analyte, an image of at least 10 particles is analyzed by analog counting, wherein the analog counting determines the presence or amount of the non-cellular analyte in the sample from the optical signal. The amplitude of all beads in the relevant sample area. In certain embodiments, the device or method further includes two or more images of a common area / volume of the sample layer, wherein the common area of ​​the sample layer is an area of ​​the sample containing at least one particle of one or more particles. In certain embodiments, the device or method further includes two or more images, wherein at least one image is a dark field image, and at least one is a bright field image. In certain embodiments, the thickness of the sample layer is sufficient to produce a monolayer of target cells between the plates. In certain embodiments, the thickness of the sample layer is sufficient to produce no substantial overlap between one or more target cells between the plates. In certain embodiments, one or more particles have a geometry (shape and size) sufficient to (i) produce a monolayer of particles between plates and (ii) there is no substantial overlap between particles between plates. In certain embodiments, one or more particles have a density sufficient to (i) produce a monolayer of particles between plates and (ii) there is no substantial overlap between particles between plates. In certain embodiments, before the sample is deposited in the device, one or more particles are mixed with the sample. In certain embodiments, before the sample is deposited, one or more particles are placed on at least one of the first plate and the second plate, and then mixed with the sample when the sample is deposited on the device. In certain embodiments, one or more particles have various shapes. In certain embodiments, the maximum size of one or more particles is 0.05 μm to 100 μm. In certain embodiments, a non-cellular analyte is detected using a competitive assay, the step comprising providing a step of providing a labeled competitive detection agent that competes with the non-cellular analyte, if present, which is combined with the capture agent of the analyte. In certain embodiments, non-cellular analytes are detected using a non-competitive assay, the step comprising providing a labeled detection agent that specifically binds to another binding site of the non-cellular analyte, wherein the other binding site is a binding group. Different from the binding site of the capture agent. In certain embodiments, one of the images is a direct image, which includes information on the position of the topological structure (i.e., geometry) and particles in a common area. Another image is a signal image, which is configured to include a signal from a labeled competitive detection agent as the main signal of the image. In certain embodiments, the sample holder includes a first plate and a second plate, and the first plate and the second plate can be moved relative to each other into different configurations, including an open configuration and a closed configuration.

[0017] In certain embodiments, the present disclosure provides a method for diagnosing a viral infection or a bacterial infection in a subject, comprising: obtaining a device comprising a first plate, a second plate, and a plurality of particles, the plurality of particles comprising one or more capture agents coupled thereto, wherein the plurality of particles are substantially arranged along at least one monolayer of the first plate and the second plate, wherein the one or more capture agents are capable of associating with an analyte, and wherein the device comprises: an open structure, wherein the first plate and the second plate are at least partially separated to expose a sample contact area comprising the plurality of particles, the sample contact area being arranged on at least one of the first plate and the second plate; a closed structure, wherein the first plate and the second plate are substantially parallel and include a space between them. Bringing the device into contact with a sample comprising the analyte and one or more cells, wherein the device is in the open structure; placing the device in the closed structure so that at least a portion of the sample is contained within the space, wherein the thickness of the at least a portion of the sample is substantially uniform within the space, and wherein, in the closed structure, the one or more cells within the space are substantially arranged as a single layer; and capturing one or more images of a common area of ​​the thin sample layer, wherein the common area of ​​the sample layer is a sample area comprising at least one of: one of the plurality of particles; and one of the one or more cells.

[0018] In certain embodiments, the method further comprises determining, using a mobile electronic device: (i) one or more first values ​​corresponding to the amount of the analyte associated with the one or more capture agents; (ii) one or more second values ​​corresponding to the number of the one or more cells within the sample. In certain embodiments, the plurality of particles are removably coupled to at least one of the first plate and the second plate. In certain embodiments, the plurality of particles are selected from glass beads, magnetic beads, polystyrene beads, latex particles, reporter molecules, fluorescent molecules, dye molecules, non-selective dye molecules, and redox-reactive molecules. In certain embodiments, the plurality of particles comprises a first plurality of particles and a second plurality of particles, wherein the first plurality of particles comprises one or more first capture agents coupled thereto, and wherein the second plurality of particles comprises one or more second capture agents coupled thereto. In certain embodiments, the one or more first capture agents are capable of associating with a first epitope on the analyte, and wherein the one or more second capture agents are capable of associating with a second epitope on the analyte. In certain embodiments, the plurality of first particles are disposed along the first plate, and the plurality of second particles are disposed along the second plate. In certain embodiments, the first plurality of particles are selected from glass beads, magnetic beads, polystyrene beads, and latex particles. In certain embodiments, the second plurality of particles are selected from reporter molecules, fluorescent molecules, dye molecules, non-selective dye molecules and redox-responsive molecules. In certain embodiments, at least one of the one or more first capture agents and the one or more second capture agents is capable of specifically associating with the analyte. In certain embodiments, the device further comprises one or more spacers disposed within the space and along at least one of the first plate or the second plate. In certain embodiments, the one or more spacers determine the distance between the first plate and the second plate. In certain embodiments, the length of the one or more spacers along the direction from the first plate to the second plate is at most about 500 microns, at most about 250 microns, at most about 100 microns, at most about 90 microns, at most about 80 microns, at most about 70 microns, at most about 60 microns, at most about 50 microns, at most about 40 microns, at most about 30 microns, at most about 25 microns, at most about 20 microns, at most about 15 microns, at most about 10 microns, at most about 5 microns, at most about 4 microns, at most about 3 microns, at most about 2 microns, at most about 1 micron, at most about 0.5 microns, or at most about 0.1 microns. In certain embodiments, the length of the one or more spacers along the direction from the first plate to the second plate is less than or approximately equal to the average diameter of the one or more cells in the sample.In certain embodiments, the thickness of at least a portion of the sample in the space is at most about 500 microns, at most about 250 microns, at most about 100 microns, at most about 90 microns, at most about 80 microns, at most about 70 microns, at most about 60 microns, at most about 50 microns, at most about 40 microns, at most about 30 microns, at most about 25 microns, at most about 20 microns, at most about 15 microns, at most about 10 microns 10 microns, at most about 5 microns, at most about 4 microns, at most about 3 microns, at most about 2 microns, at most about 1 micron, at most about 0.5 micron, at most about 0.1 micron, or at most about 0.05 micron. In certain embodiments, the plurality of particles have various shapes. In some embodiments, the maximum size of the plurality of particles is about 500 microns, about 250 microns, about 100 microns, about 90 microns, about 80 microns, about 70 microns, about 60 microns, about 50 microns, about 40 microns, about 100 microns. 30 microns, about 25 microns, about 20 microns, about 15 microns, about 10 microns, about 5 microns, about 4 microns, about 3 microns, about 2 microns, about 1 micron, about 0.5 microns, about 0.1 microns or about 0.05 microns. In some embodiments, the one or more cells in the space are substantially arranged as a non-confluent monolayer. In some embodiments, the one or more cells in the space are in a suspended state. In some embodiments, the determination includes obtaining one or more images of an area of ​​at least a portion of the sample included in the space, wherein the imaging area includes at least one of the plurality of particles. In some embodiments, the one or more first values ​​correspond to the intensity of the energy emitted by the particle. In some embodiments, the method further includes contacting the sample with a cell dye capable of associating with the one or more cells. In certain embodiments, the one or more second values ​​correspond to the intensity of the energy emitted by the cell dye. In certain embodiments, the method also includes using an algorithm to determine the relationship between the one or more first values ​​and the one or more second values. In certain embodiments, the method further includes classifying the infection level of the subject from which the sample is obtained based on the relationship between the one or more first values ​​and the one or more second values. In certain embodiments, the infection is a viral infection, a bacterial infection, or a combination thereof. In certain embodiments, the one or more cells are selected from leukocytes, leukocytes, granulocytes, granulocytes, myeloid cells, lymphoid cells, neutrophils, eosinophils, basophils, lymphocytes, T cells, B cells, natural killer cells and monocytes. In certain embodiments, the analyte is selected from polypeptides, proteins, marker proteins, fusion proteins, antibodies, small molecules, viral particles, bacteria, C-reactive protein and any fragment thereof.In certain embodiments, the method further includes contacting at least a portion of the sample with a competitive detection agent capable of associating with the one or more capture agents. In certain embodiments, the competitive detection agent competitively binds to the one or more capture agents with the analyte. In certain embodiments, the method further includes contacting at least a portion of the sample with a supplementary detection agent capable of associating with the analyte. In certain embodiments, the one or more capture agents can associate with a first epitope on the analyte, and wherein the supplementary detection agent can associate with a second epitope on the analyte. In certain embodiments, the method is performed in less than about 10 seconds, less than about 20 seconds, less than about 30 seconds, less than about 40 seconds, less than about 50 seconds, less than about 60 seconds, less than about 120 seconds. Less than about 180 seconds, less than about 240 seconds, less than about 300 seconds, less than about 400 seconds, less than about 500 seconds, less than about 1000 seconds or less than about 2000 seconds. In certain embodiments, the method does not include contacting at least a portion of the sample with a wash buffer. In certain embodiments, the method is completed without any washing steps. In certain embodiments, the first plate and the second plate are pressed together by hand. In certain embodiments, the non-cellular analyte is a polypeptide, a nucleic acid or a metabolite. In certain embodiments, the sample is a body fluid. In certain embodiments, the body fluid is amniotic fluid, aqueous humor, vitreous humor, blood, whole blood, fractionated blood, plasma, serum, breast milk, cerebrospinal fluid, chyle, endolymph, perilymph, feces, gastric acid, gastric juice, lymph, mucus, nasal drainage, sputum, pericardial fluid, peritoneal fluid, pleural fluid, pus, rhubarb, saliva, sebum, semen, sputum, sweat, synovial fluid, tears, vomitus, urine and exhaled breath condensate. In certain embodiments, the method further includes diagnosing a disease based on the result. In certain embodiments, the disease is cancer, inflammatory disease or infectious disease. In certain embodiments, the method detects a variety of different non-cellular analytes.

[0019] In certain embodiments, the present disclosure provides a kit for determining target cells and non-cellular analytes in a liquid sample, comprising: a first plate, a second plate, one or more particles and a capture agent, wherein: the first plate and the second plate can be moved relative to each other into different configurations, including an open configuration and a closed configuration; each plate has a sample contact area on its respective surface for contacting a sample containing one or more target cells and non-cellular analytes. The particle has a size of 0.2 μm to 100 μm, and has a capture agent attached to its surface, wherein the capture agent specifically binds to the non-cellular analyte. Wherein, the open configuration is such a configuration, wherein: the two plates are separated, and the sample is deposited on one or both plates; wherein the closed configuration is a configuration configured in an open configuration after the sample is deposited; in the closed configuration: at least a portion of the sample is compressed into a layer of substantially uniform thickness by the two plates, and is substantially stagnant relative to the plates, and at least one particle is within at least a portion of the sample; and wherein the uniform thickness and geometry of the particles are configured such that in the closed configuration, (i) one or more target cells do not substantially overlap each other, and (ii) the particles do not substantially overlap each other.

[0020] In certain embodiments, the present disclosure provides a kit comprising: a device comprising a first plate; a second plate; a sample contact area disposed on one or both of the first plate and the second plate for contacting a sample comprising one or more particles having a capture agent attached to their surface; wherein the first plate and the second plate are movable relative to each other into an open configuration or a closed configuration, wherein the open configuration is that the first plate and the second plate are at least partially separated so that the sample can be deposited between them; wherein the closed configuration is that the first plate is placed on top of the second plate, thereby compressing at least a portion of the sample between the first plate and the second plate into a layer having a uniform thickness.

[0021] In certain embodiments, the kit further includes instructions for using the device. In certain embodiments, the sample includes one or more target cells. In certain embodiments, the sample includes one or more non-cellular analytes. In certain embodiments, the diameter of one or more particles is 0.2 μm to 100 μm. In certain embodiments, the capture agent is combined with the non-cellular analyte. In certain embodiments, at least a portion of the sample is compressed into a layer with a substantially uniform thickness by the first plate and the second plate. In certain embodiments, at least a portion of the sample is substantially stagnant relative to the plate. In certain embodiments, the sample thickness in a closed configuration is configured to (i) make one or more target cells have a monolayer between the plates, and (ii) there is no substantial overlap between one or more target cells. In certain embodiments, one or more particles have a geometry (shape and size) configured to (i) make particles have a monolayer between the plates and (ii) there is no substantial overlap between the particles. In certain embodiments, one or more particles have a density configured to (i) make particles have a monolayer between the plates and (ii) there is no substantial overlap between the particles. In certain embodiments, the device also includes a spacer on one or both of the first plate and the second plate. In certain embodiments, the device further comprises a labeled competitive detection agent for competing with the analyte (if present) so as to bind to the capture agent of the analyte in a competitive assay. In certain embodiments, the device further comprises a labeled detection agent that specifically binds to the other binding site of the analyte in a non-competitive assay, wherein the other binding site is a binding site different from the binding site of the capture agent. In certain embodiments, the device further comprises an imager that images target cells and non-cellular analytes when the sample is between the two plates and the plate is in a closed configuration.

[0022] Incorporated by Reference

[0023] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0024] BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Those skilled in the art will appreciate that the drawings described below are for illustrative purposes only. In some figures, the drawings are drawn to scale. In the figures providing experimental data points, the lines connecting the data points are only used to guide the data viewing without other means. For clarity, some elements are enlarged when shown in the drawings. It should be noted that these drawings are not intended to strictly illustrate these elements. The size of the elements should be delineated from the description provided herein and incorporated by reference. The drawings are not intended to limit the scope of the present invention in any way.

[0026] Figure 1A , Figure 1B Schematic diagrams illustrating some exemplary methods of the present invention are provided.

[0027] Figure 2 Schematic diagrams showing some exemplary methods of the invention are provided.

[0028] Figure 3 Schematic diagrams showing some exemplary methods of the invention are provided.

[0029] Figure 4 Schematic diagrams are provided showing some exemplary methods of the invention.

[0030] Detailed description

[0031] The following detailed description illustrates certain embodiments of the present invention by way of example and not limitation. The section headings and any subheadings used herein, if any, are for organizational purposes only and should not be construed as limiting the described invention in any way. The content under a section heading and / or subheading is not limited to the section heading and / or subheading, but applies to the entire description of the present invention.

[0032] The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present claims are not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

[0033] definition

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present teachings, some exemplary methods and materials are now described.

[0035] As used herein, the term "monolayer" may refer to a cell layer that is 1 cell thick, or a molecular layer that is 1 molecule thick. In some embodiments, a monolayer or cells or molecules may be confluent. In some embodiments, a monolayer of cells or molecules (e.g., analytes) may be sub-confluent. In some embodiments, a monolayer of cells or molecules may be sparse.

[0036] The terms "link", "bind", "couple" and "attach" are used interchangeably and refer to covalent interactions (e.g., by chemical coupling) or non-covalent interactions (e.g., ionic interactions, hydrophobic interactions, hydrogen bonds, hybridization, etc.), or the binding of a molecule or cell to a surface. The terms "specific", "specifically" or "specific" refer to preferential recognition, contact and formation of a stable complex between a first molecule and a second molecule as compared to the first molecule and any one of a plurality of other molecules (e.g., there is little to no recognition, contact or formation of a stable complex between a first molecule, such as a capture agent, and any one of a plurality of other molecules, such as an analyte).

[0037] The analyte or target analyte can be, but is not limited to, a polypeptide, a protein (eg, C-reactive protein), a protein fragment, a labeled protein, a fusion protein, an antibody, an antibody fragment, a small molecule, a viral particle, a cell or any fragment thereof.

[0038] The subjects described herein can be of any age and can be adults, infants, or children. In some cases, the subject is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55 , 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 years old, or within a certain range (e.g., between 2 and 20 years old, between 20 and 40 years old, or between 40 and 90 years old). A particular class of subjects that can benefit is a subject suffering from or suspected of having an infection. Another special class of subjects that can benefit is a subject at a higher risk of infection. In addition, the subject treated by any method or composition described herein can be male or female. Any method, device or kit disclosed herein can also be performed on non-human subjects such as laboratory or farm animals. Non-limiting examples of non-human subjects include dog, goat, guinea pig, hamster, mouse, pig, non-human primate (eg, gorilla, ape, orangutan, lemur, or baboon), rat, sheep, cow, or zebrafish.

[0039] As used herein, "virus" may refer to a live but non-cellular obligate intracellular parasite that consists of DNA or RNA and a protein coat. The diameter of a virus can range from about 20 to about 300 nm. The genome of a Class I virus (Baltimore classification) may have double-stranded DNA. Class II viruses may have single-stranded DNA as their genome. Class III viruses may have double-stranded RNA as their genome. The genome of a Class IV virus may have positive single-stranded RNA, while the genome itself is mRNA. The genome of a Class V virus may be used as a template for mRNA synthesis, and its negative single-stranded RNA may be negative. Class VI and Class VI viruses may have positive single-stranded RNA genomes, but have DNA intermediates not only in replication but also in mRNA synthesis. Viruses can be identified by the diseases they cause in plants, animals, and prokaryotes. Prokaryotic viruses are also called bacteriophages.

[0040] As used herein, "fungi" may refer to a division of eukaryotic organisms that grow in irregular clumps, have no roots, stems or leaves, and do not contain chlorophyll or other pigments capable of photosynthesis. Each fungal organism may be unicellular to filamentous, and have a branching somatic structure (e.g., hyphae) surrounded by a cell wall containing glucan or chitin or both, and containing a true nucleus.

[0041] As used herein, "infection" may refer to the invasion of the body of a multicellular organism by an organism with the potential to cause disease.

[0042] The term "embossing" means that the spacers and the board are integrally fixed by stamping (ie, embossing) a piece of material to form the spacers on the board surface. The material can be a single layer of material or multiple layers of material.

[0043] The term "etched" means that the spacers and the board are integrally secured by etching a piece of material to form the spacers on the board surface. The material may be a single layer of material or multiple layers of material.

[0044] The term "fused to" means that the spacer and the board are integrally fixed by bonding the spacer and the board together, original materials for the spacer and the board are fused to each other, and there is a clear material boundary fusion between the two materials afterwards.

[0045] The term "bonded to" means that the spacer and the plate are integrally fixed by bonding the spacer and the plate through adhesion.

[0046] The term "attached to" means that the spacer and the plate are connected together.

[0047] It will be apparent to those skilled in the art after reading this disclosure that each individual embodiment described and illustrated herein has discrete components and features that can be easily separated or combined with the features of any of the other several embodiments. From the scope or spirit of this teaching. Any narrated method can be performed in the order of events or any other order that is logically possible. It will be understood by those skilled in the art that the application of the present invention is not limited to the details of the steps set forth in the description or drawings of this article, the arrangement of components, the selection of categories, weights, predetermined signal limits, or the description of this article. The present invention can have other embodiments and can be practiced or performed in many different ways.

[0048] Rapid, homogeneous, parallel staining of cellular and non-cellular analytes

[0049] A large proportion of antibiotics are used to treat diseases for which antibiotics have little or no effect. Excessive and inappropriate use of antibiotics has led to the emergence of increasingly resistant bacteria, which has become a serious problem worldwide. Individual healthcare practitioners are often faced with the difficult problem of how to identify patients who need (and especially those who do not need) antibiotic treatment.

[0050] Imaging and / or analyzing cells and non-cellular analytes in parallel can be a useful tool for determining whether a subject suffers from a disease (e.g., a virus or bacterial infection). In some embodiments, cells and non-cellular analytes can be imaged and / or analyzed simultaneously (e.g., a single image or visual field can be used to determine the amount of cells and non-cellular analytes). In some embodiments, cells and non-cellular analytes can be imaged and / or analyzed in series (e.g., the number of cells can be determined using a first image or a first visual field, and the number of non-cellular analytes can be determined using a second picture or a second visual field). In some embodiments, a single image or visual field can be used to determine one or more amounts corresponding to cells and / or non-cellular analytes. In some embodiments, two or more images or visual fields can be used to determine one or more amounts corresponding to cells and / or non-cellular analytes.

[0051] Typically, the amount of a cell or non-cellular analyte can correspond to the amount of the cell or non-cellular analyte in the sample. However, in some embodiments, the amount of a cell or non-cellular analyte can correspond to the concentration of the cell or non-cellular analyte in the sample. In some embodiments, the amount of a cell or non-cellular analyte can correspond to a portion of the cell or non-cellular analyte (e.g., a portion of cells having a given size or shape).

[0052] Analyte can generally be any molecule related to the presence or absence of a disease.Non-limiting examples of analytes include alpha-fetoprotein, prostate-specific antigen, cardiac troponin, C-reactive protein (CRP) or human chorionic gonadotropin, carcinogens, influenza virus markers, HPV, HBV, HCV, EBV, HIV, HSV, FeLV, FIV, Hanta virus, HTLV I, HTLVII or CMV infection, bacterial infection markers, fungal infection markers or parasitic infection markers. Cells can be any cells of the body. In some embodiments, cells can refer to cells of the immune system.Non-limiting examples of immune system cells include leukocytes, leukocytes, granulocytes, granulocytes, myeloid cells, lymphoid cells, neutrophils, eosinophils, basophils, lymphocytes, T cells, B cells, natural killer cells and monocytes.

[0053] Embodiments of the present disclosure may include detecting or determining (i) the number of cells in a sample and (ii) the number of non-cellular analytes. In some embodiments, the method may further include determining whether the subject suffers from a condition based on the amount of cells and the amount of non-cellular analytes in the sample. In some embodiments, the condition will be a condition that causes a change in the amount of cells or non-cellular analytes in the sample. In some embodiments of the present disclosure, analyzing cells and non-cellular analytes in a sample can be used to determine whether the analyzed sample is obtained from a subject suffering from an immune system disease. As used herein, "immune system disease or condition" may refer to a pathological condition caused by a defect in the immune system. If a person is born with a severe defect in the immune system, they may die from a viral, bacterial, fungal, or parasitic infection. In severe combined immunodeficiency, a lack of enzymes may mean that toxic waste accumulates within immune system cells, killing them, thereby destroying the immune system. A lack of immune system cells is also the basis of DiGeorge syndrome: improper development of the thymus may mean a reduced production of T cells. Most other immune diseases are caused by excessive immune responses or "autoimmunity." For example, asthma, familial Mediterranean fever, and Crohn's disease (inflammatory bowel disease) are all caused by an overreaction of the immune system, while autoimmune polyglandular syndrome and certain aspects of diabetes are caused by the immune system attacking "self" cells and molecules. A key part of the immune system's role is to distinguish between invaders and the body's own cells, and if this distinction cannot be made, the response against "self" cells and molecules can lead to autoimmune diseases.

[0054] In some embodiments of the present disclosure, cellular and non-cellular analytes in the analyzed sample can be used to determine whether the analyzed sample was obtained from a subject suffering from a metabolic disease. As used herein, "metabolic disease or disorder" can refer to a pathological condition caused by an error in the metabolic process. Metabolism can be the way the human body synthesizes energy from fats, carbohydrates, and proteins in food through enzymatic reactions of minerals and vitamins and synthesizes other molecules required. The system has a high tolerance for errors: generally, a mutation in one enzyme does not mean that the person can suffer from a certain disease. Many different enzymes may compete to modify the same molecule, and for multiple metabolic intermediates, there may be more than one way to achieve the same end result. Disease can occur if key enzymes lose function or the control mechanisms of metabolic pathways are affected.

[0055] In some embodiments of the present disclosure, analyzing cellular and non-cellular analytes in a sample can be used to determine whether the analyzed sample was obtained from a subject suffering from a muscle or bone disease. As used herein, "muscle and bone disease or disorder" can refer to a pathological condition caused by defects in genes important for the formation and function of muscle and connective tissue. Connective tissue is used herein as a broad term to include bones, cartilage, and tendons. For example, defects in fibrillin (a connective tissue protein that is important for making tissues strong yet flexible) can cause Marfan syndrome, while catastrophic dysplasia is caused by defects in sulfate transporters in cartilage.

[0056] In some embodiments of the present disclosure, analyzing cellular and non-cellular analytes in a sample can be used to determine whether the analyzed sample was obtained from a subject suffering from a neurological disease. Neurological diseases can refer to pathological conditions caused by defects in the nervous system, including the central nervous system, i.e., the brain and the peripheral nervous system. The brain and nervous system form a complex network of electrical signals responsible for coordinating muscles, sensations, speech, memory, thoughts, and emotions. Several diseases that directly affect the nervous system have a genetic component: some are due to mutations in a single gene, while others have been shown to have a more complex inheritance pattern.

[0057] In some embodiments of the present disclosure, cells (e.g., leukocytes) and non-cellular analytes (e.g., carcinogens) in the analysis sample can be used to determine whether the analyzed sample is obtained from a subject suffering from cancer. The subject can suffer from any type of cancer. Examples of cancer can include, but are not limited to, adrenal cancer, anal cancer, basal cell carcinoma, bile duct cancer, bladder cancer, blood cancer, bone cancer, brain tumor, breast cancer, bronchial cancer, liver cancer. Cardiovascular system, cervical cancer, colon cancer, colorectal cancer, digestive system cancer, endocrine system cancer, endometrial cancer, esophageal cancer, eye cancer, gallbladder cancer, gastrointestinal tumors, kidney cancer, hematopoietic system malignancies, laryngeal cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, mesothelioma, muscular system cancer, myelodysplastic syndrome (MDS), myeloma, nasal cancer, nasopharyngeal cancer, nervous system cancer, lymphatic system cancer, oral cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penis cancer, pituitary tumor, prostate cancer, rectal cancer, renal pelvis cancer, reproductive system cancer, reservoir cancer of the respiratory system, sarcoma, salivary gland cancer, skeletal system cancer, skin cancer, small intestine cancer, stomach cancer, testicular cancer, laryngeal cancer, thymus cancer, thyroid cancer, tumor, urinary system cancer, uterine cancer, vaginal cancer, or vulvar cancer. The term "lymphoma" may refer to any type of lymphoma, including B-cell lymphoma (e.g., diffuse large B-cell lymphoma, follicular lymphoma, small lymphocytic lymphoma, mantle cell lymphoma, marginal zone B-cell lymphoma, Burkitt's lymphoma, lymphoplasmacytic lymphoma, hairy cell leukemia, or primary central nervous system lymphoma) or T-cell lymphoma (e.g., precursor T-lymphocytic lymphoma or peripheral T-cell lymphoma). The term "leukemia" may refer to any type of leukemia, including acute leukemia or chronic leukemia. Types of leukemia include acute myeloid leukemia, chronic myeloid leukemia, acute lymphocytic leukemia, acute undifferentiated leukemia, or chronic lymphocytic leukemia. In some cases, a cancer patient does not have a specific type of cancer. For example, in some cases, a patient may have a cancer other than breast cancer.

[0058] The capture agent with reporter molecules can be used to detect cells and / or non-cellular analytes. For example, apidin orange can be used for leukocytes in dyeing samples. The non-limiting examples of reporter molecules include cyanine 5 (Cy5), histidine (His), V5, FLAG, influenza hemagglutinin (HA), Myc, VSV-G and thioredoxin (Trx), glutathione-S-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT) β-galactosidase, β-glucuronidase, luciferase, green fluorescent protein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP) and automatic fluorescent protein, including blue fluorescent protein (BFP). For example, anti-CRP antibodies (such as capture agents) conjugated with GFP can be used to determine the presence or amount of CRP in a sample.

[0059] A staining agent (e.g., dye) can be used to make cells (e.g., nucleated cells) detectable. For example, a sample can be stained with a pyridine orange (AO), which is a dye that can stain living cells and certain components of the cytoplasm. The absorption peak of pyridine orange is at 490nm, and it emits at 520nm when combined with DNA. Other fluorescent dyes can be used, such as Hoechst 33258 and Hoechst 33342. Generally, any non-specific fluorescent dyes for staining cells, cytoplasm, cell nucleic acid materials, or nuclei themselves can be used. These dyes are referred to as "non-specific fluorescent dyes" in this article. In some embodiments, a capture agent or staining agent can be applied to one or more surfaces of the first plate and / or the second plate. It is contemplated that a capture agent or staining agent can be applied to one or more surfaces of the first plate or the second plate to reduce the number of steps performed by the user in order to analyze the sample. For example, if the first plate is coated with a cell staining agent, the user only needs to deposit the sample on a QMAX card or in a Q quality card (e.g., in the sample contact area). After contacting the cell staining agent, the cells in the sample can be stained automatically (e.g., without user intervention). Reducing the number of steps performed by the user can reduce errors (such as human errors), improve the accuracy of data analysis, and reduce the time required for analyzing samples. In some embodiments, the capture agent can be coated on one or more surfaces of the first plate, and the stain can be coated on one or more surfaces of the second plate. In another embodiment, the capture agent and the stain can be coated on one or more surfaces of the first plate or the second plate. In yet another embodiment, the capture agent and the stain can be coated on one or more surfaces of the first plate and the second plate.

[0060] In one example, CRP (C-reactive protein) is a protein that is normally present in very low concentrations (<1 μg / mL) in the blood of healthy people. In bacterial infections, CRP concentrations increase significantly (>10 μg / mL), while viral infections usually cause only very modest CRP elevations or no elevations at all. Therefore, CRP can be used as a biomarker to clinically distinguish bacterial from viral infections. Other biomarkers in this category include white blood cell (WBC) counts, lymphocyte (LYM) counts, and PCT (calcitonin), among others.

[0061] The number of analyzing cells and non-cellular analytes can include determining the number of cells (e.g., leukocytes), determining the number of non-cellular analytes (e.g., CRP), and comparing these values ​​with one or more reference values ​​to determine whether the subject from which the analysis sample is obtained suffers from a disease. In some embodiments, the amount of analyzing cells and non-cellular analytes can include determining the ratio of the amount of cells to the amount of non-cellular analytes, and comparing the ratio with a reference value to determine whether the sample obtained from the analysis of the subject is conditional. In some embodiments, the measurement results (e.g., the number of cells or the number of non-cellular analytes) obtained from healthy subjects or subjects with a disease can be stored in a database and used to determine a reference value for comparison with future measurements.

[0062] Example 1

[0063] Rapid, homogeneous, parallel staining of cellular and non-cellular analytes

[0064] Here we describe a one-step combined blood test that reports CRP, WBC and LYM levels or counts within 60 seconds. The combination of the three tests (CRP, WBC and LYM) yields high sensitivity and specificity to differentiate bacterial from viral infections and is therefore very valuable for antibiotic administration.

[0065] In the local read method, such as Figure 1A , Figure 1B As shown, one or more particles will be measured for the following two measurements: (a) The signal from the particle area (SP). It can be from the entire particle area or from a specified area of ​​the particle area. (b) The signal from the area around the particle (local background SB). It can be from the entire area around the particle or a specified area. "Surrounding" can be defined as a distance from the outer surface of the particle of 0.01D, 0.1D, 0.2D, 0.5D, 1D, 2D, 5D, 10D, 50D or a range between any two values. Where "D" is the average diameter of the particle. The true measurement signal (SA) of each particle can be determined as SA = SP-SB. The measurement signal from each CROF (SCROF) can be the average of multiple particles. It can be all particles on the entire CROF or all particles in a specified area of ​​the CROF (for example, SCROF = average value (SA1, SA2, SA3...SAn))

[0066] In this experiment, the device for immunoassay includes a first plate and a second plate. Figure 3As shown in Panel 1, capture antibodies are first conjugated to magnetic beads. Next, as shown in Panel 2, a piece of antibody-coupled beads mixed with WBC staining reagent is coated on one x-plate. As shown in Panel 3, a second x-plate is then coated with labeled anti-CRP detection antibody. In Panel 4, after each x-plate is prepared, the Q card is ready to receive a drop of blood. As shown in Picture 5, after a drop of whole blood is applied, the x-plates are quickly pressed together to dissolve the reagents and start the assay. Subsequently, as shown in Picture 6, after 30 seconds, the assay can be analyzed for WBC count and CRP concentration.

[0067] -The experiments were conducted using the following materials:

[0068] - Anti-human CRP rabbit polyclonal antibody (Abcam)

[0069] -PureProteome NHS FlexiBind Magnetic Beads (Millipore)

[0070] - Human CRP Analyte (Fitzgerald)

[0071] -4% bovine serum albumin

[0072] -PBS (Phosphate Buffered Saline)

[0073] -PBS-Tween 20 (PBST)

[0074] -X-plate with 10μm spacer

[0075] -Whole blood

[0076] -Cr-Orange

[0077] The experiment was performed according to the following procedure:

[0078] 1. Conjugation of anti-CRP capture antibody to beads. NHS-activated beads (10 μm diameter micropores) were coupled to anti-CRP mouse monoclonal capture antibody (Fitzgerald) according to the manufacturer's protocol.

[0079] 2. Bead blocking treatment. Block the antibody-coupled beads overnight using 4% BSA in PBS. The next day, wash the beads 6 times with PBST before use.

[0080] 3. First plate coating. 1 μL of antibody-conjugated beads (bead concentration of 107–108 / mL) from step 2 with or without 25 μg / mL cridine orange (AO, WBC staining reagent) was dropped onto the Q card and air-dried at room temperature.

[0081] 4. Homogenized BEST assay. 1 μL of fresh whole blood and 1 μL of Cy5-labeled anti-CRP mouse monoclonal detection antibody (Fitzgerald) were mixed together and then dropped onto the same area of ​​the Q card that was occupied by the dried, antibody-conjugated magnetic beads. The mixture was immediately covered by a second X-plate containing 10 μm columns. The assay was incubated for 30 seconds before imaging occurred.

[0082] 5. Imaging. Without washing, use the iPhone 6s to take two fluorescent images at different wavelengths to observe the staining signal of WBC or the signal from the anti-CRP detection antibody.

[0083] In another experiment, a device for immunoassay includes a first plate and a second plate. Figure 4 As shown in the first picture, the capture antibody is first conjugated to the magnetic beads. Next, as shown in the second picture, one x-plate is coated with the antibody-conjugated beads. As shown in the third picture, the second x-plate is then coated with the labeled anti-CRP detection antibody. In the fourth picture, after each x-plate is prepared, the Q card is ready to receive a drop of blood mixed with the WBC staining reagent. As shown in the fifth picture, after adding a drop of whole blood mixed with the WBC staining reagent, the x-plates are quickly pressed together to dissolve the reagent and start the assay. Subsequently, as shown in the sixth picture, after 30 seconds, the assay can be analyzed for WBC count and CRP concentration.

[0084] QMAX System

[0085] A) QMAX card

[0086] Details of the QMAX card are described in detail in various publications including International Application No. PCT / US2016 / 046437 (Essenlix File No. ESSN-028WO), which is incorporated herein by reference for all purposes.

[0087] I. plate

[0088] In the present invention, the plates of the CROF are generally made of any material that (i) is capable of adjusting the thickness of a portion or the entire volume of the sample in conjunction with the spacers, and (ii) does not have a significant adverse effect on the sample, assay, or goal to be achieved by the plate. However, in certain embodiments, the specific materials used for the plates (and therefore their properties) can achieve certain goals.

[0089] In certain embodiments, the two panels have the same or different parameters for each of the following parameters: panel material, panel thickness, panel shape, panel area, panel flexibility, panel surface properties, and panel optical transparency.

[0090] (i) Material of the plate. The plate is made of a single material, a composite material, multiple materials, a multi-layered material, an alloy or a combination thereof. Each material used for the plate is an inorganic material, an organic material or a mixture, wherein examples of the materials are given in the paragraphs of Mat-1 and Mat-2.

[0091] Mat-1: The inorganic materials of the sheet include but are not limited to glass, quartz, oxides, silicon dioxide, silicon nitride, hafnium oxide (HfO), aluminum oxide (AIO), semiconductors: (Si, GaAs, GaN, etc.), metals (such as gold, silver, copper, aluminum, Ti, Ni, etc.), ceramics or any combination thereof.

[0092] Mat-2: Organic materials used for insulators include but are not limited to polymers (such as plastics) or amorphous organic materials. The polymer material for the spacer includes, but is not limited to, acrylate polymers, vinyl polymers, olefin polymers, cellulosic polymers, non-cellulosic polymers, polyester polymers, nylon, cyclic olefin copolymer (COC), polymethyl methacrylate (PMMA), polycarbonate (PC), cyclic olefin polymer (COP), liquid crystal polymer (LCP), polyamide (PA), polyethylene (PE), polyimide (PI), polypropylene (PP), polyphenylene ether (PPE), polystyrene (PS), polyoxymethylene (POM), polyetheretherketone (PEEK), polyethersulfone (PES), polyethylene phthalate (PET), polytetrafluoroethylene (PTFE), polyvinyl chloride (PVC), polyvinylidene fluoride (PVDF), polybutylene terephthalate (PBT), fluorinated ethylene propylene (FEP), perfluoroalkoxyalkane (PFA), polydimethylsiloxane (PDMS), rubber or any combination thereof.

[0093] In some embodiments, the plates are each independently made of at least one of glass, plastic, ceramic, and metal. In some embodiments, each plate independently includes at least one of glass, plastic, ceramic, and metal.

[0094] In some embodiments, one plate is different from another plate in lateral area, thickness, shape, material or surface treatment. In some embodiments, one plate is the same as another plate in lateral area, thickness, shape, material or surface treatment.

[0095] The material of the panels is rigid, flexible or anything in between. Rigidity (ie, stiffness) or flexibility is relative to a given pressure used to bring the panels into a closed configuration.

[0096] In certain embodiments, the choice of a rigid or flexible plate is determined by the need to control the uniformity of sample thickness in a closed configuration.

[0097] In some embodiments, at least one of the two plates is transparent (to light). In some embodiments, at least a portion or portions of one or both plates are transparent. In some embodiments, the plates are opaque.

[0098] (ii) Plate Thickness. In certain embodiments, at least one plate has an average thickness of 2 nm or less, 10 nm or less, 100 nm or less, 500 nm or less, 1000 nm or less, 2 μm (micrometers) or less, 5 μm or less, 10 micrometers or less, 20 micrometers or less, 50 micrometers or less, 100 micrometers or less, 150 micrometers or less, 200 micrometers or less, 300 micrometers or less, 500 micrometers or less, 800 micrometers or less, 1 mm (millimeter) or less, 2 mm or less, 3 mm or less, or a range between any two values.

[0099] In certain embodiments, at least one plate has an average thickness of at most 3 mm (millimeters), at most 5 mm, at most 10 mm, at most 20 mm, at most 50 mm, at most 100 mm, at most 500 mm, or a range between two values.

[0100] In some embodiments, the thickness of the plate is not uniform across the plate. Using different plate thicknesses at different locations can be used to control plate bending, folding, sample thickness adjustment, etc.

[0101] (iii) Shape and area of ​​the plate. In general, the plate can have any shape as long as the shape allows for compressed open flow of the sample and adjustment of the sample thickness. However, in certain embodiments, a specific shape may be advantageous. The shape of the plate can be circular, oval, rectangular, triangular, polygonal, annular, or any superposition of these shapes.

[0102] In some embodiments, the two plates can have the same size or shape, or different. The area of ​​the plates depends on the application. The area of ​​the plates can be up to 1 square millimeter, up to 10 square millimeters, up to 100 square millimeters, up to 1 square centimeter, up to 5 square centimeters, up to 10 square centimeters, up to 100 square centimeters. Up to 500 square centimeters, up to 1000 square centimeters, up to 5000 square centimeters, up to 10,000 square centimeters, or up to 10,000 square centimeters, or any permutation between the two values. The shape of the plates can be rectangular, square, circular, or other shapes.

[0103] In some embodiments, at least one of the panels is in the form of a strip (or strip) having a width, a thickness, and a length. The width is at most 0.1 cm, at most 0.5 cm, at most 1 cm, at most 5 cm, at most 10 cm, at most 50 cm, at most 100 cm, at most 500 cm, at most 1000 cm, or a range between any two values. The length can be a desired length. The strip-shaped panel can be rolled into a roll.

[0104] (iv) Plate surface flatness. In many embodiments, the inner surface of the plate is flat or remarkably flat, flat. In certain embodiments, the two inner surfaces are parallel to each other in the closed configuration. A flat inner surface can facilitate quantification and / or control of sample thickness by simply using a predetermined spacer height in the closed configuration. For a non-flat inner surface of the plate, not only the spacer height needs to be known, but also the exact topology of the inner surface needs to be known to quantify and / or control the sample thickness in the closed configuration. Knowing the surface topology requires additional measurements and / or corrections, which can be complex, time consuming, and expensive.

[0105] The flatness of the panel surface is relative to the final sample thickness (the final thickness is the thickness in the closed configuration) and is often characterized in terms of "relative surface flatness," which is the ratio of the final sample thickness to the final sample thickness.

[0106] In some embodiments, the relative surface is less than 0.01%, 0.1%, less than 0.5%, less than 1%, less than 2%, less than 5%, less than 10%, less than 20%, less than 30%, less than 50%, less than 70%, less than 80%, less than 100%, or a range between any two of these values.

[0107] (v) Plate surface parallelism. In some embodiments, the two surfaces of the plate are substantially parallel to each other. In some embodiments, the two surfaces of the plate are not parallel to each other.

[0108] (vi) Flexibility of the plates. In some embodiments, the plates are flexible under the compression of the CROF process. In some embodiments, both plates are flexible under the compression of the CROF process. In some embodiments, one plate is rigid and the other plate is flexible under the compression of the CROF process. In some embodiments, both plates are rigid. In some embodiments, both plates are flexible, but have different flexibilities.

[0109] (vii) Optical transparency of the panels. In some embodiments, the panels are optically transparent. In some embodiments, both panels are optically transparent. In some embodiments, a panel is optically transparent and the other panel is opaque. In some embodiments, both panels are opaque. In some embodiments, both panels are optically transparent, but have different light transmittances. The optical transparency of a panel may refer to a portion or the entire area of ​​the panel.

[0110] (viii) Surface wetting properties. In certain embodiments, the inner surface of the plate wets (i.e., the contact angle is less than 90 degrees) the sample, transfers the liquid, or has both functions. In certain embodiments, both plates have inner surfaces that wet the sample, transfer the liquid, or both. Have the same or different wettability. In certain embodiments, the plate has an inner surface that wets the sample, transfers the liquid, or both. The inner surface of the other plate is not wet (i.e., the contact angle is equal to or greater than 90 degrees). The wetting of the inner surface of the plate can refer to a portion or the entire area of ​​the plate.

[0111] In some embodiments, the inner surface of the plate has other nano- or micro-structures to control the lateral flow of the sample during CROF. The nano- or micro-structures include, but are not limited to, channels, pumps, etc. Nano- and micro-structures are also used to control the wetting properties of the inner surface.

[0112] II. Spacer

[0113] (i) Function of the spacer. In the present invention, the spacer is configured to have one or any combination of the following functions and characteristics: the spacer is configured to (1) control the thickness of the sample or the relevant volume of the sample together with the plate (preferably, the thickness control is precise, or uniform, or both within the relevant area); (2) allow the sample to have a compressed regulated open flow (CROF) on the surface of the plate; (3) do not occupy a significant surface area (volume) in a given sample area (volume); (4) reduce or increase the effect of particle or analyte sedimentation in the sample; (5) change and / or control the wettability of the inner surface of the plate; (6) determine the position, size ratio and / or information related to the plate, or (7) any combination of the above.

[0114] (ii) The structure and shape of the spacer. In order to achieve the desired sample thickness reduction and control, in certain embodiments, the spacers are fixed to their respective plates. Generally, the spacers can have any shape as long as the spacers can adjust the sample thickness during the CROF process, but in order to achieve certain functions (such as better uniformity, degree of overshoot of pressing, etc.), certain shapes are preferably used.

[0115] The spacer is a single spacer or a plurality of spacers. (e.g., an array). Certain embodiments of the plurality of spacers are arrays of spacers (e.g., pillars) where the distances between the spacers are periodic or aperiodic, or are periodic or aperiodic in certain areas of the plate, or have different distances in different areas of the plate.

[0116] There are two types of spacers: open spacers and closed spacers. Open spacers are spacers that allow sample flow through the spacer (i.e. the sample flows through and through the spacer. For example, a column acts as a spacer.), and closed spacers are spacers that block sample flow. (i.e. the sample cannot flow through the spacer. For example, a ring spacer, the sample is inside the ring.). Both types of spacers use their height to adjust the final sample thickness in a closed structure.

[0117] In some embodiments, the spacers are only open spacers. In some embodiments, the spacers are only closed spacers. In some embodiments, the spacers are a combination of open spacers and closed spacers.

[0118] The term "columnar spacer" means that the spacer has a columnar shape, and the columnar shape may refer to an object having a height and side shape that allows a sample to flow around it during compressed open flow.

[0119] In some embodiments, the lateral shape of the columnar spacer is a shape selected from the following group: (i) circle, oval, rectangle, triangle, polygon, ring, star, letter shape (e.g., L shape, C shape, letters from A to Z), number shape (e.g., 0, 1, 2, 3, 4... to 9); (ii) a shape in the (i) group having at least one rounded corner; (iii) a shape in the (i) group having a jagged or rough edge; (iv) any superposition of (i), (ii) and (iii). For multiple spacers, different spacers may have different lateral shapes and sizes and different distances from adjacent spacers.

[0120] In some embodiments, the spacers can be and / or include columns, posts, beads, spheres, and / or other suitable geometric shapes. The lateral shape and dimensions of the spacers (i.e., the dimensions transverse to the surface of the corresponding plates) can be anything, except that in some embodiments, the following limitations are excluded: (i) the geometry of the spacers does not cause significant errors in measuring sample thickness and volume; (ii) the geometry of the spacers does not prevent sample from flowing out between the plates (i.e., it is not closed form). However, in some embodiments, they require some spacers to be closed spacers to restrict sample flow.

[0121] In some embodiments, the shape of the spacer has rounded corners. For example, a rectangular spacer has one, several, or all chamfered corners (e.g., rounded instead of 90 degree angles). Rounded corners generally make the spacer easier to manufacture and, in some cases, less damaging to the biological material.

[0122] The sidewalls of the pillars can be straight, curved, angled, or different shapes in different portions of the sidewalls. In some embodiments, the spacers are pillars with various lateral shapes, sidewalls, and pillar height to pillar side area ratios. In a preferred embodiment, the spacers have a pillar shape for allowing open flow.

[0123] (iii) Material of the spacer. In the present invention, the spacer is generally made of any material that can be used with the two plates to adjust the thickness of the relevant volume of the sample. In some embodiments, the material used for the spacer is different from the material used for the plates. In some embodiments, the material used for the space is at least the same as a portion of the material used for at least one of the plates.

[0124] The spacer is made of a single material, a composite material, multiple materials, a multi-layer material, an alloy or a combination thereof. Each material used for the spacer is an inorganic material, an organic material or a mixture, where examples of materials are given in the paragraphs of Mat-1 and Mat-2. In a preferred embodiment, the spacer is made of the same material as the plate used in the CROF.

[0125] (iv) Mechanical strength and flexibility of the spacer. In some embodiments, the mechanical strength of the spacer is strong enough so that during compression and in the closed configuration of the board, the height of the spacer is the same or significantly the same as the height of the board in the open configuration. In some embodiments, the difference between the open configuration and the closed configuration of the spacer can be characterized and predetermined.

[0126] The material of the spacer is rigid, flexible, or anything in between. Rigidity is relative to the applied pressure used to bring the plates into the closed configuration: if the spacer deforms by no more than 1% of its height under pressure, the spacer material is considered rigid, otherwise it is considered flexible. When the spacer is made of a flexible material, the final sample thickness in the closed configuration can still be determined based on the pressing force and mechanical properties of the spacer.

[0127] (v) Spacers within the sample. In order to achieve the desired reduction and control of sample thickness, in particular to obtain good sample thickness uniformity, in certain embodiments, a spacer is placed within the sample or within the relevant volume of the sample. In certain embodiments, there are one or more spacers within the sample or within the relevant volume of the sample, with appropriate spacing between the spacers. In certain embodiments, at least one spacer is within the sample, at least two spacers are within the sample or within the relevant volume of the sample, or at least "n" spacers are within the sample or within the relevant volume of the sample, where "n" can be determined by the sample thickness uniformity or the desired sample fluidity during CROF.

[0128] (vi) Spacer height. In some embodiments, all spacers have the same predetermined height. In some embodiments, the spacers have different predetermined heights. In some embodiments, the spacers can be divided into groups or regions, where each group or region has its own spacer height. And in some embodiments, the predetermined height of the spacers is the average height of the spacers. In some embodiments, the spacers have approximately the same height. In some embodiments, a certain number of spacers have the same height.

[0129] The height of the spacer is selected by the desired specified final sample thickness and the residual sample thickness. The spacer height (predetermined spacer height) and / or sample thickness is 3 nm or less, 10 nm or less, 50 nm or less, 100 nm or less, 200 nm or less, 500 nm or less, 800 nm or less, 1000 nm or less, 1 μm or less, 2 μm or less, 3 μm or less, 5 μm or less, 10 μm or less, 20 μm or less, 30 μm or less, 50 μm or less, 100 μm or less, 150 μm or less, 200 μm or less, 300 μm or less, 500 μm or less, 800 μm or less, 1 mm or less, 2 mm or less, 4 mm or less, or a range between two values.

[0130] The spacer height and / or sample thickness is from 1 nm to 100 nm in one preferred embodiment, from 100 nm to 500 nm in another preferred embodiment, from 500 nm to 1000 nm in a separate preferred embodiment, from 1 μm (i.e., 1000 nm) to 2 nm, from μm in another preferred embodiment, from 2 μm to 3 μm in another preferred embodiment, from 3 μm to 5 μm in another preferred embodiment, from 5 μm to 10 μm in another preferred embodiment, from 10 μm to 50 μm in another preferred embodiment, and from 50 μm to 100 μm in a separate preferred embodiment.

[0131] In certain embodiments, the spacer height and / or sample thickness is (i) equal to or slightly larger than the smallest dimension of the analyte, or (ii) equal to or slightly larger than the largest dimension of the analyte. "Slightly larger" means that it is approximately 1% to 5% larger, and any number between the two values ​​is possible.

[0132] In certain embodiments, the spacer height and / or sample thickness is larger than the smallest dimension of the analyte (eg, the analyte has an anisotropic shape), but smaller than the largest dimension of the analyte.

[0133] For example, a red blood cell has a disc shape with a minimum dimension of 2 μm (disc thickness) and a maximum dimension of 11 μm (disc diameter). In one embodiment of the present invention, the spacer is selected so that the inner surface spacing of the plate in the relevant area is 2 μm (equal to the minimum dimension) in one embodiment, 2.2 μm in another embodiment, or 3 (50) 1% larger than the minimum dimension, but smaller than the maximum dimension of the red blood cell in another embodiment. Such an embodiment has certain advantages in blood cell counting. In one embodiment, for red blood cell counting, by making the inner surface spacing 2 or 3 μm, and making any number between the two values, the undiluted whole blood sample is averaged to limit the spacing of each red blood cell (RBC) without overlapping with other cells, so that the red blood cells can be accurately counted visually. (Too much overlap between RBCs can lead to serious errors in counting).

[0134] In certain embodiments of the invention, when the plate is in a closed configuration, it uses the plate and spacers to adjust not only the thickness of the sample, but also the orientation and / or surface density / configuration of the analytes / entities in the sample. When the plate is in a closed configuration, the thinner the sample, the less analytes / entities per unit surface area (i.e., the lower the surface concentration).

[0135] (vii) Spacer lateral dimensions. For an open spacer, its lateral dimensions can be characterized by lateral dimensions in two orthogonal directions, x and y (sometimes referred to as width). The lateral dimensions of the spacer in each direction may be the same or different. In some embodiments, the lateral dimension in each direction (x or y) is...

[0136] In some embodiments, the ratio of the lateral dimension in the x-direction to the y-direction is 1, 1.5, 2, 5, 10, 100, 500, 1000, 10,000, or a range between any two of these values. In some embodiments, different ratios are used to adjust the flow direction of the sample; for example, the larger the ratio, the larger the direction of the flow (larger in the dimension direction).

[0137] In certain embodiments, the different lateral dimensions of the spacers in the x and y directions serve as a means of (a) using the spacers as scale marks to indicate the orientation of the plate, (b) using the spacers to create more sample flow in a preferred direction, or both.

[0138] In a preferred embodiment, period, width and height.

[0139] In some embodiments, all spacers have the same shape and size. In some embodiments, each spacer has a different lateral size.

[0140] For a closed spacer, in some embodiments, the internal lateral shape and dimensions are selected based on the total volume of the sample to be enclosed by the closed spacer, where the volume size has been described in the present disclosure; in some embodiments, the external shape and dimensions are selected based on the required strength to support the pressure of the liquid against the spacer and the compressive pressure of the compression plate.

[0141] (viii) Aspect ratio of height to average lateral dimension of the pillar spacers. In certain embodiments, the aspect ratio of the height to average lateral dimension of the pillar spacers is 100,000, 10,000, 1,000, 100, 10, 1, 0.1, 0.01, 0.001, 0.0001, 0.0001, 00001, or a range between any two values.

[0142] (ix) Spacer height accuracy. The height of the spacer should be precisely controlled. The relative accuracy of the spacer (i.e., the ratio of the deviation from the desired spacer height) is less than or equal to 0.001%, less than or equal to 0.01%, less than or equal to 0.1%, less than or equal to 0.5%, less than or equal to 1%, less than or equal to 2%, less than or equal to 5%, less than or equal to 8%, less than or equal to 10%, less than or equal to 15%, less than or equal to 20%, less than or equal to 30%, less than or equal to 40%, less than or equal to 50%, less than or equal to 60%, less than or equal to 70%, less than or equal to 80%, less than or equal to 90%, less than or equal to 99.9%, or a range between any values.

[0143] (x) Spacer spacing. The spacer can be a single spacer or multiple spacers in the relevant area of ​​the plate or sample. In some embodiments, the spacers on the plate are configured and / or arranged in an array, and the array is periodic, aperiodic, or periodic in some locations of the plate and aperiodic in other locations.

[0144] In certain embodiments, the periodic array of spacers has a lattice of squares, rectangles, triangles, hexagons, polygons, or any combination thereof, where combination means that different locations of the plate have different lattices of spacers.

[0145] In certain embodiments, the inter-spacer distance of the spacer array is periodic in at least one direction of the array (ie, uniform inter-spacer distance). In certain embodiments, the distance between the spacers is configured to improve uniformity between the plate spacings in the closed configuration.

[0146] The distance between adjacent spacers (i.e., the distance between spacers) is 1 μm or less, 5 μm or less, 10 μm or less, 20 μm, 30 μm or less, 40 μm or less, 50 μm or less, 60 μm or less, 70 μm or less, 80 μm or less, 90 μm or less, 100 μm or less, 200 μm or less, 300 μm or less, 400 μm or less, or between two values.

[0147] In some embodiments, the distance between the spacers is 400, 500, 1 mm, 2 mm, 3 mm, 5 mm, 7 mm, 10 mm, or a range between any two values. In some embodiments, the distance between the spacers is 10 mm, 20 mm, 30 mm, 50 mm, 70 mm, 100 mm, or any range between two values.

[0148] The distance between adjacent spacers (i.e., the distance between the spacers) is selected so that for given characteristics of the plate and the sample, in certain embodiments, in the closed configuration of the plate, the variation in sample thickness between two adjacent spacers is, at most, 0.5%, 1%, 5%, 10%, 20%, 30%, 50%, 80%, or any range between the two values; or in certain embodiments, at most 80%, 100%, 200%, 400%, or a range between any two values.

[0149] Obviously, in order to maintain a given sample thickness variation between two adjacent spacers, a closer spacer spacing is required when using a more flexible plate.

[0150] Specifies the precision of the interval spacing distance.

[0151] In a preferred embodiment, the spacers are a periodic square array, wherein the spacers are pillars having a height of 2 to 4 μm, an average lateral dimension of 5 to 20 μm, and a spacer spacing of 1 μm to 100.

[0152] In a preferred embodiment, the spacers are a periodic square array, wherein the spacers are pillars having a height of 2 to 4 μm, an average lateral dimension of 5 to 20 μm, and a spacer spacing of 100 μm to 250 μm.

[0153] In a preferred embodiment, the spacers are a periodic square array, wherein the spacers are pillars having a height of 4 to 50 μm, an average lateral dimension of 5 to 20 μm, and a spacer spacing of 1 μm to 100.

[0154] In a preferred embodiment, the spacers are a periodic square array, wherein the spacers are pillars having a height of 4 to 50 μm, an average lateral dimension of 5 to 20 μm, and a spacer spacing of 100 μm to 250 μm.

[0155] The period of the spacer array is from 1 nm to 100 nm in one preferred embodiment, from 100 nm to 500 nm in another preferred embodiment, from 500 nm to 1000 nm in a separate preferred embodiment, from 1 μm (i.e., 1000 nm) to 2 μm in another preferred embodiment, from 2 μm to 3 μm in a separate preferred embodiment, from 3 μm to 5 μm in another preferred embodiment, from 5 μm to 10 μm in a separate preferred embodiment, from 10 μm to 50 μm in another preferred embodiment, from 50 μm to 100 μm to 175 μm in a separate preferred embodiment, from 100 μm to 175 μm in a separate preferred embodiment, and from 175 μm to 300 μm in a separate preferred embodiment.

[0156] (xi) Spacer density. Spacers are spaced at 1 μm 2 Greater than 1, every 10μm 2 Greater than 1, every 100 μm 2 Greater than 1, every 500μm 2 Greater than 1, every 1000μm 2 Greater than 1, every 5000μm 2 Greater than 1, every 0.01mm 2 Greater than 1, every 0.1mm 2 Greater than 1, every 1mm 2 Greater than 1, every 5mm 2 Greater than 1, every 10mm 2 Greater than 1, every 100mm 2 Greater than 1, every 100mm 2 Greater than 1,1000mm 2 Greater than 1, every 10000mm 2 A surface density greater than 1, or in a range between two values, is arranged on each plate.

[0157] (3) the spacer is configured so as not to occupy a large surface area (volume) in a given sample area (volume);

[0158] (xii) Ratio of spacer volume to sample volume. In many embodiments, the ratio of the spacer volume (i.e., the volume of the spacer) to the sample volume (i.e., the volume of the sample), and / or the ratio of the volume of the spacer within the relative volume of the container. The relative volume of the sample to the sample is controlled to obtain certain advantages. Advantages include, but are not limited to, uniformity of sample thickness control, uniformity of analyte, sample flow characteristics (i.e., flow rate, flow direction, etc.).

[0159] In some embodiments, the ratio of the spacer volume (r) to the sample volume, and / or the ratio of the volume of the spacer within the relevant volume of the sample to the relevant volume of the sample is at most 100%, at most 99%, at most 70%, at most 50%, at most 30%, at most 10%, at most 5%, at most 3%, at most 1%, at most 0.1%, at most 0.01%, at most 0.001%, or a range between any one value.

[0160] (xiii) Spacers fixed to the panels. The spacing distance between the spacers and the orientation of the spacers, which are critical in the present invention, are preferably maintained during the process of moving the panels from the open configuration to the closed configuration, and / or are preferably predetermined before changing the panels from the open configuration to the closed configuration.

[0161] In certain embodiments of the present disclosure, a spacer is secured to one of the panels prior to closing the panels into a closed configuration. The term "a spacer is secured to its respective panel" means that the spacer is attached to the panel and remains attached during use of the panel. An example of "a spacer is secured to its respective panel" is that the spacer is integrally made of one material of the panel and the position of the spacer relative to the panel surface does not change. An example of "a spacer is not secured to its respective panel" is that the spacer is bonded to the panel by an adhesive, but when the panel is in use, the adhesive is unable to maintain the spacer in its original position on the panel surface (that is, the spacer moves from its original position on the panel surface).

[0162] In some embodiments, at least one spacer is fixed to its respective plate. In some embodiments, two spacers are fixed to their respective plates. In some embodiments, most spacers are fixed with their respective plates. In some embodiments, all spacers are fixed with their respective plates.

[0163] In certain embodiments, the spacers are integrally secured to the panels.

[0164] In certain embodiments, the spacers are secured to their respective plates by one or any combination of the following methods and / or configurations: attached, bonded, fused, embossed, and etched.

[0165] The term "embossing" means that the spacers and the board are integrally fixed by stamping (ie, embossing) a piece of material to form the spacers on the board surface. The material can be a single layer of material or multiple layers of material.

[0166] The term "etched" means that the spacers and the board are integrally secured by etching a piece of material to form the spacers on the board surface. The material may be a single layer of material or multiple layers of material.

[0167] The term "fused to" means that the spacer and the board are integrally fixed by bonding the spacer and the board together, original materials for the spacer and the board are fused to each other, and there is a clear material boundary fusion between the two materials afterwards.

[0168] The term "bonded to" means that the spacer and the plate are integrally fixed by bonding the spacer and the plate through adhesion.

[0169] The term "attached to" means that the spacer and the plate are connected together.

[0170] In some embodiments, the spacers and the plates are made of the same material. In other embodiments, the spacers and the plates are made of different materials. In another embodiment, the spacers and the plates are formed as one piece. In another embodiment, one end of the spacer is fixed to its corresponding plate, while the end is open to accommodate different configurations of the two plates.

[0171] In other embodiments, each spacer is independently at least one of attached to, bonded to, fused to, imprinted in, and etched in the respective plate. The term "independently" means that one spacer is fixed to its respective plate by the same or different methods selected from the group consisting of attaching, bonding, fusing, imprinting, and etching on the respective plate.

[0172] In some embodiments, the distance between at least two spacers is predetermined ("predetermined spacer distance" means that the distance is known when a user uses the board).

[0173] In certain embodiments of all methods and apparatus described herein, in addition to the fixed spacers, other spacers are present.

[0174] (xiv) Specific Sample Thickness. In the present invention, it is observed that a larger plate holding force (i.e., the force holding the two plates together) can be achieved by using a smaller plate spacing (for a given sample area) or a larger sample area (for a given sample), or both.

[0175] In certain embodiments, at least one plate is transparent in an area surrounding the associated area, each plate has an inner surface configured to contact the sample in a closed configuration; the inner surfaces of the plates are substantially parallel to each other in the closed state; the inner surfaces of the plates are substantially planar except for locations with spacers; or any combination thereof.

[0176] The spacers can be fabricated on the board in various ways using lithography, etching, imprinting (nanoimprinting), deposition, stripping, fusion or a combination thereof. In some embodiments, the spacers are directly imprinted or embossed on the board. In some embodiments, the spacers are imprinted into a material (e.g., plastic) deposited on the board. In some embodiments, the spacers are fabricated by directly imprinting the surface of the CROF board. Nanoimprinting can be done using a roll-to-roll technique using a roller imprinter, or it can be rolled into a planar nanoimprint. This method has great economic advantages, thus reducing costs.

[0177] In some embodiments, the spacer is deposited on the board. The deposition can be evaporation, sticking or stripping. In the sticking process, the spacer is first made on a carrier and then the spacer is transferred from the carrier to the board. In the lift-off, a removable material is first deposited on the board and a hole is created in the material; the bottom of the hole exposes the board surface, and then an insulating material is deposited into the hole, and then the removable material is removed, leaving only the spacer on the board surface. In some embodiments, the spacer deposited on the board is fused to the board. In some embodiments, the spacer and the board are made in a single process. A single process includes imprinting (i.e., embossing, molding) or synthesis.

[0178] In certain embodiments, at least two of the spacers are secured to the respective plates by different fabrication methods, and optionally, wherein the different fabrication methods include at least one of deposition, bonding, fusion, imprinting, and etching.

[0179] In certain embodiments, one or more spacers are secured to the respective plates by a manufacturing method of bonding, fusing, embossing or etching or any combination thereof.

[0180] In certain embodiments, the manufacturing method for forming such an integrated spacer on the board includes a method of being bonded, fused, stamped or etched or any combination thereof.

[0181] (xv)ISD^4 / hE

[0182] In certain embodiments of the present disclosure, a device for forming a thin fluid sample layer having a uniform predetermined thickness by pressing may include a first plate. In certain embodiments of the present disclosure, a device for forming a thin fluid sample layer having a uniform predetermined thickness by pressing may include a second plate. In certain embodiments of the present disclosure, a device for forming a thin fluid sample layer having a uniform predetermined thickness by pressing may include a spacer. In certain embodiments, the plates may be moved relative to each other into different configurations. In certain embodiments, one or both plates are flexible. In certain embodiments, each plate includes an inner surface having a sample contact area for contacting a fluid sample. In certain embodiments, each plate includes a force area on its respective outer surface for applying pressure to press the plates together. In certain embodiments, one or both of the two plates include a spacer that is permanently fixed to the inner surface of each plate. In certain embodiments, the spacer has a predetermined substantially uniform height equal to or less than 200 microns, and a predetermined fixed spacing. In certain embodiments, the fourth power of the spacer distance (ISD) divided by the thickness (h) and Young's modulus (E) of the flexible plate (ISD4 / (hE)) is 5x10 6 μm 3 / GPa. In some embodiments, at least one spacer is within the sample contact area. In some embodiments, one configuration is an open configuration, wherein: the two plates are partially or completely separated, the spacing between the two plates is not limited by the spacer, and the sample is deposited on one or both plates. In some embodiments, another configuration is a closed configuration, which is constructed after the sample is deposited in an open configuration and the plates are forced to a closed configuration by applying pressure on the force area. In the closed state, at least a portion of the sample is compressed by the two plates into a highly uniform layer and is substantially stagnant relative to the plates, wherein the uniform thickness of the layer is limited by the contact area of ​​the sample. Two plates, regulated by the plates and the spacer.

[0183] In certain embodiments of the present disclosure, a method for forming a thin fluid sample layer having a uniform predetermined thickness by extrusion may include obtaining a device of the present disclosure. In certain embodiments of the present disclosure, a method for forming a thin fluid sample layer having a uniform predetermined thickness by pressing, the method may include: depositing a fluid sample on one or two plates when the plates are configured to be in an open configuration. In certain embodiments, the open configuration is a configuration in which two plates are partially or completely separated and the spacing between the plates is not limited by a spacer. In certain embodiments of the present disclosure, a method for forming a thin fluid sample layer having a uniform predetermined thickness by pressing may include forcing two plates into a closed configuration, wherein: at least a portion of the sample is compressed by the two plates. A layer of substantially uniform thickness is made, wherein the uniform thickness of the layer is limited by the sample contact surface of the plate and regulated by the plate and the spacer.

[0184] In certain embodiments of the present disclosure, the device for analyzing a fluid sample may include a first plate. In certain embodiments of the present disclosure, the device for analyzing a fluid sample may include a second plate. In certain embodiments of the present disclosure, the device for analyzing a fluid sample may include a spacer. In certain embodiments, the plates may be moved relative to each other into different configurations. In certain embodiments, one or both plates are flexible. In certain embodiments, each plate has a sample contact area for contacting a fluid sample on its respective inner surface. In certain embodiments, one or both of the plates include a spacer, and the spacer is fixed on the inner surface of the corresponding plate. In certain embodiments, the spacer has a predetermined substantially uniform height equal to or less than 200 microns, and the distance between the spacers is predetermined. In certain embodiments, the Young's modulus of the spacer multiplied by the filling factor of the spacer is at least 2MPa. In certain embodiments, at least one spacer is within the sample contact area. In certain embodiments, a configuration is an open configuration, wherein: the two plates are partially or completely separated, the interval between the two plates is not limited by the spacer, and the sample is deposited on one or both plates. In some embodiments, another configuration is a closed configuration, which is configured after the sample is deposited in an open configuration; in the closed state: at least a portion of the sample is compressed by two plates into a highly uniform layer, wherein the uniform thickness of the layer is limited by the sample contacting surfaces of the plates and by the plates and spacers.

[0185] In certain embodiments of the present disclosure, a method for forming a thin fluid sample layer having a uniform predetermined thickness by extrusion may include obtaining a device of the present disclosure. In certain embodiments of the present disclosure, a method for forming a thin fluid sample layer having a uniform predetermined thickness by pressing, the method may include: when the plate is configured to an open structure, depositing the fluid sample on one or two plates. In certain embodiments, the open structure is a structure in which the two plates are partially or completely separated and the spacing between the plates is not limited by a spacer. In certain embodiments of the present disclosure, a method for forming a thin fluid sample layer having a uniform predetermined thickness by extrusion may include forcing two plates into a closed structure. In certain embodiments, at least a portion of the sample is compressed by two plates into a layer of substantially uniform thickness, wherein the uniform thickness of the layer is limited by the sample contact surface of the plate and regulated by the plate and the spacer.

[0186] In certain embodiments of the present disclosure, a device for analyzing a fluid sample may include a first plate. In certain embodiments of the present disclosure, a device for analyzing a fluid sample may include a second plate. In certain embodiments, the plates may be movable relative to each other into different configurations. In certain embodiments, one or both plates are flexible. In certain embodiments, each plate has a sample contact area on its respective surface for contacting a sample containing an analyte. In certain embodiments, one or both of the plates include a spacer that is permanently fixed to the plate in the sample contact area, wherein the spacer has a predetermined substantially uniform height and a predetermined fixed spacer distance that is at least about 2 times larger than the size of the analyte, up to 200 μm, and wherein at least one spacer is within the sample contact area. In certain embodiments, one of the configurations is an open configuration, wherein: the two plates are separated, the spacing between the two plates is not limited by the spacer, and the sample is deposited on one or both plates. In certain embodiments, another of the configurations is a closed configuration, which is configured in an open configuration after sample deposition. In the closed state: at least a portion of the sample is compressed by the two plates into a highly uniform layer, wherein the uniform thickness of the layer is limited by the sample contact surface of the plate and is regulated by the plate and the spacer.

[0187] In certain embodiments of the present disclosure, a method of forming a thin fluid sample layer having a uniform predetermined thickness by extrusion may include obtaining an apparatus of the present disclosure. In certain embodiments of the present disclosure, a method of forming a thin fluid sample layer having a uniform predetermined thickness by pressing may include: depositing a fluid sample on one or both of two plates. When the plates are configured into an open configuration, wherein the open configuration is a configuration in which the two plates are partially or completely separated and the spacing between the plates is not limited by a spacer. In certain embodiments of the present disclosure, a method of forming a thin fluid sample layer having a uniform predetermined thickness by pressing may include forcing two plates into a closed configuration, wherein: at least a portion of the sample is compressed by the two plates into a layer having a substantially uniform thickness, wherein the uniform thickness of the layer is limited by the sample contacting surface of the plates and regulated by the plates and the spacer.

[0188] In certain embodiments of the present disclosure, a device for forming a thin fluid sample layer having a uniform predetermined thickness by pressing may include a first plate. In certain embodiments of the present disclosure, a device for forming a thin fluid sample layer having a uniform predetermined thickness by pressing may include a second plate. In certain embodiments of the present disclosure, a device for forming a thin fluid sample layer having a uniform predetermined thickness by pressing may include a spacer. In certain embodiments, the plates may be moved relative to each other into different configurations. In certain embodiments, one or both plates are flexible. In certain embodiments, each plate includes a sample contact area on its respective inner surface for contacting and / or compressing a fluid sample. In certain embodiments, each plate includes an area on its respective outer surface for applying a force to press the plates together. In certain embodiments, one or both of the two plates include a spacer that is permanently fixed to the inner surface of each plate. In certain embodiments, the spacer has a predetermined substantially uniform height equal to or less than 200 microns, a predetermined width, and a predetermined fixed spacing spacing. In certain embodiments, the ratio of the distance between the spacers to the width of the spacers is 1.5 or greater. In certain embodiments, at least one spacer is within the sample contact area. In certain embodiments, one configuration is an open configuration, wherein: the two plates are partially or completely separated, the spacing between the two plates is not limited by a spacer, and the sample is deposited on one or both plates. In certain embodiments, another configuration is a closed configuration, which is configured in an open configuration after sample deposition. In the closed state, at least a portion of the sample is compressed by the two plates into a highly uniform layer and is substantially stagnant relative to the plates, wherein the uniform thickness of the layer is limited by the contact area of ​​the sample. The two plates are regulated by the plates and the spacer.

[0189] In certain embodiments of the present disclosure, a method for forming a thin fluid sample layer having a uniform predetermined thickness by pressing with imprecise pressure may include obtaining a device of the present disclosure. In certain embodiments of the present disclosure, a method for forming a thin fluid sample layer having a uniform predetermined thickness by pressing with imprecise pressure may include obtaining a fluid sample. In certain embodiments of the present disclosure, a method for forming a thin fluid sample layer having a uniform predetermined thickness by pressing with imprecise pressure may include: depositing the sample on one or two plates; or depositing the sample on one or two plates. When the plates are configured in an open configuration, wherein the open configuration is a configuration in which the two plates are partially or completely separated and the spacing between the plates is not limited by a spacer. In certain embodiments of the present disclosure, a method for forming a thin fluid sample layer having a uniform predetermined thickness by pressing with imprecise pressure may include forcing two plates into a closed configuration, wherein: at least a portion of the sample is compressed by the two plates into a layer of substantially uniform thickness, wherein the uniform thickness of the layer is limited by the sample contact surface of the plates and regulated by the plates and the spacer.

[0190] In some embodiments, the spacer has a columnar shape with its foot fixed in one plate and a flat top surface for contacting another plate. In some embodiments, the spacer has a columnar shape with its foot fixed in one plate and a flat top surface for contacting another plate having a substantially uniform cross-section. In some embodiments, the spacer has a columnar shape with its foot fixed in one plate and a flat top surface for contacting another plate, wherein the flat top surface of the column has a variation of less than 10nm. In some embodiments, the spacer has a columnar shape with its foot fixed in one plate and a flat top surface for contacting another plate, wherein the flat top surface of the column has a variation of less than 50nm. In some embodiments, the spacer has a columnar shape with its foot fixed in one plate and a flat top surface for contacting another plate, wherein the flat top surface of the column has a variation of less than 50nm. In some embodiments, the spacer has a pillar shape with its feet fixed to one plate and having a flat top surface for contacting another plate, wherein the flat top surface of the pillar has a variation within a range of less than 10nm, 20nm, 30nm, 100nm, 200nm, or any two values.

[0191] In certain embodiments, the Young's modulus of the spacer multiplied by the filling factor of the spacer is at least 2 MPa. In certain embodiments, the sample includes an analyte, and the predetermined constant spacing spacing is at least about twice the size of the analyte, up to 200 μm. In certain embodiments, the sample includes an analyte, the predetermined constant spacing spacing is at least about 2 times larger than the size of the analyte, up to 200 μm, and the Young's modulus of the spacer multiplied by the filling factor of the filler is at least 2 MPa.

[0192] In certain embodiments, the fourth power of the spacer distance (ISD) divided by the thickness (h) and Young's modulus (E) of the flexible board (ISD^4 / (hE)) is 5x10^6μm^3 / GPa or less. In certain embodiments, the fourth power of the spacer distance (ISD) divided by the thickness and Young's modulus of the flexible board (ISD^4 / (hE)) is 1x10^6μm^3 / GPa. In certain embodiments, the fourth power of the spacer distance (ISD) divided by the thickness and Young's modulus of the flexible board (ISD^4 / (hE)) is 5x10^5μm^3 / GPa. In certain embodiments, the Young's modulus of the spacer multiplied by the filling factor of the spacer is at least 2MPa, and the fourth power of the spacer distance (ISD) divided by the thickness and Young's modulus of the flexible board (ISD^4 / (hE)) is 1x10^5μm^3 / GPa. In some embodiments, the Young's modulus of the spacer multiplied by the filling factor of the spacer is at least 2 MPa, and the fourth power of the inter-spacer distance (ISD) divided by the thickness of the flexible board and the Young's modulus (ISD^4 / (hE)) is 1x10^4μm^3 / GPa. In some embodiments, the Young's modulus of the spacer multiplied by the filling factor of the spacer is at least 20 MPa.

[0193] In certain embodiments of the present disclosure, the ratio of the spacing distance of the spacers to the average width of the spacers is 2 or greater. In certain embodiments, the ratio of the spacing distance of the spacers to the average width of the spacers is 2 or greater, and the Young's modulus of the spacer multiplied by the filling factor of the spacer is at least 2MPa. In certain embodiments, the distance between the spacers is at least about 2 times the size of the analyte, up to 200μm. In certain embodiments, the ratio of the distance between the spacers to the width of the spacers is 1.5 or greater. In certain embodiments, the width of the spacer is 1 or greater. In certain embodiments, the width of the spacer is 1.5 or greater. In certain embodiments, the width of the spacer is 2 or greater. In certain embodiments, the width of the spacer is greater than 2, 3, 5, 10, 20, 30, 50, or within the range of any two of the values.

[0194] In certain embodiments, the force that squeezes two plates into a closed structure is an inaccurate squeezing force. In certain embodiments, the force that presses two plates into a closed structure is an inaccurate pressing force provided by a human hand. In certain embodiments, forcing two plates to compress at least a portion of the sample into a layer of substantially uniform thickness includes using a compliant pressing, pressing the area of ​​at least one plate in parallel or sequentially to press the plates together to form a closed structure, wherein the compliant pressing produces a substantially uniform pressure on the plate on at least a portion of the sample, and the pressing expands at least a portion of the sample laterally between the sample contact surfaces of the plate. And wherein the closed structure is a structure that adjusts the spacing between the plates in the layer of the uniform thickness region by a spacer. And wherein the reduced sample thickness reduces the time for mixing the reagent on the storage position with the sample. In certain embodiments, pressing force is an inaccurate force, whose size is (a) unknown and unpredictable when applying force, or (b) unknown and cannot be predicted within equal accuracy or better than 20% of the average pressing force applied. In some embodiments, the pressing force is an imprecise force whose magnitude is (a) unknown and unpredictable at the time the force is applied, or (b) unknown and cannot be predicted within equal precision or better than 30% of the average pressing force applied. In some embodiments, the pressing force is an imprecise force whose magnitude is (a) unknown and unpredictable at the time the force is applied, or (b) unknown and cannot be predicted within equal precision or better than 30% of the average pressing force applied; and wherein the uniform thickness variation of the highly uniform thickness layer is 20%. In some embodiments, the pressing force is an imprecise force whose magnitude cannot be determined at the time the force is applied with a precision equal to or better than 30%, 40%, 50%, 70%, 100%, 200%, 300%, 500%, 1000%, 2000%, or a range between two values.

[0195] In certain embodiments of the present disclosure, the thickness of the flexible plate is in the range of 10 μm to 200 μm. In certain embodiments, the thickness of the flexible plate is in the range of 20 μm to 100 μm. In certain embodiments, the thickness of the flexible plate is in the range of 25 μm to 180 μm. In certain embodiments, the thickness of the flexible plate is in the range of 200 μm to 260 μm. In certain embodiments, the thickness of the flexible plate is equal to or less than 250 μm, 225 μm, 200 μm, 175 μm, 150 μm, 125 μm, 100 μm, 75 μm, 50 μm, 25 μm, 10 μm, 5 μm, 1 μm or a range between two values. In certain embodiments, the viscosity of the sample is 0.1 to 4 (mPa·s). In certain embodiments, the thickness of the flexible plate is in the range of 200 μm to 260 μm. In certain embodiments, the flexible sheet has a thickness in a range of 20 μm to 200 μm, and a Young's modulus in a range of 0.1 to 5 GPa.

[0196] In certain embodiments of the present disclosure, sample deposition is deposition directly from a subject to a plate without using any transfer device. In certain embodiments, during deposition, the amount of an unknown sample deposited on a plate. In certain embodiments, the method also includes an analysis of an analysis sample. In certain embodiments, the analysis includes calculating the volume of the relevant sample volume by measuring the lateral area of ​​the relevant sample volume and calculating the volume from the lateral area and a predetermined spacer height. In certain embodiments, the pH value at the sample position between two plates in a closed configuration is determined by the volume of the position and by analyzing the image acquired from the position. In certain embodiments, the determination made by analyzing the image uses artificial intelligence and machine learning.

[0197] In certain embodiments, the analysis step (e) comprises measuring: i. imaging; ii. luminescence selected from photoluminescence, electroluminescence and electrochemiluminescence, iii. surface Raman scattering, iv. electrical impedance selected from resistance, capacitance and inductance, or any combination of vi-iv. In certain embodiments, the analysis comprises reading, image analysis or counting analytes, or a combination thereof. In certain embodiments, the sample comprises one or more analytes, and one or two plate sample contact surfaces comprise one or more binding sites, each binding site and fixing the corresponding analyte. In certain embodiments, one or two plate sample contact surfaces comprise one or more storage sites, each storage site storing one or more reagents, wherein one or more reagents are dissolved and diffused in the sample. In certain embodiments, one or two plate sample contact surfaces comprise one or more amplification sites, each amplification site being capable of amplifying a signal from an analyte or a marker of an analyte when the analyte or marker is within 500 nm of the amplification site. In certain embodiments, i. one or both plate sample contact surfaces contain one or more binding sites, each binding site binds and fixes a corresponding analyte; or ii. one or both plate sample contact surfaces include one or more storage locations, each storing one or more reagents; wherein the one or more reagents are dissolved and diffused in the sample, and wherein the sample contains one or more analytes; or iii. one or more amplification sites, each capable of amplifying a signal from the analyte or the marker of the analyte when the analyte or the marker is 500nm away from the amplification site; or iv. any combination of i to iii.

[0198] In certain embodiments, the liquid sample is a biological sample selected from amniotic fluid, aqueous humor, vitreous humor, blood (e.g., whole blood, fractionated blood, plasma or serum), breast milk, cerebrospinal fluid (CSF), skin (cerumen), chyle, bells, endolymph, perilimph, feces, breath, gastric acid, gastric juice, lymph, mucus (including nasal drainage and sputum), pericardial fluid, peritoneal fluid, pleural fluid, pus, rhubarb, saliva, exhaled breath condensate, sebum, semen, sputum, sweat, synovial fluid, tears, vomitus and urine.

[0199] In certain embodiments, the layer of uniform thickness in the closed configuration is less than 150 μm. In certain embodiments, the compression is provided by a pressurized liquid, a compressed gas, or a conformal material. In certain embodiments, the analysis includes counting cells in a layer of uniform thickness. In certain embodiments, the analysis includes measuring in a layer of uniform thickness. In certain embodiments, in certain embodiments, the measurement is a binding measurement or a biochemical measurement. In certain embodiments, the total volume of the deposited sample is less than 0.5 uL. In certain embodiments, multiple drops of sample are deposited on one or two plates.

[0200] In some embodiments, the distance between the spacers is in the range of 1 μm to 120 μm. In some embodiments, the distance between the spacers is in the range of 120 μm to 50 μm. In some embodiments, the distance between the spacers is in the range of 120 μm to 200 μm. In some embodiments, the thickness of the flexible board is in the range of 20 μm to 250 μm, and the Young's modulus is in the range of 0.1 to 5 GPa. In some embodiments, for the flexible board, the thickness of the flexible board multiplied by the Young's modulus of the flexible board is in the range of 60 to 750 GPa-μm.

[0201] In some embodiments, a sample layer of uniform thickness is uniform over a lateral area of ​​at least 1 mm 2. In some embodiments, a sample layer of uniform thickness is uniform over a lateral area of ​​at least 3 mm 2. In some embodiments, a sample layer of uniform thickness is uniform over a lateral area of ​​at least 5 mm 2. In some embodiments, in some embodiments, a sample layer of uniform thickness is uniform over a lateral area of ​​at least 10 mm 2. In some embodiments, a sample layer of uniform thickness is uniform over a lateral area of ​​at least 20 mm 2. In some embodiments, a sample layer of uniform thickness is uniform over a lateral area of ​​20 mm 2 to 100 mm 2. In some embodiments, a sample layer of uniform thickness has a thickness uniformity of up to + / -5% or better. In some embodiments, a sample layer of uniform thickness has a thickness uniformity of up to + / -10% or more. In some embodiments, a sample layer of uniform thickness has a thickness uniformity of up to + / -20% or more. In some embodiments, a sample layer of uniform thickness has a thickness uniformity of up to + / -30% or more. In certain embodiments, a sample layer of uniform thickness has a thickness uniformity of up to + / - 40% or better. In certain embodiments, a sample layer of uniform thickness has a thickness uniformity of up to + / - 50% or better.

[0202] In some embodiments, the spacer is a pillar having a cross-sectional shape selected from a circle, a polygon, a circle, a square, a rectangle, an ellipse, an ellipse, or any combination thereof. In some embodiments, the spacer has a columnar shape, has a substantially flat top surface and has a substantially uniform cross-section, wherein, for each spacer, the ratio of the lateral dimension of the spacer to its height is at least 1. In an embodiment, the distance between the spacers is periodic. In some embodiments, the spacer has a packing ratio of 1% or more, wherein the packing ratio is the ratio of the spacer contact area to the total plate area. In some embodiments, the Young's modulus of the spacer multiplied by the packing factor of the spacer is equal to or greater than 20 MPa, wherein the packing factor is the ratio of the spacer contact area to the total plate area. In some embodiments, the spacing between the two plates in the closed configuration is less than 200 μm. In some embodiments, the spacing between the two plates in the closed configuration is a value selected from 1.8 μm to 3.5 μm. In some embodiments, the spacing is fixed to the plate by direct embossing of the plate or injection molding of the plate. In some embodiments, the material of the plates and spacers is selected from polystyrene, PMMA, PC, COC, COP or another plastic. In some embodiments, the spacers have a cylindrical shape and the sidewall corners of the spacers have a rounded shape with a radius of curvature of at least 1 μm. In some embodiments, the spacers have a density of at least 1000 / mm2. In some embodiments, at least one of the plates is transparent. In some embodiments, the mold used to make the spacers is made by a mold containing features that are made by: (a) directly performing reactive ion etching or ion beam etching, or (b) by replicating or multiple replicating reactive feature ion etching or ion beam etching.

[0203] In some embodiments, the spacers are configured such that the filling factor is in the range of 1% to 5%. In some embodiments, the surface variation is relative to the spacer height, and the ratio of the columnar flat top surface variation to the spacer height is less than 0.5%, 1%, 3%, 5%, 7%, 10%, 15%, 20%, 30%, 40%, or a range between two values. The preferred flat column top flatness has a ratio of the column flat top surface variation to the spacer height of less than 2%, 5%, or 10%. In some embodiments, the spacers are configured such that the filling factor is in the range of 1% to 5%. In some embodiments, the spacers are configured such that the filling factor is in the range of 5% to 10%. In some embodiments, the spacers are configured such that the filling factor is in the range of 10% to 20%. In some embodiments, the spacers are configured such that the filling factor is in the range of 20% to 30%. In some embodiments, the spacers are configured such that the filling factor is 5%, 10%, 20%, 30%, 40%, 50%, or in the range of any two values. In some embodiments, the spacers are configured such that the fill rate is 50%, 60%, 70%, 80%, or within a range of any two values.

[0204] In some embodiments, the spacer is configured so that the filling factor multiplied by the Young's modulus of the spacer is in the range of 2MPa to 10MPa. In some embodiments, the spacer is configured so that the filling factor multiplied by the Young's modulus of the spacer is in the range of 10MPa and 20MPa. In some embodiments, the spacer is configured so that the filling factor multiplied by the Young's modulus of the spacer is in the range of 20MPa to 40MPa. In some embodiments, the spacer is configured so that the filling factor multiplied by the Young's modulus of the spacer is in the range of 40MPa to 80MPa. In some embodiments, the spacer is configured so that the filling factor multiplied by the Young's modulus of the spacer is in the range of 80MPa and 120MPa. In some embodiments, the spacer is configured so that the filling factor multiplied by the Young's modulus of the spacer is in the range of 120MPa to 150MPa.

[0205] In certain embodiments, the device also includes a dry reagent coated on one or two plates. In certain embodiments, the device also includes a dry binding site with a predetermined area on one or two plates, wherein the dry binding site is combined with and fixed on the analyte in the sample. In certain embodiments, the device also includes a releasable dry reagent and a release time control material on one or two plates, and the material delays the time when the releasable dry reagent is released into the sample. In certain embodiments, the release time control material delays the time when the dry reagent starts to be released into the sample by at least 3 seconds. In certain embodiments, the reagent includes an anticoagulant and / or a staining agent. In certain embodiments, the reagent includes a cell lysis reagent. In certain embodiments, the device also includes one or more dry binding sites and / or one or more reagent sites on one or two plates. In certain embodiments, analytes include molecules (such as proteins, peptides, DNA, RNA, nucleic acids or other molecules), cells, tissues, viruses and nanoparticles with different shapes. In certain embodiments, analytes include leukocytes, erythrocytes and platelets. In certain embodiments, analytes are stained.

[0206] In certain embodiments, the spacers that adjust the layer of uniform thickness have a filling factor of at least 1%, where the filling factor is the ratio of the area of ​​the spacer in contact with the layer of uniform thickness to the total board area in contact with the layer of uniform thickness. The layer thickness is uniform. In certain embodiments, for the spacers that adjust the layer of uniform thickness, the Young's modulus of the spacer multiplied by the filling factor of the spacer is equal to or greater than 10 MPa, where the filling factor is the ratio of the area of ​​the spacer in contact with the layer. Effect of uniform thickness on the total board area in contact with the layer of uniform thickness. In certain embodiments, for flexible boards, the thickness of the flexible board multiplied by the Young's modulus of the flexible board is in the range of 60 to 750 GPa-μm. In certain embodiments, for flexible boards, the fourth power of the inter-spacer distance (ISD) divided by the thickness of the flexible board (h) and the Young's modulus (E) of the flexible board, ISD4 / (hE), is equal to or less than 10 6 μm 3 / GPa.

[0207] III. Formation of a uniform thin fluid layer by inaccurate pressure

[0208] In certain embodiments of the present invention, a uniform thin fluid sample layer is formed by using compression with an imprecise force. The term "imprecise pressing force" then adds a definition of imprecise pressing force without adding details. As used herein, the term "imprecise" in the context of a force (e.g., "imprecise pressing force") refers to a force that:

[0209] (a) its magnitude is not known or cannot be accurately predicted at the time the force is applied; (b) the pressure is in the range of 0.01 kg / cm2 (square centimeter) to 100 kg / cm2; (c) the magnitude of the force varies from one application of force to the next; (d) the uncertainty (i.e., variation) of the force in (a) and (c) is at least 20% of the total force actually applied.

[0210] Imprecise forces may be applied by a person's hand, for example, by pinching the subject together between the thumb and index finger, or by pinching and rubbing the subject together between the thumb and index finger.

[0211] In some embodiments, the imprecise force generated by hand pressure has a pressure of 0.01 kg / cm 2 , 0.1kg / cm 2 , 0.5kg / cm 2 , 1kg / cm 2 , 2kg / cm 2 ,kg / cm 2 , 5kg / cm 2 、10kg / cm 2 , 20kg / cm 2 、30kg / cm 2 , 40kg / cm 2 , 50kg / cm 2 、60kg / cm 2 , 100kg / cm 2 、150kg / cm 2 , 200kg / cm 2 Or any range between two values; preferably in the range of 0.1kg / cm 2 Up to 0.5kg / cm 2 , 0.5kg / cm 2 Up to 1kg / cm 2 , 1kg / cm 2 Up to 5kg / cm 2 , 5kg / cm 2 Up to 10kg / cm 2 (pressure).

[0212] B) Adapter

[0213] Details of the adapter are described in detail in various publications including International Application No. PCT / US2018 / 017504 (Essenlix Case No. ESXPCT18F04), which is incorporated herein by reference for all purposes.

[0214] The invention described herein solves this problem by providing a system comprising an optical adapter and a smartphone. The optical adapter device can be mounted on a smartphone and transform it into a microscope, which can take fluorescence and brightfield images of samples. The system can be conveniently and reliably operated by an average person in any position. The optical adapter utilizes the existing resources of a smartphone, including a camera, light source, processor, and display, thereby providing a low-cost solution that enables users to perform brightfield and fluorescence microscopy.

[0215] In the present invention, the optical adapter device includes a holder frame mounted on the upper part of a smartphone and an optical box attached to the holder having a sample container slot and an illumination optical device. In some references (U.S. Patent No. 2016 / 029091 and U.S. Patent No. 2011 / 0292198), the optical adapter design is a whole, including both clip-on mechanical parts that can be mounted on a smartphone and functional optical elements. The problem with this design is that they need to redesign the entire optical adapter for each specific model of smartphone. However, in the present invention, the optical adapter is divided into a bracket frame that is only used to mount the smartphone and a universal optical box that contains all functional components. For smartphones of different sizes, as long as the relative positions of the camera and the light source are the same, only the bracket frame needs to be redesigned, which can save a lot of design and manufacturing costs.

[0216] The optical box of the optical adapter includes: a receiving slot that receives the sample and places it in a sample slide within the field of view and focal length of the smartphone camera; a brightfield illumination optical device for capturing a brightfield microscopy image of the sample; a fluorescence illumination optical device for capturing a fluorescence microscopy image of the sample; and a lever that switches between the brightfield illumination optical device and the fluorescence illumination optical device by sliding inward and outward in the optical box.

[0217] The socket slot has a rubber door that completely covers the slot to prevent ambient light from entering the optical box to be collected by the camera. In US Patent 2016 / 0290916, the sample slot is always exposed to ambient light, which does not cause much problem because it can only perform bright field microscopy. But the present invention can take advantage of this rubber door when performing fluorescence microscopy, because ambient light will bring a lot of noise to the camera's image sensor.

[0218] To capture good fluorescence microscopy images, it is ideal that almost no excitation light enters the camera and the camera only collects the fluorescence emitted by the sample. However, for all common smartphones, due to the large divergence angle of the light beam emitted by the light source and infrared light source, the filter placed in front of the camera cannot well block the undesired wavelength range emitted by the smartphone light source. Optical filters do not work well for uncollimated light beams. Collimating optics can be designed to collimate the light beam emitted by the smartphone light source to solve this problem, but this approach increases the size and cost of the adapter. In contrast, in the present invention, the fluorescence illumination optics enable the excitation light to illuminate the sample at a large oblique incidence angle, partially from the waveguide inside the sample slide and partially from the back side of the sample side, so that the excitation light is almost not collected by the camera to reduce the noise signal entering the camera.

[0219] Brightfield illumination optics in the adapter receive and redirect the beam from the light source so that the sample is back-illuminated at normal incidence.

[0220] Typically, the optical box also includes a lens mounted therein that aligns with the smartphone's camera and can magnify the image captured by the camera. The image captured by the camera can be further processed by the smartphone's processor and the analysis results can be output on the smartphone's screen.

[0221] In order to realize both brightfield illumination and fluorescence illumination optics in the same optical adapter, in the present invention, a slidable lever is used. The optical element of the fluorescence illumination optical element is mounted on the lever, and when the lever is fully slid into the optical box, the fluorescence illumination optical element blocks the light path of the brightfield illumination optical element and switches the illumination optical element to the fluorescence illumination optical element. And when the lever is slid out, the fluorescence illumination optical element mounted on the lever moves out of the light path and switches the illumination optical system to the brightfield illumination optical system. This lever design enables the optical adapter to work in both brightfield and fluorescence illumination modes without designing two different single-mode optical boxes.

[0222] The lever comprises two planes at different planes at different heights.

[0223] In some embodiments, the two planes can be connected together with a vertical rod and moved in or out of the optical box together. In some embodiments, the two planes can be separated and each plane can be moved in or out of the optical box separately.

[0224] The upper lever plane includes at least one optical element, which may be, but is not limited to, an optical filter. The upper lever plane moves below the light source, and a preferred distance between the upper lever plane and the light source is 0 to 5 mm.

[0225] A portion of the bottom lever plane is non-parallel to the image plane. The surface of the non-parallel portion of the bottom lever plane has a mirror finish with a reflectivity greater than 95%. The non-parallel portion of the bottom lever plane moves under the light source and deflects light emitted from the light source to back-illuminate the sample area directly under the camera. The preferred tilt angle of the non-parallel portion of the bottom lever plane is in the range of 45 degrees to 65 degrees, and the tilt angle is defined as the angle between the non-parallel bottom plane and the vertical plane.

[0226] A portion of the bottom lever plane is parallel to the image plane and is located below the sample and 1mm to 10mm away from the sample. The surface of the parallel portion of the bottom lever plane has a high light absorption rate, and the light absorption rate is greater than 95%. This absorptive surface is to eliminate reflections on the sample at small incident angles.

[0227] In order to slide in and out to use the lever to switch the illumination optics, a plug design including a spherical plunger and a groove on the lever is used to stop the lever in a predetermined position when pulled outward from the adapter. This allows the user to pull the lever with any force but have it stop in a fixed position where the operating mode of the optical adapter is switched to bright illumination.

[0228] The sample slider fits into the socket slot to accommodate the QMAX device and place the sample within the field of view and focus of the smartphone camera in the QMAX device.

[0229] The sample slide consists of a fixed track frame and a movable arm:

[0230] The frame rail is fixedly mounted in the slot of the optical box. The rail frame has a sliding rail slot that fits the width and thickness of the QMAX device, so the QMAX device can slide along the rail. The width and height of the rail slot are carefully configured to allow the QMAX device to deviate less than 0.5mm in the direction perpendicular to the sliding direction in the sliding plane and less than 0.2mm in the thickness direction of the QMAX device.

[0231] The frame track features an open window under the field of view of the smartphone camera to allow light to illuminate the sample backwards.

[0232] The movable arm is pre-built into the sliding track slot of the track frame and moves with the QMAX device to guide the movement of the QMAX device in the track frame.

[0233] The movable arm is equipped with a stop mechanism with two predefined stop positions. For one position, the arm will stop the QMAX device at a position where the fixed sample area on the QMAX device is directly under the smartphone camera. For the other position, the arm will stop the QMAX device at a position where the sample area on the QMAX device is not in the field of view of the smartphone and the QMAX device can be easily removed from the track slot.

[0234] The movable arm switches between the two stop positions by pressing the QMAX device and the movable arm together to the end of the track groove and then releasing it.

[0235] The movable arm can indicate whether the QMAX device is inserted in the correct orientation. The shape of one corner of the QMAX device is configured differently than the other three right angles. The shape of the movable arm matches the shape of the specially shaped corner, so the QMAX device can slide into the correct position in the track slot only when it is in the correct orientation.

[0236] C) Smartphone / Detection System

[0237] Details of the smartphone / detection system are described in detail in various publications, including International Application (IA) PCT / US2016 / 046437 filed on August 10, 2016, IA PCT / US2016 / 051775 filed on September 14, 2016, U.S. Provisional Application 62 / 256065 filed on February 7, 2017, U.S. Provisional Application 62 / 456287 and 62 / 456590 filed on February 8, 2017, U.S. Provisional Application 62 / 456504 filed on February 8, 2017, U.S. Provisional Application 62 / 459,544 filed on February 15, 2017, and U.S. Provisional Application 62 / 460075 and 62 / 459920 filed on February 16, 2017, which are incorporated herein by reference in their entirety for all purposes.

[0238] The devices / apparatus, systems and methods disclosed herein may include or use a Q card for sample detection, analysis and quantification. In some embodiments, the Q card is used with an adapter that can connect the Q card to a smartphone detection system. In some embodiments, the smartphone includes a camera and / or an illumination source. In some embodiments, the smartphone includes a camera that can be used to capture an image or sample when the sample is placed in the camera's field of view (e.g., via an adapter). In some embodiments, the camera includes a set of lenses (e.g., as in an iPhone TM 6). In some embodiments, the camera includes at least two sets of lenses (e.g., as in the iPhone TM7) In some embodiments, the smartphone includes a camera, but the camera is not used for image capture.

[0239] In some embodiments, the smartphone includes a light source such as, but not limited to, an LED (light emitting diode). In some embodiments, the light source is used to provide illumination to the sample when the sample is in the field of view of the camera (e.g., through an adapter). In some embodiments, the light from the light source is enhanced, amplified, altered, and / or optimized by the optical components of the adapter.

[0240] In certain embodiments, the smartphone includes a processor configured to process information from the sample. The smartphone includes software instructions that, when executed by the processor, can enhance, amplify, and / or optimize signals (e.g., images) from the sample. The processor can include one or more hardware components, such as a central processing unit (CPU), an application specific integrated circuit (ASIC), an application specific instruction set processor (ASIP), a graphics processing unit (GPU), a physical processing unit (PPU), a digital signal processor (DSP), a field programmable gate array (FPGA), a programmable logic device (PLD), a controller, a microcontroller unit, a reduced instruction set computer (RISC), a microprocessor, etc., or any combination thereof.

[0241] In some embodiments, the smartphone includes a communication unit configured and / or used to transmit data and / or images related to the sample to another device. By way of example only, the communication unit may use a cable network, a wired network, an optical fiber network, a telecommunications network, an intranet, the Internet, a local area network (LAN), a wide area network (WAN), a wireless local area network (WLAN), a metropolitan area network (MAN), a wide area network (WAN), a public switched telephone network (PSTN), a Bluetooth network, a ZigBee network, a near field communication (NFC) network, or the like, or any combination thereof. In some embodiments, the smartphone is an iPhone TM , Android TM Phone or Windows TM Telephone.

[0242] D) Manufacturing method

[0243] Details of the manufacturing methods are described in detail in various publications, including International Application PCT / US2018 / 057873 filed on October 26, 2018, which is incorporated herein by reference for all purposes.

[0244] The device of the present disclosure can be manufactured using techniques well known in the art. The choice of manufacturing technology will depend on the material used for the device and the size of the spacer array and / or the size of the spacer. Exemplary materials for manufacturing the device of the present invention include glass, silicon, steel, nickel, polymers, such as polymethyl methacrylate (PMMA), polycarbonate, polystyrene, polyethylene, polyolefins, silicones (e.g., poly(dimethylsiloxane)), polypropylene, cis-polyisoprene (rubber), polyvinyl chloride (PVC), polyvinyl acetate (PVAc), polychloroprene (chloroprene), polytetrafluoroethylene (Teflon), polyvinylidene chloride (SaranA) and cyclic olefin polymers (COP)) and cyclic olefin copolymers (COC) and combinations thereof. Other materials are known in the art. For example, deep reactive ion etching (DRIE) is used to manufacture silicon-based devices with small gaps, small spacers, and large aspect ratios (the ratio of spacer height to lateral dimensions). For example, thermoforming of plastic devices (embossing, injection molding) can also be used when the smallest lateral features are >20 microns and the aspect ratio of these features is ≤10.

[0245] Other methods include lithography (e.g., stereolithography or X-ray lithography), molding, embossing, silicon micromachining, wet or dry chemical etching, grinding, diamond cutting, photolithography and electroforming (LIGA), and electroplating. For example, for glass, traditional silicon manufacturing techniques of photolithography can be used, followed by wet (KOH) or dry etching (reactive ion etching with fluorine or other reactive gases). For plastic materials with high photon absorption efficiency, techniques such as laser micromachining can be used. Due to the continuous nature of the process, this technology is suitable for lower-volume manufacturing. For mass-produced plastic devices, thermoplastic injection molding and compression molding may be suitable. Conventional thermoplastic injection molding used for mass-production of optical discs (which maintains the fidelity of submicron features) can also be used to manufacture the devices of the present invention. For example, device features are replicated onto a glass master by conventional photolithography. The glass master is electroformed to produce a tough, thermal shock-resistant, thermally conductive, hard mold. The mold is used as a master template for injection or compression molding the features into a plastic device. Depending on the plastic material used to make the device and the requirements for optical quality and yield of the finished product, compression molding or injection molding can be selected as the manufacturing method. Compression molding (also known as hot embossing or relief embossing) has the advantage of being compatible with high molecular weight polymers, which are very good for small structures and can replicate high aspect ratio structures, but the cycle time is longer. Injection molding is suitable for low aspect ratio structures and is best suited for low molecular weight polymers.

[0246] A device may be made in one piece or in multiple pieces and then assembled. The layers of the device may be bonded together by clamps, adhesives, heating, anodic bonding, or reactions between surface groups (e.g., wafer bonding). Alternatively, a device with channels or gaps in more than one plane may be fabricated as a single piece, such as using stereolithography or other three-dimensional manufacturing techniques.

[0247] In order to reduce the non-specific adsorption of compounds released by cells or lysed cells on the device surface, one or more surfaces of the device can be chemically modified to make it non-adhesive or repellent. The surface can be coated with a thin film coating (e.g., a monolayer) of a commercially available non-stick reagent, such as those used to form a hydrogel. Other examples of chemicals that can be used to modify the surface of the device include oligoethylene glycols, fluorinated polymers, organosilanes, thiols, polyethylene glycols, hyaluronic acid, bovine serum albumin, polyvinyl alcohol, mucin, polyHEMA, methacrylate PEG and agarose. Charged polymers can also be used to repel oppositely charged substances. The type of chemical used for repulsion and the method of attaching to the surface of the device will depend on the properties of the repelled substance and the properties of the surface and substance to which it is attached. Such surface modification techniques are well known in the art. The surface can be functionalized before or after assembling the device. The surface of the device can also be coated to capture materials in the sample, such as membrane fragments or proteins.

[0248] In certain embodiments of the present disclosure, a method for manufacturing any Q-card of the present disclosure may include injection molding of a first plate.

[0249] In certain embodiments of the present disclosure, the method for manufacturing any Q-card of the present disclosure may include nanoimprinting or extrusion printing of the second plate.

[0250] In certain embodiments of the present disclosure, a method for manufacturing any Q card of the present disclosure may include laser cutting a first plate. In certain embodiments of the present disclosure, a method for manufacturing any Q card of the present disclosure may include nanoimprinting or extrusion printing of a second plate. In certain embodiments of the present disclosure, a method for manufacturing any Q card of the present disclosure may include injection molding and laser cutting a first plate. In certain embodiments of the present disclosure, a method for manufacturing any Q card of the present disclosure may include nanoimprinting or extrusion printing of a second plate. In certain embodiments of the present disclosure, a method for manufacturing any Q card of the present disclosure may include nanoimprinting or extrusion printing to manufacture a first plate and a second plate. In certain embodiments of the present disclosure, a method for manufacturing any Q card of the present disclosure may include: manufacturing a first plate or a second plate using injection molding, laser cutting a first plate, nanoimprinting, extrusion printing, or a combination of the above. In certain embodiments of the present disclosure, a method for manufacturing any Q card of the present disclosure may include the step of attaching hinges to the first plate and the second plate after manufacturing the first plate and the second plate.

[0251] E) Sample type and subjects

[0252] Details of the samples and subjects are described in detail in various publications, including International Application (IA) PCT / US2016 / 046437 filed on August 10, 2016, IA PCT / US2016 / 051775 filed on September 14, 2016, IA No. PCT / US201 / 017307 filed on February 7, 2018, and IA No. PCT / US2017 / 065440 filed on December 8, 2017, which are hereby incorporated by reference for all purposes.

[0253] The sample can be obtained from a subject. The subject described herein can be of any age and can be an adult, infant, or child. In some cases, the age of the subject is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47 , 48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96 97,98 or 99 years old, or within a certain range (e.g., between 2 and 20 years old, between 20 and 40 years old, or between 40 and 90 years old). A particular class of subjects that can benefit is a subject suffering from or suspected of having an infection (e.g., a bacterial and / or viral infection). Another special class of subjects that can benefit is a subject at a higher risk of infection. In addition, the subject treated by any method or composition described herein can be male or female. Any method, device or kit disclosed herein can also be performed on non-human subjects such as laboratory or farm animals. Non-limiting examples of non-human subjects include dogs, goats, guinea pigs, hamsters, mice, pigs, non-human primates (e.g., gorillas, apes, orangutans, lemurs, or baboons), mice, sheep, cows or zebrafish.

[0254] The devices, apparatuses, systems and methods disclosed herein can be used for samples such as, but not limited to, diagnostic samples, clinical samples, environmental samples and food samples.

[0255] For example, in certain embodiments, the devices, apparatuses, systems and methods disclosed herein are used for samples comprising cells, tissues, body fluids and / or mixtures thereof. In certain embodiments, the sample comprises a human body fluid. In certain embodiments, the sample comprises at least one of cells, tissues, body fluids, feces, amniotic fluid, aqueous humor, vitreous humor, blood, whole blood, fractionated blood, plasma, serum, breast milk, cerebrospinal fluid, uterus, chyle, cerumen, endolymph, perilymph, feces, gastric acid, gastric juice, lymph, mucus, nasal drainage, sputum, pericardial fluid, peritoneal fluid, pleural fluid, pus, rhubarb, saliva, sebum, semen, sputum, sweat, synovial fluid, tears, vomitus, urine and exhaled breath condensate. .

[0256] In certain embodiments, the apparatus, devices, systems and methods disclosed herein are used for environmental samples obtained from any suitable source, such as, but not limited to: rivers, lakes, ponds, oceans, glaciers, icebergs, rainwater, snow, sewage, reservoirs, tap water, drinking water, etc.; solid samples from soil, compost, sand, rocks, concrete, wood, bricks, sewage, etc.; in certain embodiments, environmental samples are freshly obtained from the source; and gaseous samples from air, underwater vents, industrial exhaust, vehicle exhaust, etc. In certain embodiments, the environmental sample is processed. For example, the sample in non-liquid form is converted into liquid form before applying the apparatus, devices, systems and methods of the present invention.

[0257] In certain embodiments, the apparatus, devices, systems and methods disclosed herein are used for food samples that are suitable or have the potential to become suitable for animal consumption, such as human consumption. In certain embodiments, food samples include raw materials, cooked or processed foods, plant and animal food sources, pre-processed foods, and partially or completely processed foods, etc. In certain embodiments, samples in non-liquid form are converted to liquid form prior to application of the apparatus, devices, systems and methods of the present invention.

[0258] Subject devices, apparatus, systems and methods may be used to analyze samples of any volume. Examples of volumes include, but are not limited to, 10 mL or less, 5 mL or less, 3 mL or less, 1 microliter (uL, also referred to herein as "uL") or less, 500 uL or less, 300 uL or less, 250 uL or less, 200 uL or less, 170 uL or less, 150 uL or less, 125 uL or less, 100 uL or less, 75 uL or less, 50 uL or less, Equal to 25uL, less than or equal to 20uL, less than or equal to, less than or equal to 15uL, less than or equal to 10uL, less than or equal to 5uL, less than or equal to 3uL, less than or equal to 1uL, less than or equal to 0.5uL, less than or equal to 0.1uL, less than or equal to 0.05uL, less than or equal to 0.001uL, less than or equal to 0.0005uL, less than or equal to 0.0001uL, less than or equal to 10pL, less than or equal to 1pL, or a range between two values.

[0259] In certain embodiments, the volume of the sample includes, but is not limited to, less than or equal to 100uL, less than or equal to 75uL, less than or equal to 50uL, less than or equal to 25uL, less than or equal to 20uL, less than or equal to 15uL, less than or equal to 10uL, less than or equal to 5uL, less than or equal to 3uL, less than or equal to 1uL, less than or equal to 0.5uL, less than or equal to 0.1uL, less than or equal to 0.05uL, less than or equal to 0.001uL, less than or equal to 0.0005uL, less than or equal to 0.0001uL, less than or equal to 10pL, less than or equal to 1pL, or a range between two values. In certain embodiments, the volume of the sample includes but is not limited to less than or equal to 10uL, less than or equal to 5uL, less than or equal to 3uL, less than or equal to 1uL, less than or equal to 0.5uL, less than or equal to 0.1uL, less than or equal to 0.05uL, less than or equal to 0.001uL, less than or equal to 0.0005uL, less than or equal to 0.0001uL, less than or equal to 10pL, less than or equal to 1pL, or a range between two values.

[0260] In some embodiments, the amount of the sample is about one drop of liquid. In some embodiments, the amount of the sample is the amount collected from a punctured finger or fingertip. In some embodiments, the amount of the sample is the amount collected from a microneedle, micropipette, or intravenous aspiration.

[0261] F) Machine Learning

[0262] Details of the network are described in detail in various publications, including International Application (IA) PCT / US2018 / 017504, filed on February 8, 2018, and PCT / US2018 / 057877, filed on October 26, 2018, which are referenced herein for all purposes.

[0263] One aspect of the present invention provides a framework for machine learning and deep learning for analyte detection and localization. A machine learning algorithm is an algorithm that can learn from data. A more rigorous definition of machine learning is: "If the performance of a computer program in T tasks (measured by P) improves with experience E, then it can be said that it is a computer program that learns from experience E about some class of tasks T and performance measure P." It explores the study and construction of algorithms that can learn from data and make predictions - such algorithms make data-driven predictions or decisions by building models based on sample inputs, thereby overcoming static program instructions.

[0264] Deep learning is a special type of machine learning that is based on a set of algorithms that try to model high-level abstractions in data. In a simple case, there might be two groups of neurons: one that receives input signals and one that sends output signals. When the input layer receives input, it passes a modified version of the input to the next layer. In a deep network, there are many layers between the input and output (these layers are not made up of neurons, but they help us think that way), allowing the algorithm to use multiple layers of processing that consist of multiple linear and non-linear transformations.

[0265] One aspect of the present invention is to provide two analyte detection and localization methods. The first method is a deep learning method, and the second method is a combination of deep learning and computer vision methods.

[0266] (i) Deep Learning Approach. In the first approach, the disclosed analyte detection and localization workflow consists of two phases: training and prediction. We describe the training and prediction phases in the following paragraphs.

[0267] (a) Training phase

[0268] During the training phase, the annotated training data is input into a convolutional neural network. Convolutional neural networks are specialized neural networks for processing data, with a grid-like feedforward layered network topology. Examples of data include time series data, which can be viewed as a 1D grid sampled at fixed time intervals, and image data, which can be viewed as a 2D grid of pixels. Convolutional networks have been successful in real-world applications. The name "convolutional neural network" indicates that the network employs a mathematical operation called convolution. Convolution is a special type of linear operation. Convolutional networks are simply neural networks that use convolution instead of general matrix multiplication in at least one of their layers.

[0269] The machine learning model receives one or more sample images containing analytes acquired by the imager on a sample-mounted QMAX device as training data. The training data is annotated with the analytes to be analyzed, wherein the annotation indicates whether the analytes are in the training data and their locations in the image. The annotation can be in the form of a tight bounding box, which completely contains the analyte or the center location of the analyte. In the latter case, the center location is further converted to a circle covering the analyte or a Gaussian kernel in the dot map.

[0270] Training machine learning models faces two challenges when the amount of training data is large: annotation (usually done by humans) is time-consuming, and training is computationally expensive. To overcome these challenges, the training data can be divided into smaller patches, and then these patches or parts of patches are annotated and trained. The term "machine learning" can refer to algorithms, systems, and devices in the field of artificial intelligence, which typically use statistical techniques and artificial neural networks that are trained from data without being explicitly programmed.

[0271] The annotated images are fed to the Machine Learning (ML) training module, and the Model Trainer in the Machine Learning module will train an ML model from the training data (annotated sample images). The input data will be fed to the Model Trainer in multiple iterations until a specific stopping condition is met. The output of the ML training module is the ML model - a computational model that is built from the training process of machine learning based on data that enables the computer to perform certain tasks on its own (e.g., detect and classify subjects).

[0272] The trained machine learning model is applied by a computer in the prediction (or inference) phase. Examples of machine learning models include ResNet, DenseNet, etc., which are also called "deep learning models" due to the depth of the connected layers in their network structure. In some embodiments, the Caffe library with a fully convolutional network (FCN) is used for model training and prediction, and other convolutional neural network architectures and libraries such as TensorFlow may also be used.

[0273] The training phase generates a model that will be used in the prediction phase. The model can be reused in the prediction phase to analyze the input. Therefore, the compute unit only needs access to the generated model. It does not need access to the training data, nor does the training phase need to be run again on the compute unit.

[0274] (b) Prediction stage

[0275] In the prediction / inference phase, the detection component is applied to the input image, which is then fed into the prediction (inference) module pre-loaded with the training model generated from the training phase. The output of the prediction phase can be a bounding box containing the detected analytes and their center locations, or a dot map indicating the location of each analyte, or a heat map containing information about the detected analytes.

[0276] When the output of the prediction phase is a list of bounding boxes, the number of analytes in the sample image to be detected is characterized by the number of bounding boxes detected. When the output of the prediction phase is a spot map, the number of analytes in the sample image for analysis is characterized by the integral of the spot map. When the output of the prediction is a heat map, the localization component is used to identify the locations, and the number of detected analytes is characterized by the entries of the heat map.

[0277] One embodiment of the localization algorithm is to sort the heat map values ​​from the highest value to the lowest value into a one-dimensional ordered list. Then select the pixel with the highest value and remove the pixel and its neighboring pixels from the list. Repeat this process to select the pixel with the highest value in the list until all pixels are removed from the list.

[0278] In the detection component using heatmap, the input image is fed into a convolutional neural network along with the model generated from the training phase and the output of the detection phase is pixel-wise predictions in the form of a heatmap. The heatmap can be of the same size as the input image or a scaled-down version of the input image and it is the input to the localization component. We disclose an algorithm to localize the center of the analyte. The main idea is to iteratively detect local peaks from the heatmap. After the peak is localized, we calculate the local area around the peak, but with smaller values. We remove that area from the heatmap and find the next peak from the remaining pixels. The process is repeated to remove all pixels only from the heatmap.

[0279] In certain embodiments, the present invention provides a localization algorithm to sort the heat map values ​​from the highest value to the lowest value into a one-dimensional ordered list. The pixel with the highest value is then selected and the pixel and its neighboring pixels are removed from the list. This process is repeated to select the pixel with the highest value in the list until all pixels are removed from the list.

[0280]

[0281] After sorting, the heatmap is a one-dimensional sorted list where the heatmap values ​​are ordered from highest to lowest. Each heatmap value is associated with its corresponding pixel coordinate. The first item in the heatmap is the item with the highest value, which is the output of the pop(heatmap) function. A disk is created whose center is the pixel coordinate of the disk with the highest heatmap value. All heatmap values ​​whose pixel coordinates are inside the disk are then removed from the heatmap. The algorithm repeatedly pops the maximum value in the current heatmap and removes the disks around it until all items are removed from the heatmap.

[0282] In an ordered list heatmap, each item has knowledge of the ongoing and subsequent items. When removing an item from an ordered list, we make the following changes:

[0283] ··Assume that the deleted item is x r, whose continuation term is x p , and its subsequent term is x f .

[0284] ··For ongoing projects x p , redefines its next item to be the item after the deleted item. Therefore, x p The following term is now x f .

[0285] ··For deleting item x r , undefine its continuation and successor to remove it from the ordering list.

[0286] ··For the following items x f , redefines its processing item as the processing item of the deleted item. Therefore, x f The progressive term is now x p .

[0287] After all items are removed from the ordered list, the localization algorithm is complete. The number of elements in the set locus will be the number of analytes and the position information is the pixel coordinates of each s in the set locus.

[0288] Another embodiment searches for local peaks, which is not the local peak with the highest heatmap value. To detect each local peak, we start from a random starting point and search for the local maximum. Once a peak is found, we calculate the local area around the peak, but with a smaller value. We remove that area from the heatmap and find the next peak from the remaining pixels. This process is repeated, only to remove all pixels from the heatmap.

[0289]

[0290] This is a breadth-first search algorithm starting from s, where the conditions for visiting points are changed: the neighbor p of the current position q is added only if heatmap[p]>0 and heatmap[p]<=heatmap[q]. Therefore, every pixel in the overlay has a non-descending path to the local peak s.

[0291]

[0292]

[0293] (ii) A hybrid of deep learning and computer vision approaches. In the second approach, detection and localization are achieved by computer vision algorithms, and classification is achieved by deep learning algorithms, where the computer vision algorithm detects and localizes possible candidates for the analyte, and the deep learning algorithm classifies each possible candidate as a true analyte or a false analyte. The locations of all true analytes (along with the total number of true analytes) will be recorded as output.

[0294] (a) Detection. Computer vision algorithms detect possible candidates based on the characteristics of the analyte, including but not limited to intensity, color, size, shape, distribution, etc. Preprocessing schemes can improve the detection results. Preprocessing schemes include contrast enhancement, histogram adjustment, color enhancement, noise reduction, smoothing, defocusing, etc. After preprocessing, the input image is sent to the detector. The detector informs the presence of possible analytes and gives an estimate of their location. Detection can be based on the structure of the analyte (such as edge detection, line detection, circle detection, etc.), connectivity (such as blob detection, connected components, contour detection, etc.), intensity, color, shape using schemes such as adaptive thresholding, etc.

[0295] (b) Localization. After detection, a computer vision algorithm localizes each possible analyte candidate by providing its boundaries or a tight bounding box containing the boundaries. This can be achieved through subject segmentation algorithms such as adaptive thresholding, background subtraction, flood filling, mean shift, thresholding, etc. Many times, localization can be combined with detection to produce a detection result and the location of each analyte candidate subject.

[0296] (c) Classification. Deep learning algorithms such as convolutional neural networks achieve state-of-the-art visual classification. We employ deep learning algorithms to classify each possible analyte. Various convolutional neural networks can be used for analyte classification, such as VGGNet, ResNet, MobileNet, DenseNet, etc.

[0297] Given each possible analyte candidate, the deep learning algorithm computes layers of neurons through convolutional filters and nonlinear filters to extract high-level features that distinguish analytes from non-analytes. Fully convolutional network layers combine the high-level features into a classification result, indicating whether it is a true analyte, or a likelihood of being an analyte.

[0298] G) Applications, biological / chemical biomarkers and health conditions

[0299] Applications of the present invention include, but are not limited to (a) detection, purification and quantification of compounds or biomolecules associated with certain disease stages, such as infectious and parasitic diseases, injuries, cardiovascular diseases, cancer, mental illness, neuropsychiatric diseases and organic diseases, such as lung disease, kidney disease, (b) detection, purification and quantification of microorganisms (such as viruses, fungi and bacteria) in the environment (such as water, soil or biological samples), tissues, body fluids, (c) detection of compounds or biological samples that pose a threat to food safety or national security, quantification of toxic waste, anthrax; (d) quantification of important parameters in medical or physiological monitors, such as glucose, blood oxygen levels, total blood cell count; (e) detection and quantification of specific DNA or RNA from biological samples (such as cells, viruses); (f) sequencing and comparison of gene sequences in chromosomes and mitochondrial DNA for genome analysis; or (g) detection of reaction products during, for example, drug synthesis or purification.

[0300] Detection can be performed in various sample matrices, such as cells, tissues, body fluids and feces. Target body fluids include, but are not limited to, amniotic fluid, aqueous humor, vitreous humor, blood (e.g., whole blood, fractionated blood, plasma, serum, etc.), breast milk, cerebrospinal fluid (CSF), uterus (ear wax), chyle, chi, endolymph, perilymph, feces, gastric acid, gastric juice, lymph, mucus (including nasal drainage and sputum), pericardial fluid, peritoneal fluid, pleural fluid, pus, rhubarb, saliva, sebum (skin oil), semen, sputum, sweat, synovial fluid, tears, vomitus, urine and exhaled condensate. In some embodiments, the sample comprises a human body fluid. In some embodiments, the sample comprises at least one of cells, tissue, body fluid, feces, amniotic fluid, aqueous humor, vitreous humor, blood, whole blood, fractionated blood, plasma, serum, breast milk, cerebrospinal fluid, uterine, chyle, cerumen, endolymph, peripheral lymph, feces, gastric acid, gastric juice, lymph, mucus, nasal drainage, sputum, pericardial fluid, peritoneal fluid, pleural fluid, pus, rhubarb, saliva, sebum, semen, sputum, sweat, synovial fluid, tears, vomitus, urine, and exhaled condensate.

[0301] In embodiments, the sample is at least one of a biological sample, an environmental sample, and a biochemical sample.

[0302] The apparatus, systems and methods of the present invention can be used in a variety of different applications in a variety of fields where it is necessary to determine the presence and / or quantification of one or more analytes in a sample. For example, the subject methods can be used to detect proteins, peptides, nucleic acids, synthetic compounds, inorganic compounds, etc. The various fields include, but are not limited to, human, veterinary, agricultural, food, environmental, drug testing, etc.

[0303] In certain embodiments, the methods of the present invention can be used to detect nucleic acids, proteins or other biomolecules in a sample. The method may include detecting a set of biomarkers in a sample, such as two or more different protein or nucleic acid biomarkers. For example, the method can be used for rapid, clinical detection of two or more disease biomarkers in a biological sample, such as can be used to diagnose a disease condition in a subject or for ongoing treatment or therapy. As described above, communication with a physician or other health care provider can better ensure that the physician or other health care provider is aware of and aware of possible hidden dangers, and therefore more likely to take appropriate measures.

[0304] Applications of the apparatus, systems and methods of the present invention using CROF devices include, but are not limited to (a) detection, purification and quantification of compounds or biomolecules associated with certain disease stages, such as infectious and parasitic diseases, injuries, cardiovascular diseases, cancer, mental illness, neuropsychiatric diseases and organic diseases, such as lung disease, kidney disease, (b) detection, purification and quantification of microorganisms (such as viruses, fungi and bacteria) in environments such as water, soil or biological samples such as tissues, body fluids, (c) detection of compounds or biological samples that pose a threat to food safety or national security, quantification of toxic waste, anthrax; (d) quantification of important parameters in medical or physiological monitors, such as glucose, blood oxygen levels, total blood cell count; (e) detection and quantification of specific DNA or RNA from biological samples (such as cells, viruses); (f) sequencing and comparison of gene sequences in chromosomes and mitochondrial DNA for genomic analysis; or (g) detection of reaction products in, for example, drug synthesis or purification processes. Some specific applications of the apparatus, systems and methods of the present invention are now described in further detail.

[0305] Applications of the present invention include, but are not limited to (a) detection, purification and quantification of compounds or biomolecules associated with certain disease stages, such as infectious and parasitic diseases, injuries, cardiovascular diseases, cancer, mental illness, neuropsychiatric diseases and organic diseases, such as lung disease, kidney disease, (b) detection, purification and quantification of microorganisms (such as viruses, fungi and bacteria) in the environment (such as water, soil or biological samples), tissues, body fluids, (c) detection of compounds or biological samples that pose a threat to food safety or national security, quantification of toxic waste, anthrax; (d) quantification of important parameters in medical or physiological monitors, such as glucose, blood oxygen levels, total blood cell count; (e) detection and quantification of specific DNA or RNA from biological samples (such as cells, viruses); (f) sequencing and comparison of gene sequences in chromosomes and mitochondrial DNA for genome analysis; or (g) detection of reaction products during, for example, drug synthesis or purification.

[0306] Implementation of the devices, systems and methods of the present invention may include: a) obtaining a sample, b) applying the sample to a CROF device containing a capture agent that binds to a target analyte under conditions suitable for analyte binding. The sample is placed in the capture agent; c) cleaning the CROF device, and d) reading the CROF device to obtain a measurement of the amount of the analyte in the sample. In some embodiments, the analyte can be a biomarker, an environmental marker, or a food marker. In some cases, the sample is a liquid sample and can be a diagnostic sample (e.g., saliva, serum, blood, sputum, urine, sweat, tears, semen, or mucus); an environmental sample obtained from a river, ocean, lake, rainwater, snow, sewage, sewage treatment runoff, agricultural runoff, industrial runoff, tap water, or drinking water; or a food sample obtained from tap water, drinking water, prepared food, processed food, or raw food.

[0307] In any embodiment, the CROF device can be placed in a microfluidic device, and applying step b) can include applying the sample to the microfluidic device including the CROF device.

[0308] In any embodiment, the reading step d) may include detecting a fluorescent or luminescent signal from the CROF device.

[0309] In any embodiment, the reading step d) may comprise reading the CROF device using a handheld device configured to read the CROF device. The handheld device may be a mobile phone, such as a smart phone.

[0310] In any embodiment, the CROF device can include a labeling agent that can bind to the analyte-capture agent complex on the CROF device.

[0311] In any embodiment, the devices, systems and methods of the present invention may further comprise the step of applying a labeling agent of the analyte-capture agent complex bound to the CROF device to the CROF device between steps c) and d), and then washing the CROF device.

[0312] In any embodiment, the reading step d) may include reading an identifier of the CROF device. The identifier may be an optical barcode, a radio frequency ID tag, or a combination thereof.

[0313] In any embodiment, the devices, systems and methods of the present invention can further include applying a control sample to a CROF control device containing a capture agent that binds to the analyte, wherein the control sample includes a known detectable amount of the analyte, and reading the control CROF device to obtain a control measurement value of the known detectable amount of the analyte in the sample.

[0314] In any embodiment, the sample can be a diagnostic sample obtained from a subject, the analyte can be a biomarker, and the measured amount of the analyte in the sample can be diagnostic of a disease or condition.

[0315] In any embodiment, the devices, systems and methods of the present invention may further include receiving or providing a report to the subject indicating the absence or low measured amount of the biomarker and the measured value range of the biomarker in the individual at risk for a disease or condition, wherein the measured amount of the biomarker relative to the measured value range is diagnostic of the disease or condition.

[0316] In any embodiment, the devices, systems and methods of the present invention may also include diagnosing the subject based on information including the measured amount of the biomarker in the sample. In some cases, the diagnosing step includes sending data including the measured amount of the biomarker to a remote location and receiving a diagnosis based on information including the measured value from the remote location.

[0317] In any embodiment, applying step b) can include isolating miRNA from the sample to produce an isolated miRNA sample, and applying the isolated miRNA sample to a disk-coupled post-on-point antenna (CROF device) array.

[0318] In any embodiment, the method can include receiving or providing a report indicating the safety or harmfulness of the subject's exposure to the environment from which the sample was obtained.

[0319] In any embodiment, the method may include: sending data including the measured amounts of the environmental markers to a remote location; and receiving a report indicating the safety or harmfulness of the subject's exposure to the environment from which the sample was obtained.

[0320] In any embodiment, the CROF device array can include a plurality of capture agents that each bind to an environmental marker, and wherein the reading step d) can include obtaining a measure of the amount of the plurality of environmental markers in the sample.

[0321] In any embodiment, the sample can be a food sample, wherein the analyte can be a food marker, and wherein the amount of the food marker in the sample can be correlated to the safety of the food for consumption.

[0322] In any embodiment, the method may include receiving or providing a report indicating the safety or harmfulness of consumption by the subject of the food from which the sample was obtained.

[0323] In any embodiment, the method may include transmitting data including the measured amounts of the food markers to a remote location and receiving a report indicating the safety or harmfulness of consumption by the subject of the food from which the sample was obtained.

[0324] In any embodiment, the CROF device array can include a plurality of capture agents each bound to a food marker, wherein said obtaining can include obtaining a measure of the amount of the plurality of food markers in the sample, and wherein the plurality of food markers in the sample can be correlated to the safety of the edible food.

[0325] Also provided herein are kits useful in practicing the devices, systems and methods of the invention.

[0326] The sample size may be about one drop of sample. The sample size may be the amount collected from a punctured finger or fingertip. The sample size may be the amount collected from a microneedle or intravenous aspiration.

[0327] After obtaining the sample from the source, the sample can be used without further processing, or it can be processed to, for example, enrich the target analyte, remove large particulate matter, dissolve or resuspend solid samples, etc.

[0328] Any suitable method of applying the sample to the CROF device may be used. Suitable methods may include using a pipette, dropper, syringe, etc. In certain embodiments, when the CROF device is in the form of a dipstick on a support, as described below, the sample may be applied to the CROF device by dipping the sample into the sample, with the receiving area of ​​the dipstick entering the sample.

[0329] Samples may be collected once or multiple times. Samples collected over time may be aggregated and / or processed separately (by applying to a CROF device and obtaining measurements of the amount of analyte in the sample, as described herein). In some cases, measurements obtained over time may be aggregated and used for longitudinal analysis over time to facilitate screening, diagnosis, treatment, and / or disease prevention.

[0330] As described above, the CROF device may be washed in any convenient manner to remove unbound sample components. In certain embodiments, the surface of the CROF device is washed with a binding buffer to remove unbound sample components.

[0331] The detectable labeling of the analyte can be accomplished by any convenient method. The analyte can be labeled directly or indirectly. In direct labeling, the analyte in the sample is labeled before the sample is applied to the CROF device. In indirect labeling, as described below, the unlabeled analyte in the sample is labeled after the sample is applied to the CROF device to capture the unlabeled analyte.

[0332] Samples from subjects, health conditions of subjects, and other applications of the present invention are further described below. Exemplary samples, health conditions, and applications are also disclosed in, for example, U.S. Publication Nos. 2014 / 0154668 and 2014 / 0045209, which are incorporated herein by reference.

[0333] The present invention can be used in a variety of applications, wherein these applications are generally analyte detection applications, wherein the presence of a specific analyte in a given sample is detected qualitatively, if not quantitatively. The protocol for performing analyte detection assays is well known to those skilled in the art and need not be described in detail herein. Typically, a sample suspected of containing a target analyte is contacted with the surface of a subject nanosensor under conditions sufficient to bind the analyte to its respective capture agent bound to the sensor. The capture agent has a highly specific affinity for the target targeting molecule. This affinity can be an antigen-antibody reaction in which an antibody binds to a specific epitope on an antigen, or a DNA / RNA or DNA / RNA hybridization reaction with sequence specificity between two or more complementary chains. Therefore, if the target analyte is present in the sample, it is likely to bind to the sensor at the location of the capture agent and form a complex on the sensor surface. That is, the captured analyte is fixed to the sensor surface. After removing the unbound analyte, the presence of the bound complex on the sensor surface (i.e., the fixed target analyte) is then detected, for example, using a labeled second capture agent.

[0334] Target specific analyte detection applications include hybridization assays using nucleic acid capture agents and protein binding assays in which polypeptides such as antibodies are used. In these assays, a sample is first prepared, and after the sample is prepared, the sample is contacted with a subject nanosensor under specific binding conditions, thereby forming a complex between a target nucleic acid or polypeptide (or other molecule) complementary to the capture agent bound to the sensor surface.

[0335] In one embodiment, the capture oligonucleotide is a synthetic single-stranded DNA of 20-100 bases in length, one end of which is thiolated. These molecules are immobilized on the surface of the nanodevice to capture the target single-stranded DNA (at least 50 bp in length) with a complementary sequence to the fixed capture DNA. After the hybridization reaction, the detection single-stranded DNA (20–100 bp in length) is added, whose sequence is complementary to the unoccupied nucleic acid of the target DNA, thereby hybridizing with the target. One end of the detection DNA is coupled to a fluorescent marker, and the emission wavelength of the fluorescent marker is within the plasma resonance range of the nanodevice. Therefore, by detecting the fluorescent emission emitted from the surface of the nanodevice, the targeted single-stranded DNA can be accurately detected and quantified. The length of the capture and detection DNA determines the melting temperature (the nucleotide chains will separate above the melting temperature) and the degree of mismatch (the longer the chain, the lower the degree of mismatch).

[0336] One consideration in selecting the length of complementary binding depends on the need to minimize mismatches while keeping the melting temperature as high as possible. In addition, the total length of the hybridization length is determined to achieve optimal signal amplification.

[0337] The subject sensor can be used in a method for diagnosing a disease or condition, comprising: (a) obtaining a liquid sample from a patient suspected of having a disease or condition, (b) contacting the sample with a subject nanosensor, wherein a reagent that captures the nanosensor specifically binds to a biomarker of the disease, and wherein the contact is performed under conditions suitable for the biomarker to specifically bind to the capture agent; (c) removing any biomarker that is not bound to the capture agent; (d) reading an optical signal from the biomarker that is still bound to the nanosensor, wherein the optical signal indicates that the patient has a disease or condition, wherein the method further comprises labeling the biomarker with a luminescent tag before or after binding to the capture agent. As will be described in more detail below, the patient may be suspected of having cancer, and the antibody binds to the cancer biomarker. In other embodiments, the patient is suspected of having a neurological disease, and the antibody binds to a biomarker of a neurological disease.

[0338] Applications of subject sensors include, but are not limited to (a) detection, purification and quantification of chemical compounds or biomolecules associated with certain disease stages, such as infectious and parasitic diseases, injuries, cardiovascular diseases, cancer, psychiatric diseases, neuropsychiatric diseases and organic diseases, such as lung disease, kidney disease, (b) detection, purification and quantification of microorganisms (such as viruses, fungi and bacteria) in the environment (such as water, soil or biological samples), tissues, body fluids, (c) detection of compounds or biological samples that pose a threat to food safety or national security, quantification of toxic waste, anthrax; (d) quantification of important parameters in medical or physiological monitors, such as glucose, blood oxygen levels, total blood cell count; (e) detection and quantification of specific DNA or RNA from biological samples (such as cells, viruses); (f) sequencing and comparison of gene sequences in DNA in chromosomes and mitochondria for genome analysis; or (g) detection of reaction products during, for example, drug synthesis or purification.

[0339] Detection can be performed in various sample matrices (e.g., cells, tissues, body fluids, and feces). Target body fluids include, but are not limited to, amniotic fluid, aqueous humor, vitreous humor, blood (e.g., whole blood, fractionated blood, plasma, serum, etc.), breast milk, cerebrospinal fluid (CSF), cerumen (ear wax), chyle, cerumen, endolymph, perilymph, feces, gastric acid, gastric juice, lymph, mucus (including nasal drainage and sputum), pericardial fluid, peritoneal fluid, pleural fluid, pus, rhubarb, saliva, sebum (skin oil), semen, sputum, sweat, synovial fluid, tears, vomitus, urine, and exhaled condensate.

[0340] In some embodiments, a subject biosensor can be used to diagnose pathogen infection by detecting a target nucleic acid from a pathogen in a sample. The target nucleic acid can be, for example, selected from human immunodeficiency virus 1 and 2 (HIV-1 and HIV-2), human T-cell leukemia virus 1 and 2 (HTLV-1 and HTLV-2), respiratory syncytial virus (RSV), adenovirus, hepatitis B virus (HBV), hepatitis C virus (HCV), Epstein-Barr virus (EBV), human papillomavirus (HPV), varicella zoster virus (VZV), cytomegalovirus (CMV), herpes simplex virus 1 and 2 (HSV-1 and HSV-2), human herpes virus 8 (HHV-8, also known as Kaposi's sarcoma herpesvirus) and flavivirus, including yellow fever virus, dengue virus, Japanese encephalitis virus, West Nile virus and Ebola virus. However, the invention is not limited to the detection of nucleic acids, for example DNA or RNA sequences, from the abovementioned viruses, but can be applied without problems to other pathogens of importance for veterinary and / or human medicine.

[0341] Human papillomavirus (HPV) is further subdivided into more than 70 different types based on their DNA sequence homology. These types cause different diseases. HPV types 1, 2, 3, 4, 7, 10 and 26-29 cause benign warts. HPV types 5, 8, 9, 12, 14, 15, 17 and 19-25 and 46-50 cause lesions in patients with weakened immune systems. Types 6, 11, 34, 39, 41-44 and 51-55 cause benign condyloma acuminatum on the mucous membranes of the genitals and respiratory tract. HPV types 16 and 18 are of special medical importance because they cause epithelial dysplasia of the genital mucosa and account for a large proportion of invasive carcinomas of the cervix, vagina, vulva and anal canal. DNA integration of human papillomavirus is considered to be decisive for the carcinogenicity of cervical cancer. Human papillomavirus can be detected from the DNA sequences of, for example, the capsid proteins L1 and L2. Therefore, the method of the present invention is particularly suitable for detecting DNA sequences of HPV types 16 and / or 18 in tissue samples to assess the risk of cancer occurrence.

[0342] In some cases, nanosensors can be used to detect biomarkers present at low concentrations. For example, nanosensors can be used to detect cancer antigens in readily accessible body fluids (e.g., blood, saliva, urine, tears, etc.), to detect biomarkers of tissue-specific diseases in readily accessible body fluids (e.g., biomarkers for neurological diseases (e.g., Alzheimer's disease antigens), to detect infections (especially detection of low titer latent viruses such as HIV), to detect fetal antigens in maternal blood, and to detect exogenous compounds (e.g., drugs or pollutants).

[0343] The table below provides a list of protein biomarkers and their associated diseases that can be detected using the subject nanosensors (when used in conjunction with appropriate monoclonal antibodies). One potential source of the biomarker is also indicated in the table (e.g., "CSF"; cerebrospinal fluid). In many cases, the subject biosensors can detect those biomarkers in a body fluid different from the indicated body fluid. For example, a biomarker present in CSF can be identified in urine, blood, or saliva.

[0344] H) Practicality

[0345] The method of the present invention can be used in a variety of different applications where it is necessary to determine the presence and / or quantification of one or more analytes in a sample. For example, the subject method can be used to detect proteins, peptides, nucleic acids, synthetic compounds, inorganic compounds, etc.

[0346] In certain embodiments, the methods of the present invention can be used to detect nucleic acids, proteins or other biomolecules in a sample. The method may include detecting a set of biomarkers in a sample, such as two or more different protein or nucleic acid biomarkers. For example, the method can be used for rapid, clinical detection of two or more disease biomarkers in a biological sample, such as can be used to diagnose a disease condition in a subject or for ongoing treatment or therapy. As described above, communication with a physician or other health care provider can better ensure that the physician or other health care provider is aware of and aware of possible concerns, and therefore can be more likely to take appropriate measures.

[0347] Applications of the apparatus, systems and methods of the present invention using CROF devices include, but are not limited to (a) detection, purification and quantification of compounds or biomolecules associated with certain disease stages, such as infectious and parasitic diseases, injuries, cardiovascular diseases, cancer, mental illness, neuropsychiatric diseases and organic diseases, such as lung disease, kidney disease, (b) detection, purification and quantification of microorganisms, such as viruses, fungi and bacteria, environments, such as water, soil or biological samples, such as tissues, body fluids, (c) detection of compounds or biological samples that pose a threat to food safety or national security, quantification of toxic waste, anthrax; (d) quantification of important parameters in medical or physiological monitors, such as glucose, blood oxygen levels, total blood cell count; (e) detection and quantification of specific DNA or RNA from biological samples (such as cells, viruses); (f) sequencing and comparison of gene sequences in chromosomes and mitochondrial DNA for genomic analysis; or (g) detection of reaction products, such as in drug synthesis or purification processes. Some specific applications of the apparatus, systems and methods of the present invention are now described in further detail.

[0348] I) Diagnostic Methods

[0349] In certain embodiments, the methods of the present invention can be used to detect biomarkers. In some embodiments, the devices, systems and methods of the present invention using CROF are used to detect the presence or absence of specific biomarkers, as well as the increase or decrease in the concentration of specific biomarkers in blood, plasma, serum or plasma. Other body fluids or excretions, such as but not limited to urine, blood, serum, plasma, saliva, semen, prostatic fluid, nipple aspirate, tears, sweat, feces, cheek swabs, cerebrospinal fluid, cell lysate samples, amniotic fluid, gastrointestinal fluid, biopsy tissue, etc. Therefore, samples such as diagnostic samples can include various fluids or solid samples.

[0350] In some cases, the sample can be a body fluid sample from a subject to be diagnosed. In some cases, a solid or semi-solid sample can be provided. The sample can include tissues and / or cells collected from a subject. The sample can be a biological sample. Examples of biological samples can include, but are not limited to, blood, serum, plasma, nasal swabs, nasopharyngeal washes, saliva, urine, gastric juice, spinal fluid, tears, feces, mucus, sweat, earwax, oil, glandular secretions, cerebrospinal fluid, tissue, semen, vaginal fluid, tissue fluid derived from tumor tissue, eye fluid, spinal fluid, throat swabs, breath, hair, nails, skin, biopsy, placental fluid, amniotic fluid, umbilical cord blood, lymph, cavity fluid, sputum, pus, microbiota, meconium, breast milk, exhaled condensate and / or other excreta. The sample can include nasopharyngeal washes. Nasal swabs, throat swabs, stool samples, hair, nails, earwax, breath and other solid, semi-solid or gas samples can be processed in extraction buffers, such as fixed time or variable time for their analysis. If desired, the extraction buffer or an aliquot thereof may then be processed similarly to other fluid samples.Examples of tissue samples from a subject may include, but are not limited to, connective tissue, muscle tissue, neural tissue, epithelial tissue, cartilage, cancerous samples, or bone.

[0351] In some cases, the subject from which the diagnostic sample is obtained may be a healthy individual, or may be an individual who is at least suspected of having a disease or health condition. In some cases, the subject may be a patient.

[0352] In certain embodiments, the CROF device includes a capture agent configured to specifically bind to a biomarker in a sample provided by a subject. In certain embodiments, the biomarker may be a protein. In certain embodiments, the biomarker protein is specifically bound by an antibody capture agent present in the CROF device. In certain embodiments, the biomarker is an antibody that specifically binds to an antigen capture agent present in the CROF device. In certain embodiments, the biomarker is a nucleic acid that specifically binds to a nucleic acid capture agent that is complementary to one or both strands of a double-stranded nucleic acid biomarker or complementary to a single-stranded biomarker. In certain embodiments, the biomarker is a nucleic acid that is specifically bound by a nucleic acid binding protein. In certain embodiments, the biomarker is specifically bound by an aptamer.

[0353] The presence or absence of a biomarker or a significant change in the concentration of a biomarker can be used to diagnose disease risk, the presence of a disease in an individual, or to adjust the treatment of a disease in an individual. For example, the presence of a specific biomarker or a group of biomarkers can affect the choice of a drug treatment or dosing regimen given to an individual. In evaluating potential drug therapies, biomarkers can be used as surrogate indicators for natural endpoints (such as survival or irreversible morbidity). If a treatment changes a biomarker that is directly related to improved health, the biomarker can be used as a surrogate endpoint to evaluate the clinical benefit of a specific treatment or dosing regimen. Therefore, personalized diagnosis and treatment based on a specific biomarker or group of biomarkers detected in an individual are facilitated by the subject method. In addition, as described above, the high sensitivity of the devices, systems, and methods in the present invention facilitates early detection of biomarkers associated with the disease. Due to the ability to detect multiple biomarkers using mobile devices (such as smartphones) combined with sensitivity, scalability, and ease of use, the method disclosed in the present invention can be used in portable and point-of-care or near-patient molecular diagnostics.

[0354] In certain embodiments, the methods of the present invention can be used to detect biomarkers of diseases or disease states. In some cases, the methods of the present invention can be used to detect biomarkers to characterize cell signaling pathways and intracellular communications for drug discovery and vaccine development. For example, the subject method can be used to detect and / or quantify the amount of biomarkers in diseased, healthy or benign samples. In certain embodiments, the methods of the present invention can be used to detect biomarkers of infectious diseases or disease states. In some cases, biomarkers can be molecular biomarkers, such as but not limited to proteins, nucleic acids, carbohydrates, small molecules, etc.

[0355] The subject methods can be used in diagnostic assays, such as, but not limited to, the following: detecting and / or quantifying biomarkers, as described above; screening tests, in which samples from asymptomatic subjects are examined periodically; prognostic assays, in which the presence and / or amount of biomarkers is used to predict the likely course of a disease; stratified analyses, in which a subject's response to different drug treatments can be predicted; efficacy analyses, in which the efficacy of a drug treatment is monitored; and the like.

[0356] In some embodiments, a subject biosensor can be used to diagnose pathogen infection by detecting a target nucleic acid from a pathogen in a sample. The target nucleic acid can be, for example, selected from human immunodeficiency virus 1 and 2 (HIV-1 and HIV-2), human T-cell leukemia virus 1 and 2 (HTLV-1 and HTLV-2), respiratory syncytial virus (RSV), adenovirus, hepatitis B virus (HBV), hepatitis C virus (HCV), Epstein-Barr virus (EBV), human papillomavirus (HPV), varicella zoster virus (VZV), cytomegalovirus (CMV), herpes simplex virus 1 and 2 (HSV-1 and HSV-2), human herpes virus 8 (HHV-8, also known as Kaposi's sarcoma herpesvirus) and flavivirus, including yellow fever virus, dengue virus, Japanese encephalitis virus, West Nile virus and Ebola virus. However, the invention is not limited to the detection of nucleic acids, for example DNA or RNA sequences, from the abovementioned viruses, but can be applied without problems to other pathogens of importance for veterinary and / or human medicine.

[0357] Human papillomavirus (HPV) is further subdivided into more than 70 different types based on their DNA sequence homology. These types cause different diseases. HPV types 1, 2, 3, 4, 7, 10 and 26-29 cause benign warts. HPV types 5, 8, 9, 12, 14, 15, 17 and 19-25 and 46-50 cause lesions in patients with weakened immune systems. Types 6, 11, 34, 39, 41-44 and 51-55 cause benign condyloma acuminatum on the mucous membranes of the genitals and respiratory tract. HPV types 16 and 18 are of special medical importance because they cause epithelial dysplasia of the genital mucosa and account for a large proportion of invasive carcinomas of the cervix, vagina, vulva and anal canal. DNA integration of human papillomavirus is considered to be decisive for the carcinogenicity of cervical cancer. Human papillomavirus can be detected from the DNA sequences of, for example, the capsid proteins L1 and L2. Therefore, the method of the present invention is particularly suitable for detecting DNA sequences of HPV types 16 and / or 18 in tissue samples to assess the risk of cancer occurrence.

[0358] Other pathogens that can be detected in diagnostic samples using the devices, systems and methods of the present invention include, but are not limited to, varicella zoster; Staphylococcus epidermidis, Escherichia coli, methicillin-resistant Staphylococcus aureus (MSRA), Staphylococcus aureus, Staphylococcus hominis, Enterococcus faecalis, Pseudomonas aeruginosa, Staphylococcus keratitis, Staphylococcus aureus, Streptococcus pneumoniae, Klebsiella pneumoniae, Klebsiella pneumoniae, gonorrhea (Neisseria gonorrhoeae), syphilis (Treponema pallidum), Chlamydia trachomatis (Chlamydia trachomatis), nongonococcal urethritis (Ureaplasma urealyticum), nephrotic syndrome ...nephrotic syndrome (Neisseria gonorrhoeae), nephrotic syndrome (Neisseria gonorrhoeae), nephrotic syndrome (Neisseria urealyticum), nephrotic syndrome (Neisseria urealyticum), nephrotic syndrome (Neisseria urealyticum), nephrotic syndrome (Neisseria gonorrhoeae), nephrotic syndrome (Neisseria urealyticum), nephrotic syndrome (Neisseria urealyticum), nephrotic syndrome (Neisseria urealyticum), nephrotic syndrome (Neisseria urealyticum), nephrotic syndrome (Neisseria urealyticum), nephrotic syndrome (Neisseria urealyticum), nephrotic syndrome (Ne Meningitis (Haemophilus influenzae), Trichomonas (trichomoniasis); Pseudomonas aeruginosa, methicillin-resistant Staphylococcus aureus (MSRA), Klebsiella pneumoniae, Haemophilus influenzae, Staphylococcus aureus, Stenotrophomonas maltophilia, Haemophilus parahaemolyticus, Escherichia coli, Haemophilus enterococci, Enterococcus, Enterococcus, Enterococcus, Citrobacter freundii, Enterococcus faecalis, Klebsiella aerogenes, Pseudomonas fluorescens, Neisseria meningitidis, Streptococcus pyogenes, Pneumocystis carinii, Klebsiella pneumoniae, Legionella pneumococcus, Mycoplasma pneumoniae, Mycoplasma, etc. .

[0359] In some cases, CROF devices can be used to detect low concentrations of biomarkers. For example, CROF devices can be used to detect cancer antigens in readily accessible body fluids (e.g., blood, saliva, urine, tears, etc.), to detect biomarkers of tissue-specific diseases in readily accessible body fluids (e.g., a biomarker of a neurological disease (e.g., Alzheimer's antigen), to detect infection (especially detection of low titer latent viruses such as HIV), to detect fetal antigens in maternal blood, and to detect exogenous compounds (e.g., drugs) or pollutants).

[0360] One potential source of the biomarker is also indicated in the table (e.g., "CSF"; cerebrospinal fluid). In many cases, the subject biosensor can detect those biomarkers in a body fluid different from the indicated body fluid. For example, a biomarker present in CSF can be identified in urine, blood, or saliva. It will also be clear to one of ordinary skill in the art that the CROF device of the present invention can be configured to capture and detect more biomarkers known in the art for diagnosing a disease or health condition.

[0361] Unless otherwise specified, a biomarker can be a protein or nucleic acid (e.g., mRNA) biomarker. Unless otherwise specified, a diagnosis can be associated with an increase or decrease in the level of a biomarker in a sample. A list of biomarkers, diseases that can be used to diagnose, and samples in which the biomarkers can be detected are described in Tables 1 and 2 of U.S. Patent Application No. 62 / 234,538, filed on September 29, 2015, which is incorporated herein by reference.

[0362] In some cases, the devices, systems and methods of the present invention are used to inform the subject from whom the sample was derived of their health condition. Health conditions that can be diagnosed or measured by the devices, systems and methods, devices and systems of the present invention include, but are not limited to: chemical balance; and nutritional health; exercise; fatigue; sleep; stress; prediabetes; allergies; aging; exposure to environmental toxins, pesticides, herbicides, synthetic hormone analogs; pregnancy; menopause; and menopause. U.S. Provisional Application Serial Number Table 3 U.S. Patent Application 62 / 234,538 filed on September 29, 2015 provides a list of biomarkers that can be detected using the present CROF device (when used in conjunction with appropriate monoclonal antibodies, nucleic acids or other capture agents) and their associated health conditions.

[0363] J) Kit

[0364] As described above, aspects of the present disclosure include kits useful in performing the devices, systems and methods of the present invention. In certain embodiments, the kit includes instructions for practicing the subject method using a handheld device such as a mobile phone. These instructions may be present in the subject kit in various forms, one or more of which may be present in the kit. One form that may appear in these instructions is information printed on an appropriate medium or substrate, for example, in the packaging of the kit, in a package insert, on paper on which information is printed. The device will be a computer-readable medium, such as a disk, CD, DVD, Blu-ray, computer-readable memory, etc., on which information has been recorded or stored. Another means that may exist is a website address that can be used through the Internet to access information on a deleted site. The kit may further include software provided on a computer-readable medium for implementing a method for measuring an analyte on a device as described herein. There may be any convenient method in the kit.

[0365] In some embodiments, the kit includes a detection agent, which includes a detectable marker, such as an antioxidant. A fluorescently labeled antibody or oligonucleotide that specifically binds to the target analyte is used to label the target analyte. The detection agent can be provided in a separate container as a CROF device, or it can be provided in a CROF device.

[0366] In some embodiments, the kit includes a control sample, which includes a known detectable amount of an analyte to be detected in the sample. The control sample can be provided in a container and can be present in a solution at a known concentration, or can be provided in a dry form, such as lyophilized or freeze-dried. If provided in a dry form, the kit can also include a buffer for dissolving the control sample.

[0367] Related Documents

[0368] The present invention includes various embodiments, which can be combined in various ways as long as the various components do not contradict each other. The embodiments should be regarded as a single invention document: each document has other documents as references and is also cited as a whole for all purposes, rather than as a discrete independent document. These embodiments include not only the disclosure in the current document, but also the documents cited, incorporated or claimed priority in this document.

[0369] (1) definition

[0370] The terms used to describe the devices, systems and methods disclosed herein are defined in the present application or in PCT applications (designating the United States) PCT / US2016 / 045437 and PCT / US0216 / 051775, filed on August 10, 2016 and September 14, 2016, respectively, U.S. Provisional Application No. 62 / 456065, filed on February 7, 2017, U.S. Provisional Application No. 62 / 456287, filed on February 8, 2017, and U.S. Provisional Application No. 62 / 456504, filed on February 8, 2017, all of which are incorporated herein in their entirety for all purposes.

[0371] The terms "CROF card (or card)", "COF card", "QMAX card", "Q card", "CROF device", "COF device", "QMAX device", "CROF board", "COF board" and "QMAX board" are interchangeable, but in some embodiments, the COF card does not include a spacer. And the terms refer to a device that includes a first board and a second board that can be relatively moved to different configurations (including an open configuration and a closed configuration) and includes a spacer (except for certain embodiments of the COF card) to adjust the spacing between the boards. The term "X board" refers to one of the two boards in the CROF card, wherein the spacer is fixed to the board. Further description of COF cards, CROF cards and X boards is provided in U.S. patent application 62 / 456,065 filed on February 7, 2017, which is incorporated herein in its entirety for all purposes.

[0372] (2) Q-Cards, Spacers and Uniform Sample Thickness

[0373] The devices, systems and methods disclosed herein may include or use Q cards, spacers and uniform sample thickness embodiments for sample detection, analysis and quantification. In some embodiments, the Q card includes a spacer that helps to present at least a portion of the sample as a highly uniform layer. The structure, material, function, variation and size of the spacer and the uniformity of the sample layer are disclosed or listed herein, described and summarized in PCT applications (designated US) PCT / US2016 / 045437 and PCT / US0216 / 051775 filed on August 10, 2016 and September 14, 2016, respectively, U.S. Provisional Application No. 62 / 456065 filed on February 7, U.S. Provisional Application No. 62 / 456287 filed on February 8, 2017, and U.S. Provisional Application No. 62 / 456504 filed on February 8, 2017, all of which are incorporated herein as a whole for all purposes.

[0374] (3) Hinges, slots, recessed edges and slides

[0375] The devices, systems and methods disclosed herein may include or use Q cards for sample detection, analysis and quantification. In some embodiments, the Q card includes hinges, notches, recesses and slides, which help facilitate the manipulation of the Q card and the measurement of the sample. The structure, materials, functions, variations and dimensions of the hinges, notches, notches and slides are disclosed or listed here, described and summarized in PCT applications (designated US) PCT / US2016 / 045437 and PCT / US0216 / 051775 filed on August 10, 2016 and September 14, 2016, respectively, U.S. Provisional Application No. 62 / 456065 filed on February 7, 2017, U.S. Provisional Application No. 62 / 456287 (filed on February 8, 2017) and U.S. Provisional Application No. 62 / 456504, all of which are incorporated herein in their entirety for all purposes.

[0376] (4) Q-card, slider and smartphone inspection system

[0377] The devices, systems and methods disclosed herein may include or use Q cards for sample detection, analysis and quantification. In some embodiments, the Q card is used with a slider that allows a smartphone detection system to read the card. The structure, materials, functions, variations, dimensions and connections of the Q card, slider and smartphone detection system are disclosed or listed here, described and summarized in PCT applications (designated US) PCT / US2016 / 045437 and PCT / US0216 / 051775 filed on August 10, 2016 and September 14, 2016, respectively, U.S. Provisional Application No. 62 / 456065 filed on February 7, 2017, U.S. Provisional Application No. 62 / 456287 (filed on February 8, 2017) and U.S. Provisional Application No. 62 / 456504, all of which are incorporated herein in their entirety for all purposes.

[0378] (5) Detection Methods

[0379] The devices, systems and methods disclosed herein may include or be used for various types of detection methods. The detection methods are disclosed herein and in PCT applications (designating the United States) PCT / US2016 / 045437 and PCT / US0216 / 051775 filed on August 10, 2016 and September 14, 2016, respectively, U.S. Provisional Application No. 62 / 456065 filed on February 7, 2017, U.S. Provisional Application No. 62 / 456287 (filed on February 8, 2017) and U.S. Provisional Application No. 62 / 456504, all of which are incorporated herein in their entirety for all purposes.

[0380] (6) Labels, capture agents and detectors

[0381] The devices, systems and methods disclosed herein can employ various types of labels, capture agents and detectors for analyte detection. The labels herein are disclosed, listed, described and summarized in PCT applications (designating the United States) PCT / US2016 / 045437 and PCT / US0216 / 051775 filed on August 10, 2016 and September 14, 2016, U.S. Provisional Application No. 62 / 456065 filed on February 7, 2017, U.S. Provisional Application No. 62 / 456287 filed on February 8, 2017, and U.S. Provisional Application No. 62 / 456504 filed on February 8, 2017, all of which are incorporated herein in their entirety for all purposes.

[0382] (7) Analytes

[0383] The devices, systems and methods disclosed herein can be applied to the manipulation and detection of various types of analytes, including biomarkers. PCT applications (designating the United States) PCT / US2016 / 045437 and PCT / US0216 / 051775, filed on August 10, 2016 and September 14, 2016, respectively, U.S. Provisional Application No. 62 / 456065, filed on February 7, 2017, U.S. Provisional Application No. 62 / 456287, filed on February 8, 2017, and U.S. Provisional Application No. 62 / 456504, filed on February 8, 2017, disclose or list, describe and summarize these analytes, and the entire contents of all of these applications are incorporated herein for all purposes.

[0384] (8) Applications (fields and samples)

[0385] The devices, systems and methods disclosed herein can be used for various applications (field and samples). These applications are disclosed or listed, described and summarized in PCT applications (designating the United States) PCT / US2016 / 045437 and PCT / US0216 / 051775, filed on August 10, 2016 and September 14, 2016, respectively, U.S. Provisional Application No. 62 / 456065, filed on February 7, 2017, U.S. Provisional Application No. 62 / 456287, filed on February 8, 2017, and U.S. Provisional Application No. 62 / 456504, filed on February 8, 2017, all of which are incorporated herein in their entirety for all purposes.

[0386] (9) Cloud

[0387] The devices, systems, and methods disclosed herein may employ cloud technologies for data transmission, storage, and / or analysis. Relevant cloud technologies are disclosed or listed, described, and summarized in PCT Applications (designating the United States) PCT / US2016 / 045437 and PCT / US0216 / 051775, filed on August 10, 2016 and September 14, 2016, respectively, U.S. Provisional Application No. 62 / 456065, filed on February 7, 2017, U.S. Provisional Application No. 62 / 456287, filed on February 8, 2017, and U.S. Provisional Application No. 62 / 456504, filed on February 8, 2017, all of which are incorporated herein in their entirety for all purposes.

[0388] Other Implementations

[0389] Other examples of inventive subjects according to the present disclosure are described in the following enumerated paragraphs.

[0390] It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise, such as when the word "singular" is used. For example, reference to an "analyte" includes a single analyte and multiple analytes, reference to a "capture agent" includes a single capture agent and multiple capture agents, reference to a "detection agent" includes a single detection agent and multiple detection agents. And the word "agent" includes one agent and multiple agents.

[0391] Ranges can be expressed herein as from "about" a particular value and / or to "about" another particular value. When such a range is expressed, another embodiment includes from a particular value and / or to another particular value. Similarly, when a value is expressed as an approximation by using the antecedent "about", it will be understood that the particular value forms another embodiment. It will also be understood that the endpoints of each range are important relative to the other endpoint and independently of the other endpoint. The term "about" or "approximately" can mean within an acceptable error range for a particular value, as determined by a person of ordinary skill in the art, which will depend in part on how to measure or determine the value, i.e., the limit of the value. Measurement system. For example, according to the practice of the art, "about" can mean within 1 or more than 1 standard deviation. Alternatively, "about" can mean a range of up to 20%, up to 10%, up to 5% or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude of a value, within 5 times, and more preferably within 2 times. Where specific values ​​are described in the present application and claims, unless otherwise indicated, the term "about" should be assumed to mean that the specific value is within an acceptable error range. The term "about" has the meaning commonly understood by those of ordinary skill in the art. In some embodiments, the term "about" refers to ±10%. In some embodiments, the term "about" refers to ±5%.

[0392] As used herein, the terms "adapted" and "configured" refer to an element, component, or other subject that is designed and / or intended to perform a given function. Thus, the use of the terms "adapted" and "configured" should not be interpreted to mean that a given element, component, or other subject is merely "capable of" performing a given function. Similarly, a subject recited as being configured to perform a particular function may additionally or alternatively be described as being operable to perform that function.

[0393] As used herein, the phrase "for example", the phrase "as an example" and / or simply the terms "example" and "exemplary" in reference to one or more components, features, details, structures, embodiments and / or methods according to the present disclosure are intended to convey that the described components, features, details, structures, embodiments and / or methods are illustrative, non-exclusive examples of components, features, details, structures, embodiments and / or methods according to the present disclosure. Therefore, the described components, features, details, structures, embodiments and / or methods are not intended to be limiting, required or exclusive / exhaustive; and other components, features, details, structures, embodiments and / or methods that are similar and / or equivalent in structure and / or function are also within the scope of the present disclosure.

[0394] As used herein, the phrases "at least one" and "one or more" when referring to a list of more than one entity refer to any one or more entities in the list of entities, and are not limited to at least one of each entity explicitly listed in the list of entities. For example, "at least one of A and B" (or equivalently, "at least one of A or B", or equivalently "at least one of A and / or B") may refer to A alone, B alone, or A and B in combination.

[0395] As used herein, the term "and / or" placed between first and second entities means one of (1) the first entity, (2) the second entity, and (3) the first and second entities. Multiple entities listed with "and / or" should be interpreted in the same manner, i.e., "one or more" of the entities so connected. In addition to the entities expressly identified by the "and / or" clause, other entities may also optionally be present, whether related or unrelated to those specifically identified entities.

[0396] Where numerical ranges are mentioned herein, the invention includes embodiments where the endpoints are included, embodiments where both endpoints are excluded, and embodiments where one endpoint is included and the other is excluded. Unless otherwise indicated, both endpoints should be assumed to be included. Furthermore, unless otherwise indicated or obvious from the context and understanding of a person of ordinary skill in the art.

[0397] If any patent, patent application, or other reference is incorporated herein by reference and (1) defines a term in a manner that is inconsistent with one of the following, and / or (2) is inconsistent with the unincorporated portion of the following: in this disclosure or any other incorporated reference, the unincorporated portion of this disclosure shall control and a term therein or in the incorporated disclosure shall control solely to the extent that the term was defined in the reference and / or incorporated disclosure.

Claims

1. A device for analyzing target cells and non-cellular analytes in a sample, comprising: A first plate, a second plate, a plurality of spacers, a plurality of particles and a capture agent and a stain, wherein: a) the first plate and the second plate are movable relative to each other into different configurations, including an open configuration and a closed configuration; b) each of the first plate and the second plate has a sample contact area on its surface for contacting a sample containing or suspected of containing target cells and non-cellular analytes; c) the spacer is composed of a periodically arranged column array, the column height is uniform and less than or equal to 150 μm, and is fixed on the first plate or the second plate; d) a plurality of particles having a size of 0.2 μm to 100 μm, and being randomly distributed in the sample within the sample contacting area of ​​the first plate or the second plate when the device is in an open state; e) the capture agent is attached to the particle, wherein the capture agent binds the non-cellular analyte; and f) the staining agent stains the target cells; Wherein, in the open configuration, the first plate and the second plate are separated from each other, so that the sample can be deposited on the first plate and / or the second plate wherein, in the closed configuration, the first plate and the second plate are configured to compress the sample volume into a layer of substantially uniform thickness; the thickness of the layer being defined by the first plate and the second plate and adjusted by the first plate, the second plate and the spacer; Wherein, the sample volume can be a partial volume or the entire volume of the sample, and it is used to detect non-cellular analytes and target cells; wherein, in the closed configuration, the number of particles in the sample volume is configured to be at least 10; and The thickness and geometry of the particles are configured such that, in a closed configuration, there is no significant overlap between one or more target cells, between particles, and between target cells and particles within a sample volume of interest.

2. A method for measuring target cells and non-cellular analytes in a sample for non-disease detection purposes, comprising: a) having the device according to claim 1; b) depositing a sample containing or suspected of containing target cells and non-cellular analytes when the first plate and the second plate are in the open configuration; c) after b), the first plate and the second plate are in a closed state, and the sample contains a labeled detection agent; and d) using an imager and in a closed configuration of the plate, taking one or more images of the target cells and the labeled detection agent in the sample; e) analyzing the one or more images to detect the presence or amount of the target cells and non-cellular analytes.

3. A device for determining target cells and non-cellular analytes in a sample, comprising: The device according to claim 1; The imager takes at least one image of the relevant sample size; and A processor, wherein the processor is configured to process the at least one image to (i) detect a non-cellular analyte by analyzing the image of the particle, and (ii) detect a target cellular analyte.

4. A kit for determining target cells and non-cellular analytes in a sample, comprising: The device according to claim 1; and a labeled detection agent; wherein the capture agent or the detection agent or both the capture agent and the detection agent specifically bind to the non-cellular analyte.

5. The device according to claim 1, wherein: The spacers are scale markings used to indicate the orientation of the plates.

6. The device according to claim 1, wherein: In the open configuration, the particles are on the same plate to which the spacers are fixed.

7. The device according to claim 1, wherein: It also includes image analysis through machine learning techniques.

8. The apparatus of claim 1, further comprising an imager for obtaining an image of a relevant sample volume of the sample.

9. The method of claim 2, wherein the cells are selected from the group consisting of leukocytes, leukocytes, granulocytes, agranulocytes, myeloid cells, lymphocytes, neutrophils, eosinophils, basophils, T cells, B cells, natural killer cells and monocytes; and wherein the non-cellular analyte is CRP (C-reactive protein).

10. The apparatus of claim 1, further comprising a processor for processing the image, wherein the processing comprises at least one of: (i) detecting the non-cellular analyte by analyzing one or more images of at least 10 particles; and (ii) detecting the non-cellular analyte, the target cellular analyte.

11. The device according to claim 1, wherein: The one or more spacers are fixed to the surface of the second plate.

12. The device according to claim 1, wherein: The spacers are in a periodic array.

13. The device according to claim 1, wherein: The plurality of particles are randomly distributed in the sample contact area.

14. The device according to claim 1, wherein: The plurality of particles are periodically distributed in the sample contact region.

15. The device according to claim 1, wherein: Also included is a labeled detection agent dry-coated on one of the sample contacting areas.

16. The device according to claim 1, further comprising a labeled detection agent or a labeled competitive detection agent, wherein the capture agent and the labeled competitive detection agent are used for competitive assays, and the capture agent and the labeled detection agent simultaneously bind to the non-cellular analyte for non-competitive assays.

17. The device according to claim 1, wherein: The spacers are periodic and have a spacing range between 1 μm and 120 μm.

18. The device according to claim 1, wherein: One of the plates is flexible, a thickness of the flexible plate multiplied by a Young's modulus of the flexible plate is in the range of 60 to 750 GPa-μm, and a spacer distance (ISD) to the fourth power divided by a product of a thickness (h) of the flexible plate and a Young's modulus (E) of the flexible plate, ISD^4 / (hE) is equal to or less than 10^6μm^3 / GPa.

19. The device according to claim 1, wherein: The center-to-center distance of the spacers is 200 μm or less, and the Young's modulus of the spacers multiplied by the filling factor of the spacers is equal to or greater than 20 MPa, wherein the filling factor is the filling factor, which is the ratio of the spacer contact area to the total board area, and one of the boards is a flexible board, the thickness of the flexible board multiplied by the Young's modulus of the flexible board is in the range of 60 to 750 GPa-µm, and is the fourth power of the interval spacing (ISD) divided by the thickness of the flexible board (h) and the Young's modulus (E) of the flexible board, and ISD^4 / (hE) is equal to or less than 10^6μm^3 / GPa.

20. The device according to claim 1, wherein The fourth power of the spacing distance (ISD) divided by the product of the thickness of the flexible board (h) and the Young's modulus of the flexible board (E), ISD^4 / (hE) is equal to or less than 10^5μm^3 / GPa; one of the boards is a flexible board, and the thickness of the flexible board multiplied by the Young's modulus of the flexible board is in the range of 60 to 750 GPa-µm.

21. A method for determining cellular and non-cellular analytes in a liquid sample for non-disease detection purposes, comprising: a) having the device according to claim 1; b) having a sample containing or suspected of containing one or more target cells and non-cellular analytes; c) having an optical tag capable of binding to the non-cellular analyte to form a labeled non-cellular analyte; d) samples stored in the sample contact area of ​​the device in an open configuration, e) after step d), closing the device to compress at least a portion of the sample into a layer having a uniform thickness; f) capturing at least one image of a sample volume associated with the sample using an imager, wherein the imager is capable of imaging the optical label and the target cell; and g) analyzing at least one of the images to detect the presence or amount of (i) target cells and / or (ii) non-cellular analytes.

22. A method for determining cellular and non-cellular analytes in a liquid sample for non-disease detection purposes, comprising: a) having the device according to claim 1; b) having a sample containing or suspected of containing one or more target cells and non-cellular analytes; c) having an optical tag capable of binding to the non-cellular analyte to form a labeled non-cellular analyte; d) samples stored in the sample contact area of ​​the device in an open configuration, e) after step d), closing the device to compress at least a portion of the sample into a layer having a uniform thickness; f) taking at least one image of a sample volume associated with the sample using an imager, wherein the imager is capable of imaging the optical label and the target cell, and g) analyzing at least one of the images using a processor to detect the presence or amount of (i) target cells and / or (ii) non-cellular analytes; Wherein the processor is configured to process the at least one image to (i) detect the non-cellular analyte by analyzing the image of the at least 10 particles, and (ii) detect a target cellular analyte.

23. The method according to claim 2, wherein: When detecting the non-cellular analyte, the image of the at least 10 particles is analyzed by digital counting, wherein in the digital counting, the presence or quantity is determined based on the number or percentage of particles having an optical signal above a threshold value to determine the content of the non-cellular analyte in the sample, wherein the percentage is the number of particles having an optical signal above a threshold value or the proportion of particles having an optical signal below a threshold value.

24. The method according to claim 2, wherein: When detecting the non-cellular analyte, the image of the at least 10 particles is analyzed by analog counting, wherein the analog counting determines the presence or quantity; the amplitude of the light signal from all particles in the relevant sample area is analyzed for the non-cellular analyte in the sample.

25. The method according to claim 2, characterized in that The process also includes taking two or more images of a common area / volume of the sample layer, wherein the common area of ​​the sample layer is at least one of the one or more particles within the area containing the sample.

26. The device according to claim 1, characterized in that Also included is an imager for imaging the target cells and non-cellular analytes in the closed configuration for analysis and detection.

27. The device of claim 1, further comprising a capture agent and / or a stain coated on one or more surfaces of the first plate and / or the second plate.

28. The device according to claim 1, wherein The particles have a concentration sufficient to: (i) creating a monolayer of particles between the plates; and (ii) There is no overlap between particles between the plates.

29. The method according to claim 2, wherein: The particles are mixed with the sample before depositing the sample into the device.

30. The device according to claim 1, wherein The particles are disposed on at least one of the first plate and the second plate prior to depositing the sample, wherein the one or more particles are mixed with the sample when the sample is deposited onto the device.

31. The device according to claim 1, wherein The particles have various shapes.

32. The device according to claim 1, wherein: The maximum dimension of the particles is in the range of 0.05 μm to 100 μm.

33. The method of claim 2, wherein: The non-cellular analyte is detected using a competitive assay, including the step of providing a labeled competitive detection agent that competes with the non-cellular analyte (if present), the analyte bound to the capture object.

34. The method of claim 2, wherein: The non-cellular analyte is detected using a non-competitive assay, comprising the step of providing a labeled detection agent that specifically binds to another binding site of the non-cellular analyte, wherein the other binding site is a different binding site than the binding site of the capture agent.

35. The method of claim 2, wherein: One of the images is a direct image that includes topology; that is, information about the geometry and location of particles in a common area; and the other image is a signal image that is configured to include the signal from the labeled competitive detection agent as the primary signal of the image.

36. The device according to claim 1, wherein The spacers have the same shape and size.

37. The method according to claim 2, characterized in that It also includes the use of a mobile electronic device to determine: (i) one or more first values ​​corresponding to the amount of the analyte associated with the one or more capture agents; and (ii) one or more second values ​​corresponding to the number of the one or more cells within the sample.

38. The device according to claim 1, characterized in that The plurality of particles are removably coupled to the at least one of the first plate and the second plate.

39. The device according to claim 1, wherein: The plurality of particles are selected from glass particles, magnetic particles, polystyrene particles, plastic particles, latex particles, metal particles, alloy particles and the particles; including metals and dielectrics.

40. The device according to claim 1, wherein The plurality of particles comprises a first plurality of particles and a second plurality of particles, wherein the first plurality of particles comprises one or more first capture agents coupled thereto, and wherein the second plurality of particles comprises one or more second capture agents coupled thereto.

41. The device according to claim 40, wherein The one or more first capture agents are capable of associating with a first epitope on the analyte, and wherein the one or more second capture agents are capable of associating with a second epitope on the analyte.

42. The device of claim 40, wherein: The first plurality of particles are disposed along the first plate, and the second plurality of particles are disposed along the second plate.

43. The device of claim 40, wherein: The first plurality of particles is selected from the group consisting of glass particles, magnetic particles, polystyrene particles, and latex particles.

44. The device of claim 40, wherein: The second plurality of particles is selected from the group consisting of reporter molecules, fluorescent molecules, dye molecules, non-selective dye molecules and redox-reactive molecules.

45. The apparatus, device, kit and method of claim 40, wherein: At least one of the one or more first capture agents and the one or more second capture agents is capable of specifically associating with the analyte.

46. ​​The device of claim 1, further comprising one or more spacers disposed within the space and along at least one of the first plate or the second plate.

47. The apparatus of claim 1, wherein: The one or more spacers determine a distance between the first plate and the second plate.

48. The device of claim 1, further comprising a stain, wherein the capture agent comprises an antibody.

49. The method of claim 2, wherein the cells are selected from the group consisting of leukocytes, leukocytes, granulocytes, agranulocytes, myeloid cells, lymphocytes, neutrophils, eosinophils, basophils, T cells, B cells, natural killer cells, and monocytes; and wherein the non-cellular analyte is selected from alpha-fetoprotein, prostate specific antigen, cardiac troponin, c-reactive protein (CRP), or human chorionic gonadotropin, carcinogens, influenza virus markers, HPV, HBV, HCV, EBV, HIV, HSV, FeLV, FIV, Hantavirus, HTLV I, HTLV II, or CMV infection, bacterial infection markers, fungal infection markers, or parasitic infection markers.

50. The device according to claim 1, wherein The length of the one or more spacers along the direction from the first plate to the second plate is at most 100 microns, at most about 90 microns, at most about 80 microns, at most about 70 microns, at most about 60 microns, at most about 50 microns, at most about 40 microns, at most about 30 microns, at most about 25 microns, at most about 20 microns, at most about 15 microns, at most about 10 microns, at most about 5 microns, at most about 4 microns, at most about 3 microns, at most about 2 microns, at most about 1 micron, at most about 0.5 microns, or at most about 0.1 micron.

51. The apparatus of claim 1, wherein: The plurality of particles have various shapes.

52. The apparatus of claim 1, wherein: The maximum dimension of the plurality of particles is about 100 microns, about 90 microns, about 80 microns, about 70 microns, about 60 microns, about 50 microns, about 40 microns, about 30 microns, about 25 microns, about 20 microns, about 15 microns, about 10 microns, about 5 microns, about 4 microns, about 3 microns, about 2 microns, about 1 micron, about 0.5 microns, or about 0.02 microns.

53. The method of claim 2, wherein: The target cells within the layer are deposited as a non-confluent monolayer.

54. The method of claim 2, wherein: The target cells within the layer are in suspension.

55. The method of claim 37, wherein: The determining includes obtaining one or more images of an area including the at least a portion of the sample within the space, wherein the imaged area includes particles of at least one of the plurality of images.

56. The method of claim 37, wherein: The one or more first values ​​correspond to an intensity of energy emitted by the particle.

57. The method according to claim 56, characterized in that Also included is contacting the sample with a cellular dye capable of associating with the one or more cells.

58. The method of claim 37, wherein: The one or more second values ​​correspond to the intensity of energy emitted by the cellular dye.

59. The method according to claim 37, characterized in that Also included is determining, using an algorithm, a relationship between the one or more first values ​​and the one or more second values.

60. The method of claim 58, wherein: Also included is classifying the infection level of the subject from which the sample was obtained based on the relationship between the one or more first values ​​and the one or more second values.

61. The method of claim 60, wherein: The infection is a viral infection, a bacterial infection or a combination thereof.

62. The method of claim 2, wherein: The one or more cells are selected from the group consisting of leukocytes, granulocytes, myeloid cells, lymphoid cells, neutrophils, eosinophils, basophils, lymphocytes, T cells, B cells, natural killer cells and monocytes.

63. The method of claim 2, wherein: The analyte is selected from the group consisting of polypeptides, proteins, labeled proteins, fusion proteins, antibodies, small molecules, viral particles, bacteria, C-reactive protein and any fragments thereof.

64. The method according to claim 2, characterized in that Also included is contacting at least a portion of the sample with a competitive detection agent capable of associating with the one or more capture agents.

65. The method of claim 2, wherein: A competitive detection agent competes with the analyte for binding to the one or more capture agents.

66. The method according to claim 65, characterized in that Also included is contacting at least a portion of the sample with a supplemental detection agent capable of associating with the analyte.

67. The method of claim 2, wherein: The one or more capture agents are capable of associating with a first epitope on the analyte, and wherein the supplemental detection agent is capable of associating with a second epitope on the analyte.

68. The method of claim 2, wherein: The method can be performed in less than about 10 seconds, less than about 20 seconds, less than about 30 seconds, less than about 40 seconds, less than about 50 seconds, less than about 60 seconds, or less than about 120 seconds.

69. The method of claim 2, wherein: The method does not comprise contacting at least a portion of the sample with a wash buffer.

70. The method of claim 22, wherein: Steps (d) to (f) are performed without any washing steps.

71. The method of claim 2, wherein: The first and second plates are pressed together by hand.

72. The method of claim 2, wherein: The non-cellular analyte is a polypeptide, a nucleic acid or a metabolite.

73. The method of claim 2, wherein: The samples are amniotic fluid, aqueous humor, vitreous humor, blood, whole blood, fractionated blood, plasma, serum, breast milk, cerebrospinal fluid, uterine, chyle, bells, endolymph, perillymph, feces, gastric acid, gastric juice, lymph, mucus, nasal drainage, sputum, pericardial fluid, peritoneal fluid, pleural fluid, pus, rhubarb, saliva, sebum, semen, sweat, synovial fluid, tears, vomitus, urine, and exhaled breath condensate.

74. The method of claim 2, wherein: The method detects a variety of different non-cellular analytes.

75. The method of claim 2, wherein: The sample includes one or more target cells.

76. The method of claim 2, wherein: The sample comprises one or more non-cellular analytes.

77. The apparatus of claim 1, wherein: The one or more particles have a diameter between 0.2 μm and 100 μm.

Images