# US 12,181,472 B2 · Homogeneous assay > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 12,181,472 B2 - Document ID: US12181472B2 - Status: Granted - Kind: B2 - Application number: 16/621,185 - Filed: 2018-06-12 - Published: 2021-05-13 - Granted: 2024-12-31 - Earliest claimed: 2017-06-12 - Assignees: Essenlix Corporation - Applicants: Essenlix Corporation - Inventors: Stephen Y. Chou; Wei Ding; Yufan Zhang; Ji Li; Ji Qi; Jun Tian - Web page: https://patentgrep.com/patent/US12181472B2 ## CPC - B01L 3/50853: {with covers or lids (closures for test tubes B01L3/50825)} - G01N 33/54386: {Analytical elements} - B01L 3/502715: {characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces} - G01N 33/54393: {Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding} - G02B 21/34: Microscope slides, e.g. mounting specimens on microscope slides - G06T 7/0012: {Biomedical image inspection} - B01L 2200/025: Align devices or objects to ensure defined positions relative to each other - B01L 2300/0829: Multi-well plates; Microtitration plates - B01L 2300/0848: Specific forms of parts of containers - B01L 2300/0887: Laminated structure - B01L 2300/161: Control and use of surface tension forces, e.g. hydrophobic, hydrophilic - B01L 2300/168: Specific optical properties, e.g. reflective coatings - G01N 2333/4737: C-reactive protein - G06T 2207/10064: Fluorescence image - G06T 2207/30004: Biomedical image processing - B01L: CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) - G02B: OPTICAL ELEMENTS, SYSTEMS OR APPARATUS - G06T: IMAGE DATA PROCESSING OR GENERATION, IN GENERAL ## Abstract Among other things, the present invention is related to devices and methods of performing biological and chemical assays, such as but not limited to immunoassays and nucleic assay acid, particularly the homogeneous assay that does not use the step of wash and that is fast (e.g. 60 seconds from dropping a sample to displaying results). ## Claims ### Claim 1 A device for a homogeneous assay, comprising: a first plate, a second plate, spacers, a plurality of particles, and a capture agent, wherein: (i) the first and second plates are movable relative to each other into different configurations, including an open configuration and a closed configuration; (ii) each of the plates has, on its respective inner surface, a sample contact area for contacting a sample suspected of containing an analyte; (iii) the spacers are fixed on the inner surface of the first plate, at least one of the spacers is inside the sample contact area of the first plate, the spacers have a predetermined uniform height that is equal to 100 μm or less; (iv) the capture agent is immobilized on a surface of the plurality of particles, wherein the capture agent is capable of specifically binding and immobilizing the analyte; and (v) the plurality of particles are (a) distributed on the sample contact area of the first plate, except the areas occupied by the spacers, and (b) are temporarily or permanently fixed on the first plate; wherein in the open configuration, the two plates are partially or entirely separated apart, the spacing between the plates is not regulated by the spacers, and the sample is deposited on one or both of the plates; and wherein in the closed configuration, which is configured after deposition of the sample in the open configuration: at least part of the sample is compressed by the two plates into a layer of uniform thickness, the uniform thickness of the layer is confined by the inner surfaces of the plates and is regulated by the plates and the spacers. ### Claim 2 (depends on 1) The device of claim 1, wherein: (i) one or both of the first and second plates are flexible; (ii) each of the spacers has a pillar shape and a flat top surface, and the spacers have a predetermined uniform height and a predetermined inter-spacer distance; (iii) the fourth power of the inter-spacer-distance (ISD) divided by the thickness (h) and the Young's modulus (E) of the flexible plate (ISD4/(hE)) is 5×106 μm3/GPa or less; and (iv) the thickness of the plate times the Young's modulus of the flexible plate is in the range 60 to 750 GPa-μm. ### Claim 3 (depends on 1) The device of claim 1, wherein the distribution of the plurality of particles on the first plate is random. ### Claim 4 (depends on 1) The device of claim 1, wherein the plurality of particles are fixed on the plate and has periodic distribution. ### Claim 5 (depends on 1) The device of claim 1, wherein each of the spacers has a flat top. ### Claim 6 (depends on 1) The device of claim 1, wherein the plurality of particles are temporarily fixed on the first plate, and in an open configuration the sample is deposited on the first plate before the two plates are brought into the closed configuration. ### Claim 7 (depends on 1) The device of claim 1, wherein a thickness of the spacers is configured such that, in a closed configuration, for a certain concentration of the analytes in the sample, at least one area of the sample layer that contains one of the particle becomes optically distinguishable, when viewed outside of the sample layer, from its neighboring area that does not contain a particle. ### Claim 8 (depends on 1) The device of claim 1, wherein the plates are compressed by a compression force that is provided by human hand. ### Claim 9 (depends on 1) The device of claim 1, wherein the diameter of one or more of the plurality of particles is equal to the height of the spacers. ### Claim 10 (depends on 1) The device of claim 1, wherein the height of the spacers is about 10 μm. ### Claim 11 (depends on 1) The device of claim 1, wherein the height of the spacers is about 5 μm. ### Claim 12 (depends on 1) The device of claim 1, wherein the height of the spacers is between about 0.1 μm and about 15 μm. ### Claim 13 (depends on 1) The device of claim 1, wherein the height of the spacers is between about 0.1 μm and about 3 μm. ### Claim 14 (depends on 1) The device of claim 1, wherein at least a portion of the inner surface of one plate or both plates is hydrophilic. ### Claim 15 (depends on 1) The device of claim 1, wherein the inter-spacer distance is periodic. ### Claim 16 (depends on 1) The device of claim 1, wherein the sample is a deposition directly from a subject to the plate without using any transferring devices. ### Claim 17 (depends on 1) The device of claim 1, wherein the inter-spacer distance (ISD) is equal or less than about 120 μm (micrometer). ### Claim 18 (depends on 1) The device of claim 1, wherein the inter-spacer distance (ISD) is equal or less than about 100 μm. ### Claim 19 (depends on 2) The device of claim 2, wherein the inter-spacer distance (ISD) is equal or less than about 120 μm (micrometer). ### Claim 20 (depends on 2) The device of claim 2, wherein the inter-spacer distance (ISD) is equal or less than about 100 μm (micrometer). ### Claim 21 (depends on 2) The device of claim 2, wherein the fourth power of the inter-spacer-distance (ISD) divided by the thickness (h) and the Young's modulus (E) of the flexible plate (ISD4/(hE)) is 1×106 μm3/GPa or less. ### Claim 22 (depends on 2) The device of claim 2, wherein the fourth power of the inter-spacer-distance (ISD) divided by the thickness (h) and the Young's modulus (E) of the flexible plate (ISD4/(hE)) is 5×105 μm3/GPa or less. ### Claim 23 (depends on 1) The device of claim 1, wherein the spacers have pillar shape, a flat top surface, a predetermined uniform height, and a predetermined constant inter-spacer distance that is at least about 2 times larger than the size of the analyte, wherein the Young's modulus of the spacers times the filling factor of the spacers is equal or larger than 2 MPa, wherein the filling factor is the ratio of the spacer contact area to the total plate area, and wherein, for each spacer, the ratio of the lateral dimension of the spacer to its height is at least 1 (one). ### Claim 24 (depends on 1) The device of claim 1, wherein the spacers have pillar shape, a flat top surface, a predetermined uniform height, and a predetermined constant inter-spacer distance that is at least about 2 times larger than the size of the analyte, wherein the Young's modulus of the spacers times the filling factor of the spacers is equal or larger than 2 MPa, wherein the filling factor is the ratio of the spacer contact area to the total plate area, and wherein, for each spacer, the ratio of the lateral dimension of the spacer to its height is at least 1 (one), wherein the fourth power of the inter-spacer-distance (ISD) divided by the thickness (h) and the Young's modulus (E) of the flexible plate (ISD4/(hE)) is 5×106 μm3/GPa or less. ### Claim 25 (depends on 2) The device of claim 2, wherein the ratio of the inter-spacer distance of the spacers to the average width of the spacer is 2 or larger, and the filling factor of the spacers multiplied by the Young's modulus of the spacers is 2 MPa or larger. ### Claim 26 (depends on 1) A method of performing a homogeneous assay, comprising the steps of: (a) obtaining a sample suspected of containing an analyte; (b) obtaining the device of claim 1, wherein the capture agent is capable of specifically binding a binding site of the analyte; (c) having optical labels on at least a part of the sample contact areas of the device, wherein the optical labels are capable of binding to the analytes; (d) depositing the sample on one or both of the plates when the plates are in an open configuration, wherein in an open configuration; (e) after (d), bringing the two plates together and pressing the plates into a closed configuration, at least part of the sample is compressed by the two plates into a layer of highly uniform thickness, the uniform thickness of the layer is confined by the inner surfaces of the plates and is regulated by the plates and the spacers; (f) while the plates are in the closed configuration, analyzing the analyte in the layer of uniform thickness, wherein the analyzing comprises: (i) measuring, from outside of the sample layer, the total light signal from (a) a particle area that is an area of the sample layer that contains one particle and from (b) a surrounding area that is the area of the sample layer which is around the particle area, wherein the surrounding area is 50 D within the edge of the particle, wherein the D is the diameter of the particle; and (ii) measuring the total light signal from each of the particle area and the surrounding area of at least two different particle areas. ### Claim 27 (depends on 26) The method of claim 26, wherein the particle area for the total light signal measurement has the same area as the particle diameter. ### Claim 28 (depends on 26) The method of claim 26, wherein the particle area for the total light signal measurement is smaller than the area defined by the particle diameter. ### Claim 29 (depends on 26) The method of claim 26, wherein the analyzing the analyte in the uniform sample layer comprises averaging of the total light signal from each area. ### Claim 30 (depends on 26) The method of claim 26, wherein the analyzing the analyte in the uniform sample layer comprises (i) taking a ratio of the total light signal of each particle area to that of its surrounding area, and (ii) averaging the ratio of all particle area and surround area pairs. ### Claim 31 (depends on 26) The method of claim 26, wherein the time from the end of the sample deposition to the end of the plates being pressed into the closed configuration is less than 15 seconds. ### Claim 32 (depends on 26) The method of claim 26, wherein the time from the end of the sample deposition to the end of the plates being pressed into the closed configuration is less than 5 seconds. ### Claim 33 (depends on 1) An apparatus for homogenous assaying an analyte in a sample, comprising: (i) the device of claim 1; and (ii) one or more imagers that image at least a part of the sample contact area. ### Claim 34 (depends on 1) A smartphone system for homogeneous assay, comprising: (a) the device of claim 1; (b) a mobile communication device that comprises: (i) one or a plurality of cameras for detecting and/or imaging the sample; (ii) electronics, signal processors, hardware and software for receiving and/or processing the detected signal and/or the image(s) of the sample and for remote communication; and (c) an adaptor that is configured to accommodate the device that is in the closed configuration and be engageable to the mobile communication device; wherein when engaged with the mobile communication device, the adaptor is configured to facilitate the detection and/or imaging of the analyte in the sample. ### Claim 35 (depends on 34) The smartphone system, of claim 34, wherein the first and second particles are different. ### Claim 36 (depends on 34) The smartphone system, of claim 34, wherein the first and second particles are different in their sizes. ### Claim 37 (depends on 34) The smartphone system, of claim 34, wherein the first and second particles are different in their optical properties selected from the group consisting of: photoluminescence, electroluminescence, and electrochemiluminescence, light absorption, reflection, transmission, diffraction, scattering, diffusion, surface Raman scattering, and any combination thereof. ### Claim 38 (depends on 34) The smartphone system, of claim 34, wherein the first and second particles are different in their electric densities. ### Claim 39 (depends on 34) The smartphone system, of claim 34, wherein the first and second particles are the same, and wherein the signals from the first and second analytes are different. ### Claim 40 (depends on 1) A system for rapid homogeneous assay, comprising: (a) the device of claim 1; and (b) a detector that detects signals from the capture agent-bound analyte indicative of the presence and/or quantity of the analyte in the layer of uniform thickness. ### Claim 41 (depends on 1) A smartphone system for rapid homogeneous assay, comprising: (a) the device of claim 1; (b) a mobile communication device that comprises: i. one or a plurality of cameras for detecting and/or imaging the sample; ii. electronics, signal processors, hardware and software for receiving and/or processing the detected signal and/or the image of the sample and for remote communication; and (c) an adaptor that is configured to hold the closed device and engageable to mobile communication device; wherein when engaged with the mobile communication device, the adaptor is configured to facilitate the detection and/or imaging of the analyte in the sample at the closed configuration. ### Claim 42 (depends on 1) A method of performing a rapid homogeneous assay, comprising the steps of: (a) obtaining a sample suspected of containing an analyte; (b) obtaining the device of claim 1; (c) depositing the sample on one or both of the plates when the plates are in the open configuration; (d) after (c), bringing the two plates together and pressing the plates into the closed configuration; and (e) while the plates are at the closed configuration, detecting and analyzing the analyte in the layer of uniform thickness. ### Claim 43 (depends on 40) A method of analyzing the image for a rapid homogeneous assay, comprising the steps of: (a) obtaining an image of the signal in claim 40 at the closed configuration, wherein the image is selected from the group consisting of bright field image, dark field image, fluorescence image, and phosphorescence image; (b) analyzing the image, identifying particles in the image, and extracting information of particles size, signal intensity of particles, distance between particles, distribution of particles, and number of particles; and (c) deducing analyte concentration by analyzing the extracted information from step (b) and calculating parameters of the particles. ### Claim 44 (depends on 1) A method of performing a rapid homogeneous assay, comprising the steps of: (a) obtaining a sample suspected of containing an analyte; (b) obtaining the device of claim 1; (c) depositing the sample on one or both of the plates when the plates are in the open configuration; (d) after (c), bringing the two plates together and pressing the plates into the closed configuration; and (e) after step (d), incubating the assay for a time equal to or longer than an intended assay time, detecting and analyzing the analyte in the layer of uniform thickness. ### Claim 45 (depends on 44) The method of claim 44, wherein the intended assay time is in the range of 0.1-240 sec. ### Claim 46 (depends on 44) The method of claim 44, wherein the intended assay time is in the range of 1-60 sec. ### Claim 47 (depends on 44) The method of claim 44, wherein the intended assay time is equal to or less than 30 sec. ### Claim 48 (depends on 44) The method of claim 44, wherein the intended assay time is equal to or less than 10 sec. ### Claim 49 (depends on 44) The method of claim 44, wherein the intended assay time is equal to or less than 5 sec. ### Claim 50 (depends on 44) The method of claim 44, wherein the intended assay time is equal to or less than 1 sec. ### Claim 51 (depends on 1) The device of claim 1, wherein the analyte is labeled by a detection agent that selectively binds to the analyte and is associated with a label. ### Claim 52 (depends on 51) The device of claim 51, wherein the detection agent is coated on the inner surface(s) of one or both of the plates, and is configured to, upon contacting the sample, be dissolved and diffuse in the sample. ### Claim 53 (depends on 51) The device of claim 51, wherein the detection agent is pre-loaded into the sample before the sample is deposited on the plate(s). ### Claim 54 (depends on 51) The device of claim 51, wherein the capture agent and the detection agent are configured to bind to the analyte at different locations thereof and form capture agent-analyte-detection agent sandwich. ### Claim 55 (depends on 1) The device of claim 1, wherein the analyte competes with a detection agent to bind to the capture agent, and wherein the detection agent is labeled. ### Claim 56 (depends on 1) The device of claim 1, wherein one or both of the plates comprise, on the respective inner surface, a signal amplification surface that amplify a signal in proximity to the amplification surface. ### Claim 57 (depends on 1) The device of claim 1, wherein the particles are the spacers that regulate the thickness of the layer at the closed configuration. ### Claim 58 (depends on 1) The device of claim 1, wherein the particles are micro- or nano-particles having an average diameter in the range of 1 nm to 200 μm. ### Claim 59 (depends on 1) The device of claim 1, wherein the particles have an average diameter in the range of 0.1 μm to 10 μm. ### Claim 60 (depends on 1) The device of claim 1, wherein the particles have an average diameter in the range of 1 nm to 500 nm. ### Claim 61 (depends on 1) The device of claim 1, wherein the particles have an average diameter in the range of to 0.5 μm to 30 μm. ### Claim 62 (depends on 1) The device of claim 1, wherein ratio between the spacing between the plates at the closed configuration and average diameter of the particles is in the range of 1-100. ### Claim 63 (depends on 1) The device of claim 1, wherein the particles have an area density of 1 to 106 per mm2. ### Claim 64 (depends on 1) The device of claim 1, wherein the particles have an area density of 1 to 1000 per mm2. ### Claim 65 (depends on 1) The device of claim 1, wherein the particles are configured to amplify a signal in proximity to the particles and have a signal amplification factor in the range of 1 to 10000. ### Claim 66 (depends on 51) The device of claim 51, wherein the detection agent is configured to have a concentration in the layer of uniform thickness that is 1 to 1000 times higher than analyte concentration in the sample. ### Claim 67 (depends on 51) The device of claim 51, wherein the particles and the detection agent are on the same plate. ### Claim 68 (depends on 51) The device of claim 51, wherein the particles and the detection agent are on different plates. ### Claim 69 (depends on 1) The device of claim 1, wherein the analyte is selected from the group consisting of: cells, viruses, proteins, peptides, DNAs, RNAs, oligonucleotides, and any combination thereof. ### Claim 70 (depends on 1) The device of claim 1, wherein the analyte is C Reactive Protein (CRP). ### Claim 71 (depends on 1) The device of claim 1, wherein the capture agent is selected from the group consisting of: protein, peptide, peptidomimetics, streptavidin, biotin, oligonucleotide, oligonucleotide mimetics, any other affinity ligand and any combination thereof. ### Claim 72 (depends on 1) The device of claim 1, wherein the capture agent is an antibody. ### Claim 73 (depends on 1) The device of claim 1, wherein the capture agent is an anti-C Reactive Protein (CRP) antibody. ### Claim 74 (depends on 1) The device of claim 1, wherein the capture agent has a concentration that is sufficient to detect the presence and/or measure the amount of the analyte. ### Claim 75 (depends on 1) The device of claim 1, wherein the capture agent has a concentration that is sufficient to immobilize the analyte. ### Claim 76 (depends on 51) The device of claim 51, wherein the detection agent is selected from the group consisting of: protein, peptide, peptidomimetics, streptavidin, biotin, oligonucleotide, oligonucleotide mimetics, any other affinity ligand and any combination thereof. ### Claim 77 (depends on 51) The device of claim 51, wherein the detection agent is an antibody. ### Claim 78 (depends on 51) The device of claim 51, wherein the detection agent is an anti-CRP antibody. ### Claim 79 (depends on 1) The device of claim 1, wherein the particles are made of a material selected from the group consisting of: polystyrene, polypropylene, polycarbonate, PMMG, PC, COC, COP, glass, resin, aluminum, gold or other metal or any other material whose surface can be modified to be associated with the capture agent. ### Claim 80 (depends on 1) The device of claim 1, wherein the particles are treated with a protein stabilizer. ### Claim 81 (depends on 1) The device of claim 1, wherein the capture agent is conjugated with the particles. ### Claim 82 (depends on 1) The device of claim 1, wherein the particles are prepared by: (a) activating with N-Hydroxysuccinimide (NHS); (b) blocking with a BSA solution; and (c) incubating with a capture agent solution. ### Claim 83 (depends on 1) The device of claim 1, wherein the sample is made from a biological sample selected from the group consisting of: amniotic fluid, aqueous humour, vitreous humour, blood, breast milk, cerebrospinal fluid (CSF), cerumen (earwax), chyle, chime, endolymph, perilymph, feces, breath, gastric acid, gastric juice, lymph, mucus (including nasal drainage and phlegm), pericardial fluid, peritoneal fluid, pleural fluid, pus, rheum, saliva, exhaled breath condensates, sebum, semen, sputum, sweat, synovial fluid, tears, vomit, urine, and any combination thereof. ### Claim 84 (depends on 1) The device of claim 1, wherein the sample is an environmental liquid sample from a source selected from the group consisting of: river, lake, pond, ocean, glaciers, icebergs, rain, snow, sewage, reservoirs, tap water, or drinking water, solid samples from soil, compost, sand, rocks, concrete, wood, brick, sewage, and any combination thereof. ### Claim 85 (depends on 1) The device of claim 1, wherein the sample is an environmental gaseous sample from a source selected from the group consisting of: the air, underwater heat vents, industrial exhaust, vehicular exhaust, and any combination thereof. ### Claim 86 (depends on 1) The device of claim 1, wherein the sample is a foodstuff sample selected from the group consisting of: raw ingredients, cooked food, plant and animal sources of food, preprocessed food, and partially or fully processed food, and any combination thereof. ### Claim 87 (depends on 51) The device of claim 51, wherein the detection agent is a labeled agent. ### Claim 88 (depends on 51) The device of claim 51, wherein the detection agent is labeled with a fluorophore. ### Claim 89 (depends on 88) The device of claim 88, wherein the fluorophore is Cy5. ### Claim 90 (depends on 51) The device of claim 51, wherein the particles are associated with a label, and wherein the detection agent is a quencher that is configured to quench signal of the particles-associated label when the detection agent is in proximity of the label. ### Claim 91 (depends on 40) The system of claim 40, wherein the detector detects the signal emanating from the analyte concentration areas or particles indicative of the presence and/or quantity of the analyte. ### Claim 92 (depends on 40) The system of claim 40, wherein the signal is: (i) luminescence selected from photoluminescence, electroluminescence, and electrochemiluminescence; (ii) light absorption, reflection, transmission, diffraction, scattering, or diffusion; (iii) surface Raman scattering; (iv) electrical impedance selected from resistance, capacitance, and inductance; (v) magnetic relaxivity; or (vi) any combination of i-v. ### Claim 93 (depends on 34) The smartphone system of claim 34, wherein the mobile communication device is configured to communicate test results to a medical professional, a medical facility or an insurance company. ### Claim 94 (depends on 34) The smartphone system of claim 34, wherein the mobile communication device is further configured to communicate information on the subject with a medical professional, medical facility or insurance company. ### Claim 95 (depends on 34) The smartphone system of claim 34, wherein the mobile communication device is configured to receive a prescription, diagnosis or a recommendation from a medical professional. ### Claim 96 (depends on 34) The smartphone system of claim 34, wherein the mobile communication device communicates with a remote location via a wifi or cellular network. ### Claim 97 (depends on 34) The smartphone system of claim 34, wherein the mobile communication device is a mobile phone. ### Claim 98 (depends on 42) The method of claim 42, wherein the sample contact areas are not washed before step (e). ### Claim 99 (depends on 42) The method of claim 42, further comprising washing the sample contact area before step (e). ### Claim 100 (depends on 42) The method of claim 42, further comprising determining the presence of the analyte and/or measuring the amount of the analyte. ### Claim 101 (depends on 43) The method of claim 43, wherein the calculated parameters comprise average signal intensity from all the particles that are analyzed. ### Claim 102 (depends on 43) The method of claim 43, wherein the calculated parameters comprise highest signal intensity from all the particles that are analyzed. ### Claim 103 (depends on 43) The method of claim 43, wherein the calculated parameters comprise signal intensity distribution from all the particles that are analyzed. ### Claim 104 (depends on 43) The method of claim 43, wherein the calculated parameters comprise number of all the particles that are analyzed with signal intensity larger than a threshold. ### Claim 105 (depends on 43) The method of claim 43, wherein the calculated parameters comprise average signal intensity from all the particles that are analyzed in a first area of the image. ### Claim 106 (depends on 43) The method of claim 43, wherein the calculated parameters comprise highest signal intensity from all the particles that are analyzed in a first area of the image. ### Claim 107 (depends on 43) The method of claim 43, wherein the calculated parameters comprise signal intensity distribution from all the particles that are analyzed in a first area of the image. ### Claim 108 (depends on 43) The method of claim 43, wherein the calculated parameters comprise number of all the particles that are analyzed in a first area of the image with signal intensity larger than a threshold. ### Claim 109 (depends on 1) The device of claim 1, wherein the analyte is the analyte in detection of proteins, peptides, nucleic acids, synthetic compounds, and inorganic compounds. ### Claim 110 (depends on 1) The device of claim 1, wherein the sample is a biological sample selected from amniotic fluid, aqueous humour, vitreous humour, blood, breast milk, cerebrospinal fluid (CSF), cerumen (earwax), chyle, chime, endolymph, perilymph, feces, breath, gastric acid, gastric juice, lymph, mucus (including nasal drainage and phlegm), pericardial fluid, peritoneal fluid, pleural fluid, pus, rheum, saliva, exhaled breath condensates, sebum, semen, sputum, sweat, synovial fluid, tears, vomit, and urine. ### Claim 111 (depends on 1) The device of claim 1, wherein the spacers have a shape of pillars and a ratio of the width to the height of the pillar is equal or larger than one. ### Claim 112 (depends on 42) The method of claim 42, wherein the sample that is deposited on one or both of the plates has an unknown volume. ### Claim 113 (depends on 1) The device of claim 1, wherein the spacers have a shape of pillar, and the pillar has uniform cross-section. ### Claim 114 (depends on 1) The device of claim 1, wherein the sample is for the detection, purification and quantification of chemical compounds or biomolecules that correlates with the stage of certain diseases. ### Claim 115 (depends on 1) The device of claim 1, wherein the sample is related to an infectious or parasitic disease, injuries, cardiovascular disease, cancer, mental disorders, neuropsychiatric disorders, pulmonary diseases, renal diseases, or other organic diseases. ### Claim 116 (depends on 1) The device of claim 1, wherein the sample is related to the detection, purification and quantification of microorganism. ### Claim 117 (depends on 1) The device of claim 1, wherein the sample is related to virus, fungus and bacteria from an environment or a biological sample. ### Claim 118 (depends on 1) The device of claim 1, wherein the sample is related to the detection, quantification of chemical compounds or biological samples that pose hazard to food safety or national security. ### Claim 119 (depends on 1) The device of claim 1, wherein the sample is related to quantification of vital parameters in medical or physiological monitor. ### Claim 120 (depends on 1) The device of claim 1, wherein the sample is related to glucose, blood, oxygen level, total blood count. ### Claim 121 (depends on 1) The device of claim 1, wherein the sample is related to the detection and quantification of specific DNA or RNA from biosamples. ### Claim 122 (depends on 1) Device of claim 1, wherein the sample is related to the sequencing and comparing of genetic sequences in DNA in the chromosomes and mitochondria for genome analysis. ### Claim 123 (depends on 1) The device of claim 1, wherein the sample is cells, tissues, bodily fluids, and stool. ### Claim 124 (depends on 1) The device of claim 1, wherein the sample is the sample in the detection of proteins, peptides, nucleic acids, synthetic compounds, inorganic compounds. ### Claim 125 (depends on 1) The device of claim 1, wherein the sample is the sample in the fields of human, veterinary, agriculture, foods, environments, and drug testing. ### Claim 126 (depends on 1) The device of claim 1, wherein the sample is a biological sample is selected from blood, serum, plasma, a nasal swab, a nasopharyngeal wash, saliva, urine, gastric fluid, spinal fluid, tears, stool, mucus, sweat, earwax, oil, a glandular secretion, cerebral spinal fluid, tissue, semen, vaginal fluid, interstitial fluids derived from tumorous tissue, ocular fluids, spinal fluid, a throat swab, breath, hair, finger nails, skin, biopsy, placental fluid, amniotic fluid, cord blood, lymphatic fluids, cavity fluids, sputum, pus, microbiota, meconium, breast milk, exhaled condensate nasopharyngeal wash, nasal swab, throat swab, stool samples, hair, finger nail, ear wax, breath, connective tissue, muscle tissue, nervous tissue, epithelial tissue, cartilage, cancerous sample, or bone. ### Claim 127 (depends on 1) The device of claim 1, further comprising a detection agent on one of the first and second plates, wherein the detection agent is configured to bind to the analyte and form a capture agent-analyte-detection agent sandwich. ### Claim 128 (depends on 1) The device of claim 1, further comprising a labeled detection agent, wherein a unlabeled analyte competes with the labeled detection agent to bind to the capture agent. ### Claim 129 (depends on 1) The device of claim 1, further comprising a camera for imaging the sample. ### Claim 130 (depends on 1) The device of claim 1, further comprising a second set of particles that is different from the plurality of particles in the first plate in shape, size, optical property, or any combination thereof, wherein the second set of particles has a second capture agent immobilized on the surface of the second set of particles, wherein the second capture agent is capable of specifically binding and immobilizing a second analyte. ### Claim 131 (depends on 1) The device of claim 1, further comprising a detection agent, wherein the detection agent and the plurality of particles are on different plates. ### Claim 132 (depends on 1) The device of claim 1, further comprising a detector that images an optical signal of the sample, where the image comprises fluorescence image, bring field image, or a combination. ### Claim 133 (depends on 1) The device of claim 1, further comprising a label that attached to the analyte. ### Claim 134 (depends on 2) The device of claim 2, further comprising a detection agent on one of the first and second plates, wherein the detection agent is configured to bind to the analyte and form a capture agent-analyte-detection agent sandwich. ### Claim 135 (depends on 2) The device of claim 2, further comprising a labeled detection agent, wherein a unlabeled analyte competes with the labeled detection agent to bind to the capture agent. ### Claim 136 (depends on 2) The device of claim 2, further comprising a camera for imaging the sample. ### Claim 137 (depends on 2) The device of claim 2, further comprising a second set of particles that is different from the plurality of particles in the first plate in shape, size, optical property, or any combination thereof, wherein the second set of particles has a second capture agent immobilized on the surface of the second set of particles, wherein the second capture agent is capable of specifically binding and immobilizing a second analyte. ### Claim 138 (depends on 2) The device of claim 2, further comprising a detection agent, wherein the detection agent and the plurality of particles are on different plates. ### Claim 139 (depends on 2) The device of claim 2, further comprising a detector that images an optical signal of the sample, where the image comprises fluorescence image, bring field image, or a combination. ### Claim 140 (depends on 2) The device of claim 2, further comprising a label that attached to the analyte. ### Claim 141 (depends on 2) The device of claim 2, wherein the diameter of one or more of the plurality of particles is equal to the height of the spacers. ### Claim 142 (depends on 2) The device of claim 2, wherein the height of the spacers is about 10 μm. ### Claim 143 (depends on 2) The device of claim 2, wherein the height of the spacers is about 5 μm. ### Claim 144 (depends on 2) The device of claim 2, wherein the height of the spacers is between about 0.1 μm and about 15 μm. ### Claim 145 (depends on 2) The device of claim 2, wherein the height of the spacers is between about 0.1 μm and about 3 μm. ### Claim 146 (depends on 2) The device of claim 2, wherein the inter-spacer distance is periodic. ### Claim 147 (depends on 1) The device of claim 1, wherein the plurality of particles comprises first particles and second particles, and the capture agent comprises a first capture agent and a second capture agent, wherein the first and second particles have first and second capture agents immobilized thereon, respectively; and wherein the first and second capture agents are capable of binding to and immobilizing the first and second analytes, respectively. ### Claim 148 (depends on 1) The device of claim 1, wherein one or both of the plates further comprise, on the respective inner surface, a detection agent that is configured to, upon contacting the sample, be dissolved in the sample and bind to the analyte; wherein the capture agent and the detection agent are configured to bind to the analyte at different locations thereof and to form a capture agent-analyte-detection agent sandwich that is immobilized to the particle; and wherein the particles, the capture agent, and the detection agent are configured to render signal from the particle-associated capture agent-analyte-detection agent sandwich distinguishable from signal of free detection agent in the layer of uniform thickness. ### Claim 149 (depends on 26) The method of claim 26, wherein the spacers have a height that is equal to or less than 3 times of a diffusion parameter, wherein the diffusion parameter is square root of an intended assay time multiplying diffusion constant of the analyte in the sample and wherein the intended assay time is equal to or less than 240 seconds, and wherein an average distance between two neighboring particles is equal to or less than 2 times of the diffusion parameter. ### Claim 150 (depends on 26) The method of claim 26, wherein the ratio of the spacers' height versus the diffusion parameter is in the range of 0.01-2. ### Claim 151 (depends on 149) The method of claim 149, wherein the ratio of the spacers' height versus the diffusion parameter is in the range of 0.1-1.5. ### Claim 152 (depends on 149) The method of claim 149, wherein the ratio of the spacers' height versus the diffusion parameter is in the range of 0.01-0.5. ### Claim 153 (depends on 149) The method of claim 149, wherein the ratio of the spacers' height versus the diffusion parameter is in the range of 0.01-0.2. ### Claim 154 (depends on 149) The method of claim 149, wherein the ratio of the spacers' height versus the diffusion parameter is in the range of 0.01-0.1. ### Claim 155 (depends on 149) The method of claim 149, wherein the ratio of the average distance between two neighboring particles versus the diffusion parameter is in the range of 0.01-2, and the ratio of the spacers' height versus the diffusion parameter is in the range of 0.01-1. ### Claim 156 (depends on 149) The method of claim 149, wherein the ratio of the average distance between two neighboring particles versus the diffusion parameter is in the range of 0.01-4, and the ratio of the spacers' height versus the diffusion parameter is in the range of 0.01-1. ### Claim 157 (depends on 149) The method of claim 149, wherein the ratio of the average distance between two neighboring particles versus the diffusion parameter is in the range of 0.01-0.5, the ratio of the spacers' height versus the diffusion parameter is in the range of 0.01-0.2, and the intended assay time is equal to or less than 120 sec. ### Claim 158 (depends on 149) The method of claim 149, wherein the ratio of the average distance between two neighboring particles versus the diffusion parameter is in the range of 0.01-1; the ratio of the spacers' height versus the diffusion parameter is in the range of 0.01-0.5, and the intended assay time is equal to or less than 60 sec. ### Claim 159 (depends on 149) The method of claim 149, wherein the ratio of the average distance between two neighboring particles versus the diffusion parameter is in the range of 0.01-2; the ratio of the spacers' height versus the diffusion parameter is in the range of 0.01-1; and the intended assay time is equal to or less than 30 sec. ### Claim 160 (depends on 149) The method of claim 149, wherein the ratio of the average distance between two neighboring particles versus the diffusion parameter is in the range of 0.01-4; the ratio of the spacers' height versus the diffusion parameter is in the range of 0.01-1; and the intended assay time is equal to or less than 30 sec.