Aggregation-induced assay for enhanced sensitivity

Abstract

Provided herein are systems, devices, and methods for rapid and accurate measurement of analyte particle binding-induced aggregation of reporter particles. In the presence of analyte particles of interest, the reporter particles form aggregates whose average particle size increases with increasing analyte concentration. From analysis of the average particle size determined from the sample frame, the presence and / or concentration of the analyte can be determined.

Description

Technical Field

[0001] This application claims the benefit of priority based on U.S. Provisional Patent Application No. 63 / 007,701, filed Apr. 9, 2020, the disclosure of which is hereby incorporated by reference in its entirety as if fully set forth herein.

Background Art

[0002] There is a pressing need for point-of-care diagnostics and other assays that can be performed in the field. If the delays and costs associated with sending the results of an assay, such as a diagnostic test, especially a blood test, to a dedicated testing facility for analysis could be eliminated, responses could be made more efficiently and effectively. Clinical testing facilities deliver the results of diagnostic tests by performing biochemical assays with accurate benchtop instruments. Efforts to miniaturize such instruments or reproduce their functionality with mobile electronic devices are difficult. In many cases, the results are unusable.

Summary of the Invention

Problems to be Solved by the Invention

[0003] What is needed is an inexpensive but accurate point-of-care assay, such as a diagnostic test that provides immediate and accurate results to a physician and their patient.

Means for Solving the Problems

[0004] This specification provides systems, devices, and methods for the rapid and accurate measurement of analyte particles by binding event assays. The sensor device contains one or more types of reporter particles, including macromolecular components or nanoscale components such as nanoparticles. The reporter particles (or alternatively, “reporters”) appear in the sample frame as individually detectable objects, while generating an optical signal large enough to allow imaging of individual reporter particles. In the presence of a target analyte (e.g., a molecule, viral particle, or bacterium), the reporter particles form aggregates containing multiple reporter particles, thus appearing in the sample frame as a larger object. With increasing analyte concentration, both the degree of aggregation and aggregate size increase. The presence and / or concentration of analyte can be determined from distinguishable features in the image, such as the average object size, perimeter, shape, and color, which are determined from the sample image frame. The key to this detection strategy is the application of image processing and filtering procedures designed to remove errors and noise from the image to select objects usable for analysis, and then sum the results to determine the analyte concentration.

[0005] This specification includes, A step of combining a sample with a certain amount of one or more types of reporter particles in a detector volume, The process of recording one or more frames of the detector volume, A step of identifying images of objects corresponding to the aggregate of reporter particles and optionally individual non-aggregate reporter particles in one or more frames, A process of determining one or more object properties, A step of determining the presence and / or concentration of analytes from the material properties, Includes, A method is provided for determining the presence or concentration of analyte particles in a sample, wherein the presence of analyte induces the formation of an aggregate containing at least two reporter particles and at least one analyte particle, each aggregate containing at least two reporter particles. [Brief explanation of the drawing]

[0006] [Figure 1] This document presents the design principle for a reporter particle having two binding sites. [Figure 2] This document presents the design principle for a reporter particle having four binding sites. [Figure 3] This demonstrates the detection of antigens using gold nanoparticles conjugated with C2 and C6 antibodies. [Figure 4] The procedure for conjugating antibodies with citrate-capped gold nanoparticles is described. [Figure 5] (a) Absorption spectra of gold nanoparticles conjugated to C2 antibody and (b) to C6 antibody are shown. (i) Bare GNP (gold nanoparticles), (ii) GNP-antibody conjugate, (iii) GNP-antibody conjugate + CRP (C-reactive protein). [Figure 6] Dark-field frames of GNPs are shown. (a) Control in the absence of CRP, (b) Addition of CRP showing aggregate formation. [Figure 7] An enlarged view of individual clusters is shown. [Figure 8] An example of frame processing is shown: (a) original frame, (b) after artifact removal, (c) after object segmentation, and (d) quantification of cluster size. [Figure 9] An example of the frame cleaning procedure is shown. (a) Original frame, (b) Background, (c) Cleaned grayscale. [Figure 10] An example of the border formation procedure is shown. (a) Cleaned grayscale frame, (b) Border frame, (c) B / W frame. [Figure 11] An example of a feature identification procedure is shown: (a) cleaned grayscale frame, (b) B / W frame before denoising, (c) B / W frame after denoising. [Figure 12]This shows the titration of CRP. (a) Aggregation assay by DF detection, horizontal axis = CRP concentration (mg / L), vertical axis = average aggregate size (μm2 / cluster), (b) Conventional plasmon sensing, horizontal axis = CRP concentration (mg / L), vertical axis = scattering intensity (AU). [Figure 13] Assays performed on whole blood are shown. (a) Capillaries with depths of (i) 0.1 mm and (ii) 0.05 mm. Dark-field images of blood in capillaries with thicknesses of (b) 0.1 mm, (c) 0.05 mm, and (d) 0.01 mm are also shown. [Figure 14] (a) Dark-field imaging of nanoparticles in whole blood at imaging depths of 0.11 mm, (b) 0.05 mm, and (c) 0.01 mm is shown. Intensity (brightness) profiles at imaging depths of (a) 0.11 mm, (b) 0.05 mm, and (c) 0.01 mm are also shown, with a horizontal scale representing the distance in pixels from the center of the particle and a vertical scale representing 16-bit intensity. [Figure 15] This graph shows the results of a CRP assay performed on whole blood. Horizontal axis = CRP concentration (mg / dL), vertical axis = average cluster size (pixels). [Figure 16] (iii) schematically illustrates the design principle for detecting SARS-CoV-2 antibodies. (i) and (v) correspond to PEG linkers from Au nanoparticles to (ii) anti-human antibody and (iv) SARS-CoV-2 spike RBD, respectively. [Figure 17] This shows a SARS-CoV-2 assay performed on whole blood. (a) Microscopic observation of the aggregate, (b) Calibration curve: horizontal axis = SARS-CoV-2 antibody (mg / dL), vertical axis = aggregation size (pixels). [Figure 18] Components for a direct virus detection assay are shown. (a)(i) Details of coronavirus with numerous spike proteins highlighted. (b) Top view and (c) Side view of the interaction between the 3-part nanobody (ii) and the spike protein (i). [Figure 19]Shows the detection of the SARS-CoV-2 virus. (a) Design principle: (i) SARS-CoV-2 spike nanobody, (ii) PEG linker, (iii) Au nanoparticles. (b) and (c) Microscopic observation of aggregates at various levels of virus concentration. The aggregation mechanism described elsewhere for smaller biomolecules is clearly effective for intact viruses. [Figure 20] Shows a flowchart for image processing.

Mode for Carrying Out the Invention

[0007] The present disclosure aims to provide a method for performing biochemical assays with improved speed, accuracy, precision, and affordability. The method utilizes an aggregation process that induces aggregation into macromolecular assemblies by binding of target analyte particles to a plurality of reporter particles, facilitating direct imaging by an easily available optical device.

[0008] There is a great need to perform biochemical assays on mobile electronic devices. This has the potential to improve the speed, accuracy, sensitivity, and affordability of diagnostic tests. There are substantial technologies for capturing 2D (X / Y) images of 3D objects. Technologies have advanced from film cameras to video cameras, image plates, charge-coupled devices, and complementary metal oxide (「CMOS」) chips. Such images are projections of 3D objects onto a 2D plane. Generally, a 2D frame is subdivided into picture units called pixels for processing and downstream operations, which can be appropriately referenced via a two-coordinate addressing system. As an example, a square frame can be subdivided into a 512×512 array of tiles, each of which can be addressed by an ordered pair of integers (x,y) where both x and y can be in the range of 1 to 512, inclusive of the end values.

[0009] The observation of normal "small molecule" organic compounds and smaller biomolecules in solution is generally not feasible for two reasons: (a) the spatial resolution of the detector is much coarser than the size of such small molecules, so each pixel captures signals from multiple solute molecules, and (b) the signal from individual molecules of this size is generally too weak to exceed the noise for detection. Although studies have been conducted for imaging individual molecules, the procedures to date have generally been slow, cumbersome, and / or expensive. In contrast, techniques are available for imaging solutes above the nanometer scale, because they combine large particle sizes with enhanced signals often obtained from such larger particles.

[0010] The present disclosure solves the problem of insufficient signal-to-noise for analyte particles by utilizing reporter particles that contain macromolecular components or nanoscale components such as nanoparticles. Reporter particles (alternatively "reporters") are large enough to be imaged as individual reporter particles, and in some embodiments, appear within the sample frame as particles of finite size. In the presence of the target analyte particles, the reporter particles form aggregates containing multiple reporters and thus appear within the sample frame as larger particles. As the analyte concentration increases, both the degree of aggregation and the aggregate size increase. The presence and / or concentration of the analyte can be determined from the average particle size determined from the sample frame.

[0011] Embodiment 1 provided herein: combining a sample with an amount of one or more types of reporter particles in a detector volume; recording one or more frames of the detector volume; identifying images of objects corresponding to aggregates of reporter particles and optionally individual non-aggregated reporter particles in the one or more frames; determining one or more object characteristics; A step of determining the presence and / or concentration of analytes from the material properties, Includes, A method for determining the presence or concentration of analyte particles in a sample, wherein the presence of analyte induces the formation of an aggregate containing at least two reporter particles and at least one analyte particle, each aggregate containing at least two reporter particles.

[0012] Furthermore, the following embodiments are also provided.

[0013] Embodiment 2: The method according to Embodiment 1, wherein the particle images correspond to aggregate and individual non-aggregate reporter particles of the reporter particle.

[0014] Embodiment 3: The method according to Embodiment 1, wherein the image of the particles corresponds to the aggregate of reporter particles.

[0015] Embodiment 4: The method according to any one of Embodiments 1 to 3, wherein each of one or more frames consists of an array of pixels.

[0016] Embodiment 5: The method according to any one of Embodiments 1 to 4, wherein aggregate formation is caused by binding between a binding site on at least one of one or more types of reporter particles and a binding site on an analyte particle.

[0017] Embodiment 6: The method according to Embodiment 1, wherein each of one or more types of reporter particles comprises a nanoparticle or a quantum dot.

[0018] Embodiment 7: The method according to Embodiment 6, wherein each of one or more types of reporter particles comprises a nanoparticle.

[0019] Embodiment 8: The method according to Embodiment 7, wherein each of one or more types of reporter particles comprises gold nanoparticles ("GNP").

[0020] Embodiment 9: The method according to any one of Embodiments 1 to 8, wherein each of one or more types of reporter particles contains two or more binding sites.

[0021] Embodiment 10: The method according to Embodiment 9, wherein each of one or more types of reporter particles contains two or more binding sites of the same type.

[0022] Embodiment 11: The method according to Embodiment 9, wherein at least one of one or more types of reporter particles contains two or more non-identical binding sites.

[0023] Embodiment 12: The method according to either Embodiment 10 or 11, wherein at least one type of binding site is located on an antibody or nanobody contained in a reporter particle.

[0024] Embodiment 13: The method according to Embodiment 12, wherein at least one type of binding site is located on the antibody contained in the reporter particle.

[0025] Embodiment 14: The method according to either Embodiment 12 or 13, wherein at least one type of binding site is located on a nanobody contained in the reporter particle.

[0026] Embodiment 15: The method according to Embodiment 9, wherein one or more types of reporter particles are a single type of reporter particle.

[0027] Embodiment 16: The method according to Embodiment 15, wherein a single type of reporter particle contains a single type of binding site.

[0028] Embodiment 17: The method according to Embodiment 16, wherein a single type of binding site is located on an antibody or nanobody contained in a single type of reporter particle.

[0029] Embodiment 18: The method according to Embodiment 17, wherein a single type of binding site is located on an antibody contained in a single type of reporter particle.

[0030] Embodiment 19: The method according to Embodiment 17, wherein a single type of binding site is located on a nanobody contained in a single type of reporter particle.

[0031] Embodiment 20: The method according to any one of Embodiments 15 to 19, wherein a single type of reporter particle can simultaneously bind to two or more analyte particles.

[0032] Embodiment 21: The method according to any one of Embodiments 15 to 20, wherein the analyte particle can simultaneously bind to two or more reporter particles.

[0033] Embodiment 22: The method according to Embodiment 9, wherein one or more types of reporter particles are two types of reporter particles.

[0034] Embodiment 23: The method according to Embodiment 22, wherein each of one or more types of reporter particles contains one or more binding sites of the same type.

[0035] Embodiment 24: The method according to Embodiment 23, wherein each of the two types of reporter particles contains a different type of binding site than the other type of reporter particle.

[0036] Embodiment 25: The method according to Embodiment 24, wherein each of the two different types of binding sites is located on an antibody or nanobody contained in each of the two types of reporter particles.

[0037] Embodiment 26: The method according to Embodiment 25, wherein at least one of two different types of binding sites is located on an antibody contained in one of two types of reporter particles.

[0038] Embodiment 27: The method according to Embodiment 26, wherein two different types of binding sites are located on antibodies contained in two types of reporter particles.

[0039] Embodiment 28: The method according to either Embodiment 25 or 26, wherein at least one of two different types of binding sites is located on a nanobody contained in one of two types of reporter particles.

[0040] Embodiment 29: The method according to Embodiment 25, wherein two different types of binding sites are located on nanobodies contained in two types of reporter particles.

[0041] Embodiment 30: The method according to any one of Embodiments 22 to 29, wherein each of the two types of reporter particles is capable of simultaneously binding to two or more analyte particles.

[0042] Embodiment 31: The method according to any one of Embodiments 22 to 30, wherein the analyte particle can simultaneously bind to one or more of each of two types of reporter particles.

[0043] Embodiment 32: The method according to any one of Embodiments 13, 18, and 26, wherein at least one binding site is located on a human antibody contained in a reporter particle.

[0044] Embodiment 33: The method according to any one of Embodiments 13, 18, and 26, wherein at least one binding site is located on an anti-human antibody contained in a reporter particle.

[0045] Embodiment 34: The method according to any one of Embodiments 13, 18, and 26, wherein at least one binding site is located on an anti-C-reactive protein contained in a reporter particle.

[0046] Embodiment 35: The method according to Embodiment 34, wherein at least one binding site is located on a C2 anti-C reactive protein contained in the reporter particle.

[0047] Embodiment 36: The method according to Embodiment 34, wherein at least one binding site is located on a C6 anti-C reactive protein contained in the reporter particle.

[0048] Embodiment 37: The method according to any one of Embodiments 9 to 36, wherein at least one binding site is located on a biomolecule contained in the reporter particle.

[0049] Embodiment 38: The method according to Embodiment 37, wherein the biomolecule is derived from the viral protein viral envelope.

[0050] Embodiment 39: The method according to Embodiment 38, wherein the biomolecule is selected from membrane proteins, envelope proteins, and spike proteins.

[0051] Embodiment 40: The method according to Embodiment 39, wherein the biomolecule is a viral protein, specifically a spike protein derived from the viral envelope.

[0052] Embodiment 41: The method according to any one of Embodiments 38 to 40, wherein the virus is a coronavirus.

[0053] Embodiment 42: The method according to Embodiment 41, wherein the virus is SARS-CoV-2.

[0054] Embodiment 43: The method according to any one of Embodiments 1 to 42, wherein the analyte particle is an antibody.

[0055] Embodiment 44: The method according to Embodiment 43, wherein the analyte is an antibody against a biomolecule in the viral envelope of the virus.

[0056] Embodiment 45: The method according to Embodiment 44, wherein the analyte is an antibody against a biomolecule in the viral envelope of a virus selected from membrane proteins, envelope proteins, and spike proteins.

[0057] Embodiment 46: The method according to Embodiment 45, wherein the analyte is an antibody against the spike protein.

[0058] Embodiment 47: The method according to Embodiment 46, wherein the analyte is an antibody against the spike protein of SARS-CoV-2.

[0059] Embodiment 48: The method according to any one of Embodiments 1 to 36, wherein the analyte is a hematological protein.

[0060] Embodiment 49: The method according to Embodiment 48, wherein the hematological protein is C-reactive protein ("CRP").

[0061] Embodiment 50: The method according to any one of Embodiments 1 to 42, wherein the analyte is selected from viruses, archaea, and bacteria.

[0062] Embodiment 51: The method according to Embodiment 50, wherein the analyte is a virus.

[0063] Embodiment 52: The method according to Embodiment 51, wherein the analyte is a coronavirus.

[0064] Embodiment 53: The method according to Embodiment 52, wherein the analyte is SARS-CoV-2. In certain embodiments, the binding site binds to the spike protein.

[0065] Embodiment 54: The method according to any one of Embodiments 1 to 42, wherein the analyte is a bacterium.

[0066] Embodiment 55: The method according to Embodiment 54, wherein the binding site binds to a surface glycoprotein on a bacterium.

[0067] Embodiment 56: The method according to any one of Embodiments 1 to 55, wherein the analyte particles contain two or more binding sites.

[0068] Embodiment 57: The method according to Embodiment 56, wherein the analyte particles contain two or more identical binding sites.

[0069] Embodiment 58: The method according to Embodiment 56, wherein the analyte particles contain two or more non-identical binding sites.

[0070] Embodiment 59: The method according to any one of Embodiments 1 to 58, wherein object properties enable the separation of noise, artifacts, unusable image features, and / or background from the signal corresponding to the analyte by setting predetermined limits in the algorithm used to process and filter the image of the object.

[0071] Embodiment 60: The method according to Embodiment 59, wherein object properties enable the separation of noise from the signal corresponding to the analyte by setting predetermined limits in the algorithm used to process and filter the image of the object.

[0072] Embodiment 61: The method according to either Embodiment 59 or 60, wherein object properties enable the separation of artifacts from the signal corresponding to the analyte by setting predetermined limits in the algorithm used to process and filter the image of the object.

[0073] Embodiment 62: The method according to any one of Embodiments 59 to 61, wherein object properties enable the separation of unusable image features from the signal corresponding to the analyte by setting predetermined limits in the algorithm used to process and filter the image of the object.

[0074] Embodiment 63: The method according to any one of Embodiments 59 to 62, wherein object properties enable the separation of background from the signal corresponding to the analyte by setting predetermined limits in the algorithm used to process and filter the image of the object.

[0075] Embodiment 64: The method according to any one of Embodiments 1 to 63, wherein the object properties enable the removal of one or more images of non-analyte particles, one or more unusable images, and / or one or more background features by setting predetermined limits in the algorithm used to process and filter images of the object.

[0076] Embodiment 65: The method according to Embodiment 64, wherein the object properties allow for the removal of one or more images of non-analyte particles by setting predetermined limits in the algorithm used to process and filter images of the object.

[0077] Embodiment 66: The method according to either Embodiment 64 or 65, wherein object properties enable the removal of one or more unusable images by setting predetermined limits in an algorithm used to process and filter images of objects.

[0078] Embodiment 67: The method according to any one of embodiments 64 to 66, wherein object properties enable the removal of one or more background features by setting predetermined limits in an algorithm used to process and filter images of objects.

[0079] Embodiment 68: The method according to any one of Embodiments 1 to 67, wherein the material properties increase monotonically with the aggregate size.

[0080] Embodiment 69: The method according to Embodiment 68, wherein the object properties increase in proportion to the aggregate size.

[0081] Embodiment 70: The method according to either Embodiment 68 or 69, wherein the material properties are related to the median size of the aggregate.

[0082] Embodiment 71: The method according to either Embodiment 68 or 69, wherein the object properties are related to the average size of the aggregate.

[0083] Embodiment 72: The method according to either Embodiment 68 or 69, wherein the material properties are related to the RMS size of the aggregate.

[0084] Embodiment 73: The method according to any one of Embodiments 1 to 72, wherein the average aggregate size can be determined from the material properties using a calibration curve.

[0085] Embodiment 74: The method of Embodiment 73, wherein the method further includes the step of obtaining a calibration curve.

[0086] Embodiment 75: The method according to any one of Embodiments 1 to 74, wherein the object properties are selected from the directly observed or estimated size of the aggregate, the directly observed or estimated perimeter of the aggregate, the directly observed or estimated area of ​​the aggregate, the shape of the aggregate, the color of the aggregate, the brightness of the aggregate, the reflectance of the aggregate, the fluorescence of the aggregate, and the phosphorescence of the aggregate.

[0087] Embodiment 76: The method according to Embodiment 75, wherein the object properties are selected from the size of the aggregate as directly observed or estimated, the perimeter of the aggregate as directly observed or estimated, and the area of ​​the aggregate as directly observed or estimated.

[0088] Embodiment 77: The method according to Embodiment 75, wherein the object properties are selected from aggregate brightness, aggregate fluorescence, and aggregate phosphorescence.

[0089] Embodiment 78: The method according to Embodiment 77, wherein the object property is the brightness of the aggregate.

[0090] Embodiment 79: The method according to any one of Embodiments 1 to 78, wherein one or more types of reporter particles provide an optical signal.

[0091] Embodiment 80: The method according to Embodiment 79, wherein the optical signal is selected from UV / Vis absorbance, fluorescence emission, and phosphorescence emission.

[0092] Embodiment 81: The method according to Embodiment 79, wherein the optical signal is substantially invariant due to the binding of the reporter particle to the analyte particle.

[0093] Embodiment 82: The method according to Embodiment 79, wherein the optical signal is generated by surface plasmon resonance ("SPR").

[0094] Embodiment 83: The method according to Embodiment 82, wherein the surface plasmon resonance is localized surface plasmon resonance.

[0095] Embodiment 84: The method according to any one of Embodiments 1 to 83, further comprising the step of removing background features.

[0096] Embodiment 85: The method according to Embodiment 84, further comprising the step of removing background features larger than a given maximum size.

[0097] Embodiment 86: The method according to either Embodiment 84 or 85, further comprising the step of removing background features smaller than a given minimum size.

[0098] Embodiment 87: The following: The process of downloading images, The process of reconstructing the image, The process of dilating the image, The method according to any one of embodiments 1 to 86, further comprising:

[0099] Embodiment 88: The method according to any one of Embodiments 1 to 87, further comprising one or more steps of border formation on one or more frames of a detector volume.

[0100] Embodiment 89: A method of the method according to any one of Embodiments 1 to 88, wherein the sample is a biological fluid.

[0101] Embodiment 90: The method according to Embodiment 89, wherein the sample is a diluted, concentrated, and / or pre-treated biological fluid.

[0102] Embodiment 91: The method according to either Embodiment 89 or 90, wherein in a particular embodiment the sample is derived from blood.

[0103] Embodiment 92: The method according to Embodiment 91, wherein the sample contains intact blood cells.

[0104] Embodiment 93: The method according to Embodiment 92, wherein the sample contains intact red blood cells.

[0105] Embodiment 94: The method according to any one of Embodiments 1 to 93, wherein the sample is analyzed under a microscope.

[0106] Embodiment 95: The method according to Embodiment 94, wherein the sample is analyzed with a microscope having dark-field imaging capabilities.

[0107] Embodiment 96: The method according to either Embodiment 94 or 95, wherein the microscope has bright-field imaging capability.

[0108] Embodiment 97: The method according to any one of Embodiments 94 to 96, wherein the microscope has an autofocus function.

[0109] Embodiment 98: The method according to any one of Embodiments 94 to 97, wherein the microscope includes a fluorescent illuminator.

[0110] Embodiment 99: The method according to any one of Embodiments 94 to 98, wherein the fluorescent illuminator includes a fly-eye lens.

[0111] Embodiment 100: The method according to any one of Embodiments 1 to 99, wherein the optical path length is 0.1 mm or less in thickness.

[0112] Embodiment 101: The method according to Embodiment 100, wherein the optical path length is 0.05 mm or less in thickness.

[0113] Embodiment 102: The method according to Embodiment 101, wherein the optical path length is 0.02 mm or less in thickness.

[0114] Embodiment 103: The method according to Embodiment 102, wherein the optical path length is 0.01 mm or less in thickness.

[0115] Embodiment 104: The method according to any one of Embodiments 1 to 103, wherein the sample is processed in a microfluidic apparatus.

[0116] Embodiment 105, also provided: • Image sensor and, • A screen with image display capabilities, • Microprocessor and • Memory and, Image analysis software, which is stored in memory and executed by a processor, has the function of analyzing data captured by an image sensor and the function of digitally classifying the data. Optionally, a communication interface and An apparatus or system for carrying out the method described in any one of embodiments 1 to 104, including the method described in any one of embodiments 1 to 104.

[0117] Furthermore, the following embodiments are also provided.

[0118] Embodiment 106: The apparatus according to Embodiment 105, further including a communication interface.

[0119] Embodiment 107: The apparatus according to Embodiment 106, wherein the communication function is wireless.

[0120] Embodiment 108: The apparatus according to either Embodiment 106 or 107, wherein the apparatus or system is capable of communicating with a smartphone or tablet computer.

[0121] Embodiment 109: The apparatus according to either Embodiment 106 or 107, wherein the apparatus or system is capable of communicating with a computer, smartphone, or tablet computer.

[0122] Embodiment 110: The apparatus according to any one of Embodiments 105 to 109, wherein the image sensor can operate as part of a dark-field microscope.

[0123] Embodiment 111: The apparatus according to any one of Embodiments 105 to 110, wherein the image sensor can operate as part of a bright-field microscope.

[0124] Embodiment 112: The apparatus according to any one of Embodiments 105 to 111, wherein the image sensor includes a camera.

[0125] Embodiment 113: The apparatus according to Embodiment 112, wherein the camera is a complementary metal-oxide-semiconductor (CMOS) camera.

[0126] Embodiment 114: The apparatus according to any one of Embodiments 105 to 113, further comprising a light source or other electromagnetic radiation source.

[0127] Embodiment 115: The apparatus according to Embodiment 114, wherein the light source or other electromagnetic source includes a light-emitting diode (LED).

[0128] Embodiment 116: The apparatus according to either Embodiment 114 or 115, wherein the light source or other electromagnetic source includes a fisheye lens.

[0129] Embodiment 117: The apparatus according to any one of Embodiments 105 to 116, further comprising an optionally removable sample chamber.

[0130] Embodiment 118: The apparatus according to any one of Embodiments 105 to 116, further comprising a reporter surface made of glass or polymer, on which reporter particles containing plasmon nanoparticles functionalized by a capture element are immobilized.

[0131] Embodiment 119: The apparatus according to Embodiment 118, further comprising a waveguide suitable for dark-field microscopy observation that contacts the opposite side of the reporter surface.

[0132] Embodiment 120: The apparatus according to either Embodiment 118 or 119, wherein the reporter particle is an optical reporter particle.

[0133] Embodiment 121: The apparatus according to Embodiment 120, wherein each fixed optical reporter particle is spatially resolvable.

[0134] Embodiment 122: The apparatus according to Embodiment 121, wherein the fixed optical reporter particles are randomly arrayed.

[0135] Embodiment 123: The apparatus according to Embodiment 121, wherein the fixed optical reporter particles are arrayed in a grid or substantially grid shape.

[0136] Embodiment 124: The apparatus according to any one of Embodiments 121 to 123, wherein each fixed optical reporter particle is resolvable as one pixel of a recording device.

[0137] Embodiment 125: The capture element is One or more nanobodies bonded to the analyte, One or more nucleotide sequences that bind to analytes, and Antibodies or fragments thereof that bind to analytes. An apparatus according to any one of embodiments 118 to 124, selected from the above.

[0138] Embodiment 126: The apparatus according to any one of Embodiments 105 to 125, wherein the apparatus or system can be used to carry out the method described herein for determining the presence or concentration of analytes in a sample.

[0139] Embodiment 127: The apparatus according to Embodiment 126, wherein the apparatus or system can be used to carry out the method described herein for determining the presence of an analyte in a sample.

[0140] Embodiment 128: The apparatus according to Embodiment 126, wherein the apparatus or system can be used to carry out the method described herein for determining the concentration of analyte in a sample.

[0141] Embodiment 129: The apparatus according to any one of Embodiments 126 to 128, wherein the analyte is an antibody.

[0142] Embodiment 130: The apparatus according to Embodiment 129, wherein the analyte is an antibody against a virus.

[0143] Embodiment 131: The apparatus according to Embodiment 130, wherein the analyte is an antibody against coronavirus.

[0144] Embodiment 132: The apparatus according to Embodiment 131, wherein the analyte is an antibody against SARS-CoV-2.

[0145] Embodiment 133: The apparatus according to any one of Embodiments 126 to 128, wherein the analyte is the antigen.

[0146] Embodiment 134: The apparatus according to any one of Embodiments 126 to 128, wherein the analyte is selected from viruses, archaea, and bacteria.

[0147] Embodiment 135: The apparatus according to Embodiment 134, wherein the analyte is a virus.

[0148] Embodiment 136: The apparatus according to Embodiment 136, wherein the analyte is a coronavirus.

[0149] Embodiment 137: The apparatus according to Embodiment 135, wherein the analyte is SARS-CoV-2.

[0150] Embodiment 138: The apparatus according to any one of Embodiments 126 to 128, wherein the analyte is a nucleotide sequence.

[0151] Embodiment 139: The device or system is as follows: Downloading images Reconstructing the image, and Dilating an image, An apparatus according to any one of embodiments 105 to 138, which can be used to carry out one or more of the following.

[0152] Embodiment 140: The apparatus or system is as follows: Downloading images Reconstructing the image, and Dilating an image, The apparatus according to Embodiment 139, which can be used to perform two or more of the following.

[0153] Embodiment 141: The apparatus or system is as follows: Downloading images Reconstructing the image, and Dilating an image, The apparatus described in Embodiment 140 is usable to carry out all of the above.

[0154] Embodiment 142: The apparatus according to any one of Embodiments 105 to 141, wherein the apparatus or system can be used to perform one or more border formation steps in an image.

[0155] Embodiment 143: The apparatus according to any one of Embodiments 105 to 142, wherein the apparatus or system is entirely contained within a single device.

[0156] Embodiment 144: The apparatus according to Embodiment 143, wherein the apparatus or system is entirely contained within a single portable device.

[0157] Embodiment 145: The apparatus according to either Embodiment 143 or 144, further comprising a case for stably positioning a smartphone, a sample chamber, and a light source in very close proximity to each other.

[0158] The embodiments disclosed herein are further combinable with respect to each other, provided that the combinations are not mutually exclusive.

[0159] Terms and Definitions Unless otherwise defined, all scientific and technical terms used herein have the same meanings as those commonly understood by those skilled in the art in the field to which this invention pertains.

[0160] As used herein, the following terms have the meanings set forth below.

[0161] When a range of values ​​is disclosed and the notation "n1~n2" is used (where n1 and n2 are numbers), unless otherwise specified, this notation is intended to include the numbers themselves and the range between them. This range may be integers between them, including the endpoints, or it may be continuous between them. For example, since carbon appears in integer units, the range "2~6 carbons" is intended to include 2, 3, 4, 5, and 6 carbons. Compare this to the range "1~3 μM (micromoles)". In this case, it is intended to include 1 μM, 3 μM, and all in between to any number of significant digits (e.g., 1.255 μM, 2.1 μM, 2.9999 μM, etc.).

[0162] When used herein, the term "approximately" is intended to acknowledge that the numerical value it modifies represents a value that can vary within an acceptable margin of error. Unless a specific margin of error, such as a standard deviation relative to the mean, is given in a figure or table of data, the term "approximately" should be understood to mean the range encompassing the given value, and the range that would be included if the numerical value were rounded up or down, taking significant figures into consideration.

[0163] As used herein, the term “accuracy,” either alone or in combination, refers to the degree of approximation of a reported or estimated value to a true value. An inaccurate measurement, observation, or estimate deviates from the true value. An accurate measurement, observation, or estimate does not deviate from the true value.

[0164] As used herein, either alone or in combination, the term “aggregate” is used to describe an assembly comprising at least one reporter particle and at least one analyte particle. In some embodiments, the term “aggregate” is used for an assembly comprising five or more total particles (reporter particle and analyte particle). In some embodiments, the term “aggregate” is used for an assembly comprising ten or more total particles. In some embodiments, the term “aggregate” is used for an assembly comprising twenty or more total particles. In some embodiments, the term “aggregate” is used for an assembly comprising fifty or more total particles.

[0165] As used herein, either alone or in combination, the term “analyte molecule” is used to describe molecules or particles whose presence or absence or quantity in a sample is initially unknown, and for which knowledge of their presence or absence or quantity in the sample may be useful. Examples of analyte molecules include biomolecules, e.g., peptides, proteins, cytokines, and prions; antibodies and their fragments; nucleic acids (DNA / RNA) and particles containing them, e.g., histones; small organic and bioinorganic molecules, e.g., carbohydrates, lipids, hormones, and metabolic intermediates and products; macromolecules, e.g., macrocycles; biopolymers (e.g., oligosaccharides, polyphenols, and plastics); and viruses, viral particles, and viral products (e.g., willokine).

[0166] As used herein, either alone or in combination, the term “analyte particle” is used to encompass the “analyte molecules” and larger particles described above, whose presence or absence or quantity in a sample is initially unknown and for which it may be useful to know their presence or absence or quantity in the sample. Examples of analyte particles include viruses, prokaryotes (including bacteria and archaea), and protists (including amoebas, choanoflagellates, ciliates, diatoms, dinoflagellates, Giardia, Plasmodium, and oomycetes).

[0167] Furthermore, analyte particles can be categorized as biomarkers, i.e., compositions and / or molecules or complexes of compositions and / or molecules that are associated with the biological state of an organism (e.g., diseased or non-diseased states) and can report the presence of disease, injury, or damage to cells or organisms. When such a marker is bound to an antibody or a fragment thereof, it can be called an antigen. Significant (e.g., normal and abnormal) levels of analyte particles disclosed herein and detected by aggregation assays will be known to those skilled in the art.

[0168] The term "analyte" can refer to a specific type of substance composed of analyte particles, such as codeine, leukotrienes, and PSA. The term "analyte" can also refer to the quantity or concentration of analyte particles. The term "analyte" can also refer to several analyte particles. In certain usages, as will be clear from the context, the term "analyte" can refer to a single analyte particle.

[0169] As used herein, either alone or in combination, the term “area detector” refers to a recording device capable of recording an image of a source, that is, a recording device capable of recording not only the incident optical signal intensity but also the optical signal source. Typical examples of area detectors are television cameras, digital SLR cameras, and cell phone cameras.

[0170] As used herein, either alone or in combination, the term “bonding” refers to a non-covalent interaction between a reporter particle and an analyte particle. In some embodiments, the bonding is due to hydrogen bonding. In some embodiments, the bonding is due to van der Waals interactions. In some embodiments, the bonding is due to electrostatic and / or dipole interactions. In some embodiments, the bonding is due to a combination of one or more of the above-described interactions. In some embodiments, the bonding corresponds to a dissociation constant of 1 mM or less. In some embodiments, the bonding corresponds to a dissociation constant of 1 μM or less. In some embodiments, the bonding corresponds to a dissociation constant of 1 nM or less.

[0171] As used herein, either alone or in combination, the term “binding isotherm” refers to the binding behavior of a reporter particle / analyte particle system. In systems where both the reporter particle and the analyte have single binding sites (and therefore aggregate formation is not possible in this case), the term “binding isotherm” simply refers to the ratio of bound reporter particles to total reporter particles. It will be understood that this ratio increases with increasing analyte particle concentration and eventually approaches 1, as almost all reporter particles bind to the analyte particles. This behavior will also be obtained when either the reporter particle or the analyte has single binding sites, because aggregation (which requires both reporter and analyte particles to have multiple bindings) is not possible. When both reporter and analyte particles have multiple binding sites, the number and particle size are also included in the binding isotherm.

[0172] As used herein, either alone or in combination, the term “binding site” refers to a feature of either a reporter particle or an analyte particle that is capable of forming a non-covalent bond with a binding partner and thus holds the particle and the binding partner in close proximity. In some embodiments, a binding site on a reporter particle will exclusively bind to a binding site on an analyte particle. In some embodiments, a binding site on an analyte particle will exclusively bind to a binding site on a reporter particle. A particular reporter particle and a particular analyte particle contain a single binding site. A particular reporter particle and a particular analyte particle contain two binding sites. A particular reporter particle and a particular analyte particle contain two or more binding sites. A particular reporter particle and a particular analyte particle contain two identical binding sites. A particular reporter particle and a particular analyte particle contain two non-identical binding sites. A particular reporter particle and a particular analyte particle contain two or more identical binding sites. Certain reporter particles and certain analyte particles contain two or more non-identical binding sites. In some embodiments, either the reporter particle or the analyte contains multiple binding sites. In some embodiments, the binding behavior of each of the multiple binding sites does not correlate with each of the remaining binding sites. In other words, the possibility of one binding site forming a non-covalent bond with a partner is not affected by the presence or absence of non-covalent bonds formed by other binding sites on the same particle.

[0173] As used herein, the term “binning,” either alone or in combination, means combining signals from two or more pixels into a single signal. Binning is usable when spatial resolution can be sacrificed to improve signal-to-noise ratio. For example, “2x2 binning” means grouping pixels into 2x2 squares and taking the sum of the signals from the pixels contained in each square.

[0174] As used herein, either alone or in combination, the term "biomolecule" includes, but is not limited to, peptides, proteins, nucleic acids, sugars, monosaccharides and polysaccharides, lipids, lipoproteins, whole cells, and any type of organic or bioinorganic molecule for which detection (either qualitative or quantitative) is desired.

[0175] As used herein, the term "camera" refers to an image sensor that records visual images, for example, as digital frames. A "megapixel camera" is a camera capable of recording one million or several million pixels per frame. Many smartphone cameras include 10 megapixels or more.

[0176] As used herein, the term “communication interface” means a means for transferring data from one device or system to another. Examples of wireless communication interfaces include those used in wireless devices such as mobile phones, such as cellular, Wi-Fi, and Bluetooth technologies.

[0177] As used herein, either alone or in combination, the term “clinical use” is used to describe methods for analyzing samples containing unknown concentrations of analyte particles. The term “clinical use” is intended to be distinguished from “non-clinical use.” Clinical use includes, but is not limited to, use in a medical setting for determining analyte particle concentrations in patients. As used herein, either alone or in combination, the term “non-clinical use” is used to describe methods for analyzing samples containing known concentrations of analyte particles. Non-clinical use includes, but is not limited to, sample measurements for determining the behavior of devices and methods at clearly defined analyte particle concentrations. Non-clinical use encompasses both pre-clinical and post-clinical use.

[0178] As used herein, the term “concentration,” either alone or in combination, means the amount of solute in a solution per unit volume of solution. Concentration can be specified in molar units, i.e., the number of moles of solute per liter of solution, or number concentration, i.e., the number of solute molecules per liter of solution, or alternatively, the number of solute particles per liter of solution. Molar and number concentrations are readily interchangeable. As used herein, the term “concentration” is extended to include systems other than the traditional definition of “solution,” such as molecules tethered to a solid carrier, and particles other than the traditional definition of “molecule,” i.e., systems containing viruses and microorganisms.

[0179] In this specification, the terms “detect” or “detect,” used alone or in combination, are used to describe a method for determining the presence, existence, or nature of analyte particles in a sample.

[0180] The term "divergence" refers to the deviation from the perpendicular included in the recording device. An idealized area detector recording device would only receive rays perpendicular to the detector's plane. A real area detector would tolerate rays arriving at a certain angle from the perpendicular. This characteristic can increase the signal-to-noise ratio (because the detector receives more rays), but in addition, it reduces the spatial resolution, depending on the magnitude of the allowed divergence angle, the pixel size of the area detector, and the distance between the area detector and the sample plane.

[0181] As used herein, either alone or in combination, the term “frame” means substantially the entirety of an image captured by a recording device. A particular camera captures one frame at a given point in time. A particular camera captures a series of frames over a period of time.

[0182] As used herein, either alone or in combination, the term “image” means a feature in a camera signal resulting from a specific object captured by a recording device. For example, an image of the moon can be captured by a camera mounted on a telescope, and an image of a customer can be captured by an ATM camera. In some contexts, the term “image” means the entire camera signal, regardless of the origin of the signal. The meaning of the term will become clear from the context.

[0183] As used herein, either alone or in combination, the term “incubate” is used to describe the process of exposing a reporter particle to a sample that may potentially contain an analyte molecule.

[0184] As used herein, either alone or in combination, the term “oblong” is used to describe a volume having unequal dimensions. Examples of oblong volumes include prisms or cylinders where the distance between end faces is either significantly longer or significantly shorter than the dimension parallel to the end faces. A further example of an oblong volume is an ellipsoid where one axis is either significantly longer or significantly shorter than the other.

[0185] As used herein, either alone or in combination, the term “optical path” is used to describe the path from the reporter particle to the detector.

[0186] As used herein, either alone or in combination, the terms “optical reporter particle” or synonymously “optical reporter” are used to describe reporter particles capable of reporting the presence or absence, or the amount or concentration of analyte particles, via an optical signal. The presence or absence of analyte particles in contact with an optical reporter particle (optionally, in certain assay schemes, other optical reporter particles may be used in combination) induces a change in the optical signal. An optical reporter particle bound to an analyte particle ("bound optical reporter particle") will emit a different signal than an optical reporter particle not bound to an analyte particle ("unbound optical reporter particle").

[0187] As used herein, either alone or in combination, the term “optical signal” is used to describe a signal originating from an optical reporter particle. The optical signal may be contained within or outside the visible region of the spectrum. The signal may, for example, • Wavelength of light, • Signal intensity, • Brightness, • The shape of the signal or spectrum, • Presence or absence of spectral bands, • Absorption coefficient of the absorption band, • λ of the cerebrospinal band max , · Quantum yield of emission band, or • Fluorescence anisotropy of emission bands It is possible.

[0188] In some embodiments, the optical signal of the reporter particle changes when an analyte particle binds to it. In some embodiments, the optical signal of the reporter particle remains unchanged even when an analyte particle binds to it.

[0189] As used herein, either alone or in combination, the term “pixel” refers to an area on an area detector, such as an image sensor, in which a signal can be measured independently of other pixels. An area detector is typically divided into pixels in a two-dimensional grid, with each pixel size and the number of pixels in each direction determined by the area detector manufacturer.

[0190] As used herein, the term “accuracy,” either alone or in combination, means an estimate of the error associated with a reported or estimated value. A low-accuracy measurement, observation, or estimate involves a high degree of uncertainty regarding the approximation of the numerical value to the actual value. A high-accuracy measurement, observation, or estimate involves a low degree of uncertainty regarding the approximation of the numerical value to the actual value. Accuracy can often be quantified using error bars on a graph or a range of values. For example, an estimate reported as 10.5 ± 0.1 suggests that the true value is very likely to be between 10.4 and 10.6, and that there is a small, though not zero, chance that the true value is outside this range.

[0191] The terms “protein,” “polypeptide,” “peptide,” and “oligopeptide” are used synonymously herein and include any composition comprising two or more amino acids linked together by peptide bonds. It will be understood that polypeptides may contain amino acids other than the 20 amino acids commonly referred to as the 20 natural amino acids. Furthermore, polypeptides may contain one or more amino acids (whether natural or unnatural) modified by any means known in the art, including terminal amino acids. Examples of polypeptide modifications include glycosylation or other post-translational modifications. Modifications that may be present in the polypeptides of this disclosure include, but are not limited to, acetylation, acylation, ADP-ribosylation, amidation, covalent bonding of flavin, covalent bonding of heme moiety, covalent bonding of polynucleotides or polynucleotide derivatives, covalent bonding of lipids or lipid derivatives, covalent bonding of phosphotidylinositol, crosslinking, cyclization, disulfide bond formation, demethylation, covalent crosslinking, cystine formation, pyroglutamate formation, formylation, γ-carboxylation, glycation, glycosylation, GPI anchor formation, hydroxylation, iodization, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer RNA-mediated addition of amino acids to proteins, such as arginylation and ubiquitination.

[0192] As used herein, either alone or in combination, the term “qualitative analysis” is used to describe a method for determining the presence or absence of analyte particles in a sample. In some embodiments, the qualitative analysis method reports the presence or absence of a single analyte particle in the sample. In some embodiments, the qualitative analysis method improperly reports the absence of analyte in a sample containing analyte molecules at levels below a certain threshold. In some embodiments, the qualitative analysis method improperly reports the absence of analyte in a sample containing analyte particles at levels below a certain threshold.

[0193] As used herein, either alone or in combination, the term "quantitative analysis" is used to describe a method for determining the amount of analyte particles in a sample.

[0194] As used herein, either alone or in combination, the term “recording device” means a device for recording optical signals. In certain embodiments, the optical signals are converted into electrical signals. In certain embodiments, the recording device is a charge-coupled ("CCD") device. In certain embodiments, the recording device is a complementary metal-oxide-semiconductor ("CMOS") device.

[0195] As used herein, either alone or in combination, the term “reporter particle” is used to describe a molecule, supramolecular particle, or nanoscale particle that binds to a target analyte particle and, upon binding to the analyte, forms an aggregate containing multiple reporter particles.

[0196] The term "reporter" can refer to a type of substance composed of reporter particles. The term "reporter" can also refer to the quantity or concentration of reporter particles. In certain usages, as will be clear from the context, the term "reporter" can refer to a single reporter particle.

[0197] As used herein, either alone or in combination, the term “reporter volume” is used to describe the volume of the measuring device in which the reporter particle is located. The reporter volume may be substantially identical to the sample compartment, or it may be smaller. In certain embodiments, the dimensions of the reporter volume parallel to the optical path to the reporter particle will be small. In certain embodiments, the reporter volume will constitute a single layer.

[0198] As used herein, the term “sample,” either alone or in combination, is used to describe compositions containing the analyte particles of interest. Samples are often in a fluid state, such as an aqueous solution. Samples can be chemical or biological. Examples of biological samples include blood, plasma, or water derived from the source of interest, or extracts derived from plant, animal, or human tissue samples. Chemical samples may be those that do not contain biological substances, such as water samples containing petrochemical or industrial waste. Biological samples taken from living organisms include, but are not limited to, blood, serum, plasma, urine, mucus, saliva, sputum, feces, and other physiological secretions, as well as tissue extracts and / or any other component of the body that may contain the target particles of interest. Other similar specimens, such as cell or tissue cultures or culture broths, are also included.

[0199] The biological sample may be fresh or stored (for example, blood or blood fractions stored in a blood bank). The biological sample may be a body fluid specially obtained for the assay of the present invention, or a body fluid obtained for other purposes that can be subsampled for the assay of the present invention. In one embodiment, the biological sample is whole blood. Whole blood may be obtained from a subject using standard clinical procedures. In another embodiment, the biological sample is plasma. Plasma may be obtained from a whole blood sample by centrifugation of anticoagulated blood. Such a process provides a buffy coat of leukocyte components and a plasma supernatant. In another embodiment, the biological sample is serum. Serum may be obtained by centrifugation of a whole blood sample collected in an anticoagulant-free tube. The blood may be coagulated before centrifugation. The yellowish-red fluid obtained by centrifugation is serum. In another embodiment, the sample is urine. Samples may be pretreated by dilution in a suitable buffer solution as required, heparinized, concentrated if desired, or fractionated by one of several methods, including but not limited to ultracentrifugation, high-performance liquid chromatography (FPLC) fractionation, or precipitation of apolipoprotein B-containing proteins with dextran sulfate, or by other methods. Any of several standard aqueous buffer solutions at physiological pH, such as phosphate buffer or Tris buffer, can be used.

[0200] As used herein with respect to the binding phenomenon, the term "saturation" refers to a state in which almost all reporter particles are bound to the analyte particles. A characteristic of the saturated state is that an increase in the concentration of analyte particles results in a small increase in the degree of binding of reporter particles.

[0201] As used herein, the term “smartphone” means a handheld personal computer equipped with a mobile operating system and an integrated mobile broadband cellular network connection for voice, SMS, and internet data communication, typically Wi-Fi.

[0202] As used herein, the terms “tablet computer” or “tablet” typically mean a thin, flat, portable personal computer having a mobile operating system, an LCD touchscreen display, a rechargeable battery, and a wireless (optionally cellular) communication interface.

[0203] Aggregate formation To facilitate aggregate formation, the reporter particle contains multiple binding sites, each capable of recognizing and binding to one of the multiple binding sites on the target analyte particle. Since both the reporter particle and the analyte contain multiple binding sites, both can simultaneously bind to more binding partners to form a multinuclear aggregate. The requirements of this design are (a) that a single analyte particle can simultaneously bind to two or more reporter particles, and (b) that a single reporter particle can simultaneously bind to two or more analyte particles.

[0204] The principle of this detection system is illustrated in Figures 1(a) to 1(d). The target analyte particle contains two identical binding sites, schematically represented in Figure 1(a) as an oval shape mounted on two triangles. The analyte particle is exposed to two reporter particles drawn above the reaction arrows, each containing a triangular binding site. The analyte particle and the two reporter particles combine to form a supramolecular ternary 1:2 analyte particle:reporter particle complex. Importantly, the two reporter particles become close together by the formation of a supramolecular aggregate. Figure 1(b) shows a similar system. The difference is that the analyte particle has two different binding sites (triangular and rectangular), and the medium contains two types of reporter particles (containing triangular and rectangular binding sites).

[0205] Figure 1(c) details the system of Figure 1(a), wherein the system contains reporter particles having two identical (triangular) binding sites, each of which can bind to a binding site in an analyte particle. The reaction product contains two reporter particles and two analyte particles. However, since both the reporter particles and analyte particles have free binding sites, additional reporter particles and analyte particles can be further bound to the supramolecular structure.

[0206] Figure 1(d) is based on the system in Figure 1(b), in that the system contains reporter particles having two different (triangular and rectangular) binding sites, each of which can bind to binding sites in analyte particles. Similar to the system in Figure 1(c), the disclosed product contains two reporter particles and two analyte particles, and additional reporter and analyte particles can further bind to the supramolecular structure.

[0207] Details of the design principle are shown in Figures 2(a) and 2(b). Here, each reporter particle has four binding sites and is intended to bind to an analyte particle having two binding sites. Figure 2(a) shows a reporter particle that, with this design, simultaneously binds to four analyte particles, each having two identical binding sites. Each of the analyte particles is then bound to an additional reporter particle. Each of these four additional reporter particles can then bind to three additional analyte particles, and it will be apparent that this forms an assembly of five reporter particles and eight analyte particles in total. This process (not shown for brevity) can be further expanded by binding to further analyte particles.

[0208] The principle can be expanded by introducing a second reporter particle with different binding sites. Figure 2(b) shows a pair of non-identical reporter particles with different binding sites, intended to bind to an analyte particle at two different binding sites. The assembly shown on the right is nucleated by the formation of a 1:4 aggregate of the first reporter particle and four analyte particles, each bound at one of two binding sites. Each of the analyte particles is then bound to one of the second type of reporter particle. As before, each of these four additional reporter particles can then bind to three additional analyte particles, forming an assembly of five reporter particles (one of the first type and four of the second type) and eight analyte particles in total. This process can also be further expanded by the binding of further analyte particles.

[0209] The presence of analyte particles facilitates the assembly of aggregates containing several reporter particles. For simplicity, assuming that chromophores on reporter particles do not interact with each other, the responses of multiple reporter particles in a single aggregate will be additive. That is, two reporter particles in an assembly will provide twice the spectral signal of a single reporter particle. Furthermore, for the purposes of this disclosure, an assembly containing two or more reporter particles will necessarily be at least twice the size of a single reporter particle.

[0210] The binding phenomena of simpler systems in which reporter particles and analyte particles each possess a single binding site and can only form 1:1 complexes have been extensively studied and modeled in chemistry and biochemistry. The degree of binding between two particles and the formation of 1:1 complexes are governed by the strength of intermolecular interactions. Given a fixed concentration of all reporter particles, the concentration of unbound reporter particles asymptotically approaches zero as the analyte particle concentration increases.

[0211] If either the reporter particle or the analyte particle contains multiple binding sites, it is possible to form complexes with stoichiometric ratios greater than 1:1. Assuming that the multiple binding sites operate independently, mathematical modeling of the binding behavior remains manageable. For example, a reporter with three equivalent binding sites can form 1:1, 1:2, and 1:3 reporter:analyte complexes. Furthermore, under the condition that 90% of all reporter binding sites are occupied by analyte particles (and 10% of all reporter binding sites are not occupied by analyte particles), 99% of the reporter particles will bind to at least one analyte. From the occupancy rate of each reporter binding site, the proportions of various types (i.e., free reporter particles, as well as 1:1, 1:2, and 1:3 complexes) can be statistically estimated.

[0212] If both the reporter particle and the analyte particle contain multiple binding sites, the mathematical prediction of the proportion of each possible complex will become more complicated and less accurate. Binding behavior can be predicted by utilizing certain simplifications and assumptions. More practically, a calibration curve can be constructed from the analysis of solutions at known analyte particle concentrations. Such calibration curves can be obtained from either a prototype device or a specific device in the field. Furthermore, in certain embodiments, the calibration curve can be re-determined after repeated use of the device or after extended service time, especially when the reporter particle degrades over time.

[0213] Reporter particle The measurement device and system include a reporter particle, and the methods disclosed herein utilize it. The requirements for the reporter particle are: (a) multiple binding sites for binding to multiple analyte particles; (b) the ability to provide an optical signal to enable imaging; and (c) being large enough to allow imaging of individual reporter particles. Thus, the term “reporter particle” may encompass assemblies that may be larger than those encompassed by the alternative definition of “molecule.” The reporter particle provides an optical signal in the presence and optionally in the absence of analyte particles.

[0214] A reporter particle includes multiple binding sites so that a single reporter particle can bind to multiple analyte particles. In certain embodiments, a reporter particle includes multiple identical binding sites, each of which is capable of binding to one of multiple identical binding sites on an analyte particle. In certain embodiments, a reporter particle includes multiple substantially identical binding sites, each of which is capable of binding to one of multiple identical or substantially identical binding sites on an analyte particle. In certain embodiments, a reporter particle includes multiple non-identical binding sites, each of which is capable of binding to multiple identical, substantially identical, or non-identical binding sites on an analyte particle.

[0215] The binding sites of reporter particles can be provided by different types of molecular functionality. Reporter particles may include antibodies (or fragments thereof), nucleic acids, proteins, and peptides, all of which can be chemically or biochemically modified and all can provide binding interactions with analyte particles. Modern immunochemical methods enable the synthesis of reporter particles suitable for the methods of this disclosure. Antibodies can be produced for various target analyte particles, and for larger analyte particles in particular, different antibodies can be identified that can bind to different regions of the analyte particle and simultaneously bind to the analyte particle. Alternatively, reporter particles may contain synthetic motifs, such as those designed by host / guest or supramolecular chemistry and / or synthesized by organic chemistry that can bind to a particular analyte particle.

[0216] The reporter particle provides an optical signal in the presence and optionally in the absence of binding. In some embodiments, the optical signal is provided by ultraviolet-visible ("UV / vis") absorption. In some embodiments, the optical signal is provided by fluorescence or phosphorescence emission. In certain embodiments, the optical signal of the reporter particle is substantially invariant when one or more analyte particles are bound. In certain embodiments, the optical signal of the reporter particle is substantially invariant when a single analyte particle is bound, but changes when multiple analyte particles are bound. In certain embodiments, the optical signal of the reporter particle changes when one or more analyte particles are bound. In some embodiments, the change in the optical signal is a change in the extinction coefficient. In some embodiments, the change in the optical signal is a change in the quantum yield. In some embodiments, the change in the optical signal is a change in the absorption wavelength. In some embodiments, the change in the optical signal is a change in the emission wavelength. In some embodiments, the change in the optical signal is a change in fluorescence anisotropy.

[0217] The reporter particle may contain a chromophore. In certain embodiments, the chromophore is covalently attached to the reporter particle; alternatively, it may be attached to the reporter particle via functionalization, for example, on the surface of a quantum dot or plasmon nanoparticle. In certain embodiments, the chromophore absorbs electromagnetic radiation. In certain embodiments, the chromophore absorbs electromagnetic radiation in a spectral region selected from the visible and ultraviolet. Alternatively, the chromophore may scatter electromagnetic radiation. In certain embodiments, the chromophore is luminescent. In certain embodiments, the chromophore is fluorescent. In certain embodiments, the chromophore is phosphorescent.

[0218] In some embodiments, the reporter particle is large enough to allow imaging of individual reporter particles without further modification. In some embodiments, the reporter particle may be a chimeric particle comprising a biochemical portion and a synthetic portion. Examples include antibody-functionalized plasmon nanoparticles or quantum dots and nucleotide-functionalized plasmon nanoparticles or quantum dots. The synthetic portion can provide the optical signal and / or size required to allow imaging of individual reporter particles. In some embodiments, the reporter particle comprises nanoparticles or quantum dots. In some embodiments, the nanoparticles or quantum dots are conjugated to a binding site via covalent bonds. In some embodiments, the nanoparticles or quantum dots are conjugated to a binding site via ester or amide bonds. In some embodiments, the nanoparticles or quantum dots are conjugated to a binding site via thioether or thiol ester bonds.

[0219] object properties This disclosure measures the properties of an aggregate that can provide information relating to the degree of aggregation and, consequently, information relating to the presence or concentration of analytes. Therefore, the term “object properties” means properties of the aggregate relating to the size of the aggregate. In some embodiments, object properties relate to the median size of the aggregate. In some embodiments, object properties relate to the average size of the aggregate. In some embodiments, object properties relate to the RMS size of the aggregate. In some embodiments, object properties are directly proportional to the (median / average / RMS) size of the aggregate. In some embodiments, object properties are monotonically related to the (median / average / RMS) size of the aggregate. In some embodiments, the relationship between object properties and the (median / average / RMS) size of the aggregate can be estimated using a calibration curve. In some embodiments, the relationship between object properties and the (median / average / RMS) size of the aggregate can be estimated using the Newton-Raphson least squares fitting method. In some embodiments, the object properties are selected from the (median / average / RMS) size of the aggregate as directly observed or estimated, the (median / average / RMS) perimeter of the aggregate as directly observed or estimated, the shape of the aggregate, the color of the aggregate, the brightness of the aggregate, the reflectance of the aggregate, and the emission (fluorescence / phosphorescence) of the aggregate. In some embodiments, the estimation of the particle size (median / average / RMS) is determined from a combination of two or more such object properties.

[0220] Image processing In certain embodiments, the detector volume frame is evaluated "raw" without any further image processing. In certain embodiments, the detector volume frame is filtered to separate noise, errors, or background features from the true particle aggregate. In certain embodiments, the detector volume frame is filtered to separate errors from the true particle aggregate. In certain embodiments, the detector volume frame is filtered to separate noise from the true particle aggregate.

[0221] Inert reporter particles The methods described herein address a specific fraction of inert reporter particles, where the optical signal from these inert reporter particles is either absent or substantially different from that of bulk reporter particles. This behavior may result from (a) the reporter particles being unable to bind to the analyte particles, or (b) the reporter particles being able to bind to the analyte particles but failing to produce an optical signal or producing an optical signal substantially different from that of the rest of the reporter particles.

[0222] The presence of both types of inactive reporter particles will not complicate the aggregation-based assays of this disclosure any more than other methods. In the first case, reporter particles that do not bind to analyte particles will simply remain as individual particles in the assay without participating in the aggregate formation process. In the second case, since the role of the optical signal in aggregation-based assays is to image the aggregate and estimate its size, it is desirable to avoid disruption of the optical signal by the binding of reporter particles to analyte particles. For these reasons, reporter particles that maintain an unchanged optical signal even when bound to analyte particles may be preferable. In contrast, if the change in the optical signal is not uniform among reporter particles, many assays that rely on reporter / analyte binding will not perform well or at all.

[0223] In certain embodiments of this disclosure, the inactive reporter particles include aggregated individual proteins (including antibodies), peptides or proteins that have not been properly folded, and peptides or proteins that contain improper residues or missing chromophores.

[0224] The number of inactive reporter particles can be maintained substantially constant throughout the operating life of the measuring device, especially if the inactive reporter particles have a missing composition. However, the number of inactive particles may also increase during the operating life of the measuring device due to chemical degradation of the reporter particles, specifically photochemical degradation caused by repeated high-intensity exposure to a light source, or aggregation of proteins that form part of the reporter particles.

[0225] Inert reporter particles can be identified by a change in their optical behavior, specifically by either their inability to generate an optical signal upon exposure to analyte particles, or by the generation of an optical signal significantly different from that of bulk reporter particles upon exposure to analyte particles.

[0226] Signal processing In a particular embodiment, each of one or more frames of the detector volume consists of an array of pixels. Each of one or more frames can be processed into a digital record using methods known in the art.

[0227] In certain embodiments, the recording of one or more images includes intensity information for each pixel. In some embodiments, the intensity information for each pixel is determined by an analog-to-digital converter ("ADC") that converts the intensity information observed at each pixel of the detector into a digital format. The dynamic range of the intensity information can correlate with the properties of the ADC; for example, the use of a 12-bit ADC allows for a range of 0 to (2) for each pixel. 12 -1) or an intensity value in the range of 0 to 4095 is provided. In some embodiments, a color signal is obtained. In some embodiments, the color signal is converted to grayscale. In some embodiments, a grayscale signal is obtained.

[0228] Processing of computer recordings may include steps to remove false noise. In certain embodiments, features in computer recordings smaller than a user-supplied threshold are considered noise and removed from processing. In certain embodiments, features in computer recordings larger than a user-supplied threshold are considered noise and removed from processing. In certain embodiments, features in computer recordings not as strong as a user-supplied threshold are considered noise and removed from processing.

[0229] The processing of computer recordings may include the step of identifying features in the image that correspond to aggregates. The computer can identify the boundaries between features and the background. These boundaries in a frame can be identified by positioning regions in the frame using large light-dark gradients that correspond to the transition between the (bright) aggregate and the (dark) background.

[0230] The processing of computer recordings may include the step of calculating the area of ​​each feature on the frame. The processing may further include the step of calculating the size of each aggregate corresponding to each feature on the frame.

[0231] The processing of computer records may also include a step of analyzing information regarding the size of each aggregate, from which the analyte concentration can be estimated. In some embodiments, the analysis may be based on the properties of the coupled isotherm of the reporter particle / analyte particle pair. In some embodiments, the analysis may be based on a comparison with a control having a predetermined amount of reporter and analyte particles. In some embodiments, the comparison may be made using the average aggregate size in the solution. In some embodiments, the comparison may be made using the entire observed aggregate size distribution. In some embodiments, the comparison is performed using least squares fitting. In some embodiments, the least squares fitting is weighted by the estimation error of each particle size estimate.

[0232] A flowchart for performing the analysis is provided in Figure 20. Briefly, the sample is incubated with detector particles in solution, and then time frames of the solution are recorded. Images of individual particles and aggregates are identified. Individual images are retained or discarded using selection criteria that depend on the specific assay. Background subtraction is achieved by masking the selected images and subtracting the resulting pixels from the entire frame. Then, individual images particularly suitable for analysis are selected, and hard edges are generated using an established algorithm. Next, particle size is estimated from the binary images. Then, feedback analysis is applied to the original frames to identify and remove any artifacts. From the properties determined from the set of selected and quantified images (average size, perimeter, count, etc.), the presence and / or concentration of analytes can be determined.

[0233] Purpose The aggregation assay methods, systems, and devices disclosed herein are useful in a variety of fields and applications. Specifically, aggregation assays will be useful in “field” or portable environments. For example, aggregation assays will be useful for medical evaluation and diagnosis and pathogen detection, especially in remote areas, i.e., areas with inadequate or difficult-to-access services (e.g., due to violent conflict), areas affected by infectious diseases, and other areas where access to conventional assay equipment and / or experts is limited. They will also be useful in hospitals or clinics, or in home visit settings where they can be performed at the point of care or bedside.

[0234] Aggregation assays will be useful in veterinary settings as well as medical settings, whether in a veterinary practice room, a pasture or farm, or wherever animals requiring testing are located. They can also be used in horticultural or agricultural applications to test for plant or soil pathogens or symbiotic microorganisms, or to detect other target genotypes and phenotypes.

[0235] Aggregation assays can also be used to test water for contamination by bacteria, algae, or fungi, or their toxic products, by petroleum or its products and by-products, and by industrial waste. Such assays will be useful for food safety testing and agricultural applications, such as testing for pathogens, toxins, impurities, contaminants, and pests, either in-situ or at treatment facilities.

[0236] Assay Many types of biochemical assays are adaptable to the aggregation assay schemes disclosed herein. Examples include immunoassays in which an antigen is captured and bound by an antibody or fragment thereof; hybridization assays in which one or more DNA or RNA segments complementary to the target analyte particle DNA / RNA are used to capture the analyte particle; and ligand binding assays in which a binding partner for a receptor, enzyme, or other protein, or vice versa, is used as a capture agent for a partner analyte particle (e.g., a protein or fragment thereof).

[0237] In some embodiments, heterogeneous assay protocols utilizing two or more types of reporter particles are used. The presence of multiple types of reporter particles can simplify the synthesis and aggregation challenges. Furthermore, since this method images the entire aggregate containing multiple reporter particles, only a single type of reporter particle needs to provide an optical signal to provide an image of the aggregate.

[0238] The methods disclosed herein can be used to identify target phenotypic or genotypic conditions associated with clinically diagnosed disease conditions. Such disease conditions include, for example, cancer, cardiovascular disease, inflammatory diseases, autoimmune diseases, neurological disorders, infectious diseases, and pregnancy-related disorders. Alternatively, health conditions can be detected using markers.

[0239] Cancer phenotypes are included in several aspects of the present invention. Examples of cancers herein, but are not limited to, breast cancer, skin cancer, bone cancer, prostate cancer, liver cancer, lung cancer, brain cancer, laryngeal cancer, gallbladder, pancreas, rectum, parathyroid gland, thyroid gland, adrenal gland, nerve tissue, head and neck, colon, stomach, bronchi, kidney, basal cell carcinoma, squamous cell carcinoma of both ulcerative and papillary types, metastatic skin cancer, osteosarcoma, Ewing's sarcoma, reticular cell sarcoma, myeloma, giant cell tumor, small cell lung tumor, non-small cell lung cancer, gallstones, islet cell tumor, primary brain tumor, acute and chronic lymphocytic and granulocytic tumors, hairy cell tumor, adenoma, hyperplasia, medullary carcinoma, pheochromocytoma, mucosal neuroma, enteric ganglion neuroma, hyperplastic corneal neuroma, Marfan syndrome-like tumor, Wilms' tumor, seminomas, ovarian tumors, leiomyoma tumors, cervical dysplasia and in Examples include situ carcinoma, neuroblastoma, retinoblastoma, soft tissue sarcoma, malignant carcinoid, focal skin lesions, mycosis fungoides, rhabdomyosarcoma, Kaposi's sarcoma, osteogenic and other sarcomas, malignant hypercalcemia, renal cell tumors, polycythemia vera, adenocarcinoma, glioblastoma multiforme, leukemia, lymphoma, malignant melanoma, epidermal carcinoma, and other carcinomas and sarcomas.

[0240] Cardiovascular diseases may be included in other applications of the present invention. Examples of cardiovascular diseases include, but are not limited to, congestive heart failure, hypertension, arrhythmias, atherosclerosis, cholesterol, Wolff-Parkinson-White syndrome, long QT syndrome, angina pectoris, tachycardia, bradycardia, atrial fibrillation, ventricular fibrillation, myocardial ischemia, myocardial infarction, cardiac tamponade, myocarditis, pericarditis, arrhythmogenic right ventricular dysplasia, hypertrophic cardiomyopathy, Williams syndrome, valvular heart disease, endocarditis, bacterial diseases, pulmonary atresia, aortic stenosis, Raynaud's disease, cholesterol embolism, Wallenberg syndrome, Hippel-Lindau disease, and telangiectasia.

[0241] Inflammatory and autoimmune diseases may be included in other embodiments of the present invention. Examples of inflammatory and autoimmune diseases include, but are not limited to, rheumatoid arthritis, nonspecific arthritis, inflammatory laryngeal disease, inflammatory bowel disorder, psoriasis, hypothyroidism (e.g., Hashimoto's hyperthyroidism), colitis, type 1 diabetes mellitus, pelvic inflammatory disease, inflammatory central nervous system disease, temporal arteritis, polymyalgia rheumatica, ankylosing spondylitis, polyarteritis nodosa, Reiter's syndrome, scleroderma, systemic lupus, and lupus erythematosus.

[0242] The compositions and methods disclosed herein will be useful for detecting viruses. As shown elsewhere, viruses include surface macromolecules, generally proteins, that are involved in the entry of the virus into cells. These surface macromolecules include recognition elements that interact with corresponding features on the invaded cell. A well-known example of such surface macromolecules is the spike protein of coronaviruses. These surface molecules can be utilized as binding sites for suitably functionalized reporter particles.

[0243] The methods and compositions of the present invention also include adenovirus, Bordetella pertussis, Chlamydia pneumoniae virus, Chlamydia trachomatis, cholera toxin, cholera toxin β, Campylobacter jejuni, cytomegalovirus, diphtheria toxin, Epstein-Barr NA, Epstein-Barr EA, Epstein-Barr VCA, and Helicobacter pylori. Pylori), Hepatitis B virus (HBV) core, Hepatitis B virus (HBV) envelope, Hepatitis B virus (HBV) surface (Ay), Hepatitis C virus (HCV) core, Hepatitis C virus (HCV) NS3, Hepatitis C virus (HCV) NS4, Hepatitis C virus (HCV) NS5, Hepatitis A virus, Hepatitis D virus, Hepatitis E virus (HEV) orf2 3KD, Hepatitis E virus (HEV) orf2 6KD, Hepatitis E virus (HEV) orf3 3KD, Human immunodeficiency virus (HIV)-1 p24, Human immunodeficiency virus (HIV)-1 gp41, Human immunodeficiency virus (HIV)-1 gp120, Human papillomavirus (HPV), Herpes simplex virus HSV-1 / 2, Herpes simplex virus HSV-1 gD, Herpes simplex virus HSV-2 gG, Human T-cell leukemia virus (HTLV)-1 / 2, Influenza A virus, Influenza A H3N2 virus, Influenza B virus, Leishmania donovani, Lyme disease virus, Mumps virus, M. pneumoniae virus, M. tuberculosis, Parainfluenza 1 virus, Parainfluenza 2 virus, Parainfluenza 3 virus, Poliovirus, Respiratory syncytial virus (RSV), Rubella virus, Measles virus, Streptolysin O, Tetanus toxin, T. pallidum 15kd, T. pallidum p47, T. cruzi (T.We can provide laboratory information on markers for infectious diseases, including markers for Toxoplasma (cruzi), Toxoplasma species, and varicella-zoster virus.

[0244] Furthermore, it will be evident that the aforementioned recognition feature, which enables aggregation-based detection of viruses, is also applicable to larger organisms such as archaea, bacteria, and other microorganisms. For example, bacteria often contain surface glycoproteins that can be used as recognition sites.

[0245] The methods disclosed herein can be used to detect gene mutations. Gene mutations herein include, but are not limited to, one or more substitutions, inversions, insertions, deletions, or mutations of nucleotide sequences (e.g., DNA and RNA) and proteins (e.g., peptides and proteins), one or more microdeletions, one or more rare alleles, polymorphisms, single nucleotide polymorphisms (SNPs), large gene polymorphisms such as inversions and translocations, differences in abundance and / or copy number of one or more nucleotide molecules (e.g., DNA) (e.g., copy number variants CNVs), trisomy, monosomy, and genomic rearrangements. In some embodiments, gene mutations may be associated with the metastasis, presence, absence, and / or risk of disease such as cancer, pharmacokinetic variability, drug toxicity, adverse events, recurrence, and / or the presence, absence, or risk of organ transplant rejection in subjects. For example, copy number variations in the HER2 gene may affect whether breast cancer patients respond to Herceptin treatment. Similarly, detecting an increase in the copy number of chromosome 21 (or chromosomes 18 or 13 or sex chromosomes) in the blood of a pregnant woman can be used as a non-invasive diagnostic tool for Down syndrome (or Patau syndrome or Edwards syndrome) in a prenatal child. Another example is the detection of alleles of transplanted organs that are not present in the recipient genome; monitoring the frequency or copy number of these alleles can identify signs of potential organ rejection.

[0246] Measurement devices and systems The aggregation assay methods described herein utilize a measuring device or system, both of which include components necessary for the analysis of the sample. The measuring device includes a sample compartment into which the sample is introduced, either by direct addition of the sample or by insertion of a cuvette or slide containing the sample itself. The sample compartment further provides a component containing a reporter particle having the function of binding to the target analyte particle to form an aggregate. In designs relying on luminescence, the measuring device provides an illumination device for exciting the chromophores contained in the reporter particle. The measuring device includes a recording device (e.g., an image sensor, such as a digital camera) for detecting and recording the optical signal of the reporter particle. Finally, the measuring device may include additional components, such as means for controlling the operation, a device for displaying or reporting the analysis results, and an interface with an external computer. The existence of various components and their custom versions may vary depending on the design of the measuring device.

[0247] The system or device as a whole may be equipped with a mount that changes the orientation of the device for convenient sample addition or removal. The system may be linked with a mobile computing device, which may be a smartphone, handheld computer, tablet computer, or similar portable computing device. In some examples, the mobile computing device includes all the necessary components, such as a display, processor, memory, and program instructions stored in memory and executable by the processor, to enable highly automated execution of processes such as (i) sample introduction, (ii) optical excitation, (iii) optional sample pre-screening to evaluate sample quality and optimal exposure time, recording of frames by a recording device, (iv) reduction of detector bias as required, (v) digitization of the detector signal, (vi) recording of the digital signal to non-volatile memory, (vii) recycling of the detector as required, and (Viii) processing of the digital signal. The functionality may further include determining the results of the aggregation assay and communicating those results to the end user in a visible manner.

[0248] Using smartphones or other mobile computing devices as detection instruments for aggregation assays enables the execution of assays in the field, i.e., outside of a laboratory, through inexpensive, portable, and multifunctional systems. Applications include point-of-care diagnostic systems for measuring viral load, nutritional status, disease biomarkers, or environmental contaminants without the need to transport samples to a central laboratory. Such tests can be performed in private homes, law enforcement facilities, global health facilities, and medical clinics. Since mobile computing devices are internet-connected, it will be possible to combine sensor data with patient information and geographical location. The ability to connect to external computing facilities can be provided for data interpretation, geographical and demographic mapping, database construction and maintenance, and the delivery of notifications to remote medical professionals and relevant authorities. Compact, field-operable digital measurement devices will eliminate the need for skilled technicians in laboratories from the assays. Instead, these assays can be performed by anyone thanks to the size and affordability of the detection systems.

[0249] Sample compartment The measuring device provides a sample compartment suitable for introducing the target sample. Measuring devices utilizing optical measurement techniques will benefit from oblong sample compartments with short dimensions. The optical signal path from the reporter particle to the recording device will be aligned parallel to the short dimensions. This arrangement will minimize absorption and dispersion of the optical signal, which may be problematic with longer optical paths. This measure allows for the use of either prismatic or cylindrical sample compartments.

[0250] Reporter Volume The sample compartment includes a component called a reporter volume, which contains reporter particles. This component avoids the need to add reporter particles to the sample under consideration and instead allows for the recycling of the reporter particles. In some embodiments, the reporter volume is defined by a physical enclosure that holds the reporter particles within itself. The physical enclosure may be porous to allow analytes to flow into the reporter volume and come into contact with the reporter particles. In some embodiments, the reporter volume is not defined by a physical enclosure. Instead, other means can be provided for holding the reporter particles within the reporter volume.

[0251] The degree of optical path overlap can be estimated from a few parameters defining the receptor volume, including the specific distribution of reporter particles (random, semi-random, aggregated, ordered), the concentration and effective size of the reporter particles, and the thickness of the reporter volume. In certain embodiments of this disclosure, substantially the entire optical path between the reporter particle and the recording device does not encounter other reporter particles. In certain embodiments, substantially the entire optical path between the reporter particle and the recording device encounters at most one other reporter particle.

[0252] In some embodiments, the reporter volume is thin enough to allow a single layer of reporter particles. In this design, optical path overlap is impossible because all reporter particles are substantially located in a plane perpendicular to the optical path and parallel to the recording device.

[0253] Recording devices and microscopes A recording device is provided for recording an optical signal from a reporter particle. In certain embodiments, the optical signal from the reporter particle passes through a transparent window in the sample compartment. In certain embodiments, the recording device may be an image sensor, such as a camera. For example, a CMOS (complementary metal-oxide-semiconductor) camera is useful because each pixel can be read individually, and also because CMOS cameras consume very little power, thus lasting longer than when used as part of a device in the field. Almost all smartphone cameras have CMOS cameras, many with resolutions exceeding 10 megapixels, and are therefore useful for the methods, systems, and devices disclosed herein.

[0254] In certain embodiments of this disclosure, the recording device enables observation of one or more signals from a sample compartment. In certain embodiments, each of the multiple signals originates from a different region of the sample compartment. In certain embodiments, each of the multiple signals originates from a single pixel of a regular geometric grid that extends throughout the sample compartment.

[0255] In certain embodiments, the pixels of the recording device are arranged in a rectangular or square array. In certain embodiments, the pixels of the recording device are arranged in a 512×512 square array, a 1024×1024 square array, a 2048×2048 square array, or a 4096×4096 square array. In certain embodiments, the signal from each pixel is recorded independently of all other pixels. In certain embodiments, the signals from 2×2 sets of pixels are binned together.

[0256] The measurement device can enable the observation of multiple signals from multiple reporter particles in different regions. In certain further embodiments, the multiple reporter particles in different regions are arranged in a regular grid. Alternatively, individual optical signals from substantially all reporter particles can be observed without being affected by interference from any other reporter particles.

[0257] When used in combination with a magnifying lens, the recording device can further detect smaller signals. Such lenses are well known in the art. In certain embodiments, the recording device can capture individual pixels and / or individual reporter particles. Using plasmonic nanoparticles / quantum dots as the substrate on which the capture element is functionalized facilitates this detection.

[0258] The recording device can use any technique known in the art to detect and quantify the reporter particle / analyte particle complex. The recording device can use methods of light absorption and emission that are paired in the reporter particle design.

[0259] Alternatively, the light output can include fluorescence emission from either a fluorophore on the reporter particle excited by a light source or a fluorophore associated with the reporter particle. Then, the presence and amount of the analyte particle will be reported by the intensity of the fluorescence emission. The fluorophore can be placed in proximity to a surface such as a photonic crystal so that the fluorescence emission is enhanced. The fluorescence signal can be adjusted using multiple fluorophores to obtain the desired outcome. In that case, the optical signal can be modulated by excitation transfer between two or more fluorophores.

[0260] The quantum yields, λ max shifts, and anisotropies of fluorescence and phosphorescence are contemplated in the present disclosure. In anisotropic measurements, a polarizer can be introduced into the optical path of either the excitation or the emission or both. The light source can be linked to the emission method. The light source can be a conventional broadband light source, a light-emitting diode, or a laser, and can be delivered to the sample either directly or via a wavelength selection device such as a grating to optimize the excitation. The light is directed through a total internal reflection component incorporating a waveguide and forming the base of the reporter volume, providing dark field excitation.

[0261] Identification of Inert Reporter Particles A method for identifying inert reporter particles, referred to herein as the "identification method", is provided. In a particular system, two solutions, a first solution free of analyte particles and a second solution of high concentration of analyte particles, are sequentially applied to the reporter volume. It will be appreciated that these two solutions respectively cause the absence of analyte particles bound to the reporter particles and the substantially saturation of the analyte particles bound to the reporter particles. Frames are recorded using a recording device, and a comparison is made between the frame in the analyte particle-free state and the frame in the analyte particle-saturated state. Reporter particles that do not meet the selected criteria are marked as inert.

[0262] In the case of inactivation due to nanoparticle aggregation, the identification of inert reporter particles will be easy. The formation of aggregates will be revealed by visual inspection of the frames from the recording device. Also, the "analyte particle-free" and "analyte particle-saturated" procedures outlined above will not be required.

[0263] The identification method maintains a record of the positions of the reporter particles in the measurement device. The positions of the reporter particles can be referenced by x / y coordinates, for example, based on an appropriate geometric grid in the measurement device or based on pixel coordinates on the recorder device. The record of the inert reporter particles can be maintained in non-volatile computer memory.

[0264] The identification method will provide criteria for tagging reporter particles as inactive. The criteria will be set to balance eliminating the use of malfunctioning reporter particles with maintaining a sufficient number of reporter particles to obtain a specific accuracy and sensitivity for the measurement device. Numerical thresholds can be selected based on the type of optical signal observed to eliminate bias and enable automatic tagging. For example, certain reporter particles emit λ upon binding to an analyte particle. max This can cause a shift, and in this example, only 20 nanometers (nm) of λ relative to the bulk compound. max This can cause a shift. A 5nm shift threshold can be selected for this particular example.

[0265] To meet accuracy and sensitivity requirements, a numerical threshold can be selected to exclude a certain fraction of reporter particles. Referring to the previous example, 95% of the reporter particles have a λ of 12 nm. max A shift may be observed. In that case, 95% of the reporter particles are kept active and 5% are discarded as inactive, using a 12 nm λ. max A threshold can be selected.

[0266] Various criteria can be applied to assign an inactive state. Importantly, any criterion can be selected to assign a reporter particle as inactive. Since the binding of any one reporter particle does not depend on any of the other reporter particles, removing a reporter particle from the pool of active reporter particles does not affect the behavior of the remaining particles.

[0267] If necessary, the identification method described above can be repeated periodically throughout the operational life of the measuring device. This would be particularly beneficial for reporter devices whose performance is prone to degradation over time. Ideally, the identification method would require minimal operator intervention if the measuring device performs all required steps automatically. In the case of nanoparticle-based reporter particles, frames can be recorded periodically, and any aggregations that may occur over time can be identified by pattern matching software.

[0268] The identification method may also involve exposing the measuring device to one or more solutions containing intermediate concentrations of analyte particles. This would be particularly important for the quantitative measurement of analyte particles where a range of reporter particle saturation is assumed. By using several solutions across a range of analyte particle concentrations, a calibration curve can be constructed to better match the optically reported data with the analyte particle concentrations.

[0269] The main advantage of using spatially resolved signals from reporter particle regions is that it is possible to flag and discard problematic recording device regions in subsequent analysis. This includes not only inactive reporter particles, i.e., improperly folded antibodies, but also any region that presents challenges. This may include overlapping spots of two or more nearby reporter particles for whatever reason, or reporter particles that are poorly distinguishable between free and bound states. Since the binding of each individual reporter particle does not depend on any other, discarding a small set of optical signals may improve accuracy or precision with only a slight impact on sensitivity.

[0270] Accuracy / Precision / Sensitivity The accuracy of the disclosed digital measurement method is expected to be at least as good as that of conventional analog methods. The digital measurement method will minimize or eliminate several sources of error that are, by definition, sources of low accuracy. For example, latent errors arise from inactive reporter particles, i.e., those that do not bind to analyte particles or that bind to analyte particles but do not provide the expected optical signal. As discussed elsewhere, the presence of reporter particles that do not bind to analyte particles simply represents a fraction of reporter particles that do not form aggregates and can be taken into account by calibration of the analyte sample at known concentrations. Furthermore, aggregation assay designs can employ reporter particles with relatively poor sensitivity of the chromophore to guest binding, as disruption of the optical signal is avoided even if analyte binds. For both of these reasons, inactive particles present fewer accuracy and precision challenges than many other host / guest-based analytical systems.

[0271] The accuracy of the disclosed digital measurement method is expected to be at least as good as that of conventional analog measurements. Conventional methods for observing the bulk signal from the entire reporter particle can, in most cases, provide accurate estimates using various statistical and numerical methods, but not always.

[0272] In contrast, aggregation assays, especially in systems and conditions that result in relatively small aggregate formation, can be considered "digital" in that each aggregate consists of a very small integer number of reporter particles. The observed particle size (specified by the pixel spacing or physical size of the sample volume, for example) can be attributed to the "digital" aggregate size containing an integer number of reporter particles. In some embodiments, it may be preferable to intentionally form small aggregates by either weak reporter particle / analyte binding or pre-dilution of the sample.

[0273] bond isotherm The relationship between the number and particle size of analyte particle concentrations (referred to herein as the "binding isotherm") is complex and indirect. Simply put, given a fixed reporter particle concentration, the ratio of binding sites to fully binding sites based on the reporter particle asymptotically increases to 1 as the total analyte particle concentration increases. Higher affinity reporter particles will bind to a higher proportion of analyte particles at any given analyte particle concentration. Importantly, the total reporter particle concentration considers only active reporter particles.

[0274] For many of the uses envisioned by the disclosed method, thresholds or cutoffs corresponding to critical values ​​have been established. These thresholds or cutoffs are either compliant with regulatory levels set by environmental laws or correspond to critical biomarker levels for specific health conditions. The measurement method is adjustable in terms of recommended scan parameters and dilution levels to provide appropriate measurement conditions for the intended use.

[0275] In certain embodiments, the measuring device can provide an automated sample dilution mechanism. This can be achieved by taking a fraction of an existing sample and introducing a solute for dilution. This can also be achieved by introducing a new sample that has been pre-diluted with a solute.

[0276] In certain embodiments, the recommended dilution level can be calculated by a computing device either integrated with or connected to the measuring device. The computing device can provide the recommended dilution level to the operator via an interface (e.g., a display, printout, or synthesized voice report). The computing device can also perform any process requiring automated analysis of the diluted sample without user intervention by directly controlling the measuring device.

[0277] In certain embodiments, the threshold or cutoff can be pre-set by a computing device in the form of firmware or software that can be optionally updated when the threshold or cutoff is changed. Additionally, the operating software can prompt the user for further input of the samples to be measured. For example, in the case of biomarker measurement, the user can input subject history parameters such as age, weight, gender, etc., which can change the threshold or cutoff and thus affect the accuracy required for a given measurement.

[0278] As will be demonstrated in the examples, an important feature in the aggregate assay is that the average particle size can increase smoothly over a range of analyte concentrations. This behavior is due to the statistically low probability of forming larger aggregates that require multiple simultaneous binding interactions. This behavior is in contrast to the conventional 1:1 binding behavior, which often exhibits sigmoid on-off behavior over a narrow range of analyte concentrations. In other words, the 1:1 binding isotherm results in a situation where the reporter particles are either substantially bound or substantially unbound for the overwhelming majority of an analyte concentration range. For these reasons, conventional 1:1 binding is of limited value in determining analyte concentration (in contrast to the presence / absence of the analyte). In contrast, the smooth relationship between analyte concentration and particle size is sufficient to accurately determine the analyte concentration.

[0279] Abbreviations The following abbreviations are used in the present disclosure. Other abbreviations recognized by those skilled in the art may also be used.

[0280] PEG: Poly(ethylene glycol), EDC: 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, NHS: N-Hydroxysuccinimide, DDI water: Double deionized water, PBS: Phosphate buffered saline, RBD: Receptor binding domain of the SARS-CoV-2 spike protein

Examples

[0281] The present invention is further illustrated by the following examples.

[0282] Example 1: Carboxylated Nanoparticles A suspension of 100 nM Au nanoparticles ("GNP", NanoComposix, San Diego, CA) in 1 mL of EtOH was mixed with a 1 mL solution (10 mM) of 3-mercaptopropionic acid in EtOH. The resulting mixture was incubated in a centrifuge tube at rt for several hours with intermittent shaking and sonication. After final sonication, the mixture was centrifuged at approximately 5000 RCF for 5 minutes or until the particles had settled completely. The supernatant was carefully removed, taking care not to disturb the solid pellet. EtOH rinsing was performed as follows: 1 mL of EtOH was added, and the mixture was sonicated to resuspend the particles. The mixture was centrifuged at approximately 5000 RCF for 5 minutes or until the particles had settled completely. The EtOH rinsing was repeated two more times for a total of three EtOH rinsings. Water rinsing was performed using water instead of EtOH, following the EtOH rinsing procedure, for a total of three water rinsings.

[0283] Example 2: C2 anti-CRP antibody conjugate nanoparticles To an aqueous suspension of carboxy-functionalized nanoparticles (1 mL), 20 μL of aqueous solution containing 10 mg / mL EDC was added, followed by 40 μL of aqueous solution containing 10 mL / mg sulfo-NHS. The mixture was incubated for 30 minutes with rotation, and then centrifuged at 5000 RCF for 5 minutes. The supernatant was carefully removed, taking care not to disturb the solid pellet. 1 mL of 0.1 × PBS was added to the pellet, and the particles were resuspended while vortexing and / or sonicating (<30 sec). 20 μG of C2 anti-CRP (C-reactive protein) antibody (Abcam, Cambridge, UK) was added to the suspension, and the mixture was incubated at rt for 2 hours, and then centrifuged. The supernatant was carefully removed, taking care not to disturb the solid pellet. PBS rinsing was performed as follows: 1 mL of 0.1 × PBS was added to the pellet, and the particles were resuspended while vortexing and / or sonicating (<30 sec). This was repeated two more times with PBS, for a total of three rinses. For the third and final rinse, 1 mL of 0.1 × PBS + 0.5% (v / v) Tween 20 was used. The obtained material was stored at 4°C.

[0284] Example 3: C6 anti-CRP antibody conjugate nanoparticles The procedure in Example 2 was followed using a C6 anti-CRP antibody (Abcam, Cambridge, UK) instead of a C2 anti-CRP antibody.

[0285] Example 4: Optical absorption test of conjugate Figure 5 shows the UV-Vis absorption spectra of suspensions of GNP (gold nanoparticles) conjugated to C2 antibody (panel (a)) and C6 antibody (panel (b)). For each conjugate, a slight redshift is observed from bare GNP (i) to the GNP-antibody conjugate (ii). This is likely due to dielectric disturbance in GNP resulting in a change in localized surface plasmon resonance (LSRP). For each conjugate, the addition of CRP causes a further change in optical absorbance (iii), confirming that the antibody retains the activity to capture CRP in solution. A larger change in optical absorbance was observed with the C6 antibody (panel (b), trace (iii)) than with the C2 antibody (panel (a), trace (iii)).

[0286] Example 5: Aggregation Test GNP conjugated with C2 and C6 antibodies was combined in a 1:1 ratio and suspended in PBS + 0.05% TWEEN® 20. A surfactant was added to stabilize the particle suspension. Next, a solution of CRP (10 μL) and a solution of GNP-antibody conjugate (20 μL) were mixed and incubated at 37°C for a certain period of time. After incubation, 15 μL of the CRP / GNP-antibody conjugate mixture was sandwiched between two microscope glass plates to form a thin liquid film. Aggregation behavior was observed using an upright optical microscope in DF mode, typically with a 40× objective lens. A full-size DF frame typically contains 2,000 to 10,000 particles within a 250 mm × 200 mm area. Higher concentrations are expected to increase the degree of overlap in aggregate frames and complicate signal processing.

[0287] Figures 6 and 7 show DF micrographs from control and positive experiments. In the absence of CRP, individual GNPs are imaged (Figure 6(a)). Addition of CRP results in the formation of GNP clusters clearly visible in the micrograph (Figure 6(b)). Individual clusters are imaged in Figures 7(a)–7(c).

[0288] A typical experiment consists of 10 frames and requires the analysis of 20,000 to 100,000 particles. The need to analyze such a large number of particles was the driving force behind the development of the image processing methods described in Examples 6-9, which automate the procedure and avoid laborious manual evaluation. Factors considered for signal processing included bright circular defects in the frame due to dust / debris outside the focal plane, dimmer optical signals from individual GNPs, and heterogeneous aggregates.

[0289] An overview of the signal processing method is illustrated in Figure 8. The original frame, converted to grayscale (panel (a)), is processed to remove artifacts (panel (b)). After this process, the frame is segmented into regions corresponding to individual particles or particle aggregates (panel (c)). The size of each segment is then quantified (panel (d)).

[0290] Example 6: Subtraction of signal background The following MATLAB® code removes the background from a frame. The code operates on frame I, indexed by j, and requires a user-supplied bglvl parameter. se=strel('disk',bglvl);% Create a structured element Ie=imerode(I{j,1},se);% Using structured elements % Identify large background features % Next, a series of functions to optimize the background frame. Iobr=imreconstruct(Ie,I{j,1}); Iobrd = imdilate(Iobr,se); Iobrcbr=imreconstruct(imcomplement(Iobrd),imcomplement(Iobr)); Iobrcbr=imcomplement(Iobrcbr);% final background frame % Subtract background frames i = I{j,1} - Iobrcbr;

[0291] The effect of this procedure is shown in Figure 9. The original frame of panel (a) is processed to generate a background frame (panel (b)), which is then subtracted from the original frame to give background subtraction panel (c).

[0292] Example 7: Border Formation The MATLAB® code below constructs a frame from a cleaned grayscale frame containing B / W features corresponding to individual aggregates. The procedure creates borders at high gradient locations (i.e., rapid transitions from black to white). The gradient magnitude threshold is modified by the user-supplied parameter FudgeFactor. For most analyses, a value of FudgeFactor=1.05 was used. To accommodate features not completely enclosed by the borders generated by this process, the border size is diluted. Then, the dilation is reversed by filling in the areas fully enclosed by the borders. [~,threshold]=edge(i,'sobel');%Find the threshold for the edge BWs=edge(i,'sobel',threshold*fudgeFactor); % Draw a border around the object BWsdil=imdilate(BWs,strel('disk',1)); Dilute the border by % % Merge nearby objects % Connects most borders BWdfill=imfill(BWsdil,'holes');% Fill the enclosed area BWnobord=imclearborder(BWdfill,4);% Remove objects from edges Bwfinal=imerode(Bwnobord,strel('disk',1)); % Restore the previous dilation

[0293] Figure 10 shows an example of the border formation procedure. The frame shown in panel (a) of the panel cleaned from the background subtraction process is subjected to the border formation procedure. Panel (b) shows those that have successfully identified the borders of four features on this frame. Next, the borders are filled in to provide a B / W frame corresponding to the four features (panel (c)).

[0294] Example 8: Feature Identification The following MATLAB® code identifies the features corresponding to individual aggregates. The feature vectors are created from the B / W frame using the MATLAB® bwconncomp function. Features having fewer pixels than the user-supplied parameter T are considered noise and sent back to the background. s = size(Bwfinal); BWfinal = BWfinal(ceil(s(1)) / 8):(floor(7*s(1) / 8)-1), ceil(s(2)) / 8):floor(7*s(2) / 8-1); % Such distorted regions on the frame % Remove the first and last 1 / 8 CC = bwconncomp(BWfinal,4); % Build a list of objects numPixels = cellfun(@numel,CC.PixelIdxList); [~,idx] = find(numPixels<T); % Elements with <T pixels for k = 1:length(idx) BWfinal(CC.PixelIdxList{idx(k)}) = 0; end

[0295] Figure 11 shows an example of the feature identification procedure. Panel (a) is the cleaned grayscale frame before processing. Panel (b) shows the B / W frame from the border formation procedure. Panel (c) is the B / W frame after denoising. You will notice that the artifacts circled in all three panels have been successfully removed.

[0296] Example 9: Frame Padding The MATLAB® code below identifies frames and pads them to their original size for easier comparison. It constructs a label matrix with elements corresponding to each feature. It calculates and stores the area of ​​each feature. LL=bwlabel(BWfinal,4);% Converts B / W frame to label frame ss=(s-size(LL)) / 2; X.label{j,1}=padarray(LL,floor(ss),0,'both'); % Restore array to its original size x=regionprops(X.label{j},'Area'); Areas{j.1}=cell2mat({x.Area});

[0297] Example 10: CRP titration Figure 12 illustrates a CRP assay using the aggregation method of this disclosure compared to conventional plasmon sensing methods. The polydispersity of particle clusters results in a noisy response in ensemble mean analysis. In contrast, particle-versus-particle analysis provides detailed information about particle aggregation and results in improved sensitivity and dynamic range. Panel (a) shows a calibration curve derived from the analysis of cluster size distributions at several CRP concentrations. The linear relationship between mean cluster size and CRP concentration is noteworthy and contrasts with conventional plasmon sensing methods shown in Panel (b), which exhibit a saturated isotherm typical of many assays. Importantly, the linear relationship in Panel (a) shows clearly significant different mean cluster sizes for CRP concentrations of 2–3 mg / mL (corresponding to the risk threshold for cardiac disease) and 10 mg / mL (corresponding to the normal level). Conventional plasmon sensing methods could not distinguish between these two levels.

[0298] Example 11: Detection of C-reactive protein (CRP) in whole blood The detection of particles in whole blood is shown in Figure 13. Capillaries with depths of (i) 0.1 mm and (ii) 0.05 mm are shown in panel (a). Dark-field images showing particles in whole blood using capillaries with thicknesses of 0.1 mm, 0.05 mm, and 0.01 mm are shown in panels (b), (c), and (d), respectively.

[0299] Images of individual nanoparticles in whole blood at various depths are shown in Figure 14. The nanoparticles are depicted in media at depths of 0.1 mm, 0.05 mm, and 0.01 mm in panels (a), (b), and (c), respectively. 16-bit intensity (brightness) plots of these images are shown in panels (a), (b), and (c), respectively.

[0300] The performance of the CRP assay in whole blood is shown in the plot in Figure 15, where the horizontal axis = CRP concentration (mg / dL) and the vertical axis = mean cluster size (pixels). As seen in Figure 12, the assay takes advantage of the linear relationship between analyte concentration and aggregate size.

[0301] Example 12: Synthesis of anti-human antibody nanoparticles Au nanoparticles functionalized with anti-human antibodies were prepared using the method described in Example 2.

[0302] Example 13: Synthesis of RBD-linked nanoparticles RBD-linked Au nanoparticles for intact SARS-CoV-2 virus detection are prepared using the following protocol.

[0303] Gold nanoparticles, 1 mL PEGylated 80 nm gold nanoparticle solution (20 OD = 1.3E11 particles / mL). (Nanocomposix, Bio ready, Carboxyl, 20 OD, SKU: AUXR80-5M) Scaling as needed.

[0304] RBD Thermo Fisher Scientific, SARS-CoV-2 spike protein (S-RBD) (aa319-541), His Tag recombinant protein.

[0305] Other chemicals: EDC, NHS, DDI water, PBS buffer, KH2PO4, 20kDa PEG (recommended), glycine (or Tris buffer), Tween20.

[0306] A solution containing 10 mg / mL of EDC was prepared by dissolving 1 to 10 mg of EDC in water. Similarly, a solution containing 10 mg / mL of sulfo-NHS was prepared by dissolving 1 to 10 mg of sulfo-NHS in water. For both solutions, 100 μL of H2O was used per 1 mg of solute, and the solutions were prepared immediately before use.

[0307] 1 mL of -COOH-terminated particles (suspended in DDI water) was mixed with 7 μL of EDC solution, followed by 14 μL of sulfo-NHS solution. The solutions were thoroughly mixed and then incubated on a tube rotator at room temperature for 30 minutes. The solution was centrifuged at 5000 RCF for 5 minutes. The supernatant was carefully removed and 1 mL of reaction buffer (5 mM KH2PO4 + 0.5% (v / v) 20 kDa PEG at pH 7.4) was added. The mixture was vortexed and sonicated (<30 seconds) to resuspend the particles. 20 μL of 1 mg / mL RBD protein was added to the mixture, the mixture was vortexed and mixed, and then incubated at room temperature for 2 hours. 5 μL of saturated glycine (in DDI water) was then added to the mixture, and the contents were mixed and incubated for 10 minutes.

[0308] The particles were washed with the reaction buffer as follows: the mixture was centrifuged at 5000 RCF for 5 minutes, the supernatant was removed, and the pellet was resuspended in 1 mL of reaction buffer using sonication for less than 30 seconds. The reaction buffer rinse was repeated two more times, for a total of three rinses. In the third and final rinse, the pellet was resuspended in 1 mL of 0.1 × PBS + 0.5% (v / v) Tween 20. The resulting material was stored at 4°C.

[0309] To avoid nonspecific aggregation, PEG was repacked onto the particle surface using the following sequence: A solution of 2 mg of SH-PEG-COOH (MW6000) in 50 mL of 0.1 × PBS was prepared. Equivolutes of this solution, the conjugate particle solution, and the PEG solution were mixed and incubated at 25°C for 6 hours with shaking, or alternatively, at 4°C overnight.

[0310] The particles were washed with the reaction buffer as follows: the mixture was centrifuged at 5000 RCF for 5 minutes, the supernatant was removed, and the pellet was resuspended in 1 mL of 0.1 × PBS + 0.5% (v / v) Tween 20. The reaction buffer rinse was repeated two more times, for a total of three rinses. The resulting material was stored at 4°C.

[0311] Example 14: Detection of SARS-CoV-2 antibodies in whole blood The following assay was developed to detect and quantify antibodies against the SARS-CoV-2 spike protein. The design principle is illustrated in Figure 16. The nanoparticles of Example 12 are shown together with an anti-human antibody (ii) linked to the nanoparticle via PEG(i). The nanoparticles of Example 13 are shown together with an RBD domain (iv) linked to the nanoparticle via PEG(v). Analytes (iii), i.e., antibodies against the SARS-CoV-2 spike protein, can be simultaneously bound to two types of functionalized nanoparticles.

[0312] Chemical product: Antibody: SARS-CoV-2 spike protein S1 chimeric recombinant human monoclonal antibody (H6) Whole blood ZenBio,Inc.,Cat No:SER-WB10ML

[0313] Whole blood samples were prepared at the desired antibody concentration. A solution containing 10 μL of RBD conjugate particles (20 OD=1.3E11 particles / mL) and 10 μL of 0.1 × PBS + 0.5% (v / v) Tween20 solution was prepared. 20 μL of the diluted particle solution (10 OD=6.5E10 particles / mL) was mixed with 5 μL of whole blood sample, the mixture was gently vortexed, and then incubated at 25°C for 1 hour.

[0314] A 5 μL solution was applied to a large glass slide (CORNING coverslip, 1.24 × 50 mm thick, Cat. No. 2975-245), and then covered with a small coverslip (CORNING coverslip, 1.18 × 18 mm thick, Cat. No. 2845-18). Dark-field images were recorded using a 20× objective lens. The images were processed using a self-developed image analysis program.

[0315] The assay results are illustrated in Figure 17. Panel (a) shows microscopic observation of the aggregate. Panel (b) provides a calibration curve: horizontal axis = SARS-CoV-2 antibody (mg / dL), vertical axis = aggregation size (pixels). Good linearity is observed at antibody concentrations of 5 mg / dL or higher. Note that the COVID-19 IgG concentration in patient serum corresponds to 0.14–420 mg / dL, ranging from 1.4–4200 μg / ml. Furthermore, 10 mg / dL (or 100 μg / mL) is the cutoff concentration for identifying convalescent serum therapy donors.

[0316] Example 15: Detection of intact SARS-CoV-2 virus Components used in the intact SARS-CoV-2 detection assay are illustrated in Figure 18. Panel (a) shows details of the coronavirus by highlighting numerous spike proteins (i) that decorate the surface of the virus and are involved in cell entry. The nanobodies were designed by others to bind to the spike proteins. Panels (b) and (c) show top and side views illustrating the interaction between the three-part nanobodies (ii) and the spike proteins (i).

[0317] Nanoparticles functionalized with nanobodies against the SARS-CoV-2 spike protein were prepared using the method described in Example 2. Details of SARS-CoV-2 detection are illustrated in Figure 19, which shows the detection of the SARS-CoV-2 virus. Panel (a) shows nanobodies functionalized nanoparticles: (i) SARS-CoV-2 spike nanobodies, (ii) PEG linkers, and (iii) Au nanoparticles. Panels (b) and (c) show microscopic observations of aggregates at various levels of virus concentration.

[0318] All U.S. or foreign references, patents, or applications cited in this application are incorporated by reference in this application as if they were described in their entirety herein. In the event of any conflict, the one actually disclosed herein shall prevail.

[0319] From the above description, those skilled in the art will be able to easily confirm the essential characteristics of the present invention, and will be able to make various changes and modifications to the present invention to suit various uses and conditions without departing from its spirit and scope.

Claims

1. A step of combining a sample with a certain amount of one or more types of reporter particles in a detector volume, A step of recording one or more frames of the detector volume, The steps include identifying images of the aggregate of reporter particles and, optionally, individual non-aggregate reporter particles within one or more frames, A step of determining at least one object property that indicates the size of the aggregate, A step of determining the presence and / or concentration of analyte particles from the aforementioned material properties, Includes, The presence of analyte particles induces the formation of aggregates, each containing at least two reporter particles and at least one analyte particle. The presence or concentration of analyte particles in the sample is determined such that the aforementioned material properties increase monotonically with the average aggregate size. A method for digitally filtering the frame of the detector volume to remove or suppress noise, errors, or background features, thereby distinguishing between true particle aggregates and non-aggregate image features.

2. The method according to claim 1, wherein aggregate formation is caused by binding between a binding site on the reporter particle and a binding site on the analyte particle.

3. The method according to claim 1, wherein the reporter particle provides an optical signal.

4. The method according to claim 1, further comprising the step of removing background features.

5. The process of downloading images, The process of reconstructing the image, The process of dilating the image, The method according to claim 4, further comprising:

6. The method according to claim 5, further comprising one or more border forming steps in one or more frames of the detector volume.

7. The method according to claim 1, wherein the reporter particle comprises nanoparticles or quantum dots.

8. The method according to claim 3, wherein the optical signal is induced by surface plasmon resonance ("SPR").

9. The method according to claim 1, wherein each of the one or more types of reporter particles present in the aforementioned amount of reporter particles contains one or more binding sites of the same type.

10. The method according to claim 1, wherein the analyte is an antibody, a hematological protein, a virus, a bacterium, a biological fluid, or derived from a biological fluid.

11. - Image sensor and, • A screen with image display capabilities, • Microprocessor and • Memory and, - Image analysis software stored in the memory and executable by the processor, having the function of analyzing data captured by the image sensor and the function of digitally classifying the data, • Communication interface, Apparatus or system for carrying out a method disclosed in any one of claims 1 to 10, including

12. - A reporter surface made of glass or polymer with optical reporter particles containing functionalized plasmon nanoparticles attached to one side using a capture element, and Waveguides suitable for dark-field microscopy that contact the opposite side of the reporter surface. It also includes, The capture element is One or more nanobodies bonded to the analyte, One or more nucleotide sequences that bind to the analyte, and The antibody or fragment thereof that binds to the aforementioned analyte. The apparatus or system according to claim 11, selected from the following.

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