Methods, systems, articles, kits and uses thereof for detecting biomolecules, bio-organelles, biological particles, cells and microorganisms

Abstract

The present disclosure relates to a method, system, article, and kit for detecting the presence of a target analyte in a sample, where the target analyte includes biomolecules, bio-organelles, biological particles, cells, and microorganisms. In certain embodiments, the method for detecting a target analyte includes the steps of: (I) providing a plurality of probe particles having a sensing element that is capable of specifically interacting with a target analyte bound to a surface coated with another sensing element, (ii) applying a mechanical force (such as a magnetic force) to remove non-specifically bound probe particles, and (iii) measuring the amount of specifically bound probe particles or non-specifically bound probe particles by particle density, aggregate size, or color density. In one embodiment, the target analyte is SARS-CoV-2.

Description

[0001] Citation of Related Applications

[0002] This application claims priority to Singapore Application No. 10202005073Y, filed on May 29, 2020, the disclosure of which is incorporated herein by reference. Technical Field

[0003] The present invention generally relates to the field of detection of biomolecules, bio-organelles, bio-particles, cells and microorganisms. Specifically, the present invention relates to the detection of target analytes using specific target analyte-probe particle interactions under mechanical force. Background Art

[0004] For virus and microorganism detection, currently available techniques based on viral RNA or DNA, such as RT-PCR-based assays (Ian M. Mackay et al., Nucleic Acids Res, 2002, 30(6):1292–1305), can be used. Currently available antibody-based detections, such as 1) enzyme-linked immunosorbent assay (ELISA, Eva Engvall and Peter Perlmann, J Immunol, 1972, 109(1):129-135), 2) immunogold-labeled assays, and 3) chemiluminescent microparticle immunoassay (Abbott Laboratories Inc.), can also be used. However, these methods may require fluorescent labeling or enzyme attachment, which may affect the results due to the labeling.

[0005] Other diagnostic methods, such as surface plasmon resonance (SPR)-based assays, rely on surface plasmon resonance signals. In SPR-based assays, interference from surface non-specific interactions may result in false positive signals.

[0006] There is a need to develop methods for the detection of biomolecules, bio-particles, bio-organelles, cells and microorganisms with different detection principles to overcome or improve one or more limitations of the prior art. Summary of the Invention

[0007] There is provided a general fluorescence-free method for detecting biomolecules, bio-particles, bio-organelles, cells and microorganisms under a wide range of environmental conditions, which has single molecule sensitivity and enhanced accuracy based on the mechanical selection of specific target analyte-probe particle interactions. This new method enables rapid, low-cost centralized or point-of-care diagnosis and home self-diagnosis of biomolecules, bio-particles, bio-organelles, cells and microorganisms in suspected human samples.

[0008] The method principles described herein are also applicable to the rapid, accurate, and sensitive detection of any monovalent or multivalent targets, such as viruses, antibodies, exosomes, antibodies, pathogen-associated molecules, and contaminants. The method can serve as a new platform for disease diagnosis, biopharmaceutical development, and environmental monitoring.

[0009] In one aspect, the present disclosure relates to a method for detecting the presence of a target analyte in a sample, the method comprising the steps of:

[0010] a) incubating the sample with a surface coated with a first sensing element, wherein the first sensing element is capable of specifically binding to the target analyte, and wherein, during or after incubation, when the target analyte is present in the sample, the target analyte binds to the first sensing element present on the coated surface;

[0011] b) incubating the coated surface of step (a) with a plurality of probe particles, wherein the probe particles are coated with a second sensing element, the second sensing element being capable of specifically interacting with the target analyte bound to the coated surface or capable of specifically interacting with the first sensing element on the coated surface;

[0012] c) applying a mechanical force to separate non-specifically bound probe particles from the coated surface of step (b); and

[0013] d) measuring a property that reflects the amount of specifically bound probe particles on the coated surface, wherein the property is selected from the group consisting of particle density, color density, aggregate size, and combinations thereof, or measuring a property that reflects the amount of non-specifically bound probe particles, wherein the property is selected from the group consisting of color density, aggregate size, and combinations thereof.

[0014] Advantageously, the method can be universal such that any virus with a known host receptor and / or an antibody against a viral surface protein, any antibody with a known antigen or any antigen can be detected by the method. The method does not require the use of fluorescent labels or enzyme attachment as in ELISA to detect a wide variety of target analytes, including microorganisms, under a wide range of environmental conditions. Thus, the method eliminates the effects of fluorescent labeling and photobleaching and avoids the equipment costs of fluorescence detection or light absorption detection (ELISA).

[0015] Further advantageously, specific target analyte-probe particle interactions can be used in the method to achieve single-target detection sensitivity, such that low concentrations of target analytes as low as femtomolar can be detected in a small volume (<30 μl), thus requiring less sample volume. These features in turn enable early diagnosis of diseases.

[0016] Even more advantageously, the method uses specific target analyte-probe particle interactions that are selected by applying mechanical force to ensure high accuracy and high specificity of detection. Thus, the likelihood of false positive results in the method can be significantly reduced compared to other detection methods. Parallel orthogonal detection can be performed using a set of probe particles or by coating the surfaces of different wells in a microplate with different first sensing elements to increase the throughput of detection. Even more advantageously, the method does not require the use of washing steps (such as those required in ELISA).

[0017] Even more advantageously, the method can be used to detect antibodies by both IgG / IgM / IgA testing and neutralization activity assessment. Thus, the method can simultaneously achieve IgG / IgM / IgA testing and neutralizing antibody (Nab) testing in the same method.

[0018] Even more advantageously, the method produces different types of readouts, allowing the method to be used for diagnosis in different clinical settings, such as for point-of-care testing in home self-diagnosis or as a laboratory test in a central laboratory.

[0019] In another aspect, the present disclosure relates to a system for detecting the presence of a target analyte in a sample, the system comprising:

[0020] a) a surface coated with a first sensing element, wherein the first sensing element is capable of specifically binding to the target analyte;

[0021] b) a plurality of probe particles, wherein the probe particles are coated with a second sensing element, the second sensing element being capable of specifically interacting with the target analyte when the target analyte is present or being capable of specifically interacting with the first sensing element on the coated surface, thereby forming specifically bound probe particles on the coated surface;

[0022] c) a mechanical force capable of separating non-specifically bound probe particles from the coated surface; and

[0023] d) a measuring device for measuring the properties of the specifically bound probe particles, wherein the properties are selected from the group consisting of: the particle density, color density, aggregate size of the specifically bound probe particles; or for measuring the properties of the non-specifically bound probe particles, wherein the properties are selected from the group consisting of: the color density, aggregate size of the non-specifically bound probe particles.

[0024] Advantageously, the system can be specifically designed for the detection of biomolecules, bio-organelles, biological particles, cells, and microorganisms for use in actual clinical trials using human samples such as serum, saliva, nasal swabs, and urine. Thus, the system can allow for the detection of target viruses in patient samples and the detection of antibodies induced by specific viral infections.

[0025] In another aspect, the present disclosure relates to an article for detecting the presence of a target analyte in a sample, the article comprising:

[0026] At least one test well comprising a bottom surface coated with a first sensing element configured to bind to the target analyte in the sample, wherein the test well is configured to receive the sample and probe particles contained in one or more liquid media, the probe particles being coated with a second sensing element configured to bind to one selected from the group consisting of the first sensing element and the target analyte, such that the probe particles specifically bind to the coated surface depending on the presence of the target analyte in the sample; and

[0027] A channel connected to the test well at a distance from the bottom surface to allow fluid communication between the channel and the test well, wherein the channel is configured to receive non-specifically bound probe particles in the test well after an external force is applied to the probe particles, the external force moving the non-specifically bound probe particles away from the bottom surface.

[0028] In another aspect, the present disclosure relates to a kit for detecting the presence of a target analyte in a sample, the kit comprising:

[0029] An article as disclosed herein;

[0030] Probe particles coated with a second sensing element, the probe particles being contained in a solution; and

[0031] An instrument configured to apply an external force to the probe particles.

[0032] Advantageously, the kit can allow for high-throughput screening, thereby rapidly detecting a large number of samples in a short period of time. The kit can also provide low-cost and rapid diagnosis of the targeted analyte or therapeutic agent, which can be applicable to applications in a clinical setting, such as central laboratory tests that require high-volume, inexpensive, and rapid diagnosis.

[0033] Further advantageously, the kit can be easy to use because, due to the specific target analyte-probe particle interaction and the specific force required to remove non-specifically bound probe particles from the coated surface, the detection of the target analyte has high specificity, thus eliminating the washing step when using the kit. Therefore, the use of the kit can be time-saving.

[0034] Further advantageously, the kit can be readily used as a central laboratory test kit or a point-of-care diagnostic kit or a home self-diagnostic kit.

[0035] Advantageously, the kit can allow for proper mixing of the probe particles and the test sample and introduction of the mixture into the test well and removal of non-specifically bound probe particles without the aid of sophisticated laboratory equipment such as a micropipette. This enables the test to be performed by non-professionals in a non-laboratory environment. In combination with smartphone-based imaging or visual inspection, the kit enables point-of-care testing and home self-testing.

[0036] In another aspect, the present disclosure relates to a method of using a kit as disclosed herein, the method comprising the steps of:

[0037] introducing a sample and probe particles contained in one or more liquid media into the article;

[0038] applying the external force to the probe particles to move the non-specifically bound probe particles away from the bottom surface of the test well and into the channel;

[0039] analyzing the probe particles in at least one selected from the group consisting of the test well and the channel to determine the presence of the target analyte in the sample.

[0040] In another aspect, the present disclosure relates to the use of a system, or an article or a kit as described herein for detecting biomolecules, bio-organelles, biological particles, cells or microorganisms.

[0041] Advantageously, the use of the system or the article or the kit can be specifically designed for the detection of biomolecules, bio-organelles, biological particles, cells or microorganisms for use in actual clinical trials using human samples such as serum and urine. Thus, the use of the system or the article or the kit can allow for the detection of a target virus in a patient sample or the detection of antibodies induced by a specific viral infection.

[0042] Further advantageously, the system, or the article or the kit can be used on a high-throughput scale to rapidly detect a large number of samples in a short period of time. If desired, the use of the system or the article or the kit can also be carried out on a large scale or can be easily scaled up to a larger scale as needed.

[0043] Definitions

[0044] The following words and terms used herein shall have the indicated meanings:

[0045] When used in connection with probe particles, the terms "non-specific binding" or "bind non-specifically" mean that the probe particles do not bind strongly (i.e., neither bind directly nor bind via an intermediate target analyte particle) to the coated surface and will thus be removed more quickly by the application of mechanical force.

[0046] When used in connection with probe particles, the terms "cross-link" or "link" mean that the probe particles bind specifically (bind directly or bind via an intermediate target analyte particle) to the coated surface and will thus be difficult to remove by the application of mechanical force within the calibration detection time.

[0047] The terms "cross-link assay" or "link assay" refer to an assay in which there is specific binding of the probe particles to the coated surface (bind directly or bind via an intermediate target analyte particle), and thus it is difficult to remove the probe particles by the application of mechanical force within the calibration time. Further, for a "cross-link assay" or "link assay", the higher the level of specifically bound probe particles present on the coated surface, the higher the concentration level of the target analyte detected in the sample.

[0048] When used in connection with probe particles, the terms "block" or "disconnect" mean that there is no specific binding between the probe particles and the coated surface due to the presence in the sample of target analytes that compete with or prevent the probe particles from binding to the coated surface.

[0049] The terms "block assay" or "un-linking assay" refer to an assay in which there is no specific binding between the probe particles and the coated surface due to the presence in the sample of target analytes that compete with or prevent the probe particles from binding to the coated surface. Further, for a "block assay" or "un-linking assay", the higher the level of specifically bound probe particles on the coated surface, the lower the concentration level of the target analyte detected in the sample.

[0050] The term "probe particle" refers to a particle that can be perturbed (attracted or repelled) when a mechanical force is applied to the particle, resulting in its dissociation or movement.

[0051] The term "non-bait molecule" refers to a biomolecule, or a biological particle, or a material or product derived from a biological organelle, virus, cell, or microorganism that, when coated on a surface, cannot cross-link one or more target analyte particles to the surface.

[0052] Unless otherwise specified, the terms "comprise", "include", "contain" and their grammatical variants are intended to be "open-ended" or "inclusive" language such that they include the recited elements but also permit the inclusion of additional, unrecited elements.

[0053] As used herein, in the context of the concentration of a component of a formulation, the term "about" generally means + / - 5% of the value, more typically + / - 4% of the value, more typically + / - 3% of the value, more typically + / - 2% of the value, even more typically + / - 1% of the value, and even more typically + / - 0.5% of the value.

[0054] Throughout this disclosure, certain embodiments may be disclosed in range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as a rigid limitation on the scope of the disclosed range. Accordingly, the range description should be considered to have specifically disclosed all possible sub-ranges as well as individual numerical values within that range. For example, a range description of, for example, 1 to 6 should be considered to have specifically disclosed sub-ranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numerical values within that range, such as 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range. Specific embodiments

[0055] Exemplary, non-limiting embodiments of a method for detecting the presence of a target analyte in a sample will now be disclosed.

[0056] The method for detecting the presence of a target analyte in a sample comprises the following steps:

[0057] a) incubating the sample with a surface coated with a first sensing element, wherein the first sensing element is capable of specifically binding to the target analyte, and wherein, during or after incubation, when the target analyte is present in the sample, the target analyte binds to the first sensing element present on the coated surface;

[0058] b) incubating the coated surface of step (a) with a plurality of probe particles, wherein the probe particles are coated with a second sensing element, the second sensing element being capable of specifically interacting with the target analyte bound to the coated surface or being capable of specifically interacting with the first sensing element on the coated surface;

[0059] c) applying a mechanical force to separate non-specifically bound probe particles of step (b) from the coated surface; and

[0060] d) measuring a property that reflects the amount of specifically bound probe particles on the coated surface, wherein the property is selected from the group consisting of particle density, color density, aggregate size, and combinations thereof, or measuring a property that reflects the amount of non-specifically bound probe particles, wherein the property is selected from the group consisting of color density, aggregate size, and combinations thereof.

[0061] A method for detecting the presence of a target analyte in a sample may include the following steps:

[0062] a) Incubating the sample with a surface coated with a first sensing element, wherein the first sensing element is capable of specifically binding to the target analyte, and wherein, during or after incubation, when the target analyte is present in the sample, the target analyte binds to the first sensing element present on the coated surface;

[0063] b) Incubating the coated surface of step (a) with a plurality of probe particles, wherein the probe particles are coated with a second sensing element, and the second sensing element is capable of specifically interacting with the target analyte bound to the coated surface or is capable of specifically interacting with the first sensing element on the coated surface;

[0064] c) Applying a mechanical force to separate non-specifically bound probe particles of step (c) from the coated surface; and

[0065] d) Measuring a property that reflects the amount of specifically bound probe particles on the coated surface, wherein the property is selected from the group consisting of particle density, color density, aggregate size, and combinations thereof.

[0066] A method for detecting the presence of a target analyte in a sample may include the following steps:

[0067] a) Incubating the sample with a surface coated with a first sensing element, wherein the first sensing element is capable of specifically binding to the target analyte, and wherein, during or after incubation, when the target analyte is present in the sample, the target analyte binds to the first sensing element present on the coated surface;

[0068] b) Incubating the coated surface of step (a) with a plurality of probe particles, wherein the probe particles are coated with a second sensing element, and the second sensing element is capable of specifically interacting with the target analyte bound to the coated surface or is capable of specifically interacting with the first sensing element on the coated surface;

[0069] c) Applying a mechanical force to separate non-specifically bound probe particles of step (b) to a second surface and aggregating the non-specifically bound probe particles into aggregates on the second surface under the application of a mechanical force to the second surface; and

[0070] e) Measuring a property that reflects the amount of non-specifically bound probe particles, wherein the property is selected from the group consisting of color density, aggregate size, and combinations thereof.

[0071] By following the above steps, both specifically bound probe particles and non-specifically bound probe particles can be measured. The measurement of non-specifically bound probe particles can be negatively correlated with the measurement of specifically bound probe particles.

[0072] The sample can be a liquid sample. The sample can be derived from or obtained from a human, an animal, a biological organelle, a virus, a cell, a microorganism, or the environment. The sample can include blood, urine, saliva, sputum, serum, a liquid derived from a cell or tissue.

[0073] The sample can be diluted or serially diluted. The sample can be filtered to remove contaminants. The sample can be pretreated to remove, reduce, or destroy molecules that will compete with the target analyte for binding to the coated surface, or molecules that will compete with the target analyte for binding to the probe particles, or molecules that will compete with the target analyte for binding to both the coated surface and the probe particles. The pretreatment can include processing steps selected from the group consisting of homogenization, lysis, extraction, vortexing, stirring, dilution, heating, pressurization, centrifugation, bioseparation, dialysis, chromatography, fractionation, purification, separation, refinement, recovery, concentration, and combinations thereof. The pretreatment can include adding one or more liquid media to the sample. The one or more liquid media can allow the target analyte to be released from the components of the sample. For example, the one or more liquid media can lyse a virus, or a microorganism or a cell, and allow the target analyte to be released from the intracellular components of the virus, microorganism, or cell. In addition, the one or more liquid media can homogenize the properties of the solution.

[0074] The surface can be a hard surface. The surface can be a non-porous surface. The surface can be selected from the group consisting of glass, borosilicate glass, quartz, polymers, and metal-coated surfaces. The surface can be selected from the group consisting of cover slips, plates, ELISA plates, microtiter plates, and multi-well plates. The surface can have lower light scattering properties for electromagnetic waves of a specific wavelength in the visible light region compared to the probe particles. The surface can be transparent.

[0075] The surface coated with the first sensing element can be considered a pre-coated surface or a surface pre-coated with the first sensing element. The first sensing element coated on the surface can be the same as or different from the second sensing element coated on the probe particles.

[0076] The first sensing element and the second sensing element can independently be selected from the group consisting of biomolecules, biological particles, materials and products derived from biological organelles, viruses, cells, or microorganisms. The first sensing element and the second sensing element can independently be selected from the group consisting of carbohydrates, polysaccharides, lipids, proteins, peptides, nucleic acids, antibodies, antigens, hormones, enzymes, and chemical compounds. The first and second sensing elements can be a pair of antibodies that recognize different epitopes of an antigen.

[0077] The target analyte can have multiple binding sites for binding to the first sensing element and the second sensing element. The target analyte is soluble in an aqueous medium or a solvent. The target analyte can be dispersed in a liquid medium before incubation with the coated surface. The liquid medium can increase the solubility of the target analyte. The liquid medium can homogenize the properties (such as pH and salt concentration) of the sample solution containing the target analyte.

[0078] The target analyte can be selected from the group consisting of: cells, viruses, bacteria, archaea, fungi, protozoa, algae, deoxyribonucleic acid (DNA), ribonucleic acid (RNA), proteins, antibodies, antigens, cells, exosomes, pathogen-associated molecules, contaminants, biomarkers, target receptors, intracellular substances, extracellular substances, products derived from biological organelles, viruses, cells, microorganisms, modified biomaterials, and combinations thereof. The target analyte can be multivalent. The target analyte can be a virus. The target analyte can be a COVID-19 virus. The target analyte can be a SARS-COV-2 receptor-binding domain protein. The target analyte can be other proteins in SARS-COV-2, such as the nucleocapsid protein. The target analyte can also be the RNA of SARS-COV-2.

[0079] The probe particles can be non-magnetic, magnetic, or superparamagnetic. The probe particles can be biological particles or non-biological particles. The probe particles can be spherical. The non-magnetic probe particles can be polymers or glass.

[0080] The magnetic or superparamagnetic probe particles can be selected from the group consisting of: iron, cobalt, nickel, their alloys, their oxides, and combinations thereof. The magnetic or superparamagnetic probe particles can be ferromagnetic, paramagnetic, or ferrimagnetic. The probe particles can be polystyrene beads encapsulated with nanoscale magnetic or superparamagnetic particles. When the probe particles are magnetic or superparamagnetic particles, they can be aggregated into aggregates by the magnetic force applied by using a permanent magnet or an electromagnet in the form of a magnetic needle.

[0081] The probe particles can be opaque, translucent, or colored. The probe particles can be capable of scattering electromagnetic waves. Compared with the coated surface and the surrounding medium, the probe particles can exhibit higher light scattering properties for electromagnetic waves of a specific wavelength in the visible light region, and thus, can be observed as colored patches that can be qualitatively or quantitatively analyzed.

[0082] The size of the probe particles can be in the range of about 8 nm to about 100,000 nm, about 10 nm to about 100,000 nm, about 100 nm to about 100,000 nm, about 1,000 nm to about 100,000 nm, about 10,000 nm to about 100,000 nm, about 8 nm to about 10 nm, about 8 nm to about 100 nm, about 8 nm to about 1,000 nm, about 8 nm to about 10,000 nm, about 10 nm to about 100 nm, about 10 nm to about 1,000 nm, about 10 nm to about 10,000 nm, about 100 nm to about 1,000 nm, about 100 nm to about 10,000 nm, or about 1,000 nm to about 10,000 nm.

[0083] When the size of the probe particles is in the range of about 100 nm to about 100,000 nm, the probe particles can be seen under a microscope. Therefore, the particle density of the probe particles can be quantitatively determined by microscopy imaging at single-particle resolution.

[0084] Mechanical forces can be generated by permanent magnets, electromagnets, centrifuges, acoustic devices, ultrasonic waves, laser beams, fluid motion, fluid buoyancy, or gravity. Mechanical forces can be generated by instruments such as atomic force spectroscopy, optical tweezers, magnetic tweezers, acoustic force spectroscopy, microneedles, centrifuges, or biomembranes. Mechanical forces can be magnetic forces generated by applying a magnetic field. Mechanical forces can be in the range of about 0.01 pN to about 100 pN, about 0.1 pN to about 100 pN, about 1 pN to about 100 pN, about 10 pN to about 100 pN, about 50 pN to about 100 pN, about 75 pN to about 100 pN, about 0.01 pN to about 0.1 pN, about 0.01 pN to about 1 pN, about 0.01 pN to about 10 pN, about 0.01 pN to about 50 pN, about 0.01 pN to about 75 pN, about 0.1 pN to about 1 pN, about 0.1 pN to about 10 pN, about 0.1 pN to about 50 pN, about 0.1 pN to about 75 pN, about 1 pN to about 10 pN, about 1 pN to about 50 pN, about 1 pN to about 75 pN, about 10 pN to about 50 pN, about 10 pN to about 75 pN, or about 50 pN to about 75 pN.

[0085] Advantageously, the generated mechanical force can be used as a selection tool to distinguish non-specifically bound probe particles from specifically bound probe particles, thus reducing the likelihood of false positives. Specific target analyte-probe particle interactions can result in cross-linking of the probe particles to the coated surface or detachment of the probe particles from the coated surface. When specific target analyte-probe particle interactions result in cross-linking of the probe particles to the coated surface, the probe particles are specifically bound to the coated surface via the target analyte. When specific target analyte-probe particle interactions result in cross-linking of the probe particles to the coated surface via the target analyte, its application can be used for virus detection. When the target analyte prevents the probe particles from specifically binding to the coated surface, the target analyte causes the probe particles to detach from the coated surface, so the probe particles are non-specifically bound. When the target analyte causes the probe particles to detach from the coated surface, its application can be used for neutralizing antibody detection. Since non-specific binding is generally much weaker than specific binding, most of the probe particles bound by non-specific binding will dissociate after being exposed to mechanical force for a calibrated time. By quantifying the remaining bound probe particles (mainly specifically bound probe particles) on the coated surface after applying mechanical force, the presence of the target analyte can be accurately determined without artificial amplification.

[0086] When the measured property is the color density, or the aggregate size, or both the color density and the aggregate size formed by specifically bound probe particles, step (d) can further include step (d0), i.e., prior to step (d), aggregating the specifically bound probe particles on the coated surface into aggregates by applying a magnetic force on the other side of the coated surface. Measurement of the aggregates formed by specifically bound probe particles can enhance the detection signal, which can be detected by the human naked eye or quantified using a smartphone camera.

[0087] Step (d0) can be carried out by placing a permanent magnet or an electromagnet in the form of a magnetic needle on the other side of the coated surface. Since the distance between the specifically bound probe particles on one side of the coated surface and the magnetic needle on the other side of the coated surface is short (equivalent to the thickness of the coated surface), the magnetic needle can generate sufficient force to attract the specifically bound probe particles together to form aggregates. The applied magnetic force can be in the range of 0.01 pN to 100 pN.

[0088] In the case of measuring the characteristics of non-specifically bound probe particles, step (c) further includes providing a second surface to contact the non-specifically bound probe particles separated from the coated surface; and aggregating the non-specifically bound probe particles into aggregates on the other side of the second surface by applying a mechanical force on one side of the second surface; wherein the mechanical force is a magnetic force. Thus, if only the characteristics of specifically bound probe particles are measured, this step is optional. The applied magnetic force can be in the range of 0.01 pN to 100 pN. The color density of the aggregates, or the aggregate size, or both the color density and the aggregate size can be measured. Measuring the aggregates formed by non-specifically bound probe particles can enhance the detection signal for the eye or a smartphone camera.

[0089] The second surface can be a hard surface. The second surface can be a non-porous surface. The second surface can be selected from the group consisting of: glass, borosilicate glass, quartz, polymers, and metal-coated surfaces. The second surface can be a coverslip, a plate, or a housing. The second surface can have lower light scattering characteristics for electromagnetic waves of a specific wavelength in the visible light region compared to the probe particles. The second surface can be transparent.

[0090] The non-specifically bound probe particles can be aggregated into aggregates on the other side of the second surface by placing a permanent magnet or an electromagnet in the form of a magnetic needle on one side of the second surface. Due to the short distance (equivalent to the thickness of the second surface) between the non-specifically bound probe particles on one side of the second surface and the magnetic needle on the other side of the second surface, the magnetic needle can generate sufficient force to attract the non-specifically bound probe particles together to form aggregates. The applied magnetic force can be in the range of 0.01 pN to 100 pN.

[0091] The aggregates formed on one side of the coated surface or one side of the second surface can exhibit strong light scattering characteristics for electromagnetic waves of a specific wavelength in the visible light region, and thus, can be seen as colored patches of aggregates on one side of the coated surface or one side of the second surface, respectively. Thus, the color density of the aggregates can be measured by observing that side of the coated surface or that side of the second surface under a camera or a smartphone camera or the naked eye, and quantifying it by software color analysis or analyzing the intensity of the scattered light in the recorded image. When using a crosslinking / linking assay or a blocking / separating assay, respectively, the color density of the aggregates formed on one side of the coated surface can be positively or negatively correlated with the concentration of the target analyte in the sample. When using a blocking / separating assay or a crosslinking / linking assay, respectively, the color density of the aggregates formed on one side of the second surface can be positively or negatively correlated with the concentration of the target analyte in the sample.

[0092] If only the aggregates formed on one side of the coated surface or on one side of the second surface are observed with the naked eye, the measurement of the color density can be a qualitative analysis. If the visualization of the aggregates formed on one side of the coated surface or on one side of the second surface is combined with the analysis of the intensity of the light scattered by the aggregates, the measurement of the color density can be a quantitative analysis. If the aggregates formed on one side of the coated surface or on one side of the second surface are not sufficient to form visible colored patches, the measurement of the color density may lack sensitivity. When measuring the color density or the aggregate size using only the naked eye and / or a smartphone camera for the method, the method can be applicable to central laboratories or home self-diagnosis or point-of-care testing.

[0093] The aggregate size of the aggregates formed on one side of the coated surface or on one side of the second surface can be measured by observing one side of the coated surface or one side of the second surface under a camera or a smartphone camera or with the naked eye and quantifying the fractional coverage area of the aggregates. When using a crosslinking / ligation assay or a blocking / separation assay separately, the aggregate size of the aggregates formed on one side of the coated surface can be positively or negatively correlated with the concentration of the target analyte in the sample. When using a blocking / separation assay or a crosslinking / ligation assay separately, the aggregate size of the aggregates formed on one side of the second surface can be positively or negatively correlated with the concentration of the target analyte in the sample. If the visualization of the aggregates formed on one side of the second surface or on one side of the second surface is combined with the quantification of the fractional coverage area of the aggregates, the measurement of the aggregate size can be a quantitative analysis. Compared with the measurement of the color density of the aggregates, the measurement of the aggregate size of the aggregates may be more accurate. When measuring the aggregate size of the aggregates using only the naked eye and / or a smartphone camera and / or a microscope for the method, the method can be applicable to central laboratories or home self-diagnosis or point-of-care testing.

[0094] The incubation time for incubating the coated surface with the sample in step (a) can be in the range of about 1 second to about 90 minutes, about 1 minute to about 90 minutes, about 10 minutes to about 90 minutes, about 50 minutes to about 90 minutes, about 70 minutes to about 90 minutes, about 1 second to about 1 minute, about 1 second to about 10 minutes, about 1 second to about 50 minutes, about 1 second to about 70 minutes, about 1 minute to about 10 minutes, about 1 minute to about 50 minutes, about 1 minute to about 70 minutes, about 10 minutes to about 50 minutes, about 10 minutes to about 70 minutes, or about 50 minutes to about 70 minutes.

[0095] The incubation time for incubating the probe particles with the coated surface in step (b) can be in the range of about 10 seconds to about 90 minutes, about 1 minute to about 90 minutes, about 10 minutes to about 90 minutes, about 50 minutes to about 90 minutes, about 70 minutes to about 90 minutes, about 10 seconds to about 1 minute, about 10 seconds to about 10 minutes, about 10 seconds to about 50 minutes, about 10 seconds to about 70 minutes, about 1 minute to about 10 minutes, about 1 minute to about 50 minutes, about 1 minute to about 70 minutes, about 10 minutes to about 50 minutes, about 10 minutes to about 70 minutes, or about 50 minutes to about 70 minutes.

[0096] When the property measured in step (d) is the particle density of specifically bound probe particles, it can be measured by observing the coated surface under a microscope, or a camera, or a smartphone camera, or the naked eye and quantifying by manually or using a software analysis counter to count the number of specifically bound probe particles on the coated surface, or analyzing the covered area fraction or the intensity of the recorded image. Quantification can be performed by using a software analysis counter to count the number of specifically bound probe particles accumulated on the bottom of a well in a coated surface such as a microtiter plate. This can be done by taking an image of the bottom of the microtiter plate and processing the image using a software analysis counter.

[0097] When using a crosslinking / ligation assay or a blocking / separation assay separately, the particle density can be positively or negatively correlated with the concentration of the target analyte in the sample. If only the naked eye is used to observe the specifically bound probe particles on the coated surface, the measurement of the particle density of specifically bound probe particles in step (d) can be a qualitative analysis. If the visualization of specifically bound probe particles on the coated surface is combined with the counting of specifically bound probe particles on the coated surface, the measurement of the particle density of specifically bound probe particles in step (d) can be a quantitative analysis.

[0098] The specifically bound probe particles in step (d) can exhibit strong light scattering properties for electromagnetic waves of a specific wavelength in the visible light region, and thus can be observed as colored patches of specifically bound probe particles on the coated surface. Therefore, the measurement of the color density of specifically bound probe particles can be completed by observing the coated surface under a smartphone camera or the naked eye and quantifying by software color analysis or analyzing the intensity of scattered light in the recorded image. When using a crosslinking / ligation assay or a blocking / separation assay separately, the color density can be positively or negatively correlated with the concentration of the target analyte in the sample.

[0099] If only the specifically bound probe particles on the coated surface are observed with the naked eye, the measurement of the color density of the specifically bound probe particles in step (d) can be a qualitative analysis. If the visualization of the specifically bound probe particles on the coated surface is combined with the analysis of the intensity of the light scattered by the specifically bound probe particles on the coated surface, the measurement of the color density of the specifically bound probe particles in step (d) can be a quantitative analysis. If the number of specifically bound probe particles on the coated surface is not sufficient to form a visible colored patch, the measurement of the color density of the specifically bound probe particles in step (d) may lack sensitivity.

[0100] Compared with measuring the color density or aggregate size, measuring the particle density characteristics in step (d) using a microscope may be more accurate. However, the use of a microscope may require access to a microscope and trained personnel. Thus, when the method is performed such that step (d) measures the particle density characteristics using a microscope, the method may be suitable for application in a central laboratory. Alternatively, if only the naked eye and / or a smartphone camera are used to measure the particle density, there is no need to use a microscope, and thus no need to use bulky or expensive instruments. Thus, when the method is performed such that step (d) measures the particle density characteristics using only the naked eye and / or only a smartphone camera, the method may be suitable for home self-diagnosis or point-of-care testing.

[0101] Compared with measuring the particle density or aggregate size, the measurement of the color density characteristics in step (d) can be more convenient. However, when the concentration of the target analyte is low, the amount of specifically bound probe particles in step (d) may be low, and thus may not present a visible colored patch on the coated surface, resulting in low sensitivity. When the method is performed such that step (d) measures the color density characteristics using only the naked eye and / or only a smartphone camera, the method may be suitable for a central laboratory or home self-diagnosis or point-of-care testing.

[0102] Steps (a) and (b) can be carried out simultaneously, thereby incubating the mixture of the sample and the plurality of probe particles with the coated surface.

[0103] Here, one or more liquid media can be added to the sample and the probe particles before mixing into the mixture. Before incubating the mixture with the coated surface, the mixture can be diluted or serially diluted. One or more liquid media can increase the solubility of the target analyte in the mixture, or homogenize the characteristics (e.g., pH and salt concentration) of the sample solution containing the target analyte. One or more liquid media can allow the target analyte to be released from the components of the sample. For example, one or more liquid media can lyse viruses, or microorganisms or cells, and allow the target analyte to be released from the intracellular components of the viruses, microorganisms or cells.

[0104] The incubation time of the incubation mixture with the coated surface can range from about 1 minute to about 60 minutes, from 10 minutes to about 60 minutes, from about 20 minutes to about 60 minutes, from about 30 minutes to about 60 minutes, from about 40 minutes to about 60 minutes, from about 1 minute to about 10 minutes, from about 1 minute to about 20 minutes, from about 1 minute to about 30 minutes, from about 1 minute to about 40 minutes, from about 10 minutes to 20 minutes, from about 10 minutes to 30 minutes, from about 10 minutes to about 40 minutes, from about 20 minutes to about 30 minutes, from about 20 minutes to about 40 minutes, or from about 30 minutes to about 40 minutes.

[0105] When steps (a) and (b) are carried out simultaneously, this can be regarded as one step, resulting in a reduction in the total incubation time, thereby increasing the throughput of the method.

[0106] The total duration of the method can range from about 20 minutes to about 90 minutes, from 40 minutes to about 90 minutes, from about 50 minutes to about 90 minutes, from about 60 minutes to about 90 minutes, from about 80 minutes to about 90 minutes, from about 20 minutes to about 40 minutes, from about 20 minutes to about 50 minutes, from about 20 minutes to about 60 minutes, from about 20 minutes to about 80 minutes, from about 40 minutes to about 50 minutes, from about 40 minutes to about 60 minutes, from about 40 minutes to about 80 minutes, from about 50 minutes to about 60 minutes, from about 50 minutes to about 80 minutes, or from about 60 minutes to about 80 minutes.

[0107] Advantageously, the method can be universal such that any virus with a known host receptor and / or an antibody against a viral surface protein, any antibody with a known antigen or any antigen can be detected by the method. The method does not require the use of fluorescent labels or enzyme attachment as in ELISA to detect a wide variety of target analytes under a wide variety of environmental conditions, including microorganisms. Thus, the method eliminates the effects of fluorescent labeling and photobleaching and avoids the equipment costs of fluorescence detection or light absorption detection (ELISA).

[0108] Even more advantageously, specific target analyte-probe particle interactions can be used in the method to achieve single-target detection sensitivity, such that low concentrations of target analytes as low as femtomolar can be detected in small volumes (<30 μl), thus requiring less sample volume. These features in turn enable early diagnosis of diseases.

[0109] Even more advantageously, the method uses specific target analyte-probe particle interactions that can be selected by applying mechanical force to ensure high accuracy and high specificity of detection. Thus, the likelihood of false positive results in this method can be significantly reduced compared to other detection methods. Parallel orthogonal detection can be performed using a set of probe particles or by coating the surfaces of different wells in a porous plate with different first sensing elements to increase the throughput of detection. Even more advantageously, the method does not require the use of washing steps (such as those required in ELISA).

[0110] Even more advantageously, the method can be used to detect antibodies by IgG / IgM / IgA testing and neutralization activity assessment. Thus, the method can simultaneously perform IgG / IgM / IgA testing and neutralizing antibody (Nab) testing in the same method.

[0111] Even more advantageously, the method produces different types of readouts, which allows the method to be used for diagnosis in different clinical settings, such as as a point-of-care test in home self-diagnosis or as a laboratory test in a central laboratory.

[0112] Exemplary, non-limiting embodiments of the system will now be disclosed.

[0113] A system for detecting the presence of a target analyte in a sample, the system comprising:

[0114] a) A surface coated with a first sensing element, wherein the first sensing element is capable of specifically binding to the target analyte;

[0115] b) A plurality of probe particles, wherein the probe particles are coated with a second sensing element, and the second sensing element is capable of specifically interacting with the target analyte or specifically interacting with the first sensing element on the coated surface when the target analyte is present, thereby forming specifically bound probe particles on the coated surface;

[0116] c) A mechanical force capable of separating non-specifically bound probe particles from the coated surface; and

[0117] d) A measuring device for measuring the properties of the specifically bound probe particles, wherein the properties are selected from the group consisting of: the particle density, color density, aggregate size of the specifically bound probe particles; or

[0118] For measuring the properties of the non-specifically bound probe particles, wherein the properties are selected from the group consisting of: the color density, aggregate size of the non-specifically bound probe particles.

[0119] The system may further include a second surface for contacting the probe particles of the non-specific binding. The second surface may be a hard surface. The second surface may be a non-porous surface. The second surface may be selected from the group consisting of: glass, borosilicate glass, quartz, polymer, and metal-coated surfaces. The second surface may be a coverslip, plate, or housing. Compared with the probe particles, the second surface may have lower light scattering characteristics for electromagnetic waves of a specific wavelength in the visible light region. The second surface may be transparent.

[0120] The sample may be a liquid sample. The sample may be derived from or obtained from a human, animal, biological organelle, virus, cell, microorganism, or the environment. The sample may include blood, urine, saliva, sputum, serum, and liquids derived from cells or tissues.

[0121] The sample may be diluted or serially diluted. The sample may be filtered to remove contaminants. The sample may be pretreated to remove, reduce, or destroy molecules that will compete with the target analyte for binding to the coated surface, or molecules that will compete with the target analyte for binding to the probe particles, or molecules that will compete with the target analyte for binding to both the coated surface and the probe particles. The pretreatment may include processing steps selected from the group consisting of: homogenization, lysis, extraction, vortexing, stirring, dilution, heating, pressurization, centrifugation, bioseparation, dialysis, chromatography, fractionation, purification, separation, refinement, recovery, concentration, and combinations thereof. The pretreatment may include adding one or more liquid media to the sample. The one or more liquid media may allow the target analyte to be released from the components of the sample. For example, the one or more liquid media may lyse a virus, or a microorganism or a cell, and allow the target analyte to be released from the intracellular components of the virus, microorganism, or cell. In addition, the one or more liquid media may homogenize the properties of the solution.

[0122] The surface may be a hard surface. The surface may be a non-porous surface. The surface may be selected from the group consisting of: glass, borosilicate glass, quartz, polymer, and metal-coated surfaces. The surface may be selected from the group consisting of: coverslip, plate, ELISA plate, microtiter plate, and multi-well plate. Compared with the probe particles, the surface may have lower light scattering characteristics for electromagnetic waves of a specific wavelength in the visible light region. The surface may be transparent.

[0123] The surface coated with the first sensing element may be considered a pre-coated surface or a surface pre-coated with the first sensing element. The first sensing element coated on the surface may be the same as or different from the second sensing element coated on the probe particles.

[0124] The first sensing element and the second sensing element can be independently selected from the group consisting of: biomolecules, biological particles, materials and products derived from biological organelles, viruses, cells or microorganisms. The first sensing element and the second sensing element can be independently selected from the group consisting of: carbohydrates, polysaccharides, lipids, proteins, peptides, nucleic acids, antibodies, antigens, hormones, enzymes and chemical compounds. The first and second sensing elements can be a pair of antibodies that recognize different epitopes of an antigen.

[0125] The target analyte can have multiple binding sites for binding to the first sensing element and the second sensing element. The target analyte is soluble in an aqueous medium or a solvent. The target analyte can be dispersed in a liquid medium before incubation with the coated surface. The liquid medium can increase the solubility of the target analyte. The liquid medium can homogenize the properties (such as pH and salt concentration) of the sample solution containing the target analyte.

[0126] The target analyte can be selected from the group consisting of: cells, viruses, bacteria, archaea, fungi, protozoa, algae, deoxyribonucleic acid (DNA), ribonucleic acid (RNA), proteins, antibodies, antigens, cells, exosomes, pathogen-associated molecules, contaminants, biomarkers, target receptors, intracellular substances, extracellular substances, products derived from biological organelles, viruses, cells, microorganisms, modified biomaterials and combinations thereof. The target analyte can be multivalent. The target analyte can be a virus. The target analyte can be the COVID-19 virus. The target analyte can be the SARS-COV-2 receptor-binding domain protein. The target analyte can be other proteins in SARS-COV-2, such as the nucleocapsid protein. The target analyte can also be the RNA of SARS-COV-2.

[0127] The probe particles can be non-magnetic, magnetic or superparamagnetic. The probe particles can be biological particles or non-biological particles. The probe particles can be spherical. The non-magnetic probe particles can be polymers or glass.

[0128] The magnetic or superparamagnetic probe particles can be selected from the group consisting of: iron, cobalt, nickel, their alloys, their oxides and combinations thereof. The magnetic or superparamagnetic probe particles can be ferromagnetic, paramagnetic or ferrimagnetic. The probe particles can be polystyrene beads encapsulated with nanoscale magnetic or superparamagnetic particles. When the probe particles are magnetic or superparamagnetic particles, they can be aggregated into aggregates by the magnetic force applied by using a permanent magnet in the form of a magnetic needle or an electromagnet.

[0129] The probe particles can be opaque, translucent, or colored. The probe particles can be capable of scattering electromagnetic waves. Compared with the coated surface and the surrounding medium, the probe particles can exhibit higher light scattering characteristics for electromagnetic waves of a specific wavelength in the visible light region, and thus, can be observed as colored patches that can be qualitatively or quantitatively analyzed.

[0130] The size of the probe particles can be in the range of about 8 nm to about 100,000 nm, about 10 nm to about 100,000 nm, about 100 nm to about 100,000 nm, about 1,000 nm to about 100,000 nm, about 10,000 nm to about 100,000 nm, about 8 nm to about 10 nm, about 8 nm to about 100 nm, about 8 nm to about 1,000 nm, about 8 nm to about 10,000 nm, about 10 nm to about 100 nm, about 10 nm to about 1,000 nm, about 10 nm to about 10,000 nm, about 100 nm to about 1,000 nm, about 100 nm to about 10,000 nm, or about 1,000 nm to about 10,000 nm.

[0131] When the size of the probe particles is in the range of about 100 nm to about 100,000 nm, the probe particles can be seen under a microscope, and thus, the particle density of the probe particles can be quantitatively determined by microscopic imaging at single particle resolution.

[0132] Mechanical force can be generated by a permanent magnet, an electromagnet, a centrifuge, an acoustic device, ultrasonic waves, a laser beam, fluid motion, fluid buoyancy, or gravity. Mechanical force can be generated by an instrument such as atomic force spectroscopy, optical tweezers, magnetic tweezers, acoustic force spectroscopy, a micro-needle, a centrifuge, or a biofilm. Mechanical force can be a magnetic force generated by applying a magnetic field.

[0133] Mechanical force can be in the range of about 0.01 pN to about 100 pN, about 0.1 pN to about 100 pN, about 1 pN to about 100 pN, about 10 pN to about 100 pN, about 50 pN to about 100 pN, about 75 pN to about 100 pN, about 0.01 pN to about 0.1 pN, about 0.01 pN to about 1 pN, about 0.01 pN to about 10 pN, about 0.01 pN to about 50 pN, about 0.01 pN to about 75 pN, about 0.1 pN to about 1 pN, about 0.1 pN to about 10 pN, about 0.1 pN to about 50 pN, about 0.1 pN to about 75 pN, about 1 pN to about 10 pN, about 1 pN to about 50 pN, about 1 pN to about 75 pN, about 10 pN to about 50 pN, about 10 pN to about 75 pN, or about 50 pN to about 75 pN.

[0134] Advantageously, the system can be specifically designed for the detection of biomolecules, bioparticles, bioorganelles, viruses, cells, and / or microorganisms for use in actual clinical trials using human samples such as serum and urine. Thus, the system can allow for the detection of target viruses in patient samples and the detection of antibodies induced by specific viral infections.

[0135] Exemplary, non-limiting embodiments of articles, kits, and methods of using the kits will now be disclosed.

[0136] Figure 12 A figure showing a perspective view of a kit 1200 for detecting the presence of a target analyte in a sample according to an exemplary embodiment is shown. The target analyte includes, but is not limited to, viruses, antibodies, exosomes, antibodies, pathogen-associated molecules, and contaminants. The sample can be a liquid sample and can include one or more components derived from or obtained from humans, animals, microorganisms, or the environment.

[0137] Kit 1200 includes an article 1202, represented in Figure 12 as a well plate 1204 and a water tank 1206. The well plate 1204 can be transparent and can be made of a material selected from the group consisting of glass and polymers. The well plate 1204 can have dimensions similar to those of a commercial multi-well plate to enable scaling. The well plate 1204 includes a plurality of test wells 1208. These test wells 1208 are coated with a first sensing element at least at the bottom surface 1210 of the test wells 1208. The first sensing element is configured to bind to the target analyte in the sample. Each test well 1208 is configured to receive the sample and probe particles contained in one or more liquid media through the opening 1212 of the test well 1208.

[0138] The water tank 1206 includes a housing 1214 and a plug 1216 that covers the holes of the housing 1214. The housing 1214 is configured to receive the well plate 1204 after introducing the liquid media containing the sample and probe particles into the test wells 1208. The plug 1216 is then inserted to cover the holes of the housing 1214 to prevent leakage of the water tank 1206 and minimize the possibility of environmental contamination.

[0139] The orifice plate 1204 includes a wall 1218 extending outwardly from an opening 1212 of the test orifice 1208. The housing 1214 includes four inner surfaces facing the orifice plate 1204 and one of these inner surfaces faces the wall 1218 and the test orifice 1208. A channel is formed outside the test orifice 1208 between the spaces defined between the inner surface and the wall 1218. When using the kit 1200, an external force is applied to the probe particles within the test orifice 1208, which moves the non-specifically bound probe particles (i.e., the probe particles that do not specifically bind to the coated bottom surface 1210) away from the bottom surface 1210 of the test orifice 1208. The channel is configured to receive these non-specifically bound probe particles from the test orifice 1208 after the application of the external force. In the case of applying a force using a magnet, the diameter of the test orifice 1208 can be less than or comparable to the height of the test orifice to apply a uniform mechanical force to the bound probe particles. 1212. The housing 1214 contains a transparent liquid 1220 to allow the non-specifically bound probe particles to move into the channel. In one embodiment, the transparent liquid 1220 can comprise phosphate buffered saline (PBS) and bovine serum albumin (BSA).

[0140] The kit 1200 further includes probe particles contained in a probe particle solution 1222. As Figure 12 shown, the probe particle solution 1222 is contained in a microcentrifuge tube 1224. It should be noted that the probe particles can be lyophilized during storage. The probe particle solution 1222 is mixed with the sample before loading into the test orifice 1208. The probe particles are magnetic or superparamagnetic particles coated with a second sensing element. The second sensing element is configured to bind to the first sensing element or the target analyte such that the probe particles specifically bind to the coated bottom surface 1210 of the test orifice 1208 depending on the presence of the target analyte in the sample. Thus, the presence of the target analyte can be determined by analyzing (e.g., counting, estimating, image processing) the specifically and / or non-specifically bound probe particles.

[0141] Specifically, if the kit 1200 is used for a crosslinking assay, the second sensing element is configured to bind to the target analyte. Thus, if the target analyte is present in the sample, the probe particles will specifically bind to the coated bottom surface 1210 through the target analyte, resulting in a larger amount of specifically bound probe particles and a smaller amount of non-specifically bound probe particles in the test orifice 1208. On the other hand, if the target analyte is not present in the sample, the probe particles will not specifically bind to the coated bottom surface 1210, resulting in no specifically bound probe particles in the test orifice 1208 and a larger amount of non-specifically bound probe particles.

[0142] Conversely, if kit 1200 is used for a blocking assay, the second sensing element is configured to bind to the first sensing element. Thus, if the target analyte is present in the sample, the probe particles will be separated from the coated bottom surface 1210, resulting in a lower amount of specifically bound probe particles and a higher amount of non-specifically bound probe particles in the test well 1208. On the other hand, if the target analyte is not present in the sample, the probe particles will specifically bind to the coated bottom surface 1210, resulting in a higher amount of specifically bound probe particles and a lower amount of non-specifically bound probe particles in the test well 1208.

[0143] The first sensing element and the second sensing element can be independently selected from the group consisting of: carbohydrates, polysaccharides, lipids, proteins, peptides, nucleic acids, antibodies, antigens, hormones, enzymes, and chemical compounds. It should be noted that the first sensing element coated on the bottom surface 1210 of the test well 1208 can be the same as or different from the second sensing element coated on the probe particles.

[0144] Kit 1200 further includes an instrument configured to apply an external force to the probe particles. The instrument is represented in Figure 12 as a magnetic needle array 1226 including a plurality of thin magnetic needles 1228. These magnetic needles 1228 are configured to apply a force to the bound probe particles and control the movement of the separated non-specifically bound probe particles into a channel in the article 1202 before the step of analyzing specifically and / or non-specifically bound probe particles to determine the presence of the target analyte.

[0145] In an alternative embodiment, the well plate 1204 can include system control wells and blank control wells that receive a liquid medium including the sample and the probe particles. The addition of the system control wells and the blank control wells ensures that kit 1200 functions properly, thereby producing accurate results. Specifically, the system control wells are coated with the target analyte, which can directly bind to the second sensing element coated on the probe particles, thus always producing a positive result regardless of whether the sample contains the target analyte. On the other hand, the blank control wells are coated with non-bait molecules that do not specifically bind to the target analyte, and thus always produce a negative result regardless of whether the sample contains the target analyte. In other words, if the system control wells do not show a positive result during the assay and the blank control wells do not show a negative result, kit 1200 may not be functioning properly and the results produced may be inaccurate.

[0146] In an alternative embodiment, the well plate 1204 may include positive control wells and negative control wells that receive a liquid medium comprising the sample and the probe particles, where the sample is prepared to include and exclude the target analyte, respectively. The addition of the positive control wells and the negative control wells ensures that the kit 1200 functions properly, thereby generating accurate results. Specifically, the positive control wells receive a liquid medium containing the target analyte such that, in the crosslinking and blocking assays, respectively, the probe particles can specifically bind to the coated bottom surface 1210 via the target analyte, or the probe particles are separated from the coated bottom surface 1210 due to the target analyte. On the other hand, the negative control wells receive a liquid medium that does not contain the target analyte such that, in the crosslinking and blocking assays, respectively, the probe particles cannot specifically bind to the coated bottom surface 1210 due to the absence of the target analyte, or the probe particles can specifically bind to the coated bottom surface 1210 due to the absence of the target analyte. In other words, if the positive control wells do not show positive results during the assay while the negative control wells do not show negative results, the kit 1200 may not be functioning properly and the results generated may be inaccurate.

[0147] In an alternative embodiment, the external force applied to the probe particles may be generated by means or methods other than the magnetic force of the magnetic needle, including but not limited to gravity, centrifugation, and fluid motion.

[0148] Advantageously, the kit 1200 includes a plurality of test wells 1208 that allow multiple tests to be performed in parallel, thereby improving time and cost efficiency. The kit 1200 is well-suited for use as a laboratory test kit in a central laboratory where inexpensive and high-throughput testing is required.

[0149] Figures 13A - 13F Shows when the kit 1200 is used in a crosslinking assay Figure 12 A schematic diagram of the kit 1200 or a part thereof.

[0150] Figure 13A Shows a side view of the well plate 1204 after the sample and the probe particles are introduced into the test wells 1208. Before the introduction step, the sample and the probe particles are contained in respective test tubes 1224 and incubated. Subsequently, the well plate 1204 is placed on a flat horizontal surface to allow the incubation of the sample and the probe particles to occur within the corresponding test wells 1208.

[0151] Figure 13B Shows a side view of the well plate 1204 during the incubation step within the test wells 1208. As Figure 13A Shown, the probe particles sink to the bottom surface 1210 in the test wells 1208, forming a visible layer 1302 adjacent to the bottom surface 1210 of the test wells 1208.

[0152] Figure 13CA side view of the orifice plate 1204 enclosed in the water tank 1206 is shown. After the incubation step within the test hole 1208 is completed, the orifice plate 1204 is inserted into the housing 1214 pre-filled with the transparent liquid 1220 such that the orifice plate 1204 is fully immersed in the transparent liquid 1220. During the insertion of the orifice plate 1204 into the housing 1214, the housing 1214 is tilted by approximately 30 degrees relative to the horizontal plane to prevent the transparent liquid 1220 from overflowing. Then, the housing 1214 is sealed with the plug 1216 to prevent the transparent liquid 1220 from leaking when the water tank 1206 is placed on a horizontal surface and the test hole 1208 faces upward (as shown by the first reference cue 1304 including the horizontal line and the upward arrow).

[0153] As Figure 13C shown, the housing 1214 includes an inner surface facing the test hole, herein denoted as the inner surface 1306. The orifice plate 1204 includes a wall 1218 extending outward from the opening 1212 of the test hole 1208. The space defined between the inner surface 1306 and the wall 1218 outside the test hole 1208 forms a channel (herein denoted as the channel 1308). As shown in this figure, the channel 1308 is connected to the test hole 1208 at a certain distance from the bottom surface 1210. Specifically, the channel 1308 is connected to the test hole 1208 adjacent to the opening 1212 of the test hole 1208, allowing for fluid communication between the channel 1308 and the test hole 1208.

[0154] Figure 13D A side view and a perspective view of the orifice plate 1204 enclosed in the water tank 1206 are shown, where both the orifice plate 1204 and the water tank 1206 face downward. After the orifice plate 1204 is inserted into the water tank 1206, the water tank 1206 is flipped 180 degrees such that the opening 1212 of the test hole 1208 faces downward, as shown by the second reference cue 1310 including the horizontal line and the downward arrow. Next, the magnetic needle array 1226 is placed below the water tank 1206 to apply a magnetic force to pull the probe particles downward towards the opening 1212 of the test hole 1208. As a result, the non-specifically bound probe particles form aggregates 1312 in the channel 1308 outside the test hole 1208 above the tip of the magnetic needle 1228.

[0155] Figure 13E A side view and a perspective view of the orifice plate 1204 enclosed in the water tank 1206 are shown, where the magnetic needle array 1226 is used to move the aggregates 1312 through the channel 1308. Here, the water tank 1206 slides above the magnetic needle array 1226 while facing downward to move the aggregates 1312 of the non-specifically bound probe particles out of the test hole 1208 into the probe particle trap 1314. The probe particle trap 1314 is a shallow dent formed on the wall 1218 between the openings 1212 of the test hole 1208 to prevent the non-specifically bound particles from moving back into the test hole 1208.

[0156] At this stage, probe particles specifically bound to the coated bottom surface 1210 in the test well 1208 can be analyzed to determine the presence of a target analyte in the sample. Detection methods include:

[0157] a) A colorimetric detection method based on the intensity of scattered light generated by specifically bound probe particles in the test well 1208. An example of interpreting the results obtained using this method is provided below with reference to Figure 14A an example of interpreting the results obtained using this method is provided below with reference to

[0158] b) A microscopic detection method by imaging the article 1202 placed on a microscope stage. An example of interpreting the results obtained using this method is provided below with reference to Figure 14B an example of interpreting the results obtained using this method is provided below with reference to

[0159] In addition to the above methods (a) and (b), the size of the probe particle aggregates in the test well 1208 can be analyzed to determine the presence of a target analyte in the sample. This method requires additional steps, as explained in more detail below with reference to Figure 13F including flipping the kit 1204 together with the water tank 1206. The probe particle trap 1314 prevents dissociated non-specifically bound particles from moving back into the test well 1208. Alternatively, the size of the aggregates of dissociated probe particles that have moved outside the test well 1208 can be analyzed to determine the presence of the target. In this case, it is not necessary to flip the kit together with the water tank, and thus the probe particle trap 1314 is not required either.

[0160] Figure 13F A side view and a top view of the well plate 1204 enclosed in the water tank 1206 are shown, with both the well plate 1204 and the water tank 1206 facing upwards. After moving the aggregates 1312 of non-specifically bound probe particles away from the test well 1208, the water tank 1206 is flipped 180 degrees and restored so that the opening 1212 of the test well 1208 faces upwards. Due to the presence of the probe particle trap 1314, after the water tank 1206 is flipped 180 degrees and restored, the dissociated non-specifically bound probe particles that have fallen into the probe particle trap 1314 are prevented from moving back into the well.

[0161] Then, a magnetic needle 1228 is placed below the bottom surface 1210 of the test well 1208 to aggregate the specifically bound probe particles into small aggregates 1316. The size of these aggregates 1316 indicates the amount of specifically bound probe particles retained on the bottom surface 1210, and it can be analyzed to determine whether a target analyte is present in the sample. An example of interpreting the results obtained using a software analysis counter is provided below with reference to Figure 14C an example of interpreting the results obtained using a software analysis counter is provided below with reference to

[0162] It should be noted that the probe particle aggregate based method can also be performed on the aggregates 1312 of non-specifically bound probe particles in the probe particle well 1314. Alternatively, when the test wells are facing downward, the aggregates 1608 of non-specifically bound probe particles can also be analyzed without the additional step of flipping the microfluidic plate 180 degrees to recover.

[0163] Advantageously, moving aggregates 1312 of non-specifically bound probe particles out of the test wells 1208 into the probe particle wells 1314 improves the distinguishability between specifically bound probe particles and non-specifically bound probe particles 1312 during analysis.

[0164] Figure 14A An image of a well plate 1204 incubated with liquid media containing different concentrations of target analytes is shown. Figure 14A The results correspond to those in Example 4. Here, the above reference Figure 13E The steps described above are followed, and images captured using a smartphone camera are analyzed to determine the sensitivity of the kit 1200 to different concentrations of the nucleocapsid antigen target analyte in the sample under different solution conditions.

[0165] Well plate 1204 includes three negative control test wells 1208. The probe particle solution 1222 contained in the four test wells in the top A of well plate 1204 is prepared using a homogenization buffer as a solution, mixed with a sample containing a target analyte and probe particles (prepared at three different concentrations (0 pM, 1 pM and 10 pM)). The probe particle solution 1222 contained in the four test wells in the middle portion B of well plate 1204 is prepared using a middle turbinate swab as a solution, mixed with a sample containing a target analyte (prepared at three different concentrations (0 pM, 1 pM and 10 pM)). The probe particle solution 1222 contained in the four test wells in the bottom C of well plate 1204 is prepared using saliva as a solution, mixed with a sample containing a target analyte (prepared at three different concentrations (0 pM, 1 pM and 10 pM)). Negative control well 1208 corresponds to 0 pM of target analyte.

[0166] like Figure 14A As shown, the scattered light intensity in the negative control well 1208 is the lowest, indicating that the test kit 1200 works properly. The scattered light intensity in the test well increases with the increase of the target analyte concentration, which indicates that more probe particles have been bound to the first sensing element coated in the bottom surface 1210 of the test well 1208 as the concentration of the nucleocapsid antigen target analyte increases.

[0167] Figure 14B 12 shows an enlarged image of the test hole 1208 observed under a microscope. Figure 13EThe described steps are analyzed using high-resolution images of test well 1208 recorded by a 4x microscope objective. As Figure 14B shown, probe particles 1404 (represented by multiple points on the image) specifically bind to the bottom surface 1210 of test well 1208. These probe particles 1404 can be quantified using software, providing a highly quantitative assessment of the target analyte in the sample.

[0168] Figure 14C A graph is shown illustrating the relationship between the normalized fraction of the number of probe particles and the normalized size of the probe particle aggregates. Here, the steps described above with reference to Figure 13F are completed, and images of aggregates 1316 taken by a smartphone camera are analyzed to determine the presence of the target analyte in the sample.

[0169] The X-axis represents the "normalized fraction of the number of probe particles" and the Y-axis represents the "normalized size of the probe particle aggregates". As shown, the number of probe particles increases as the size of aggregates 1316 increases. In other words, the size of aggregates 1316 obtained from the images taken by the smartphone camera can be used to estimate the number of probe particles retained in test well 1208. Thereafter, based on the number of probe particles retained in test well 1208, a quantitative assessment of the target analyte in the sample can be made to determine the presence of the target analyte in the sample.

[0170] Figure 15 A perspective view of a kit 1500 for detecting the presence of a target analyte in a sample according to another exemplary embodiment is shown. Target analytes include, but are not limited to, viruses, antibodies, exosomes, antibodies, pathogen-associated molecules, and contaminants. The sample can be a liquid sample and can include one or more components derived from or obtained from humans, animals, microorganisms, or the environment.

[0171] Kit 1500 includes an article represented as a microfluidic plate 1502 in Figure 15 . Microfluidic plate 1502 can be transparent and can be made of a material selected from the group consisting of glass and polymers. Microfluidic plate 1502 includes two test wells 1504. Test wells 1504 are coated with a first sensing element at least at the bottom surface 1506 of test wells 1504. The first sensing element is configured to bind to the target analyte in the sample. Each test well 1504 is configured to receive the sample and probe particles contained in one or more liquid media at the opening 1508 of test well 1504.

[0172] The microfluidic plate 1502 further includes a system control well 1510 and a blank control well 1512, which receive a liquid medium including a sample and probe particles. The addition of the system control well 1510 and the blank control well 1512 ensures that the kit 1500 functions properly, thus producing accurate results. Specifically, the system control well 1510 is coated with a target analyte, which can directly bind to a second sensing element coated on the probe particles, thus always producing a positive result regardless of whether the sample contains the target analyte. On the other hand, the blank control well is coated with a non-bait molecule that does not specifically bind to the target analyte, so it always produces a negative result regardless of whether the sample contains the target analyte. In other words, if the system control well does not show a positive result during the assay while the blank control well does not show a negative result, the kit 1500 may not be functioning properly and the results produced may be inaccurate.

[0173] The microfluidic plate 1502 further includes an inlet 1514, which allows the introduction of one or more liquid media containing a sample and probe particles. The microfluidic plate 1502 further includes a loading channel 1516, which conveys the liquid medium from the inlet 1514 to the test wells 1504, the system control well 1510, and the blank control well 1512.

[0174] The microfluidic plate 1502 further includes a removal channel 1524 connected to each well. When using the kit 1500, an external force is applied to the probe particles within the well, and this external force causes non-specifically bound probe particles (i.e., probe particles that are not specifically bound to the coated bottom surface 1506) to move away from the bottom surface 1506 of the well. The removal channel 1524 is configured to receive these non-specifically bound probe particles from the well after the external force is applied.

[0175] The microfluidic plate 1502 further includes a probe particle trap 1526 connected to the removal channel 1524. The probe particle trap 1526 is a shallow indentation formed towards the distal end of the removal channel 1524 to prevent non-specifically bound particles from moving back into the well.

[0176] The microfluidic plate 1502 further includes a waste reservoir 1528 connected to the probe particle trap 1526. An absorbent pad 1530 may be disposed within the waste reservoir 1528. The absorbent pad 1530 and the waste reservoir 1528 are configured to prevent the liquid medium from leaking out of the microfluidic plate 1502. The microfluidic plate 1502 further includes a vent 1532 connected to the waste reservoir 1528. After introducing the liquid medium through the inlet 1514, the vent 1532 provides an outlet for the compressed air within the microfluidic plate 1502.

[0177] The kit 1500 further includes probe particles contained in a probe particle solution 1534. As Figure 15As shown, the probe particle solution 1534 is contained in the dropper bottle 1536. The probe particles are magnetic or superparamagnetic particles coated with a second sensing element. The second sensing element is configured to bind to the first sensing element or the target analyte such that the probe particles specifically bind to the coated bottom surface 1506 of the test well 1504 depending on the presence of the target analyte in the sample. Thus, the presence of the target analyte can be determined by analyzing (e.g., counting, estimating, image processing) the specifically and / or non-specifically bound probe particles.

[0178] Specifically, if the kit 1500 is used for a crosslinking assay, the second sensing element is configured to bind to the target analyte. Thus, if the target analyte is present in the sample, the probe particles will specifically bind to the coated bottom surface 1506 via the target analyte, resulting in a larger amount of specifically bound probe particles and a smaller amount of non-specifically bound probe particles in the test well 1504. On the other hand, if the target analyte is not present in the sample, the probe particles will not be attached to the coated bottom surface 1506, resulting in no specifically bound probe particles and a large amount of non-specifically bound probe particles in the test well 1504.

[0179] Conversely, if the kit 1500 is used for a blocking assay, the second sensing element is configured to bind to the first sensing element. Thus, if the target analyte is present in the sample, the probe particles will be separated from the coated bottom surface 1506, resulting in a smaller amount of specifically bound probe particles and a larger amount of non-specifically bound probe particles in the test well 1504. On the other hand, if the target analyte is not present in the sample, the probe particles will specifically bind to the coated bottom surface 1506, resulting in a larger amount of specifically bound probe particles and a smaller amount of non-specifically bound probe particles in the test well 1504.

[0180] The first and second sensing elements can be selected from the group consisting of: carbohydrates, polysaccharides, lipids, proteins, peptides, nucleic acids, antibodies, antigens, hormones, enzymes, and chemical compounds. It should be noted that the first sensing element coated on the bottom surface 1506 of the test well 1504 can be the same as or different from the second sensing element coated on the probe particles.

[0181] The dropper bottle 1536 is connected to a syringe 1538 that includes a barrel 1540 and a plunger 1542 configured to transfer a liquid medium between the dropper bottle 1536 and the microfluidic plate 1502. As Figure 15 shown, the barrel 1540 contains a sample in the sample solution 1544 for testing. A filter 1546 is configured to be disposed in the barrel 1540 to remove particulates from the liquid medium.

[0182] The kit 1500 further includes an instrument configured to apply an external force to the probe particles. The instrument is shown in Figure 15 as a magnetic needle array 1548 including a plurality of thin magnetic needles 1550. These magnetic needles 1550 are configured to control the force applied to the bound probe particles and to control the movement of non-specifically bound probe particles into the removal channel 1524 in the microfluidic plate 1502 before the step of analyzing the specifically and / or non-specifically bound probe particles to determine the presence of the target analyte.

[0183] In an alternative embodiment, the external force applied to the probe particles can be generated by other means or methods other than magnetic needles, including but not limited to permanent magnets, electromagnets, centrifuges, acoustic methods, ultrasonic methods, laser beams, or gravity.

[0184] Advantageously, the kit 1500 is well-suited as a home self-diagnosis kit or a point-of-care testing (POCT) kit because results can be obtained without the use of bulky or expensive instruments (such as microscopes). Specifically, the intensity of the scattered light generated in the test well 1504 and the intensity of the aggregates formed inside the test well 1504 or the probe particle trap 1526 using the magnetic needles 1550 can be easily determined by the naked eye or analyzed based on images taken by a smartphone camera.

[0185] Figures 16A - 16F A schematic diagram of the kit 1500 or a part thereof when the kit 1500 is used in a cross-linking assay is shown Figure 15 in

[0186] Figure 16A Three images are shown, illustrating the steps involved in the preparation of a liquid medium including a sample and probe particles. In the first image, a syringe 1538 pushes a sample solution 1544 through a filter 1546 into a dropper bottle 1536, where the sample solution 1544 is mixed with a probe particle solution 1534. In the second image, the syringe 1538 and the dropper bottle 1536 are flipped upward to remove air from the dropper bottle 1536. In the third image, the liquid medium containing the probe particle solution 1534 and the sample solution 1544 is ready, and the dropper bottle 1536 can be connected to the inlet 1514 of the microfluidic plate 1502 together with the syringe 1538.

[0187] Figure 16B A side view of the microfluidic plate 1502 after introducing the liquid medium into the test well 1504 is shown. The microfluidic plate 1502 is placed on a flat horizontal surface to allow incubation of the sample and probe particles to occur inside the test well 1504. A first reference cue 1602 including a horizontal line and an upward arrow is shown in Figure 16B to indicate that the microfluidic plate 1502 is facing upward.

[0188] Figure 16C shows a side view of the microfluidic plate 1502 during the incubation step within the test well 1504. As Figure 16C shown, the probe particles sink to the bottom surface 1506 within the test well 1504, forming a visible layer 1604 adjacent to the bottom surface 1506 of the test well 1504.

[0189] Figure 16D shows a side view of the microfluidic plate 1502 face - down. After the incubation step within the test well 1504 is completed, the microfluidic plate 1502 is flipped 180 degrees so that the opening 1508 of the test well 1504 faces down, as shown by the second reference cue 1606 including a horizontal line and a downward arrow. Next, the magnetic needle array 1548 is placed beneath the microfluidic plate 1502 to apply a magnetic force to the probe particles. The dissociated non - specifically bound probe particles are pulled downward towards the opening 1508 of the test well 1504. As a result, the non - specifically bound probe particles form an aggregate 1608 above the magnetic needle tips and are then removed from the test well via the removal channel 1524 by sliding the microfluidic plate relative to the magnetic needle 1550.

[0190] Figure 16E shows a side view of the microfluidic plate 1502 face - down, where the aggregate 1608 is moved through the removal channel 1524 using the magnetic needle array 1548. Here, the microfluidic plate 1502 slides above the magnetic needle array 1548 while face - down to remove the aggregate 1608 of non - specifically bound probe particles from the test well 1504 to the probe particle trap 1526.

[0191] At this stage, the specifically bound probe particles that are bound to the coated bottom surface 1506 within the test well 1504 can be analyzed to determine the presence of the target analyte in the sample. The detection methods include:

[0192] a) A color detection method based on the intensity of the scattered light generated by the specifically bound probe particles in the test well 1504.

[0193] b) A microscopy detection method by imaging with the microfluidic plate 1502 placed on a microscope stage.

[0194] In addition to the above methods (a) and (b), the size of the probe particle aggregates in the test well 1504 or the probe particle trap 1526 can be analyzed to determine the presence of the target analyte in the sample. The method requires additional steps, as more detailedly explained below with reference to Figure 16F as more detailedly explained below with reference to

[0195] Figure 16FA side view of the microfluidic plate 1502 facing upward for the magnetic aggregation step performed in the test well 1504 is shown. After the aggregate 1608 of non-specifically bound probe particles is removed from the test well 1504, the microfluidic plate 1502 is flipped 180 degrees back so that the opening 1508 of the test well 1504 faces upward. As a result, the non-specific probe particles fall into the probe particle trap 1526, preventing them from moving back into the test well.

[0196] Then, the magnetic needle 1550 is placed below the bottom surface 1506 of the test well 1504 to aggregate the specifically bound probe particles into small aggregates 1610. The size of these aggregates 1610 represents the amount of specifically bound probe particles remaining on the bottom surface 1506, and it can be analyzed to determine whether a target analyte is present in the sample.

[0197] It should be noted that the method based on the probe particle aggregates can also be performed on the aggregate 1608 of non-specifically bound probe particles in the probe particle trap 1526. Alternatively, when the test well is facing downward, the aggregate 1608 of non-specifically bound probe particles can also be analyzed without the additional step of flipping the microfluidic plate 180 degrees back.

[0198] Advantageously, removing the aggregate 1608 of non-specifically bound probe particles from the test well 1504 to the probe particle trap 1526 improves the distinguishability between the specifically bound probe particles and the non-specifically bound probe particles 1608 during the analysis.

[0199] Exemplary, non-limiting embodiments of using a system, or an article of manufacture, or a kit will now be disclosed.

[0200] The use of the system, or an article of manufacture, or a kit as described herein for detecting biomolecules, bio-organelles, biological particles, cells, or microorganisms.

[0201] Biological particles can be viruses or virus-like particles or exosomes. The biological particles may be SARS-CoV-2. Biomolecules can be proteins, antibodies, antigens, DNA, or RNA. Microorganisms can be pathogenic.

[0202] Advantageously, the use of the system or an article of manufacture, or a kit can be specifically designed for the detection of biomolecules, bio-organelles, biological particles, cells, or microorganisms for use in actual clinical trials using human samples such as serum and urine. Thus, the use of the system or an article of manufacture, or a kit can allow for the detection of a target virus in a patient sample or the detection of antibodies induced by a specific viral infection.

[0203] Further advantageously, the system, article, or kit can be used at a high-throughput scale, thereby enabling rapid detection of a large number of samples in a short time. If desired, the use of the system or article or kit can also be carried out on a large scale, or can be easily scaled up to a larger scale as needed.

[0204] BRIEF DESCRIPTION OF THE DRAWINGS

[0205] The drawings illustrate the disclosed embodiments and are used to explain the principles of the disclosed embodiments. However, it should be understood that these drawings are designed for illustrative purposes only and not as a definition of the limitations of the present invention.

[0206] Figure 1A is a schematic diagram of the general concept of the detection method of the present invention, where (1) represents the sensing element, (2) represents the target analyte, and (3) represents the probe particles.

[0207] Figure 1B is a schematic diagram of the detection method of the present disclosure, where in the presence of the target analyte, specific target analyte-probe particle interactions cause the probe particles to crosslink to the coated surface, resulting in a positive result as shown in (a). In the absence of the target analyte, a negative result as shown in (b) will be obtained. The label (7) represents the target analyte, (8) represents the sensing element, and (9) represents the probe particles.

[0208] Figure 1C is a schematic diagram of the detection method of the present disclosure, where specific target analyte-probe particle interactions cause the probe particles to separate from the coated surface, resulting in a positive result as shown in (a). In the absence of the target analyte, a negative result as shown in (b) will be obtained. The label (11) represents the target analyte, (12) represents the sensing element, and (13) represents the probe particles.

[0209] Figure 2 is a schematic diagram of a proof-of-concept experimental procedure for detecting a mock virus target analyte. The label (15) represents the ACE2-coated surface, (16) represents the target analyte (RBD-coated mock virus), and (17) represents the superparamagnetic probe particles (ACE2-coated).

[0210] Figure 3 describes Figure 2 is an image of the experimental results of the procedure, where specific binding of superparamagnetic probe particles was found at the bottom of the multi-well plate for various mock virus target analyte concentrations.

[0211] Figure 4 illustrates in Figure 2Data on the experimental results of the number of superparamagnetic probe particles that specifically bind relative to the concentration of the simulated viral target analyte in the procedure described in: (a) two examples of analyzing images of the coated surface obtained under different simulated viral target analyte concentration conditions, (b) ten example trials of experiments with different simulated viral target analyte concentrations, (c) an example trial of an experiment with simulated viral analyte concentrations of 100 fM and 1 pM in the sample, (d) two example trials for determining the cross-reactivity level of the method.

[0212] Figure 5 is a schematic diagram of a proof-of-concept experimental procedure for detecting an antibody target analyte (CR3022, an antibody against SARS-CoV-2).

[0213] Figure 6 is a set of images depicting Figure 5 the experimental results of the procedure for multiple antibody analyte concentrations.

[0214] Figure 7 is data on the experimental results of the number of superparamagnetic probe particles that specifically bind relative to the concentration of the antibody target analyte (CR3022, an antibody against SARS-CoV-2) in the experiment for detecting the antibody target analyte. The experimental result data of the control experiment without using the antibody target analyte (i.e., 0 M) are used for comparison.

[0215] Figure 8A is an image depicting the visualization and subsequent quantification of the color density of the probe particles specifically bound on the coated surface. The label "+" indicates the wells containing positive results, while the label "-" indicates the wells containing negative results.

[0216] Figure 8B is an image depicting the visualization and subsequent quantification of the particle density of the probe particles specifically bound on the coated surface using a microscope.

[0217] Figure 8C is an image depicting the visualization and quantification of the aggregate size of the aggregates formed by the specifically bound probe particles on the coated surface. The arrows indicate the aggregates formed as spots on the coated surface.

[0218] Figure 8D is an image depicting the visualization and quantification of the aggregate size of the aggregates formed by the non-specifically bound probe particles on the second surface (the position of the aggregates is indicated by the white arrow). The black arrow indicates the position of the specifically bound probe particles on the coated surface.

[0219] Figure 9AData demonstrating the clinical validation of the method in the detection of anti-SARS-CoV-2 receptor binding domain (anti-SARS-CoV-2 RBD) antibodies, presenting the quantitative results of antibody levels detected in plasma samples from convalescent COVID-19 and dengue patients.

[0220] Figure 9B Data demonstrating the clinical validation of the method in the detection of anti-SARS-CoV-2 receptor binding domain (anti-SARS-CoV-2 RBD) antibodies, where Figure 9A the results obtained by the method of the present disclosure described therein are directly compared with the quantitative results obtained using ELISA titration on the same set of plasma samples from convalescent COVID-19 and dengue patients.

[0221] Figure 10A An image showing the preliminary test results for IgG antibodies against the SARS-CoV-2 receptor binding domain (SARS-CoV-2 RBD). The label "w / o" indicates the absence of the target analyte and serves as a negative control.

[0222] Figure 10B Data demonstrating Figure 10A the corresponding quantitative results. The label "w / o" indicates the absence of the target analyte and serves as a negative control.

[0223] Figure 11A An image showing the results of probe particles specifically bound to the coated glass surface of the wells for the detection of SARS-CoV-2 nucleocapsid antigen (target analyte). The label "w / o" indicates the absence of the target analyte and serves as a negative control.

[0224] Figure 11B Data demonstrating Figure 11A the corresponding microscopy imaging results for a specific sample. The label "w / o" indicates the absence of the target analyte and serves as a negative control.

[0225] Figure 11C Data demonstrating Figure 11B the corresponding quantitative results. The label "w / o" indicates the absence of the target analyte and serves as a negative control.

[0226] Figure 11D An image showing the detection of SARS-CoV-2 nucleocapsid antigen (target analyte) at a specified target analyte concentration using a commercially available PANBIOTM COVID-19 Ag rapid test device. The label "w / o" indicates the absence of the target analyte and serves as a negative control.

[0227] Figure 12The figure shown illustrates a perspective view of a kit for detecting the presence of a target analyte in a sample according to an example embodiment.

[0228] Figure 13A A side view of the well plate is shown after introducing the sample and probe particles into the test well.

[0229] Figure 13B A side view of the well plate is shown during the incubation step within the test well.

[0230] Figure 13C A side view of the well plate enclosed within a water tank is shown.

[0231] Figure 13D A side view and a perspective view of the well plate enclosed within a water tank are shown, where both the well plate and the water tank are face - down.

[0232] Figure 13E A side view and a perspective view of the well plate enclosed within a water tank are shown, where an aggregate is moved through a channel using a magnetic needle array.

[0233] Figure 13F A side view and a top view of the well plate enclosed within a water tank are shown, where both the well plate and the water tank are face - up.

[0234] Figure 14A A smartphone camera image of the well plate is shown after non - specifically bound beads have been separated by force and removed outside the test well. Prior to the force application step, the wells had been incubated with liquid media of different nucleocapsid protein concentrations under different solution conditions (top four wells: working buffer; middle four wells: middle turbinate sample; bottom four wells: saliva sample).

[0235] Figure 14B An enlarged image of the test well visible under a microscope is shown.

[0236] Figure 14C A graph is shown illustrating the relationship between the normalized fraction of the amount of probe particles and the normalized size of the probe particle aggregates.

[0237] Figure 15 The figure shown illustrates a perspective view of a kit for detecting the presence of a target analyte in a sample according to another example embodiment.

[0238] Figure 16A Three images shown illustrate the steps involved in preparing a liquid medium containing a sample and probe particles.

[0239] Figure 16B A side view of the microfluidic plate is shown after introducing the liquid medium into the test well.

[0240] Figure 16CShows a side view of the microfluidic plate during the incubation step within the test well.

[0241] Figure 16D Shows a side view of the microfluidic plate face-down.

[0242] Figure 16E Shows a side view of the microfluidic plate face-down, where an array of magnetic needles is used to move the aggregates through the removal channel.

[0243] Figure 16F Shows a side view of the microfluidic plate face-up for the magnetic aggregation step performed within the test well.

[0244] Detailed description of the drawings

[0245] Figure 1A Is a schematic general concept of the detection method of the present disclosure. First, the first sensing element (1) is pre-coated on a surface (such as a coverslip). Then the coated surface (4) is incubated with a sample that may contain the target analyte. If the target analyte (2) is present in the test sample, the target analyte (2) will bind to the coated surface (4). Thereafter, probe particles (3) coated with the second sensing element are loaded onto the coated surface (4) and incubated to allow specific interaction to occur between the target analyte (2) bound to the coated surface and the probe particles (3). Finally, a mechanical force is applied to the probe particles (3) as a selection step to separate the non-specifically bound probe particles from the coated surface (4). To determine the presence of the target analyte (2), the number of specifically bound probe particles on the coated surface is measured. The selected specific target analyte-binding surface can be used for other downstream analyses, such as sequencing.

[0246] Figure 1BSchematic diagram of the detection method of the present disclosure, where in the presence of a target analyte, specific target analyte-probe particle interactions cause the probe particles to crosslink to the coated surface. First, a first sensing element (8) is pre-coated on a surface (such as a coverslip). Then the coated surface is incubated with a sample that may contain the target analyte. If the target analyte (7) is present in the test sample, the target analyte (7) will bind to the coated surface. Thereafter, probe particles (9) coated with a second sensing element are loaded onto the surface and incubated to allow specific interactions to occur between the target analyte (7) bound to the coated surface and the probe particles (9). In this case, if the target analyte (7) is present in the test sample (positive result), the probe particles (9) can crosslink to the coated surface via specific target analyte-probe particle interactions, as shown in (a). In the absence of the target analyte (7) (negative result), the probe particles (9) will bind non-specifically to the coated surface, as shown in (b). Thereafter, a mechanical force is applied to the probe particles (9) as a selection step to separate the non-specifically bound probe particles from the coated surface. In the absence of the target analyte (7) as in (b), the probe particles (9) bind weakly to the coated surface due to non-specific binding and will thus be removed from the coated surface after the application of the mechanical force. In contrast, for the probe particles (9) crosslinked to the coated surface via specific target analyte-probe particle interactions as in (a), they can withstand a certain mechanical force and remain bound to the coated surface. To determine the presence of the target analyte (7), the number of specifically bound probe particles on the coated surface is measured.

[0247] Figure 1CSchematic diagram of the detection method of the present disclosure, where specific target analyte-probe particle interaction results in the separation of probe particles from the coated surface. First, the first sensing element (12) is pre-coated on a surface (such as a cover glass). Then the coated surface is incubated with a test sample that may contain the target analyte (11). If the target analyte (11) is present in the test sample, the analyte (11) will bind to the coated surface. Thereafter, probe particles (13) coated with the second sensing element are loaded onto the surface and incubated to allow specific interaction between the target analyte (11) bound to the coated surface and the probe particles (13). In this case, if the target analyte (11) is present in the sample, the target analyte (11) will prevent the probe particles (13) from cross-linking to the coated surface via specific target analyte-probe particle interaction as shown in (a), so the target analyte (11) causes the probe particles (13) to separate from the coated surface. In the absence of the target analyte (11), the probe particles (13) will cross-link with the coated surface, as shown in (b). Thereafter, a mechanical force is applied to the probe particles (13) as a selection step to separate non-specifically bound probe particles from the coated surface. In the absence of the target analyte (11) as in (b), due to the cross-linking of the probe particles (13) with the coated surface, the probe particles (13) bind strongly to the coated surface, so the probe particles (13) will not be removed from the coated surface after the mechanical force is applied. In contrast, for the probe particles (13) that are not connected to the coated surface due to the presence of the intermediate target analyte (11) as in (a), the probe particles (13) cannot withstand the mechanical force and will be removed from the coated surface. To determine the presence of the target analyte (11), the number of probe particles remaining on the coated surface is measured.

[0248] Figure 2 Schematic diagram of a proof-of-concept experimental procedure for detecting a mimic virus target analyte, where in the presence of the mimic virus target analyte, specific mimic virus target analyte-superparamagnetic probe particle interaction results in the cross-linking of superparamagnetic probe particles to the coated surface, resulting in a positive result as shown in (a). In the absence of the mimic virus target analyte, a negative result as shown in (b) will be obtained. The label (15) represents the ACE2-coated surface, (16) represents the target analyte (RBD-coated mimic virus), and (17) represents the superparamagnetic probe particles (ACE2-coated).

[0249] Figure 3 describes Figure 2Images of the experimental results of the procedure, where in an experiment detecting a mock viral target analyte at multiple mock viral target analyte concentrations (0 pM (control), 100 fM, 1 pM, and 10 pM), after applying a mechanical force in the form of a magnetic force to remove non-specifically bound superparamagnetic probe particles, specifically bound superparamagnetic probe particles are found at the bottom of the microplate. The results can be directly observed with the naked eye and distinguished by the intensity change on the surface (in this case, the increased opaque area at the bottom of the wells shown by the solid arrow (→), which is caused by the different densities of specifically bound superparamagnetic probe particles on the surface for different concentrations of the mock viral target analyte). The area without any superparamagnetic probe particles is shown by the dashed arrow (→).

[0250] Figure 4 illustrates the data of the experimental results of the number of specifically bound superparamagnetic probe particles relative to the mock viral target analyte concentration in the procedure described in Figure 2 where: (a) Two example analysis images of the coated surface obtained under 0 pM (control, left) and 10 pM (right) mock viral target analyte conditions, (b) Ten example trials of experiments using 0 pM (control) and 10 pM mock viral analytes. For each pair of box plots, as represented by the grey area under "assembled" and the dashed areas from "trial 1" to "trial 10", the left (lower) box plot represents the control and the right box plot (higher) represents the test value. All trials clearly show the detection of 10 pM mock viral target analyte in the sample, (c) Example trials of experiments clearly detecting 100 fM and 1 pM mock viral target analytes in the sample. Similar experiments were repeated more than 5 times, (d) Two example trials to determine the cross-reactivity level of the method. The results clearly show that the method can specifically detect the target mock viral analyte from other non-target analytes. "NA" represents neutravidin-coated paramagnetic beads.

[0251] Figure 5 is a schematic diagram of a proof-of-concept experimental procedure for detecting an antibody target analyte (CR3022, an antibody against SARS-CoV-2), where in the presence of the antibody target analyte, the specific antibody target analyte-superparamagnetic probe particle interaction causes the superparamagnetic probe particles to separate from the coated surface, resulting in a positive result as shown in (a). If the antibody target analyte is absent, it will result in a negative result as shown in (b).

[0252] Figure 6 depicts Figure 5A set of images of the experimental results of the procedure, where in the experiment for detecting the antibody target analyte (CR3022, an antibody against SARS-CoV-2), for different antibody target analyte concentrations ((a) 0 M, (b) 100 pM, (c) 1 nM, (d) 10 nM, (e) 100 nM, and (f) 1 μM), the specifically bound superparamagnetic probe particles after applying mechanical force in the form of magnetic force to remove non-specifically bound superparamagnetic probe particles.

[0253] Figure 7 Data showing the experimental results of the number of specifically bound superparamagnetic probe particles relative to the concentration of the antibody target analyte (CR3022, an antibody against SARS-CoV-2) in the experiment for detecting the antibody target analyte. The data of the control experiment without using the antibody target analyte (i.e., 0 M) was used for comparison.

[0254] Figure 8A Images depicting the visualization and subsequent quantification of specifically bound probe particles on a coated surface suitable for point-of-care testing, where the coated surface has multiple wells allowing the simultaneous testing of one or more samples, positive, and negative controls. Since the specifically bound probe particles form colored patches on the coated surface, the test results can be qualitatively analyzed by visually measuring the color density during the visualization process. The mark "+" indicates the well containing a positive result, while the mark "-" indicates the well containing a negative result.

[0255] Figure 8B Images depicting the visualization and subsequent quantification of specifically bound probe particles on a coated surface using a microscope. The particle density of specifically bound probe particles per unit area can be quantified. This is a form of quantitative analysis of the test results by measuring the particle density, with high accuracy and suitable for central laboratory-based testing.

[0256] Figure 8C Images depicting the visualization and quantification of aggregates formed by specifically bound probe particles on a coated surface suitable for central laboratory-based testing or point-of-care testing when used in conjunction with smartphone-based imaging, where the coated surface has multiple wells allowing the simultaneous testing of one or more samples, positive, and negative controls. This is a form of quantitative analysis by measuring the aggregate size or the color density of the aggregates, where the aggregate size and color density are positively or negatively correlated with the concentration of the target analyte in the sample when using crosslinking / ligation assays or blocking / separation assays, respectively. Arrows indicate the aggregates formed as spots on the coated surface.

[0257] Figure 8Dis an image depicting the visualization and quantification of aggregates formed by probe particles of non-specific binding on a second surface, which is applicable to tests in a central laboratory or point-of-care testing when used in conjunction with smartphone-based imaging, allowing for the simultaneous testing of one or more samples, positive, and negative controls. This is a form of quantitative analysis by measuring the aggregate size or the color density of the aggregates (white arrows indicate aggregates), where the aggregate size and color density are positively or negatively correlated with the concentration of the target analyte in the sample when using a blocking / separation assay or a crosslinking / ligation assay, respectively. Since the second surface can be in the form of a transparent outer shell surrounding the first surface, specifically bound probe particles (black arrows indicate specifically bound probe particles in the wells of the coated microplate) can also be observed through the second surface, and thus the visualization and quantification of specifically bound probe particles can also be measured.

[0258] Figure 10A is an image illustrating the preliminary results of detecting IgG antibodies against the SARS-CoV-2 receptor binding domain (SARS-CoV-2 RBD), where (top row) the images are obtained for specifically bound probe particles on a coated surface for different concentrations of the CR3022 antibody (target analyte), and (bottom row) the images are obtained for human sera containing the CR3022 antibody (target analyte) at different serial dilution levels. The label "w / o" indicates the absence of the target analyte and serves as a negative control.

[0259] Figure 10B is an illustration of Figure 10A the corresponding quantitative results, where in the experiment for IgG antibodies against the SARS-CoV-2 receptor binding domain (SARS-CoV-2 RBD), specifically bound probe particles on a coated surface of every 100x100μm 2 area were detected. The left figure shows the particle density data obtained for specifically bound probe particles on a coated surface at different concentrations of the CR3022 antibody (target analyte), and the right figure shows the particle density data obtained for human sera containing the CR3022 antibody (target analyte) at different serial dilution levels. The label "w / o" indicates the absence of the target analyte and serves as a negative control.

[0260] Figure 11A is an image illustrating the results of specifically bound probe particles on a coated glass surface of wells for detecting the SARS-CoV-2 nucleocapsid antigen (target analyte), where the SARS-CoV-2 nucleocapsid antigen was incorporated into a homogenization buffer at a specified concentration (top four wells), middle turbinate samples pretreated with the homogenization buffer (middle four wells), and saliva samples pretreated with the homogenization buffer (bottom four wells). The label "w / o" indicates the absence of the target analyte and serves as a negative control.

[0261] Figure 11B illustrates the corresponding microscopic imaging results (using a 4x magnification lens) of probe particles with specific binding to middle turbinate samples (top row) and saliva samples (bottom row) in an experiment for detecting SARS-CoV-2 nucleocapsid antigen (target analyte). The label "w / o" indicates the absence of the target analyte and serves as a negative control. Figure 11A illustrates the corresponding microscopic imaging results (using a 4x magnification lens) of probe particles with specific binding to middle turbinate samples (top row) and saliva samples (bottom row) in an experiment for detecting SARS-CoV-2 nucleocapsid antigen (target analyte). The label "w / o" indicates the absence of the target analyte and serves as a negative control.

[0262] Figure 11C illustrates Figure 11B the corresponding quantitative results, where specific binding probe particles on a coated surface with an area of every 100x100μm 2 were detected to detect SARS-CoV-2 nucleocapsid antigen (target analyte), and the SARS-CoV-2 nucleocapsid antigen was incorporated into middle turbinate samples (left figure) and saliva samples (right figure) at a specified concentration. The label "w / o" indicates the absence of the target analyte and serves as a negative control.

[0263] Figure 11D illustrates the detection of SARS-CoV-2 nucleocapsid antigen (target analyte) at a specified target analyte concentration using a commercially available PANBIOTM COVID-19Ag rapid test device. The label "w / o" indicates the absence of the target analyte and serves as a negative control.

[0264] Example

[0265] Example 1: Detection of an Analyte Simulating the SARS-CoV-2 Virus

[0266] To demonstrate the feasibility of the detection, mock SARS-CoV-2 virus particles prepared by coating microspherical polystyrene particles with a layer of SARS-CoV-2 receptor binding domain protein (RBD) were used as the target analyte (16) in the experiment as Figure 2 shown to mimic real virus bioparticles. The diameter of the mock virus particles is approximately 200nm, within the range of real virus bioparticles from 20nm to 400nm. The coated surface (15) used is a coverslip coated with a layer of the receptor protein ACE2 as a sensing element. The superparamagnetic probe particles (17) are also coated with a layer of the receptor protein ACE2 as a sensing element. The receptor protein ACE2 was selected because it is known to be able to bind to the RBD coated on the mock virus particles.

[0267] The coated coverslip (15) was incubated with 10 μl of a liquid saliva sample containing pseudovirus particles coated with RBD at concentrations of 100 fM, 1 pM, and 10 pM for 30 minutes at room temperature to allow the RBD-coated pseudovirus particles (16) to bind to the coated coverslip (15). The volume of the liquid sample used was sufficient to completely cover the surface of the coated coverslip (15). A control experiment was conducted in which the coated coverslip (15) was incubated with 10 μl of a liquid saliva sample without coated pseudovirus particles (i.e., 0 M RBD-coated pseudovirus particles).

[0268] Thereafter, a sufficient amount of ACE2-coated superparamagnetic probe particles (17) was loaded onto the coverslip and incubated for 10 minutes at room temperature. The coverslip was exposed to a pN-level selective mechanical force using a superparamagnetic array to separate non-specifically bound ACE2-coated superparamagnetic probe particles from the ACE2-coated coverslip. For the above sample incubation duration, applied mechanical force level, and duration of force application, calibration was performed for the pseudovirus target analyte and the corresponding sensing elements demonstrated in the examples. These parameters may vary when using different sensing elements or for different analytes. For each system, these values should be pre-calibrated before application.

[0269] The coated coverslip (15) that has bound the target pseudovirus target analyte (16) (i.e., RBD-coated pseudovirus particles) will crosslink with the superparamagnetic probe particles (17), resulting in a much higher density of specifically bound probe particles on the surface compared to the control when exposed to the selective mechanical force as shown in (a) of Figure 2 .

[0270] The sample wells on the coverslip can be directly observed with the naked eye as shown in Figure 3 , or observed under a microscope with a 4× objective lens to record high-resolution images. In the case of recording images with an optical microscope, the remaining superparamagnetic probe particles on the coated surface can be quantified by automatically counting the number of particles using specially written software, as shown in Figure 4 . According to the results, for target analytes with concentrations ranging from 100 fM to 10 pM, the number of superparamagnetic probe particles was significantly different from that obtained in the control. In addition, Figure 4 the results of (d) show that the method is capable of specifically detecting pseudovirus target analytes from other non-target analytes.

[0271] The results were used as a preliminary proof of concept for the present disclosure, demonstrating that the detection limit for low-concentration target analytes can be as low as the sub-picomolar level. Based on this result, the method of the present disclosure can be applied to a clinical setting for accurate diagnosis of viruses in human samples.

[0272] Example 2: Detection of an Antibody Analyte

[0273] The ability of the present disclosure in antibody detection was also tested. In this experiment, CR3022 (an antibody that can bind to the SARS-CoV-2 receptor binding domain protein (RBD)) was used as the target analyte (21) as shown in Figure 5 Figure. The coated surface (20) used was a coverslip coated with a layer of RBD as its sensing element. The superparamagnetic probe particles (22) were coated with a layer of the receptor protein ACE2 as the sensing element. The receptor protein ACE2 was selected because it is known to be able to bind to RBD. Since it is also known that the target antibody analyte CR3022 (21) can bind to RBD, the target analyte (21) will prevent the superparamagnetic probe particles (22) from binding to the coated coverslip (20).

[0274] The coated coverslip (20) was incubated with 10 μl of a liquid serum sample containing CR3022 antibodies at concentrations of 100 pM, 1 nM, 10 nM, 100 nM, and 1 μM at room temperature for 60 minutes to allow CR3022 (21) to bind to the RBD-coated coverslip (20). The volume of the liquid sample used was sufficient to completely cover the surface of the coated coverslip (20). A control experiment was conducted in which the coated coverslip (20) was incubated with 10 μl of a liquid serum sample without the antibody CR3022 (i.e., 0 M CR3022).

[0275] Thereafter, the ACE2-coated superparamagnetic probe particles (22) were loaded onto the coverslip and incubated at room temperature for 10 minutes. The coverslip was exposed to a selection mechanical force at the pN level using a magnet array to separate the non-specifically bound ACE2-coated superparamagnetic probe particles from the RBD-coated coverslip.

[0276] For the above sample incubation duration, applied mechanical force level, and duration of force application, calibration was performed for the specific sensing element used in the serum. These parameters may change when using different sensing elements or different media. For each system, these values should be pre-calibrated before application.

[0277] The coated coverslip (20) that has bound the target analyte CR3022 (21) will prevent the superparamagnetic probe particles (22) from cross-linking with the RBD-coated coverslip (20), thus resulting in a smaller amount of superparamagnetic probe particles remaining on the coated surface after the application of mechanical force. Therefore, the target analyte CR3022 (21) inhibits the stable binding of the superparamagnetic probe particles (22) to the coated coverslip (20). As shown in Figure 5As shown in (a) thereof, after exposure to the selected mechanical force, the superparamagnetic probe particles (22) are removed, resulting in a positive result indicated by a decrease in the number of superparamagnetic probe particles remaining on the surface. In a control experiment where the coated cover glass (20) has no binding of the target analyte (21), after exposure to the selected mechanical force as shown in Figure 5 (b) thereof, the superparamagnetic probe particles (22) will stably bind to the coated surface (20), which results in a negative result indicated by more superparamagnetic probe particles remaining on the surface.

[0278] The cover glass is placed under a microscope using a 4x objective lens to observe the specifically bound superparamagnetic probe particles on the coated surface, as shown in Figure 6 shown. According to these results, for the target analyte with a concentration ranging from 1 nM to 1 μM ( Figure 6 (c) to Figure 6 (f)), the number of specifically bound superparamagnetic probe particles on the coated surface is significantly lower than the number of specifically bound superparamagnetic probe particles on the control coated surface ( Figure 6 (a)).

[0279] The detailed quantification of the specifically bound superparamagnetic probe particles on the coated surface is as shown in Figure 7 shown. The results clearly show that for the target antibody analyte with a concentration ranging from 1 nM to 1 μM, the number of specifically bound superparamagnetic probe particles on the coated surface is significantly lower than the number of specifically bound superparamagnetic probe particles on the control coated surface. Therefore, as a preliminary proof of concept of the present disclosure, it is demonstrated that the detection limit of low-concentration target antibody analytes in undiluted serum can be as low as the nanomolar level.

[0280] Based on this result, the method of the present disclosure can be applied to the clinical environment for accurate diagnosis of virus antibodies in human samples.

[0281] Example 3: Detection of Anti-SARS-CoV-2 RBD Antibodies in Clinical Samples

[0282] Antibody detection: The method of the present disclosure has been applied to detect and quantify anti-receptor binding domain protein (RBD) IgG antibodies (target analytes) in human plasma (samples). A total of 22 convalescent plasma samples were used in the validation experiment. Among these samples, 18 plasma samples were collected from 17 patients who had recovered from COVID-19. One patient donated two samples on different dates, namely sample #10 and #11. Sample #11 was collected 90 days after the patient was admitted to the hospital. All other samples were collected between 29 and 59 days after admission. In addition to the COVID-19 convalescent patient samples, the anti-RBD antibody levels in 4 plasma samples (samples #19 to #22) from patients who presented with dengue symptoms and were confirmed to be SARS-CoV-2 negative were also detected using the method of the present invention. Although the patients who provided samples #20 and #22 were tested negative for dengue, these 4 samples (samples #19 to #22) were used as negative controls.

[0283] Quantification of the anti-RBD antibody levels in all samples is shown in Figure 9A . The particle density of the probe particles specifically bound on the coated surface obtained for the measured samples was compared with a commercially available SARS-CoV-2 negative human serum (Sigma Aldrich, US) with a result set to 0, and a serum (Acro Biosystems) incorporated with 1 μM SAD-S35 anti-RBD antibody with a result set to 100. According to Figure 9A the results shown, it was found that the dengue patient samples #19 to #22 were consistent, generating signals near 0, indicating the absence of anti-RBD antibodies in these patient samples. For the COVID-19 patient samples, as shown by the results of samples #1 to #18 in Figure 9A , significantly different anti-RBD antibody levels were measured. For example, it was found that the antibody intensity in patient sample #5 exceeded 1 μM SAD-S35, with a reading of approximately 127, while the readings of many other samples (such as #3, #7-11, #13, etc.) were comparable to the negative control, indicating the absence of such specific antibodies in the patient plasma.

[0284] The antibody levels in the same COVID-19 patient samples were measured using the current gold standard ELISA titer assay. The quantitative results obtained from the ELISA titer assay were represented by the half-maximal absorbance of the optical density at a wavelength of 450 nm. The data generated by the method of the present disclosure shown in Figure 9A was plotted against the results generated by the ELISA titer assay shown in Figure 9B . According to Figure 9B the results shown, it was observed that the higher the titer value, the higher the signal generated by the method. When used to quantify anti-RBD antibodies in human plasma, the method of the present disclosure showed similar accuracy to the ELISA titer assay.

[0285] Quantitative titer antibody assay: The methods of the present disclosure can be used to perform classical quantitative titer assays where a sample is serially diluted until the signal generated is below a certain preset value. The method can include a standard antibody as a reference signal for additional quantitative levels.

[0286] In this experiment, the well-characterized RBD-binding IgG antibody CR3022 (Creative Biolabs) was used as the target analyte and dissolved at different concentrations in pre-diluted human serum samples (Sigma Aldrich, US). Commercially available human serum was mixed with laboratory-prepared homogenized working buffer in a 1:49 ratio (i.e., human serum was diluted 50-fold) to prepare pre-diluted human serum. Probe particles (superparamagnetic microbeads) coated with RBD (the second sensing element) were incubated with 50 μl of the pre-diluted human serum / CR3022 mixture in a test tube for 15 minutes to allow the probe particles to bind to the CR3022 target analyte in the pre-diluted human serum. Thereafter, the pre-diluted human serum was separated from the probe particles by using a magnet to capture the superparamagnetic probe particles in the test tube and removing the pre-diluted human serum solution using a micropipette. Thereafter, the probe particles were resuspended in 50 μl of laboratory-prepared homogenized working buffer in the test tube, and 30 μl of the resuspended mixture was loaded into the test wells, incubated for 30 minutes, and then analyzed.

[0287] Based on Figure 10A the results shown in, the methods of the present disclosure showed the ability to detect and quantify the presence of the CR3022 target analyte at concentrations as low as approximately 10 pM (which is approximately 1000-fold lower than the dissociation constant of the CR3022 target analyte from the RBD sensing element). Considering that the human serum was pre-diluted 50-fold, it corresponds to approximately 500 pM CR3022 target analyte (equivalent to approximately 0.1 μg / mL CR3022 target analyte). As Figure 10B shown, the methods of the present disclosure can also be used to perform titer assays. The tests using the methods of the present disclosure take approximately 45 minutes, while typical quantitative ELISA assays take 2 to 3 hours as it involves multiple rounds of buffer exchange. In addition, the tests using the methods of the present disclosure only require 30 μl of pre-diluted human serum solution (diluted 50-fold); thus only a small amount of the original human serum is needed, which can be conveniently obtained by finger-prick extraction of a blood drop. Although the data obtained was based on a specific RBD-binding IgG antibody (target analyte) dissolved in commercially available human serum, it is expected that the same principle would apply to other RBD-binding IgG antibodies, or other antibodies against different antigens as target analytes, where the sample can be extracted from whole blood samples collected by finger-prick.

[0288] Example 4: Detection of SARS-CoV-2 Nucleocapsid Antigen in Human Saliva and Middle Turbinate-Spiked Samples

[0289] In this experiment, a pair of antibodies that recognize different epitopes of the SARS-CoV-2 nucleocapsid antigen (target analyte) were used as the first and second sensing elements, and were coated on a surface and probe particles, respectively. A homogenization buffer was prepared to homogenize saliva samples and middle turbinate samples premixed with the antigen. The samples mixed with the homogenization buffer will be referred to as pretreated samples hereinafter. The homogenization buffer can also facilitate the release of the nucleocapsid antigen from SARS-CoV-2 virions. This experiment was conducted based on saliva and middle turbinate samples obtained from healthy human donors, in which different concentrations of the nucleocapsid antigen were spiked into the samples.

[0290] Figure 11A The results shown were obtained from an experiment conducted in a 12-well plate, where the bottom glass surface was coated with an antibody belonging to the anti-SARS-CoV-2 nucleocapsid antibody pair as described above. Probe particles (superparamagnetic microbeads) (Invitrogen, US) were coated with the other antibody in the pair to be used as probe particles. The probe particles were mixed with the homogenization buffer, saliva samples pretreated with the homogenization buffer, or middle turbinate samples, and 30 μl of the mixture was loaded into each well. After incubation for 30 minutes, a mechanical force was applied to the probe particles using a magnet array to separate non-specifically bound probe particles from the coated surface. Thereafter, specifically bound probe particles on the coated surface were detected using a smartphone camera (as Figure 11A shown) or a microscope (as Figure 11B shown). For microscopic detection, the particle density of specifically bound probe particles on the coated surface for every 100×100 μm 2 area was quantified.

[0291] Figure 11A The results indicate that specifically bound probe particles can be directly observed using a smartphone camera to detect the pale yellow light scattered from the specifically bound probe particles, where the specifically bound probe particles can be observed as yellow patches. The intensity of the scattered pale yellow light can indicate the relative amount of specifically bound probe particles contained in the well. Figure 11A The results show that there are visually perceivable differences in color density for different target analyte concentrations of 10 pM, 1 pM, and 0 pM (labeled "w / o", negative control) in the homogenization buffer (top 4 wells), middle turbinate samples pretreated with the homogenization buffer (middle 4 wells), or saliva samples pretreated with the homogenization buffer (bottom 4 wells).

[0292] Figure 11BThe results show a microscopic image of the coated glass surface of the test well when using a 4x magnifying objective lens. Specific binding probe particles were observed as tiny dark spots in the microscopic image, and the particle density of the specific binding probe particles decreased as the concentration of the nucleocapsid antigen (target analyte) decreased. Based on the particle density on the coated surface per 100×100μm 2 area of the coated surface, Figure 11B The corresponding quantification of the particle density of the specific binding probe particles of Figure 11C is shown in

[0293] Figures 11A to 11C The results prove that when using a smartphone camera or a microscope to visualize and quantify saliva and middle turbinate samples for ultra-low viral loads (i.e., target analyte concentration) (as low as about 1 fM (about 10 5 / mL)), the method of the present disclosure can be used to detect the presence of ≥1 pM SARS-CoV-2 nucleocapsid antigen, provided that most of the nucleocapsid antigen is released by the homogenization buffer. This ultra-low viral load achievable is comparable to the detection limit of the current RT-qPCR detection method, where in contrast, the method of the present disclosure is faster, cheaper, and easier to operate.

[0294] In Figure 11D another comparison shown, it was observed that the commercially available PANBIO TM COVID-19 Ag rapid test device only detected >10 pM nucleocapsid antigen (target analyte) incorporated into the working buffer of the PANBIO TM antigen detection kit. Therefore, the sensitivity of nucleocapsid antigen detection based on the method of the present disclosure is at least 10 times higher than that of the PANBIO TM antigen detection kit. Although the data provided were obtained from saliva and middle turbinate samples, similar detection sensitivities are expected for nasopharyngeal swab samples.

[0295] Industrial Applicability

[0296] The methods, systems, articles, and kits disclosed herein can be used in a wide variety of diagnostic applications, such as clinical testing, environmental monitoring, central laboratory testing, and home self-diagnosis, to detect the presence of a target analyte in a sample. The methods, systems, articles, and kits provide single-target detection sensitivity using specific target analyte-probe particle interactions, such that low concentrations of target analytes in the nanomolar to femtomolar range can be detected, thus reducing the sample volume required, which in turn enables early diagnosis of diseases in clinical testing.

[0297] It will be apparent to those skilled in the art that, upon reading the foregoing disclosure, various other modifications and improvements of the invention will be apparent to those skilled in the art without departing from the spirit and scope of the invention, and it is intended that all such modifications and improvements be included within the scope of the appended claims.

Claims

1. A method for detecting the presence of a target analyte in a sample, the method comprising the steps of: (a) incubating the sample with a surface coated with a first sensing element, wherein the first sensing element is capable of specifically binding to the target analyte, and wherein, during or after incubation, when the target analyte is present in the sample, the target analyte binds to the first sensing element present on the coated surface; (b) incubating the coated surface of step (a) with a plurality of magnetic probe particles, wherein the probe particles are coated with a second sensing element, and the second sensing element is capable of specifically interacting with the first sensing element on the coated surface; (c) applying a mechanical force to separate non-specifically bound probe particles of step (b) from the coated surface; and (d) measuring a property that reflects the amount of specifically bound probe particles on the coated surface, wherein the property is selected from the group consisting of: particle density, color density, aggregate size, and combinations thereof, or measuring a property that reflects the amount of non-specifically bound probe particles, wherein the property is selected from the group consisting of: color density, aggregate size, and combinations thereof, wherein the target analyte is a biomolecule or a biological particle.

2. The method according to claim 1, wherein the first sensing element is different from the second sensing element, and wherein the first sensing element and the second sensing element are biomolecules or biological particles, or wherein the first sensing element and the second sensing element are independently selected from the group consisting of: proteins and antigens.

3. The method according to claim 2, wherein the protein is an antibody.

4. The method according to claim 1 or 2, wherein the target analyte is soluble in an aqueous medium or a solvent, and wherein the target analyte is dispersed in a liquid medium before incubation with the coated surface.

5. The method according to claim 1 or 2, wherein the size of the probe particles is in the range of 8 nm to 100,000 nm.

6. The method according to claim 1 or 2, wherein the mechanical force is generated by a permanent magnet, an electromagnet, a centrifuge, an acoustic device, an ultrasonic wave, a laser beam, fluid motion, fluid buoyancy, or gravity, and wherein the mechanical force is in the range of 0.01 pN to 100 pN.

7. The method according to claim 1, wherein when the measured property is color density, or aggregate size, or color density and aggregate size, step (d) further comprises step (d0): aggregating the specifically bound probe particles on the coated surface into aggregates by applying a magnetic force on the other side of the coated surface before step (d).

8. The method according to claim 1, wherein step (c) further comprises providing a second surface to contact the non-specifically bound probe particles separated from the coated surface; and aggregating the non-specifically bound probe particles into aggregates on the other side of the second surface by applying a mechanical force on one side of the second surface; wherein the mechanical force is a magnetic force.

9. The method according to claim 8, further comprising measuring the color density of the aggregates, or the aggregate size, or the color density and the aggregate size.

10. The method according to claim 7 or 8, wherein the magnetic force is generated by a permanent magnet in the form of a magnetic needle or an electromagnet, and wherein the magnetic force is in the range of 0.01 pN to 100 pN.

11. The method according to claim 1 or 2, wherein when steps (a) and (b) are carried out simultaneously, a mixture of the sample and the plurality of probe particles is incubated with the coated surface.

12. The method according to claim 1, wherein the magnetic probe particles are superparamagnetic probe particles.

13. A system for detecting the presence of a target analyte in a sample, the system comprising: (a) a surface coated with a first sensing element, wherein the first sensing element is capable of specifically binding to the target analyte; (b) a plurality of magnetic probe particles, wherein the probe particles are coated with a second sensing element, the second sensing element being capable of specifically interacting with the first sensing element on the coated surface, thereby forming specifically bound probe particles on the coated surface; (c) a mechanical force capable of separating non-specifically bound probe particles from the coated surface; and (d) a measuring device for measuring a property of the specifically bound probe particles, wherein the property is selected from the group consisting of: the particle density, color density, aggregate size of the specifically bound probe particles; or for measuring a property of the non-specifically bound probe particles, wherein the property is selected from the group consisting of: the color density, aggregate size of the non-specifically bound probe particles, wherein the target analyte is a biomolecule or a biological particle.

14. The system according to claim 13, further comprising a second surface for contacting the non-specifically bound probe particles; and a measuring device for measuring a property of the non-specifically bound probe particles, wherein the property is selected from the group consisting of: color density, aggregate size, and combinations thereof.

15. The system according to claim 13, wherein the magnetic probe particles are superparamagnetic probe particles.

16. An article for detecting the presence of a target analyte in a sample, the article comprising: at least one test well comprising a bottom surface coated with a first sensing element configured to bind to the target analyte in the sample, wherein the target analyte is a biomolecule or a biological particle and wherein the test well is configured to receive a sample and magnetic probe particles contained in one or more liquid media, the probe particles being coated with a second sensing element configured to bind to the first sensing element, such that the probe particles specifically bind to the coated surface depending on the presence of the target analyte in the sample; and A channel that is connected to the test hole at a certain distance from the bottom surface to allow fluid communication between the channel and the test hole, wherein the channel is configured to receive non-specifically bound probe particles in the test hole after an external force is applied to the probe particles, and the external force moves the non-specifically bound probe particles away from the bottom surface.

17. The article according to claim 16, wherein the channel includes a probe particle trap configured to accommodate the non-specifically bound probe particles received by the channel after the external force applied to the probe particles is removed.

18. The article according to claim 16 or 17, further comprising a system control hole, the system control hole including a bottom surface coated with the target analyte, wherein the system control hole is configured to receive the sample and the probe particles.

19. The article according to claim 16 or 17, further comprising a blank control hole, the blank control hole including a bottom surface coated with a non-bait molecule that prevents binding to the target analyte, wherein the blank control hole is configured to receive the sample and the probe particles.

20. The article according to claim 16 or 17, further comprising a negative control hole, the negative control hole including a bottom surface coated with the first sensing element, wherein the negative control hole is configured to receive one or more liquid media in which the target analyte is absent.

21. The article according to claim 16 or 17, wherein the channel is connected to the test hole adjacent to the opening of the test hole, and the opening is formed in the top portion of the test hole.

22. The article according to claim 21, further comprising a housing, the housing including an inner surface configured to face the test hole, and wherein the channel includes a space defined between the inner surface of the housing and a wall extending outward from the opening of the test hole.

23. The article according to claim 16, wherein the magnetic probe particles are superparamagnetic probe particles.

24. A kit for detecting the presence of a target analyte in a sample, the kit comprising: The article according to any one of claims 16 to 23; Magnetic probe particles coated with a second sensing element, the probe particles being contained in a solution; And An instrument configured to apply an external force to the probe particles, wherein the target analyte is a biomolecule or a biological particle.

25. The kit according to claim 24, wherein the instrument includes a magnetic needle.

26. The kit according to claim 24 or 25, further comprising a syringe for introducing the sample into a dropper bottle to mix with the solution containing the probe particles to form one or more liquid media and transferring the one or more liquid media to the article.

27. The kit according to claim 24, wherein the magnetic probe particles are superparamagnetic probe particles.

28. A method of using the kit according to any one of claims 24 to 27, the method comprising the following steps: Introduce the sample and the magnetic probe particles included in the one or more liquid media into the test well of the article; Apply the external force to the probe particles to move the non-specifically bound probe particles away from the bottom surface of the test well and into the channel; Analyze the probe particles in at least one selected from the group consisting of the test well and the channel to determine the presence of the target analyte in the sample, wherein the target analyte is a biomolecule or a biological particle.

29. The method according to claim 28, wherein the step of applying the external force to the probe particles comprises: Invert the article; and Use a magnetic needle to control the movement of the non-specifically bound probe particles into the channel.

30. The method according to claim 28, wherein the magnetic probe particles are superparamagnetic probe particles.

31. Use of the system according to any one of claims 13 to 15 or the article according to any one of claims 16 to 23 or the kit according to any one of claims 24 to 27 for detecting biomolecules or biological particles.

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