METHODS AND APPARATUS FOR DETECTING MOLECULES

Abstract

The present invention provides a method and an apparatus for detecting molecules. The method for detecting a signal molecule comprises the following steps: (1) providing a solution comprising microparticles, wherein the microparticles comprise microparticles binding to the signal molecule to be detected; (2) applying the microparticles in the solution to the surface and/or the interior of a solid phase support; (3) counting the microparticles in a selected field of view under a bright field; (4) counting the microparticles binding to the signal molecule in a selected field of view under a dark field; and (5) determining the concentration of the signal molecule according to the counting results obtained in step (3) and step (4). On this basis, the present invention also provides a method and an apparatus for detecting a target molecule. The methods and apparatus provided by the present invention can realize a rapid, simple and convenient detection of molecules, especially biomolecules, which are of low cost, and facilitate promotion in multiple fields including scientific research, clinical diagnosis, and epidemic prevention.

Claims

1. A method for detecting a signal molecule, comprising the following steps of: (1) providing a solution comprising microparticles, wherein the microparticles comprise microparticles binding to the signal molecule to be detected; (2) immobilizing the microparticles in the solution to the surface and/or the interior of a solid phase support; (3) counting the microparticles in a selected field of view under a bright field; and (4) counting the microparticles binding to the signal molecule in a selected field of view under a dark field; and (5) determining the concentration of the signal molecule according to the counting results obtained in steps (3) and (4); wherein step (3) comprises removing the agglomerated/overlapped microparticles in the bright field.

2. A method for detecting one or more target molecules, comprising the following steps of: (1) providing a solution comprising microparticles, wherein a target molecule forms a complex by a specific binding reaction, the microparticles is linked to the complex, and the complex is labeled with a signal molecule; (2) immobilizing the microparticles in the solution to the surface and/or the interior of a solid phase support; (3) counting the microparticles in a selected field of view under a bright field; (4) counting the microparticles binding to the complex in a selected field of view under a dark field; and (5) determining the concentration of the signal molecule according to the counting results obtained in steps (3) and (4), and further determining the concentration of the target molecule; wherein step (3) comprises removing the agglomerated/overlapped microparticles in the bright field.

3. The method according to claim 2, wherein in step (2), the solid phase support does not need to be spatially divided to achieve a random distribution.

4. The method according to claim 2, wherein an uncertain quantity of microparticles are immobilized to the surface and/or the interior of the solid support.

5. The method according to claim 2, wherein the target molecule is one or more selected from the group consisting of a protein, a polypeptide, an amino acid, an antigen, a receptor, a ligand, and a nucleic acid.

6. The method according to claim 2, wherein the target molecule is an antibody.

7. The method according to claim 2, wherein the specific binding reaction is one or more selected from the group consiting of an immune reaction, a hybridization reaction, and a receptor-ligand interaction.

8. The method according to claim 2, wherein the microparticles are magnetic microparticles.

9. The method according to claim 8, wherein the magnetic microparticles are magnetic beads have a particle size ranging from 600 nm to 10 .Math.m.

10. (canceled)

11. (canceled)

12. The method according to claim 2, wherein the signal molecule is one or more selected from the group consiting of a chromophore, a digoxin-labeled probe, a metal nanoparticle, and an enzyme or form the group consiting of an organic small molecule fluorescent probe, a quantum dot, a fluorescent bead, a three-dimensional DNA nanostructure reporter probe, an upconversion luminescent nanomaterial bead, a rolling circle amplification fluorescent molecule amplification structure, and a nucleic acid aptamer fluorescent molecule amplification structure.

13. (canceled)

14. The method according to claim 2, wherein the signal molecule is a quantum dot bead.

15. (canceled)

16. The method according to claim 2, wherein in step (2), the microparticles are immobilized to the surface and/or the interior of the solid phase support by an applied magnetic field and/or an electric field and/or a gel.

17. The method according to claim 2, wherein the solid phase support is selected from a multi-well plate, a flat plate or a flow channel.

18. The method according to claim 2, wherein the coordinates of the microparticles in the image are determined by bright field microscopic imaging.

19. The method according to claim 18, wherein the coordinates of the microparticles are determined from the difference in brightness of the microparticles, wherein the difference in brightness of the microparticles is the brightness difference of the microparticles themselves.

20. (canceled)

21. The method according to claim 18, wherein the counting in step (4) is determined by the coordinates of the microparticles in the image.

22. The method according to claim 1, wherein the method for determining the concentration of the target molecule in step (5) comprises: determining the concentration of the target molecule according to a proportional relation between the numbers of the microparticles obtained in step (3) and step (4) in combination with a standard curve.

23. The method according to claim 2, wherein the method for determining the concentration of the target molecule in step (5) comprises: determining the concentration of the target molecule according to a proportional relation between the numbers of the microparticles obtained in step (3) and step (4) in combination with a standard curve.

24. Use of the method according to claim 2 in the preparation of a diagnostic reagent for detecting a biomolecule.

25. A detection apparatus for implementing the method according to claim 2, comprising: a solid phase support capable of immobilizing microparticles, the microparticles comprising microparticles binding to a signal molecule to be detected and being dispersed at the surface and/or in the interior of the solid phase support; at least one first light source irradiating microparticles within a selected field of view of the solid phase support to form bright field signals related to the total number of microparticles, and at least one second light source irradiating microparticles within a selected field of view of the solid phase support to form dark field signals related to the total number of microparticles binding to the signal molecule to be detected; a signal acquisition unit for acquiring the bright field signals and dark field signals; and a signal processing unit for determining the concentration of the signal molecule according to the acquired bright field signals and dark field signals.

26. The apparatus according to claim 25, wherein the signal acquisition unit comprises an amplification assembly for amplifying microparticles within the selected field of view.

27. The apparatus according to claim 26, wherein the amplification assembly is an objective lens.

28. (canceled)

29. Apparatus according to claim 25, wherein the solid phase support is at least a partially optically transparent support.

30. The apparatus according to claim 29, wherein the solid phase support is removably disposed above or below the signal acquisition unit.

31. (canceled)

32. The apparatus of claim 25, wherein the solid phase support comprises at least one flow channel comprising an inlet and an outlet, and a solution comprising the microparticles is dispersed within the flow channel.

33. The apparatus of claim 25, wherein the solid phase support is a multi-well plate or a flat plate.

34. The apparatus according to claim 25, wherein the apparatus further comprises a magnetic field generating device or an electric field generating device for immobilizing and dispersing the microparticles at the surface and/or in the interior of the solid phase support.

35. The apparatus according to claim 25, wherein the solid phase support is a turntable which can rotate relative to the signal acquisition unit, wherein the turntable comprises at least one optically transparent detection site which is detected by the signal acquisition unit when the detection site is located in an optical path of the signal acquisition unit, wherein the turntable is configured to rotate sequentially between a plurality of stations in a stepwise manner and to perform the following operations on a solution to be detected at the detection site at the plurality of stations: immobilizing and rinsing microparticles in the solution, wherein the plurality of stations comprise a detection station located in an optical path of the signal acquisition unit, at least one pre-treatment station located upstream of the detection station, and at least one post-treatment station located downstream of the detection station, wherein an operation of immobilizing and dispersing microparticles in the solution is performed at the pre-treatment station, and a rinsing operation is performed at the post-treatment station.

36. (canceled)

37. (canceled)

38. The apparatus according to claim 25, wherein the signal acquisition unit comprises at least one photographing assembly.

39. The apparatus according to claim 25, wherein the apparatus further comprises a displacement mechanism for actuating the signal acquisition unit/solid phase support.

40. (canceled)

41. A non-transient computer-readable storage medium for storing a program for executing the method according to claim 2 and/or data generated by programs for executing the method and data enerated by executing the method.

42. An electronic device comprising the non-transient computer-readable storage medium according to claim 41.

43. Use of the method according to claim 1 in the preparation of a diagnostic reagent for detecting a biomolecule.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0144] The embodiments of the present invention will be described in detail in connection with the drawings, in which:

[0145] FIG. 1 is a schematic representation of a magnetic bead which is coated with capture antibodies to capture antigens and forms an immune complex on the surface thereof according to one embodiment of the present invention;

[0146] FIG. 2 is a photograph of magnetic beads taken under a bright field microscopic imaging mode according to one embodiment of the present invention;

[0147] FIG. 3 is a photograph of a solution of plated magnetic beads having different particle sizes taken in a bright field microscopic imaging mode according to one embodiment of the present invention;

[0148] FIG. 4 is a photograph of a solution of plated magnetic beads separately taken in bright field and dark field microscopic imaging modes according to one embodiment of the present invention.

[0149] FIG. 5 shows the detection result of different concentrations of Biotin-Qbeads with streptavidin-modified magnetic beads according to one embodiment of the present invention;

[0150] FIG. 6 shows the detection result of expressed and purified Spike protein according to one embodiment of the present invention;

[0151] FIG. 7 shows the detection result of pseudovirus expressing a novel coronavirus S protein on the surface according to one embodiment of the present invention;

[0152] FIG. 8 shows the detection result of IL-6 according to one embodiment of the present invention;

[0153] FIG. 9 is a schematic representation of the turntable according to one embodiment of the present invention;

[0154] FIG. 10 is a schematic representation of the apparatus for detecting molecules according to one embodiment of the present invention; and

[0155] FIG. 11 is a schematic representation of the position of the turntable relative to the objective lens according to one embodiment of the present invention;

[0156] whrerein 1-position 1; 2-position 2; 3-position 3; 4-position 4; 5-position 5; 6-position 6; 7-position 7; 8-position 8; 201-turntable; 202-blind hole; 203- rotary shaft; 301-magnetic bead solution; 302-blind hole containing a solution of magnetic beads to be detected; 303-bright field light source; 304-condenser lens; 305-objective lens; 306-fluorescent light source; 307-lens; 308-dichroic beamsplitter; 309-filter; 310-lens; 311-camera.

BEST MODES FOR CARRYING OUT THE INVENTION

[0157] The present invention will be further described in detail below in connection with the specific examples. The examples given are only for the purpose of illustrating the present invention, but not intended to limit the scope of the present invention.

Detection Apparatus

[0158] Referring to FIG. 9 to FIG. 11, the apparatus for detecting a target molecule provided by the present invention comprises: [0159] a solid phase support 201 capable of immobilizing the microparticles in a microparticle solution 301, wherein the microparticles comprise the microparticles binding to a signal molecule to be detected, and are dispersed at the surface and/or in the interior of the solid phase support; [0160] at least one first light source 303 and at least one second light source 306, wherein the microparticles in a selected field of view on the solid phase support 201 (such as the magnetic beads in a blind hole 302 of a turntable 201 containing a solution of magnetic beads to be detected) are irradiated with the light emitted by the first light source 303 through a condenser lens 304, so as to form bright field signals related to the total number of the microparticles; the microparticles in a selected field of view on the solid phase support 201 are irradiated with the fluorescence emitted by the second light source 306 sequentially through a lens 307 and a dichroic beamsplitter 308 to form dark field signals related to the total number of microparticles binding to the signal molecule to be detected; wherein the first light source 303 and the second light source 306 may be provided by LEDs; [0161] a signal acquisition unit for acquiring the bright field signals and dark field signals, respectively; the signal acquisition unit comprises an amplification assembly objective lens 305, a lens 310, a filter 309 and a photographing assembly camera 311, and the bright field signals and the dark field signals reach the camera 311 through the objective lens 305, the lens 310 and the filter 309 in sequence for microscopic imaging; and [0162] a signal processing unit (not shown) for determining the concentration of the target molecule according to the acquired bright field signals and dark field signals.

[0163] In the apparatus for detecting a target molecule of the present invention, the solid phase support is a turntable 201 capable of rotating with respect to the signal acquisition unit as shown in FIG. 9 or FIG. 10. Referring to FIG. 10, a silicate glass turntable 201 as a solid phase support is disposed above the signal acquisition unit and can be removed from above the signal acquisition unit. In other alternative embodiments, the solid phase support is a flat plate, such as a glass slide, and the microparticles in the solution 301 are randomly distributed evenly over the surface of the flat plate. In other alternative embodiments, the solid phase support has at least one flow channel comprising an inlet and an outlet. The microparticles in the solution 301 are uniformly distributed in the flow channel. At least a part of the flow channel overlaps an optical path of the light detecting unit to allow detection by the first light source 303 and the second light source 306.

[0164] The material of the turntable 201 may be quartz or glass. The turntable 201 comprises at least one optically transparent detection site which is detected by the signal acquisition unit when the detection site is located in an optical path of the signal acquisition unit. As shown in FIG. 9, the turntable 201 has eight blind holes 202 as the detection sites, and one of the blind holes 302 is a blind hole for containing a solution of the microparticles to be detected.

[0165] The turntable 201 as a solid phase support can rotate in a plane above the signal acquisition unit. FIG. 11 shows that the blind hole 302 containing a solution of the microparticles to be detected is positioned above the objective lens 305 by rotation. The turntable 201 is configured to rotate sequentially between a plurality of stations in a stepwise manner and to perform the following operations on a solution to be detected at the detection site at the plurality of stations: immobilizing, dispersing and rinsing the microparticles (e.g. magnetic beads) in the solution. The plurality of stations comprise a detection station located in an optical path of the signal acquisition unit, at least one pre-treatment station located upstream of the detection station, and at least one post-treatment station located downstream of the detection station, wherein an operation of immobilizing and dispersing microparticles in the solution is performed at the pre-treatment station, and a rinsing operation is performed at the post-treatment station.

[0166] The apparatus for detecting molecules further comprises a magnetic field generating device or an electric field generating device (not shown) for immobilizing and dispersing the microparticles at the surface and/or in the interior of the solid phase support.

[0167] In a preferred embodiment, the microparticles are magnetic beads which are one or more selected from the group consiting of paramagnetic beads and superparamagnetic beads. The magnetic beads have a particle size ranging from 600 nm to 10 .Math.m.

[0168] The apparatus of the present invention further comprises a displacement mechanism (not shown) for actuating the signal acquisition unit and/or the solid phase support, wherein the displacement mechanism is preferably one or more selected from the group consisting of a one-dimensional displacement stage, a two-dimensional displacement stage, and a three-dimensional displacement stage; for example, the displacement mechanism may be a two-dimensional displacement stage to allow bright field and dark field microscopic imaging of individual microparticles to be acquired in a planar motion manner while the individual microparticles are dispersed on the surface of the solid phase support.

[0169] Referring to FIG. 11, the reacted microparticles solution 301 with the immunocomplex of signal molecule is pipetted into the blind holes 202 of the transparent solid phase support, which at this time corresponds to position 5 of the eight blind holes 202 on the turntable 201 in FIG. 11. The rotary table drives the rotary shaft 203 to sequentially move the sample from position 5 to positions 4, 3, 2 and 1 to perform the steps of adding liquid agar, controlling the spatial distribution of the microparticles by external force, cooling the solidable agar to immobilize the microparticles, and acquiring signals, and the steps at positions 2 and 4 may be omitted. At position 1, the first light source 303 (i.e. a bright field light source) generates a bright field image of the magnetic beads through a condenser lens 304 and a blind hole 302 containing a solution of magnetic beads to be detected, and the signals are collected through an objective lens 305, and imaged on a camera 311 through a lens 310. If the signal molecule is fluorescent group or particle, the second light source (such as an excitation light source) 306 excites the signal molecule on the microparticles through the lens 307, the dichroic beamsplitter 308 and the objective lens 305, and the generated fluorescent signals are collected through the objective lens 305, transmitted through the dichroic beamsplitter 308, filtered through the filter 309 to remove background signals, and imaged on the camera 311 through the lens 310. The fluorescent signals are collectively referred to herein as dark field imaging. Bright field imaging, which provides information on the number of dispersed microparticles, and dark field imaging, which provides information on the number of the microparticles containing the signal molecule in these dispersed microparticles, are performed alternately; preferably, the concentration of target molecule can be further analyzed by any suitable algorithm, including but not limited to standard curve and Poisson distribution. After the data acquisition is finished, the sample continues to rotate from position 1, via positions 8, 7 and 6, to position 5 (i.e. performing multiple elution to recover the microparticles), during which the blind hole 202 is rinsed to perform the next cycle of detection.

[0170] It should be noted that: [0171] (1) when the microparticles are immobilized with agar, after the sample is loaded and moved from position 5 to position 4 for adding agar, the blind hole at position 6 is moved to position 5 to start loading the next sample, and so on, to ensure that each blind hole moves to this position to load a sample to realize a cyclical detection with a high-efficiency; [0172] (2) in addition to immobilizing the microparticles using agar, the microparticles solution 301 with the immunocomplex comprising a signal molecule after the reaction may be pipetted onto a transparent flat solid phase support (quartz, glass, etc.), or a transparent porous solid phase support, and then the microparticles may be plated closely on the solid phase support using a magnet, gravity, or magnetic field directly. Preferably, the solvent of the microparticles solution 301 is removed, and the microparticles are then detected directly by a two-dimensional distribution; and [0173] (3) when the biomolecule is a nucleic acid, it can also be analyzed using the same analysis apparatus with the same apparatus configuration.

Example 1 Screening of Magnetic Beads

[0174] Solutions of magnetic beads with different particle sizes (500 nm, 1 .Math.m, 2 .Math.m, 3 .Math.m) were provided. The above solutions of magnetic beads with the four particle sizes were randomly distributed on glass slides, and observed and photographed under a microscope, and the results are shown in FIG. 3.

[0175] As can be seen from FIG. 3, when the particle size of the magnetic beads was 500 nm, the magnetic beads had an agglomeration effect and could not be counted individually. The magnetic beads with a particle size larger than 10 .Math.m were easy to sink due to gravity in an incubation process, and were distributed unevenly in an incubation solution, which affected the collision probability, and reduced the detection sensitivity. The magnetic beads with a particle size of 600 nm to 10 .Math.m could avoid the two above-mentioned influencing factors. Under different particle sizes and corresponding specific magnifications, the image characteristics presented by bright field microscopic imaging are darkness in the periphery and brightness in the center (the particle size as shown in FIG. 3 is 3 .Math.m). These characteristics in combination with the sizes of magnetic beads could be used to distinguish magnetic beads from other impurities, so that the detection and analysis results are improved. The method has a high detection tolerance to a contaminated sample and a sample with complex background, such as a pharyngeal swab, saliva, a nasal swab, alveolar lavage fluid and other smear swab samples.

[0176] The magnetic beads with a particle size of 1 .Math.m and 2 .Math.m shown in FIG. 3 also exhibited the above-mentioned characteristics of darkness in the periphery and brightness in the center (not shown) at their respective specific magnifications.

Example 2 Detection of Different Concentrations of Biotin-Qbeads By Streptavidin-Modified Magnetic Beads

1. Experimental Components

[0177] Streptavidin-modified magnetic beads, biotin-modified quantum dot beads (Biotin-Qbeads), Buffer A (2% BSA in 10 mM PBS, pH 7.4), Buffer B (0.5% Tween 20 in 10 mM PBS, pH 7.4).

2. Experimental Method

[0178] Biotin-Qbeads were diluted to 0, 0.05, 0.25, 0.5, 2.5, 5, 25, 50 fM with Buffer A. Streptavidin-modified beads were diluted to 2 × 10.sup.7/mL.

[0179] 10 .Math.L of diluted biotin-Qbeads with different concentrations, 10 .Math.L of diluted streptavidin-modified magnetic beads and 80 .Math.L of Buffer A were mixed by vortexing, and reacted for 1 h at 37° C.

[0180] The supernatant was removed by rinsing six times with Buffer B.

[0181] The magnetic beads were resuspended in 20 .Math.L of PBS, and 5 .Math.L of suspension was transferred to a coverslip. The magnetic beads were attached to the bottom of the coverslip using a magnet, and single molecule imaging was performed using a fluorescence microscope.

[0182] The magnetic beads distributed on the bottom surface of the glass were imaged under a bright field and a fluorescence imaging mode respectively using a low-power objective lens to obtain two sets of data of bright field and the fluorescence. A concentration of antigen could be determined by the ratio of the number of magnetic beads containing the immune complex (i.e., the number of active beads) to the total number of magnetic beads.

[0183] Measurements were performed on a series of different concentrations, with each concentration point being repeated three times.

3. Experimental Results

[0184] The experimental results are shown in FIG. 5. The detection concentrations from 50 aM to 50 fM are within the linear range of this detection method. Therefore, the linear dynamic range of this method has 3 orders of magnitude. In the example, the limit sensitivity of the system was verified in a reaction system without an antibody, and with the participation of only two reactants with a stronger affinity, streptavidin and biotin, and the results show that if the affinity of the antibody is high enough, the method would be used for detection at a concentration as low as aM, providing a new technical means for detecting low-abundance factors.

Example 3 Detection of Purified Spike Protein of Novel Coronavirus

1. Experimental Components

[0185] Carboxyl-modified magnetic beads, capture antibody (SinoBiological 40150-D006), detection antibody (SinoBiological 40591-MM43), streptavidin-modified quantum dot beads, Spike protein (RBD, SinoBiological), N-hydroxysulfosuccinimide (S-NHS), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), PBS buffer, Buffer A (0.1% Tween 20 in 10 mM PBS, pH 7.4), Buffer B (2% BSA in 10 mM PBS, pH 7.4), Buffer C (0.5% Tween 20 in 10 mM PBS, pH 7.4), MES, Tris-HCI, NaOH, EZ-LinkNHS-PEG 4-Biotinylation Kit, and desalting column (Zeba™ Spin Desalting Columns).

2. Preparation Mthod

2.1 Covalent Coupling of Magnetic Beads With a Capture Antibody

[0186] 20 uL of the magnetic bead suspension was taken into a 1.5 mL EP tube, the EP tube was placed in a magnetic separation rack for enriching the magnetic beads, and the supernatant was removed.

[0187] 0.5 mL of H.sub.2O was taken into a centrifuge tube, the magnetic beads were mixed uniformly by vortexing for 15 s, the EP tube was placed in the magnetic separation rack for enriching the magnetic beads, and the supernatant was removed.

[0188] 0.5 mL of NaOH was added into the centrifuge tube, the magnetic beads were mixed uniformly by vortexing for 15 s, the EP tube was placed in the magnetic separation rack for enriching the magnetic beads, and the supernatant was removed. This procedure was repeated twice.

[0189] 0.5 mL of H.sub.2O was added into the centrifuge tube, the magnetic beads were mixed uniformly by vortexing for 15 s, the EP tube was placed in a magnetic separation rack for enriching the magnetic beads, and the supernatant was removed. This procedure was repeated once.

[0190] 0.5 mL of MES (pH 5.0) was added into the centrifuge tube, the magnetic beads were mixed uniformly by vortexing for 15 s, the EP tube was placed in a magnetic separation rack for enriching the magnetic beads, and the supernatant was removed. This procedure was repeated once.

[0191] 0.1 mL MES (pH 5.0) was added into the centrifuge tube, the magnetic beads were mixed uniformly by vortexing for 15 s, then 0.1 mL 100 mg/ml EDC and 50 mg/ml S-NHS were added, and the mixture was vortexed for 15 s. The EP tube was placed in a horizontal shaker, and the mixture was reacted for 40 min at room temperature. The EP tube was placed in the magnetic separation rack for enriching magnetic beads, and the supernatant was removed.

[0192] 0.5 mL of MES (pH 5.0) was added in the centrifuge tube, the magnetic beads were mixed uniformly by vortexing for 15 s, the EP tube was placed in the magnetic separation rack for enriching the magnetic beads, and the supernatant was removed. This procedure was repeated once. The beads were resuspended by adding 0.1 mL of MES (pH 5.0), and the mixture was vortexed for 15 s.

[0193] 88 .Math.g of capture antibody (40150-D006) was diluted with 0.1 mL of MES (pH 5.0) and added to the magnetic bead suspension, and the mixture was vortexed for 15 s. The EP tube was placed in a horizontal shaker, and the mixture was reacted for 1 h at room temperature.

[0194] The EP tube was placed in the magnetic separation rack for enriching magnetic beads, and the supernatant was removed. The beads were resuspended by adding 0.4 mL of Tris-HCl (pH 7.4), and the mixture was vortexed for 15 s; the EP tube was placed in the horizontal shaker, and the mixture was reacted for 1 h at room temperature.

[0195] The EP tube was placed in the magnetic separation rack for enriching magnetic beads, and the supernatant was removed. 0.5 mL of Buffer A was added, and the mixture was vortexed for 15 s to uniformly mix the magnetic beads; the EP tube was placed in the magnetic separation rack for enriching the magnetic beads, and the supernatant was emoved. This procedure was repeated twice.

[0196] 0.5 mL of PBS was added and the mixture was vortexed for 15 s for uniformly mixing the magnetic beads, the EP tube was placed in the magnetic separation rack for enriching the magnetic beads, and the supernatant was removed.

[0197] 0.15 mL of Buffer B was added, the mixture was vortexed for 15 s for uniformly mixing the beads and stored at 4° C.

2.2 Biotinylation Modification of Detection Antibody

[0198] 1 mg of detection antibody was diluted with 1 mL of 10 mM PBS to a concentration of 1 mg/mL, and was stored at 4° C. for later use.

[0199] One tube of NHS-PEG4-Biotin packed in the kit was dissolved by adding 0.17 mL of ultrapure water to obtain an NHS-PEG 4-Biotin solution with a concentration of 20 .Math.M.

[0200] 6.65.Math.L of NHS-PEG4-Biotin solution was added to the detection antibody solution, and the mixture was reacted for 1 h at room temperature.

[0201] Buffer was displaced using a desalting column (Zeba™ Spin Desalting Columns), and excess NHS-PEG 4-Biotin solution was removed from the system at the same time.

[0202] The concentration of biotinylated detection antibody was determined using Nanodrop, and the mixtuer was stored at 4° C.

3. Experimental Method

[0203] The Spike protein was diluted to concentrations of 0, 0.01, 0.1, 1, 10 and 100 pg/mL with Buffer B.

[0204] The magnetic beads labeled with the capture antibody were diluted 50-fold with Buffer B, the biotin-labeled detection antibody was diluted to a concentration of 4 .Math.g/mL, and SA-Qbeads was diluted to 1.67 nM.

[0205] 25 .Math.L of diluted Spike proteins with different concentrations, the magnetic beads labeled with the capture antibody, biotin labeled detection antibody and SA-Qbeads were taken respectively, uniformly mixed by vortexing, and reacted for 1 h at 37° C.

[0206] The mixture was rinsed six times with Buffer C, and the supernatant was removed.

[0207] The beads were resuspended by adding 20 .Math.L of PBS, and 5 .Math.L of suspension was transferred to a coverslip. The beads were attached to the bottom of the coverslip using a magnet, and single molecule imaging was performed using a fluorescence microscope.

[0208] The magnetic beads distributed on the bottom surface of the glass were imaged under a bright field and a fluorescence imaging mode respectively by using a low-power objective lens, to obtain two sets of data of bright field and fluorescence. A concentration of antigen could be determined by the ratio of the number of magnetic beads containing the immune complex (i.e., the number of active beads) to the total number of magnetic beads.

[0209] Measurements were performed on a series of Spike protein concentrations, with each concentration point being repeated three times.

4. Experimental Results

[0210] The detection results are shown in FIG. 6, and it can be seen that in this example, the detection range for Spike protein was 0.01 pg/mL to 100 pg/mL, and the lower limit of detection could reach 10 fg/mL.

Comparative Example 1: Detection of Pseudovirus Expressing Novel Coronavirus S Protein on Surface

1. Experimental Components

[0211] The detection sample was a pseudovirus expressing novel coronavirus S protein on the surface, and the remaining experimental components were identical to those in Example 3.

2. Preparation Method:

2.1 Covalent Coupling of Magnetic Beads With Capture Antibody

[0212] The procedure was conducted identically to the corresponding procedure in Example 3.

2.2 Biotinylation Modification of Detection Antibody

[0213] The procedure was conducted identically to the corresponding procedure in Example 3.

3. Experimental Method

[0214] Pseudovirus was used as the detection sample, including solutions without the pseudovirus and a series of solutions containing 2, 5, 20, 100 and 200 pseudoviruses per 100 .Math.L. The remaining steps were performed identically to the corresponding steps in Example 3.

4. Experimental Results

[0215] It can be seen from the results in FIG. 7 that the number of pseudovirus detected from 2 to 200 is within the linear range of the detection method. In the case that the number of pseudovirus is 2 or 5, although the ratio of the number of fluorescent magnetic beads to the total number of magnetic beads is lower, it can still be significantly distinguished from the control group without the virus. The current detection sensitivity of the same antibody in the chemiluminescence platform is about 50 pseudoviruses, and the result of the example is 25 times higher than that of the chemiluminescence method.

Example 4 Magnetic Bead Assay for Detection of IL-6

1. Experimental Components

[0216] In the experiment, the capture antibody is 8C9 (Cnpair Biotech Co., Ltd.), the detection antibody is 9A2 (Cnpair Biotech Co., Ltd.), the detection sample is IL-6, and the other experimental components were identical to those in Example 1.

2. Preparation Method

2.1 Covalent Coupling of Magnetic Beads With Capture Antibody

[0217] The procedure was conducted identically to the corresponding procedure in Example 1.

2.2 Biotinylation Modification of Detection Antibody

[0218] The procedure was conducted identically to the corresponding procedure in Example 1.

3. Experimental Method

[0219] IL-6 was diluted to concentrations of 0, 0.05, 0.2, 0.5, 2, 5 and 20 pg/mL with Buffer B.

[0220] The magnetic beads labeled with capture antibody were diluted 150-fold with Buffer B, the biotin-labeled detection antibody was diluted to a concentration of 4 .Math.g/mL, and SA-Qbeads were diluted to 8 nM. The remaining procedures were performed identically to the corresponding procedures in Example 1.

4. Experimental Results

[0221] The detection results are shown in FIG. 8, it can be seen that in this example, the detection range of IL-6 is from 0.05 pg/mL to 20 pg/mL, which is within the linear range of the detection method, and the lower limit of detection could reach 50 fg/mL. The current detection sensitivity of the same antibody is higher than 1 pg/ml in a chemiluminescence platform, and the result of the example is about 20 times higher than that of a chemiluminescence method.