CN112229774A

CN112229774A - Method and device for detecting molecules

Info

Publication number: CN112229774A
Application number: CN202011494318.1A
Authority: CN
Inventors: 纪伟; 谷陆生; 徐涛
Original assignee: Institute of Biophysics of CAS; Bioisland Laboratory
Current assignee: Institute of Biophysics of CAS; Bioisland Laboratory
Priority date: 2020-12-17
Filing date: 2020-12-17
Publication date: 2021-01-15
Anticipated expiration: 2040-12-17
Also published as: CN112229774B

Abstract

The invention provides a method and 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 to which a signal molecule to be detected is bound; (2) applying the microparticles in the solution to the surface and/or interior of a solid support; (3) counting particles in the selected field of view by bright field; (4) counting particles bound with signal molecules in the selected field of view by dark field; (5) and (4) determining the concentration of the signal molecule according to the counting results obtained in the steps (3) and (4). On the basis, the invention also provides a method and a device for detecting the target molecules. The method and the device provided by the invention can realize rapid and simple detection of molecules, especially biomolecules, have low cost and are convenient to popularize in fields of scientific research, clinical diagnosis, epidemic prevention work and the like.

Description

Method and device for detecting molecules

Technical Field

The invention belongs to the field of biological detection. In particular, the present invention relates to a method and apparatus for detecting molecules.

Background

In the in vitro diagnostic product (IVD) market segment, immunodiagnosis and molecular diagnosis forego-trif. The emergence of digital PCR has marked that molecular diagnostics has entered the digital era first, and the development and popularization thereof are in a rapid development stage. Immunological tests based on immunological theory and principles play an important role in the prevention, diagnosis, treatment and prognosis evaluation of clinical diseases. Taking our country as an example, 35% (about 200 hundred million RMB) of immunodiagnostics in the IVD market in 2018. The immunodiagnosis has been developed for decades and has undergone Radioimmunoassay (RIA), immunocolloidal gold technique, enzyme-linked immunosorbent assay (ELISA), time-resolved fluoroimmunoassay (TRFIA), and the like. The currently predominant technology is chemiluminescence immunoassay (CLIA), which accounts for approximately 70% of the market share. However, both the acartiridine ester luminescence and the Roche electrochemiluminescence utilize luminescence reaction, that is, quantitative analysis is realized by detecting the overall luminescence intensity of the solution, so that the detection sensitivity, dynamic range, required sample amount and the like are limited by the detection principle and methodology, and accurate quantitative detection cannot be realized on various disease-related molecules such as low-abundance nerve factors, cancer factors, immune factors, hormones and the like. The digital immunodiagnosis technology can break the bottleneck of the detection sensitivity of the existing luminescence system from the detection principle, realize digital quantitative detection and analysis by directly counting single immune complex molecules, can realize trace, high-sensitivity and high-dynamic range detection, and hopefully become the next generation immunodiagnosis technology to replace the core position of chemiluminescence.

The digital immunoassay is to capture molecules to be detected by an immunolabeling method, carry out fluorescence signal molecular labeling or enzyme-linked labeling, and realize single-molecule-level detection by direct single-molecule fluorescence counting or indirect single-molecule enzymatic reaction amplification, wherein the former needs a system with high optical detection sensitivity, and the latter needs to efficiently obtain micro-reaction space (femtoliter-picoliter) of microdroplets or micropores so as to prevent the diffusion of fluorescent substrates generated by the reaction, and finally realize the reading of digital fluorescence signals. The two methods realize the digitization of immunoassay, and the detection sensitivity of the two methods is far higher than that of the existing chemiluminescence technology platform. The enterprise and capital markets with advanced strategic eye in foreign countries have also initiated the industrial deployment of digital detection technologies in the immunodiagnostic market. At present, the digital detection devices commercialized abroad mainly include a SiMoA system (micro-space in-situ signal amplification) developed by quantrix corporation in the united states, and an SMCxPro system (high-sensitivity single-molecule detection and counting) being popularized by Merck corporation. Both techniques use the same principle of particle Capture and antigen enrichment as chemiluminescence, and in the case of double Antibody sandwich, they link the particles to Capture Antibodies (CA), which are linked to CA and Detection Antibodies (DA) via different epitopes to form Immune Complexes (ICs) attached to the particles, as shown in fig. 1, and finally, the counting and concentration determination of ICs in the form of optical signals is achieved by linking DA to Reporter molecules (reporters, enzymes or fluorescent molecules). After the IC is formed and linked to a reporter molecule, these two techniques achieve digitized optical signal readout in different ways, where:

SiMoA system: the final reaction liquid containing the microspheres is uniformly coated on a chip containing tens of thousands of micropores, the microspheres carrying the IC have beta-galactosidase (beta G), the micropores contain reaction substrates, namely resisof-beta-d-galactopyranoside (RGP), the RGP can generate fluorescent substrates after the catalysis of the enzyme, the micropores form a micro-reactor after oil sealing, and the fluorescent substrates can not diffuse out of a single reaction micropore after the reaction, and the high-concentration fluorescent substrates can cause local signal amplification. Microwells containing IC microspheres will produce a local high concentration of fluorescent substrate, and this fluorescent signal will be clearly distinguishable from microwells without IC. The IC, i.e. the concentration of the antigen to be detected, can be calculated by calculating the ratio of the number of the micropores containing the IC microspheres (the micropores with fluorescence) to the number of the micropores containing all the microspheres. The technical solution of the system is protected in US patent application No. US12/731130, which discloses a method of determining the concentration of analyte molecules or particles in a fluid sample. However, the inventors have found in long-term studies that the SiMoA system has the following disadvantages: (1) the instrument structure of a self-forming system and a microsphere enrichment and reaction method are adopted, so that the instrument cost is overhigh; (2) the detected solid phase carrier needs to be divided into spaces in advance, for example, a chip needs to be etched according to rules in advance, and the aperture needs to be matched with the particle size of the microsphere, so that the chip preparation method is difficult and high in cost; (3) the loss of the microspheres in the detection process is excessive, and the microspheres finally entering the reaction micropores only account for 5-10% of the number of the reaction microspheres, so that the sensitivity and stability of the detection of the low-abundance sample are influenced; (4) the detection signal reading process needs to wait for several minutes, the signal reading can be carried out only when the substrate fluorescence signal generated by the enzyme reaction reaches a certain intensity, the time can be tolerated for single sample detection, but the waiting time is too long for high-throughput clinical detection, and the detection flux of the system is influenced. The method is only applied to basic research at present and is difficult to apply clinically;

SMCxPro system: unlike the chemiluminescent and SiMoA systems, which treat the reacted solution with a urea solution to elute the IC from the microspheres and separate it from the microspheres, the IC is destroyed, but each fluorescent molecule in the solution corresponds to an IC, and thus the two are identical in quantity and concentration. The SMCxPro system uses a high-sensitivity optical detection system to perform random scanning detection on different positions in a solution, and performs single-molecule detection and counting on a solution containing a reporter molecule, so as to estimate the concentration of an IC, namely an antigen to be detected. However, the inventors have found in long-term studies that the SMCxPro system has the following drawbacks: (1) because of the high sensitivity optical system, the cost of the instrument and the accessory is high; (2) the former generation of instrument adopts a glass tube flow detection system, which is easy to block and causes system instability, while the new generation of system solves the problem, but can not solve the defect of local sampling only due to factors such as free molecular diffusion, and the like, and can only estimate the concentration of the whole sample through the local sample, thereby influencing the sensitivity and stability of low abundance sample detection; (3) single molecules have the stability problems of easy bleaching and quenching and the like; (4) the former generation of instrument adopts a glass tube flow detection system, the 20 microliter final reaction solution signal reading process needs more than 20 minutes, the new generation of product should be improved, but the signal detection is still based on single excitation light point excitation and photomultiplier single-point detection, the signal reading efficiency is low, a large amount of solution different positions need to be traversed to improve the accuracy, the estimated detection time is more than 1 minute, and the system detection flux is influenced.

Chinese patent application No. 201280049085.1 discloses a biomolecule analysis method and a biomolecule analysis apparatus, which realize a wide dynamic range and rapid analysis using biomolecule count in the biomolecule analysis method. The application relates to a method of biomolecule analysis, comprising: the method for detecting a biomolecule of the present invention comprises a step of immobilizing a biomolecule to be analyzed on the surface of a magnetic fine particle, a step of reacting a labeled probe molecule with the biomolecule to be analyzed, a step of collecting and immobilizing the fine particle on a support substrate, and a step of measuring the label on the support substrate. This application realizes counting of the number of biomolecules by using magnetic fine particles having one molecule, and realizes a rapid reaction by performing hybridization and a reaction between antigen and antibody in a state where fine particles having biomolecules immobilized thereon are dispersed. However, the inventors have found in long-term studies that the application has the following disadvantages: (1) it is not given how to calculate the molecule concentration when more than one molecule is carried on the magnetic bead, and when the proportion of the magnetic bead with the molecule exceeds 10%, there is a high probability that one magnetic bead has 2 or more molecules, and how to accurately determine the molecule concentration needs to introduce a statistical distribution model. (2) There are two errors that can be easily introduced, only by generating a bright spot count from the molecular fluorescence signal. Firstly, the loss degree is different in the magnetic bead processing procedure, can influence final bright spot figure, does not distinguish and reject the processing to the pollution and the impurity signal that produce the bright spot in addition, need increase the washing number of times to ensure calculation accuracy. (3) The fluorescent probes, quantum dots and other fluorescent markers with weaker fluorescent signals, which are mentioned in the application, have higher requirements on the power of a light source, the magnification factor and the numerical aperture of an objective lens and the sensitivity of a detection camera in order to improve the detection signal-to-noise ratio, and the convenience and the flux of system data acquisition are increased while the cost is increased.

Chinese patent application No. 202080000774.8 discloses a single molecule quantitative detection method and a detection system. The application utilizes in-situ signal enhancement nano particles with optical characteristics to mark molecules to be detected through chemical modification and molecular recognition technology, so that single-molecule signals can be captured and recognized by optical imaging equipment. The ultrahigh-sensitivity quantitative detection of the molecules to be detected is realized by counting the number signals of the in-situ signal enhanced nanoparticles. However, the inventors have found in long-term studies that the application has the following disadvantages: (1) the bright spot count or integrated intensity information is generated only from the molecular fluorescence signal, with two errors that are easily introduced. Firstly, the magnetic bead processing in-process loss degree is different, can influence final bright spot figure, does not distinguish and reject the processing to the pollution and the impurity signal that produce the bright spot equally in addition, consequently needs the washing flow many times to ensure calculation accuracy. (2) The in-situ signal enhanced nanoparticles mentioned in the application have the main reason that the particles are too small, which causes weak signals and cannot be detected, and the particles have too large sizes, which affects the detection sensitivity, and the reason is that the force between the magnetic beads and the particles is supported by an immunological binding method, in order to balance the two extreme cases, the application has strict requirements on the particle size, which is 180-480nm, but the size still causes antibodies with relatively weak binding capacity to be unable to effectively bind antigens, so that immune complexes are generated, and the detection is affected. (3) The light source used in the application is a laser, and the cost is high.

In summary, the prior art has the following problems: (1) the need to process the background signal, which may be high, is not explicitly disclosed, such as: firstly, free fluorescent molecules in a solution cannot be washed clean by a compound captured by particles in an elution process, the free fluorescent molecules which are not combined exist in the solution, impurities also exist in the solution and can nonspecifically adsorb the fluorescent molecules, and the dark field detection cannot distinguish the fluorescent molecules which are specifically combined on magnetic beads from the fluorescent molecules which are free in the solution or the fluorescent molecules which are nonspecifically combined by the impurities in the two aspects, so that a false positive signal (shown in figure 4) is generated, and the detection accuracy and sensitivity are influenced; (2) the magnetic beads marked with fluorescent molecules are lost in the processing process, and the number of particles finally containing signals is influenced by directly adopting dark field detection without using bright field to count lost particles, so that the accuracy is low; (3) the magnetic beads are aggregated to influence the detection accuracy; (4) the chip used for the existing digital immunoassay needs to divide the space position in advance and then divides the captured compound into a plurality of positions in space, the chip processing technology is complex, and the chip cost is high; (5) at present, the digital immunoassay basically adopts a high power lens, and the cost is high.

At present, clinical detection requirements of new coronary antigen detection, nerve factors, cancer factors and the like have urgent needs on a high-sensitivity detection method, but the cost and the detection convenience in the field influence the clinical application of a single-molecule immune method, and the urgent needs exist for developing a method for realizing effective detection of molecules quickly, simply and cheaply.

Disclosure of Invention

Based on the above problems in the prior art, the present invention provides a method and an apparatus for detecting molecules, which utilize common solid phase carriers (such as glass slides, multi-well plates, and flow channels), and analyze and detect biomolecules by random and uniform distribution of particles under bright and dark fields, thereby simplifying the detection process, improving stability, sensitivity and accuracy, reducing cost, and facilitating popularization in scientific research, clinical diagnosis, epidemic prevention and other fields.

The above object of the present invention is achieved by the following technical solutions:

in a first aspect, the present invention provides a method of detecting a signal molecule comprising the steps of:

(1) providing a solution comprising microparticles, wherein the microparticles comprise microparticles to which a signal molecule to be detected is bound;

(2) immobilizing the microparticles in the solution to the surface and/or inside a solid support;

(3) counting particles in the selected field of view by bright field;

(4) counting particles bound with signal molecules in the selected field of view by dark field;

(5) and (4) determining the concentration of the signal molecule according to the counting results obtained in the steps (3) and (4).

In a second aspect, the present invention provides a method of detecting one or more target molecules, comprising the steps of:

(1) providing a solution comprising microparticles, target molecules forming complexes by a specific binding reaction, said microparticles being attached to said complexes, said complexes being labeled with a signal molecule;

(3) counting particles in the selected field of view by bright field;

(4) counting the particles bound with the complexes in the selected field of view by dark field;

(5) and (4) determining the concentration of the signal molecule according to the counting results obtained in the steps (3) and (4), and further determining the concentration of the target molecule.

In the method of the present invention, the target molecule is selected from one or more of a protein, a polypeptide, an amino acid, an antigen, an antibody, a receptor, a ligand, or a nucleic acid.

In the method of the present invention, the specific binding reaction is selected from one or more of an immune reaction, a hybridization reaction, and a receptor-ligand interaction.

In a preferred method of the invention, the complex may be an immune complex and the specific binding reaction may be a sandwich or competition immune reaction. For example, the sandwich method immunoreaction refers to that a particle is coupled with a capture antibody, is specifically combined with a first site of a target molecule, so as to capture the target molecule, and is added with a detection antibody, so that the detection antibody is combined with a second site of the target molecule, so as to form an immune complex, wherein the detection antibody is directly or indirectly marked with a signal molecule; or the second binding site of the target molecule is bound to the detection antibody first, and the conjugate is then bound to the capture antibody to form a particle of an immune complex with a signal molecule, as shown in FIG. 1.

In the method of the present invention, the microparticles may take the form of spheres, ellipsoids, spheroids, cubes, polyhedra, cylinders, or irregular shapes. The size of the microparticles may be 600nm to 10 μm, for example 600nm, 1 μm, 2 μm, 3 μm, 5 μm, 10 μm; preferably 1 μm to 5 μm.

The surface of the microparticles may be modified with one or more reactive functional groups, which may be selected from-OH, -COOH, -NH₂-CHO and-SO₃H. In some embodiments, the capture molecule is conjugated or bound to the microparticle by physisorption or chemical conjugation (e.g., bridging by a bridge). The bridge may be selected from one or more of a protein, a label-anti-label complex or a cross-linking agent suitable for a carboxyl group and/or a primary amine. The protein may be selected from one or more of bovine serum albumin, ovalbumin, keyhole limpet hemocyanin, immunoglobulin, thyroglobulin, and polylysine. The crosslinking agent suitable for the carboxyl and/or primary amine is selected from one or more of Dicyclohexylcarbodiimide (DCC), carbodiimide (EDC), N-hydroxybenzotriazole (HOBt) and N-hydroxysuccinimide (NHS).

In the method of the present invention, the concentration of microparticles in the solution comprising microparticles may be in the range of 1 thousand to 200 ten thousand microparticles per 50. mu.l to 400. mu.l (e.g., 100. mu.l, 200. mu.l, 300. mu.l, preferably 10. mu.l to 20. mu.l) of the solution.

In the method of the present invention, the density of the individual microparticles when laid on the surface of a solid support is 1 to 2000 ten thousand/cm²E.g. 10/cm²100 pieces/cm²1000 pieces/cm²5000 ten thousand pieces/cm ²1 ten thousand/cm²10 ten thousand/cm ²15 ten thousand/cm²20 ten thousand/cm²25 ten thousand/cm²30 ten thousand/cm²50 ten thousand/cm²100 ten thousand/cm ²500 ten thousand pieces/cm²Or 1000 ten thousand/cm²。

In the method of the present invention, the particles are magnetic particles, and preferably, the magnetic particles are magnetic beads, and a magnetic substance is contained in the magnetic beads. The magnetic substance may be a metal (simple metal or alloy), a nonmetal, or a composite of a metal and a nonmetal. The metal may be, for example, iron, aluminum, nickel, cobalt, or the like; the non-metal may be, for example, a ferrite non-metal (preferably Fe)₂O₃Or Fe₃O₄Magnetic nanoparticles); the composite of metal and nonmetal may be, for example, neodymium iron boron rubber magnetic composite material.

Preferably, the magnetic beads are selected from one or more of paramagnetic and superparamagnetic. The diameter of the magnetic beads may be 600nm to 10 μm, e.g. 600nm, 1 μm, 2 μm, 3 μm, 5 μm, 10 μm; preferably 1 μm to 5 μm.

In the method of the present invention, the particles can be randomly and uniformly distributed on the surface and/or inside the solid phase carrier, and the particles are still randomly and uniformly distributed after being immobilized, so as to facilitate subsequent detection.

In the method of the present invention, the immobilized microparticles do not substantially agglomerate or overlap with each other.

In a preferred embodiment of the invention, the process of the invention further comprises the steps of: and (4) uniformly distributing the particles on the solid phase carrier by using a magnetic field or an electric field, and adsorbing the particles to the surface of the solid phase carrier. For example, placing a magnet under the solid support adsorbs the particles to the surface of the solid support.

In a preferred embodiment of the invention, the process of the invention further comprises the steps of: the solvent in the solution of microparticles is removed, for example, by blotting the water with a material having water-absorbing properties, or by air drying, or by oven drying (preferably at a temperature of 40 to 80 ℃ C., for example 50 ℃ C., 55 ℃ C., 60 ℃ C., 70 ℃ C.).

In the method of the present invention, the signal molecule is selected from one or more of a chromophore, a digoxigenin-labeled probe, a metal nanoparticle, or an enzyme.

A chromophore selected from one or more of a fluorescent molecule, a quantum dot, a chemiluminescent molecule, a luminescent compound and a dye;

an enzyme selected from the group consisting of enzymes that produce a detectable signal, such as horseradish peroxidase, alkaline phosphatase, beta-galactosidase, and glucose-6-phosphate dehydrogenase.

In the method of the present invention, wherein the signal molecule is selected from one or more of an organic small molecule fluorescent probe, a quantum dot bead, a fluorescent bead, a chemiluminescent signal molecule, a chemiluminescent molecule-coated bead, a three-dimensional DNA nanostructure reporter probe, an upconversion luminescent nanomaterial bead, a rolling circle amplification fluorescent molecule amplification structure, or an aptamer fluorescent molecule amplification structure.

Preferably, the size of the quantum dot globule is within 110 nm.

In the method of the present invention, in the step (2), the microparticles are immobilized on the surface and/or inside the solid support by an external magnetic field and/or electric field and/or gel.

In the method of the present invention, the position-fixed microparticles are distributed two-dimensionally on the solid support, or three-dimensionally in or on the surface of the solid support. When the microparticles are distributed in two dimensions on the solid support, the solid support for immobilizing the microparticles is a planar support. The particle solution is firstly dispersed in the gel solution, and then added on the solid phase carrier for solidification, thereby realizing that the particles are three-dimensionally distributed in the interior and/or on the surface of the solid phase carrier, and the solid phase carrier for fixing the particles can be a plane carrier or a pore plate, etc.

In the method of the present invention, the solid phase carrier is selected from a porous plate, a flat plate or a flow channel, and the solid phase carrier is silicate glass, transparent plastic or organic glass (such as acrylic) carrier. The solid phase carrier does not need special treatment such as space division, so that the detection cost is greatly reduced. When the solid phase carrier is a multi-well plate, the multi-well is used for detecting a plurality of samples, one sample is added into each well, and each sample is randomly and uniformly distributed in different wells, rather than performing space division on the multi-well plate according to a rule.

Fixing the microparticles in the solution to the surface and/or inside of the solid phase carrier in the step (2), wherein the number of the microparticles fixed to the surface and/or inside of the solid phase carrier is uncertain, and the determined number of the microparticles does not need to be added to a region which is divided in space in advance.

In the method of the present invention, the solid phase carrier is a multi-well plate, such as a 48-well plate, a 96-well plate, a 384-well plate, a 512-well plate, a 1024-well plate, or a 1536-well plate.

In the present invention, the solid support is at least partially light transmissive or substantially transparent to visible light. The light transmittance refers to the transmittance of visible light being greater than or equal to 10%, or greater than or equal to 20%, or greater than or equal to 30%, 40%, 50%, 60%, 70%, 80%, or 90%.

In the method according to the invention, the coordinates of the particles in the image are determined by bright field microscopy.

In the method according to the invention, the boundaries of the individual particles are determined in the bright field mode (e.g. by microscopic bright field imaging).

In the method of the present invention, the coordinates of the particles are determined based on the brightness difference of the particles. In bright field mode, the center of the particle is bright and the periphery is dark, forming a visible bright spot in the center of the bead, as shown in fig. 2, to realize the identification and counting of the bead.

In a preferred embodiment of the invention, the clustering/overlapping of the particles is determined in the bright field mode, the clustered/overlapping particles are removed, and only a single particle is counted as a basis for calculating the concentration of the molecules to be measured.

In the method of the present invention, the counting in step (4) is determined by coordinates of the particles in the image.

In the method according to the invention, the counting of the bright and dark fields is usually carried out in the same field of view.

The detection accuracy and sensitivity can be improved by the separate imaging of the bright and dark fields, and false positive signals are easily generated if the dark field detection alone cannot distinguish between specifically bound fluorescent molecules on the magnetic beads or free fluorescent molecules in the solution or non-specifically bound fluorescent molecules of impurities, for example, in the rectangular area in fig. 4, the magnetic beads are not present in the bright field, and the signals can be detected in the dark field.

In the method according to the invention, counting statistics of the particles and/or signal molecules are carried out after imaging by means of a microscope.

Preferably, the method of the present invention further comprises counting the number of particles in the bright field and the number of particles containing the signal molecule in the dark field, thereby calculating the concentration of the target molecule to be detected. The calculation methods include, but are not limited to: determining the concentration of the molecules to be detected according to the proportional relation between the number of the signal molecule-containing particles and the number of the particles under the bright field view and a test curve obtained by combining standard products with different concentrations; and determining the concentration of the molecules to be detected according to a test curve obtained by combining the average signal intensity on the particles containing the signal molecules with standard substances with different concentrations.

In the method of the invention, the target molecule may be one or more species and the particle comprises at least one capture molecule, for example, when the target molecules are two species, the particle:

at least partially coupled with two capture molecules; or

At least part of the first capture molecules are immobilized, at least part of the second capture molecules are immobilized.

The capture molecules are respectively and specifically combined with the corresponding first combination sites of the target molecules, detection molecules are added and are respectively and specifically combined with the second combination sites of the target molecules, the detection molecules are marked with signal molecules, and different target molecules are determined by detecting different shapes or particle shapes of the signal molecules marked by the detection molecules.

The methods of the invention may be for non-diagnostic purposes.

In a third aspect, the present invention provides the use of the above method for the preparation of a diagnostic reagent for the detection of biomolecules.

In the use of the invention, the diagnostic reagent is a protein or nucleic acid detection reagent, and the reagent comprises a microparticle, a capture molecule, a detection molecule and a signal molecule; optionally, the diagnostic reagent further comprises a buffer reagent, a coupling reagent, and a washing reagent; optionally, the diagnostic reagent further comprises a detection device.

In a fourth aspect, the present invention further provides a detection apparatus for implementing the method, including:

a solid phase carrier capable of immobilizing microparticles comprising microparticles to which a signal molecule to be detected is bound and dispersed on the surface and/or inside thereof;

at least one first light source and at least one second light source, the first light source illuminating particles within a selected field of view of the solid support to form a bright field signal related to the total number of particles; a second light source illuminates particles within a selected field of view of the solid support to form a dark field signal related to the total number of particles bound with signal molecules to be detected;

the signal acquisition unit is used for acquiring a bright field signal and a dark field signal;

and the signal processing unit is used for determining the concentration of the signal molecules according to the collected bright field signal and dark field signal.

In the device of the present invention, the solid phase carrier is disposed above or below the signal collection unit, and the solid phase carrier is configured to be removable from above or below the signal collection unit.

In the device of the present invention, the solid support may comprise at least one flow channel, the flow channel comprising an inlet and an outlet, and the solution containing the microparticles is dispersed in the flow channel. Preferably, the solid phase carrier includes a flow channel for sending in and out a solution containing the microparticles, wherein at least a part of the flow channel overlaps with an optical path of the light detection unit to allow detection of the first light source and the second light source. Preferably, the flow channel is selected from a glass tube or a microfluidic chip.

In the device of the present invention, the solid phase carrier may be a multi-well plate or a flat plate.

In the device of the present invention, the device further comprises a magnetic field generating device or an electric field generating device for immobilizing and dispersing the microparticles on the surface and/or inside the solid phase carrier. Preferably, the magnetic force generating means may preferably be a magnet.

In the device of the present invention, preferably, the solid phase carrier is a rotating disc capable of rotating relative to the signal acquisition unit, wherein the rotating disc comprises at least one light-permeable detection site, such as a blind hole, which is detected by the signal acquisition unit when the detection site is in the light path of the signal acquisition unit. The number of blind holes may be 1 or more, for example 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12. Preferably, the carousel is configured to rotate in a stepwise manner between a plurality of stations in sequence and to perform the following operations on the solution to be detected at the detection site at the plurality of stations: fixing the microparticles in the solution and rinsing. Preferably, the plurality of stations include a detection station located in the optical path of the signal acquisition unit, at least one pre-treatment station located upstream of the detection station, where an operation of fixedly dispersing particles in the solution is performed, and at least one post-treatment station located downstream of the detection station, where a rinsing operation is performed.

The rotating disc is preferably a circular disc or an annular disc. "annular disc" means an annular disc, typically a large disc on which the remainder of a small concentric disc is cut; in the present invention, the annular disc is not required to be completely closed, it may have one or several notches, and the outline of the ring is not limited to a circle, it may be an irregular figure, for example, a polygon, preferably a regular polygon.

Corresponding equipment required for forming the signal acquisition unit and the signal processing unit will be easily understood by those skilled in the art according to the description of the present invention. For example, the signal acquisition unit includes an amplification unit, a lens, a filter, a photographing unit, etc., and the signal processing unit includes a computer that controls image acquisition and storage. Preferably, the magnifying assembly is for magnifying particles within the selected field of view. Preferably, the magnifying assembly is an objective lens; more preferably, the objective lens is a low power objective lens, such as 4X, 10X, 20X, 40X. The low-power objective lens can detect a plurality of samples/liquid drops in one visual field relative to the high-power objective lens, so that the detection efficiency is improved, and the detection cost is reduced.

In the device of the present invention, the device further comprises a displacement mechanism for moving the signal acquisition unit and/or the solid support.

In the apparatus of the present invention, preferably, the displacement mechanism is 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.

In the apparatus of the present invention, preferably, the displacement mechanism is a two-dimensional displacement stage to allow bright-field and dark-field microscopic imaging of individual microparticles to be acquired in a planar motion while the individual microparticles are dispersed on the surface of the solid support.

In the apparatus of the present invention, preferably, the displacement mechanism is a three-dimensional displacement table to allow bright-field and dark-field microscopic imaging of individual particles to be acquired layer by layer in a spatial motion manner while the individual particles are dispersed inside the solid-phase carrier.

In a fifth aspect, the present invention provides a computer-readable storage medium for storing a program for performing the methods of the first and second aspects and/or data generated by performing the methods.

The computer storage medium is for storing a computer instruction, a program, a code set, or a set of instructions, which when run on a computer, causes the computer to perform the method for detecting a signal molecule on a microparticle, the method for analyzing a target molecule, as described above, or the method for determining a plurality of types of target molecules, as described above.

Any combination of one or more computer-readable media may be employed. The computer readable medium may be a computer readable signal medium or a computer readable storage medium. A computer readable storage medium includes, but is not limited to, an electronic, magnetic, optical, electromagnetic, infrared, or semiconductor system, apparatus, or device, or any combination of the foregoing. More specific examples of the computer readable storage medium include, but are not limited to: an electrical connection having one or more wires, a portable computer diskette, a hard disk, a Random Access Memory (RAM), a read-only memory (ROM), an erasable programmable read-only memory (EPROM or flash memory), an optical fiber, a portable compact disc read-only memory, an optical storage device, a magnetic storage device, or any suitable combination of the foregoing. In the context of this document, a computer readable storage medium may be any tangible medium that can contain, or store a program for use by or in connection with an instruction execution system, apparatus, or device.

A computer readable signal medium may include a propagated data signal with computer readable program code embodied therein, for example, in baseband or as part of a carrier wave. Such a propagated data signal may take many forms, including, but not limited to, electro-magnetic, optical, or any suitable combination thereof. A computer readable signal medium may also be any computer readable medium that is not a computer readable storage medium and that can communicate, propagate, or transport a program for use by or in connection with an instruction execution system, apparatus, or device.

Program code embodied on a computer readable medium may be transmitted using any appropriate medium, including but not limited to wireless, wireline, optical fiber cable, RF, or any suitable combination of the foregoing.

Computer program code for carrying out operations for aspects of the present invention may be written in any combination of one or more programming languages, including an object oriented programming language such as Java, Smalltalk, C + +, or the like, as well as conventional procedural programming languages, such as the C language or similar programming languages. The program code may execute entirely on the user's computer, partly on the user's computer, as a stand-alone software package, partly on the user's computer and partly on a remote computer or entirely on the remote computer or server. In the case of a remote computer, the remote computer may be connected to the user's computer through any type of network, including a Local Area Network (LAN) or a Wide Area Network (WAN), and optionally, the connection may be made to an external computer (for example, through the Internet using an Internet service provider).

In a sixth aspect, the present invention further provides an electronic device, which includes the computer-readable storage medium of the fifth aspect.

The electronic device includes: one or more processors; and

a storage device storing one or more programs,

when the one or more programs are executed by the one or more processors, the one or more processors implement the method for detecting a signal molecule on a microparticle as described above, the method for analyzing a target molecule as described above, or the method for analyzing a plurality of types of target molecules as described above (which may be any one or more steps thereof).

Alternatively, the electronic device may further comprise a transceiver. The processor is coupled to the transceiver, such as via a bus. It should be noted that the transceiver in practical application is not limited to one, and the structure of the electronic device does not constitute a limitation to the embodiments of the present application.

The processor may be a CPU, general purpose processor, DSP, ASIC, FPGA or other programmable logic device, transistor logic device, hardware component, or any combination thereof. Which may implement or perform the various illustrative logical blocks, modules, and circuits described in connection with the disclosure. A processor may also be a combination of computing functions, e.g., comprising one or more microprocessors, a DSP, and a microprocessor.

A bus may include a path that transfers information between the above components. The bus may be a PCI bus or an EISA bus, etc. The bus may be divided into an address bus, a data bus, a control bus, etc. The memory may be, but is not limited to, a ROM or other type of static storage device that can store static information and instructions, a RAM or other type of dynamic storage device that can store information and instructions, an EEPROM, a CD-ROM or other optical disk storage, optical disk storage (including compact disk, laser disk, optical disk, digital versatile disk, blu-ray disk, etc.), magnetic disk storage media or other magnetic storage devices, or any other medium that can be used to carry or store desired program code in the form of instructions or data structures and that can be accessed by a computer.

In the electronic device of the invention, preferably, the mentioned computer instructions, programs, code sets or instruction sets have a function selected from any one or more of the following:

(1) calculating the number of particles;

(2) adjusting the light intensity of the bright field to obtain a higher signal-to-noise ratio;

(3) when the signal molecule is a fluorescent substance, adjusting the wavelength and/or intensity of the excitation light to obtain a signal, and/or obtaining a higher signal-to-noise ratio;

(4) adjusting the focal length of the shooting component, and/or adjusting the distance between the shooting component and the solid phase carrier, and/or switching the shooting visual field, and/or adjusting the exposure time;

(5) carrying out image recognition on the image data of the particle bright field microscope, extracting and determining the image position information of a single particle, and comparing the image position information with the image position information of the signal molecule to determine the proportion of x, y and/or z;

(6) converting the obtained numerical value by using a standard curve obtained by a standard substance to obtain target molecule concentration information;

(7) establishing a signal molecule intensity threshold for the particles and enabling identification of particles below said threshold; and/or eliminating interference of impurities and spontaneous signaling molecules (e.g., autofluorescence) in the sample;

(8) confirming the boundaries of the particles, enabling the identification of particles that are in contact with each other, agglomerated, or overlapping each other;

(9) the aggregated or overlapping particles are removed from the population.

In the electronic device according to the present invention, preferably, in the case where the particles are two-dimensionally distributed and fixed to the solid carrier, the system collects bright field and dark field signals of all the particles two-dimensionally distributed by moving the lateral displacement stage; when the particles are distributed in three dimensions and fixed on a solid phase carrier, the system moves the axial one-dimensional displacement table or the three-dimensional displacement table to acquire the bright field and dark field signals of the particles on each layer surface in the three-dimensional space.

Term(s) for

In the context of the present invention, the term "immobilized" refers to a state in which, when a particle and a solid support are simultaneously present, the relative position of the particle on/in the solid support (e.g., the relative position between a magnetic bead and/or the relative position between a magnetic bead and a solid support) is not substantially changed in order to facilitate counting of the number of signal molecules and/or magnetic beads on the magnetic bead. In a preferred embodiment of the present invention, the solid support can interact with the molecules on the solid support through the molecules modified on the surface of the magnetic beads to make the immobilization more robust. The means of interaction may be covalent bonds, ionic bonds, van der waals forces, hydrogen bonds, gravitational forces, magnetic forces, or combinations thereof. In some embodiments, the mode of action between the microparticle and the solid support is achieved by bridging as described above.

In the context of the present invention, the terms "substantially", "essentially" and variants thereof are intended to indicate that the feature being described is equal or approximately equal to the value or description. For example, a "substantially flat" surface is intended to mean a flat or nearly flat surface. Further, as defined above, "substantially similar" is intended to mean that the two values are equal or approximately equal. In some embodiments, "substantially similar" may refer to values that are within about 10% of each other, such as within about 5% of each other or within about 2% of each other.

In the context of the present invention, the term "quantum dot globule" refers to a nanoparticle encapsulating a plurality of quantum dots.

In the context of the present invention, the term "fluorescent bead" refers to a nanoparticle encapsulating a plurality of fluorescent molecules.

In the context of the present invention, the term "dark field" refers to an environment that facilitates the detection of signal molecules. Typical forms of detection include capturing an image of the optical signal of the signal molecule, and optionally counting the presence or absence of the signal molecule and calculating the signal intensity of the signal molecule, etc. The above-mentioned photographing methods may include, but are not limited to, scattered light imaging using dense particles, up-conversion luminescence imaging, chemiluminescence signal molecule imaging, and fluorescence imaging. In a preferred embodiment of the present invention, the dark field is an environment substantially free of visible light, providing a higher signal-to-noise ratio environment for signal molecule detection. For example, when the signal molecule is a fluorescence signal, the dark field may be an environment substantially free of visible light but having excitation light, etc., so as to facilitate the capture of the fluorescence signal and improve the image signal-to-noise ratio.

Counting of particles and/or signal molecules

In a preferred embodiment of the invention, the counting of microparticles and microparticles containing signal molecules is performed under bright field and dark field mode microscopy, respectively.

In a particular field of view, the number of particles in the bright field is represented by x, and the number of particles containing signal molecules in the dark field is represented by y.

In a preferred embodiment of the invention, the method of the invention further comprises counting the sum of the signal intensities (the sum of the signal intensities is denoted z) on the particles with the signal molecules in dark field mode.

In a preferred embodiment of the invention, counting statistics of the microparticles and/or signal molecules are performed after imaging by microscopy.

In a preferred embodiment of the invention, the number x of particles is determined by electromagnetic wave imaging or acoustic wave imaging techniques.

The electromagnetic wave is formed by an electric field and a magnetic field which oscillate in phase and are perpendicular to each other, and the energy and the momentum are transmitted in space in the form of waves, and the propagation direction of the electromagnetic wave is perpendicular to the plane formed by the electric field and the magnetic field. The electromagnetic wave may be selected from one or more of radio waves, microwaves, infrared rays, visible light, ultraviolet rays, X-rays, and gamma rays. The preferred electromagnetic wave is visible light, having a wavelength of about 380nm to 780 nm. The acoustic wave is preferably an ultrasonic wave.

In a preferred embodiment of the invention, the number of particles x in a particular field of view is counted by microscopic bright field imaging. The number y of particles with signal molecules in a particular field of view and the acquisition of the sum z of the signal intensities are determined depending on the type of signal molecules. In a preferred embodiment of the invention, the number y of particles with signal molecules is determined by dark-field imaging of said signal molecules.

In a preferred embodiment of the present invention, the number of the specific fields is n, and the number x of the microparticles in each field is counted respectively_iAnd the number y of particles with signal molecules_iAnd/or signal strength sum z_iAnd calculating the value of x, y and/or z; wherein n and i are non-0 natural numbers,

，

，

。

the signal molecule concentration information can be obtained by combining three numerical calculations of x, y and z with a standard concentration curve, wherein the three numerical calculations include proportional relationships, which can be x and y, x and z, y and z, or x and y, z, or parameters obtained by multiplying the three numerical calculations by specific coefficients or deforming the three numerical calculations, and the calculation of the signal molecule intensity by the skilled person is not influenced. For example, when y/x =1, it means that all particles carry signal molecules; y/x =0, which means that all particles do not carry signal molecules, the ratio is preferably used with the detection curves of standards at different concentrations to calculate the number of signal molecules.

In a preferred embodiment of the invention, the method of determining the number of signal molecules comprises using the average intensity of the signal molecules in conjunction with a detection curve for different concentrations of standard when the ratio of y to x is greater than 10% (e.g., 20%, 30%, 40%, 50% or higher).

In a preferred embodiment of the invention, no statistical clustering/overlapping of particles is performed in the calculation of any one or more of x, y, z (e.g., x, y; x, z; y, z) during counting of particles and/or signal molecules.

In a preferred embodiment of the invention, alternatively, multi-well plates can be used to process samples in parallel to increase the detection throughput.

Target molecules

In a preferred embodiment of the invention, the target molecule is a cell, an organelle, a microorganism, a nucleic acid, a protein, a polypeptide or a small molecule compound with a molecular weight below 1000, preferably a protein, a polypeptide or a nucleic acid.

Low abundance detection substances common in the prior art include, but are not limited to: nerve factors such as: TNF alpha, IFN-gamma, etc., inflammatory factors such as: IL-1 alpha, IL-1 beta, IL-6, IL-13, etc. Cancer factors PSA, CEA, AFP, CA 19-9.

In a preferred embodiment of the invention, the microorganism comprises a virus, a bacterium, a fungal cell.

In a preferred embodiment of the invention, the virus is selected from one or more of the families adenoviridae (adenoviridae), arenaviridae (arenaviridae), astroviridae (astroviridae), bunyaviridae (bunyaviridae), caliciviridae (caliciviridae), flaviviridae (flaviviridae), hepadnaviridae (hepeviridae), mononegavirales (monoegavirales), nidoviridae (nidovirales), picornaviridae (picornaviridae), orthomyxoviridae (orthomyxoviridae), papilloma virus (papillomaviridae), parvoviridae (paraviridae), polyomaviridae (polymaviridae), poxviridae (poxviridae), enteroviridae (reoviridae), retroviridae (retroviridae), and retroviridae (polytriaceae).

In a preferred embodiment of the invention, the bacteria are selected from one or more of the genera staphylococcus, streptococcus, listeria, erysipelothrix, nephrobacter, bacillus, clostridium, mycobacterium, actinomyces, nocardia, corynebacterium, rhodococcus, and/or one or more of bacillus anthracis, erysipelothrix, tetanus, listeria, mycobacterium aerothrix, escherichia coli, proteus, dysentery bacillus, pneumonia, brucella, clostridium perfringens, haemophilus influenzae, haemophilus parainfluenzae, moraxella catarrhalis, acinetobacter, yersinia, legionella pneumophila, bordetella pertussis, bordetella parapertussis, shigella, pasteurella, vibrio cholerae and haemophilus parahaemolyticus.

In a preferred embodiment of the invention, the fungus is selected from one or more of coccidiodes immitis, pediococcus pluvialis, histoplasma capsulatum, histoplasma durnumerous, blastomyces lobbiensis, paracoccidioides brasiliensis, blastomyces dermatitidis, sporothrix schenckii, penicillium marneffei, candida albicans, candida glabrata, candida tropicalis, candida viticola, aspergillus, Exophiala jejuni, chromocor zeylani, chromocor miehei, Plasmodium poecium, Cryptococcus neoformans, Trichosporon mentagrophytes, Rhizopus oryzae, Mucor hiemalis, Absidia, Coptomyces racemosus, frogmatis, Conidiobolus, Isospora polyspora, Nostosporum occidentalis, and Sporotrichum atrox.

In the context of the present invention, the term "nucleic acid" refers to any nucleic acid-containing molecule, including but not limited to DNA or RNA. The term encompasses sequences comprising any known base analog of DNA and RNA, including but not limited to: 4-acetylcytosine, 8-hydroxy-N6-methyladenosine, aziridinylcytosine, pseudoisocytosine, 5- (carboxyhydroxymethyl) uracil, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, dihydrouracil, inosine, N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil, 1-methylguanine, 1-methylinosine, 2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine, 7-methylguanine, 5-methylaminomethyluracil, N6-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, β -D-mannosyl Q nucleoside, 5' -methoxycarbonylmethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, methyl uracil-5-oxyacetate, uracil-5-oxyacetic acid, oxybutoxythymidine (oxybutoxosine), 5-methyl-2-thiouracil, 4-thiouracil, 5-methyluracil, methyl N-uracil-5-oxyacetate, uracil-5-oxyacetic acid, pseudouracil, Q nucleoside, 2-thiocytosine and 2, 6-diaminopurine.

In a preferred embodiment of the invention, the nucleic acid is a miRNA or shRNA.

In the context of the present invention, the term "polypeptide" refers to a molecule comprising at least two amino acid residues joined by a peptide bond to form a polypeptide. The term "polypeptide" also includes domains of proteins. Small polypeptides of less than 50 amino acids may be referred to as "peptides". Preferred polypeptides are disease markers such as tumor markers.

In a preferred embodiment of the invention, the small molecule compound is a small molecule compound having a molecular weight of less than 1000 (or less than 500).

In a preferred embodiment of the invention, the capture molecule-detection molecule, capture molecule-target molecule binding modes are each independently selected from the following combinations:

biotin or its derivatives/streptavidin (streptavidin), biotin or its derivatives/avidin (avidin), biotin or its derivatives/NeutrAvidin (NeutrAvidin), biotin or its derivatives/avidin or its derivative antibodies, haptens/antibodies, antigens/antibodies, polypeptides/antibodies, receptors/ligands, digoxigenin/digoxigenin, carbohydrates/lectins, polynucleotides/complementary polynucleotides and aptamers/aptamer-recognizing substances; wherein the derivative of biotin is any one of D-biotin, activated biotin, biocytin, ethylenediamine biotin, cadaverine biotin and desthiobiotin.

In the above-mentioned binding methods, only the binding method is defined, but the state of the capture molecule, the detection molecule, the target molecule or the details thereof are not limited. Taking an antigen/antibody binding manner as an example, the capture molecule may be an antibody, and the target molecule may be an antigen; or the capture molecule may be an antigen and the target molecule may be an antibody; or the capture molecule may be a mixture coupled to or comprising antibodies and the target molecule may be a mixture coupled to or comprising antigens or antibodies; or the capture molecule and the target molecule may carry additional fusion proteins or labels.

The capture antibody/detection antibody is classified according to the specificity of the antibody, and can be one or more of polyclonal antibody, monoclonal antibody, single-chain antibody, antigen binding fragment and nano antibody. The capture antibody is classified according to the source and can be one or more of a murine antibody, a rabbit antibody, an ovine antibody and an alpaca antibody.

In a preferred embodiment of the invention, the signal molecule labeled with the detection molecule may be labeled in advance and then bound to the target molecule or the capture molecule. The signal molecule labeled with the detection molecule may be bound to the target molecule or the capture molecule before labeling the signal molecule.

In a preferred embodiment of the present invention, the concentration of the target molecule is calculated based on the detection result of the signal molecule.

The method of the invention is also suitable for detecting various types of target molecules. The species of capture molecules may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more. Accordingly, the target molecule and the detection molecule should be smaller than or equal to the species of the capture molecule.

In a preferred embodiment of the present invention, the plurality of detector molecules each carry a distinct signal molecule that can be distinguished by means including, but not limited to, different colors, different fluorescence, different molecular weights, etc.

Due to the large number of particles, when it is desired to detect a' target molecules, the corresponding a capture molecules can be dispersedly coated on the surface of different particles. For example, a may be partially fixed to the fine particles₁A capture molecule, another part of the microparticles being immobilized with a₂A capture molecule, and a further portion of the microparticles having a immobilized thereon₃A capture molecule, and a₁∪a₂∪a₃= A; or a particle comprising a capture molecule, which may be the same or different; or two or more different capture molecules are captured on one microparticle; or a single capture molecule on a portion of the particles, or multiple capture molecules on a portion of the particles.

In a preferred embodiment of the present invention, the plurality of detection molecules may have a signal molecule, different target molecules to be detected are distinguished by the shape of the particles, the particles may be spheres, nearly spheres, cubes, polyhedrons or irregular shapes, and the particles with different shapes may be coupled with different capture antibodies, so as to specifically bind different target molecules, thereby realizing the detection of various types of target molecules.

It is to be noted that the present invention is not particularly limited to the number of capture molecules immobilized per unit particle, and the number of capture molecules may be one or more even if there is only one type of capture molecule.

Drawings

Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:

FIG. 1 is a schematic view of capture of an antigen by magnetic beads coated with capture antibodies and formation of immune complexes on the surface thereof, according to one embodiment of the present invention;

FIG. 2 is a photograph of magnetic beads taken in bright field microscopy imaging mode, according to one embodiment of the present invention;

FIG. 3 is a photograph of a flat-laid solution of magnetic beads with different particle sizes, which is obtained by distribution in bright field microscopy imaging mode according to an embodiment of the present invention;

fig. 4 is a photograph of a flat-laid magnetic bead solution taken in a bright-field and dark-field microscopic imaging model, according to an embodiment of the present invention.

FIG. 5 shows the detection results of streptavidin-modified magnetic beads on Biotin-Qbeads at different concentrations according to one embodiment of the present invention;

FIG. 6 shows the results of the detection of the expression of purified Spike protein according to one embodiment of the present invention;

FIG. 7 is a graph showing the results of detecting pseudoviruses having the novel coronavirus S protein surface-expressed, according to an embodiment of the present invention

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

FIG. 9 is a schematic view of a turntable according to one embodiment of the present invention;

FIG. 10 is a schematic view of a device for detecting molecules according to an embodiment of the present invention;

FIG. 11 is a schematic diagram of the relative positions of a turret and an objective lens according to one embodiment of the invention;

wherein, 1-1 position; position 2-2; position 3-3; position 4-4; position 5-5; 6-6 position; position 7-7; position 8-8; 201-a turntable; 202-blind holes; 203-rotation axis; 301-magnetic bead solution; 302-a blind hole for accommodating a magnetic bead solution to be detected; 303-bright field light source; 304-a condenser lens; 305-an objective lens; 306-a fluorescent light source; 307-a lens; 308-a dichroic beam splitter; 309-optical filter; 310-a lens; 311-camera.

Detailed description of the preferred embodiments

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention.

Detection device

Referring to fig. 9 to 11, the present invention provides an apparatus for detecting a target molecule, comprising: a solid phase carrier 201 capable of immobilizing microparticles in a microparticle solution 301, the microparticles comprising microparticles to which a signal molecule to be detected is bound and being dispersed on the surface and/or inside the solid phase carrier;

at least one first light source 303 and at least one second light source 306, wherein light emitted by the first light source 303 is irradiated to particles (such as magnetic beads in the blind holes 302 of the rotating disc 201 containing the solution of magnetic beads to be detected) in a selected field of view on the solid-phase carrier 201 through the condenser 304 to form a bright field signal related to the total number of the particles; the fluorescent light emitted from the second light source 306 is sequentially irradiated onto the particles in the selected field of view on the solid phase carrier 201 through the lens 307 and the dichroic beam splitter 308 to form a dark field signal related to the total number of the particles combined with the signal molecules to be detected; wherein the first light source 303 and the second light source 306 may be provided by LEDs.

The signal acquisition unit is used for respectively acquiring a bright field signal and a dark field signal; the signal acquisition unit comprises an amplifying assembly objective lens 305, a lens 310, an optical filter 309 and a shooting assembly camera 311, and the bright field signal and the dark field signal respectively reach the camera 311 through the objective lens 305, the lens 310 and the optical filter 309 in sequence for microscopic imaging.

A signal processing unit (not shown) for determining the concentration of the target molecule based on the collected bright field signal and dark field signal.

In the device for detecting a target molecule of the present invention, the solid carrier is a rotating disk 201 which can be rotated relative to the signal collecting unit as shown in FIG. 9 or FIG. 10. Referring to fig. 10, a silicate glass rotary plate 201 as a solid carrier is disposed above the signal pickup unit and can be removed therefrom. In other alternative embodiments, the solid support is a plate, such as a glass slide, and the particles in solution 301 are randomly distributed uniformly over the surface of the plate. In other alternative embodiments, the solid support has at least one flow channel comprising an inlet and an outlet. The particles in the solution 301 are uniformly distributed in the flow channel. At least a portion of the flow channel overlaps with the optical path of the light detection unit to allow detection of the first light source 303 and the second light source 306.

The turntable 201 may be made of quartz, glass, or the like. The turntable 201 comprises at least one optically transparent detection site which is detected by the signal acquisition unit when the detection site is in the optical path of the signal acquisition unit. As shown in fig. 9, the turntable 201 has 8 blind holes 202 as detection sites, and one of the blind holes 302 is a blind hole for receiving a solution of particles to be detected.

The turntable 201, which is a solid carrier, can rotate in a plane above the signal acquisition unit. Fig. 11 shows that the blind hole 302 containing the solution of particles to be detected is positioned above the objective lens 305 by rotation. The carousel 201 is configured to rotate in a stepwise manner sequentially between a plurality of stations where the following operations are performed on the solution to be detected at the detection site: immobilizing and dispersing the particles (e.g., magnetic beads) in the solution and washing. The plurality of stations comprise a detection station positioned in the light path of the signal acquisition unit, at least one pretreatment station positioned at the upstream of the detection station and at least one post-treatment station positioned at the downstream of the detection station, wherein the pretreatment station is used for fixedly dispersing particles in the solution, and the post-treatment station is used for flushing.

The device for detecting molecules further comprises a magnetic field generating device or an electric field generating device (not shown), wherein the magnetic field generating device or the electric field generating device is used for fixedly dispersing the particles on the surface and/or in the solid phase carrier.

In a preferred embodiment, the particles are magnetic beads, which may be selected from one or more of paramagnetic and superparamagnetic. The magnetic beads have a particle size of 600nm to 10 μm.

The apparatus of the present invention further comprises a displacement mechanism (not shown) for moving the signal acquisition unit and/or the solid 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, and for example, can be a two-dimensional displacement stage, so as to allow bright-field and dark-field microscopic imaging of individual particles to be obtained in a planar motion manner when the individual particles are dispersed on the surface of the solid support.

Referring to FIG. 11, the reacted microparticle solution 301 of the immunocomplex with the signal molecule is sucked into the blind holes 202 of the transparent solid phase carrier, which corresponds to the number 5 of 8 blind holes 202 on the rotating disk 201 in FIG. 11. The rotating platform drives the rotating shaft 203 to sequentially move the sample from the No. 5 position to the No. 4, 3, 2 and 1 positions to carry out the steps of liquid agar adding, particle space distribution control by external force, cooling and solidification of agar fixing particles and signal acquisition, wherein the steps of the No. 2 position and the No. 4 position can be omitted. In position 1, a first light source 303 (i.e., a bright field light source) transmits through a condenser lens 304 and a blind hole 302 for receiving a solution of magnetic beads to be detected to form a bright field image of the magnetic beads, and the bright field image is collected by an objective lens 305 and imaged by a camera 311 through a lens 310. If the signal molecule is a fluorophore or a particle, the second light source (e.g., an excitation light source) 306 excites the signal molecule on the particle through the lens 307, the dichroic beam splitter 308 and the objective lens 305, and the generated fluorescence signal is collected by the objective lens 305, transmitted through the dichroic beam splitter 308, filtered by the filter 309, and imaged on the camera 311 through the lens 310. Fluorescence signals are collectively referred to herein as dark field imaging. Bright field and dark field imaging are performed alternately, the bright field signal providing information on the number of dispersed particles, and the dark field imaging providing the number of particles containing signal molecules in these dispersed particles, and preferably, the concentration of target molecules can be further analyzed by poisson distribution. After the data collection is completed, the sample continues to rotate from position 1 to position 5 through

positions

8, 7 and 6 (i.e., multiple elutions are performed to recover particles), during which the blind holes 202 are flushed for the next cycle of testing.

It should be noted that:

(1) when adopting agar to fix the particle, when the sample loaded the back and shifted to No. 4 position filling agar from No. 5, the blind hole of No. 6 position can shift to No. 5 position this moment and begin to load next sample, analogizes in proper order, guarantees that every blind hole moves this position and all loads the sample, realizes high efficiency cycle detection.

(2) In addition to the immobilization of the microparticles with agar, the microparticle solution 301 of the immunocomplex with a signal molecule after the reaction may be pipetted onto a transparent flat solid phase carrier (quartz, glass, etc.) or a light-transmitting porous solid phase carrier, and then the microparticles are closely spread on the solid phase carrier by directly using a magnet, gravity, or a magnetic field, preferably the solvent of the microparticle solution 301 is removed, and then the microparticles are directly detected by two-dimensional distribution.

(3) When the biomolecule is a nucleic acid, it can be analyzed by an analyzer having the same structure as the analyzer.

Example 1 screening of magnetic beads

Providing magnetic bead solutions with different particle sizes (the particle sizes are 500 nm, 1 mu m, 2 mu m and 3 mu m). The magnetic bead solutions with the 4 particle sizes are randomly distributed on the glass slide, and the glass slide is observed and photographed under a microscope, and the result is shown in 3.

As can be seen from fig. 3, when the particle size of the magnetic bead is 500 nm, the magnetic bead generates a clustering effect, and the number of individual particles cannot be counted. And the magnetic beads with the particle size larger than 10 mu m are easy to sink due to gravity in the incubation process, and are unevenly distributed in the incubated solution, so that the collision probability is influenced, and the detection sensitivity is reduced. The magnetic beads with the particle size of 600nm-10 mu m can avoid the two influence factors, under different particle sizes and corresponding specific magnification, bright field microscopic imaging shows image characteristics of surrounding black and bright middle (the particle size is 3 mu m as shown in fig. 3), and the characteristics are combined with the size of the magnetic beads, so that the magnetic beads and other impurities can be distinguished, and the detection and analysis result is improved. The detection tolerance of samples with complex pollution and background, such as pharyngeal swabs, saliva, nasal swabs, alveolar lavage fluid and other smear swabs, is higher.

The 1 μm and 2 μm size magnetic beads described in FIG. 3 also exhibit the above-described bright-in-dark-around characteristics (not shown) at each particular magnification.

Example 2 detection of streptavidin-modified magnetic beads on Biotin-Qbeads at different concentrations

1. Experimental Components

Streptavidin modified magnetic beads, Biotin modified quantum dot microspheres (Biotin-Qbeads), Buffer A (2% BSA in 10mM PBS pH7.4), Buffer B (0.5% Tween20 in 10mM PBS pH7.4).

2. Experimental methods

First, Biotin-Qbeads were diluted to 0, 0.05, 0.25, 0.5, 2.5, 5, 25, and 50fM using Buffer a. Dilute streptavidin-modified magnetic beads to 2X10⁷one/mL.

10 mu L of diluted biotin-Qbeads with different concentrations, 10 mu L of diluted streptavidin modified magnetic beads and 80 mu L of Buffer A are respectively taken, evenly mixed by vortex and reacted for 1h at 37 ℃.

Washing the third step with Buffer B for 6 times, and removing the supernatant.

The magnetic beads were resuspended in 20 μ L PBS, 5 μ L of the suspension was transferred to a cover slip, the magnetic beads were adsorbed to the bottom of the slide using a magnet, and single-molecule imaging was performed using a fluorescence microscope.

Fifthly, imaging is carried out on the magnetic beads distributed on the bottom surface of the glass by using a low-power objective lens in a bright field imaging mode and a fluorescence imaging mode respectively, and two sets of data of the bright field and the fluorescence are obtained. The antigen concentration is obtained by the ratio of the number of magnetic beads containing immune complexes (i.e. the number of active beads) to the total number of magnetic beads.

Sixthly, detecting a series of different concentrations, and repeating each concentration point for 3 times.

3. Results of the experiment

The results of the experiment are shown in FIG. 5, and the detection concentration from 50aM to 50fM is in the linear range of the detection method. The linear dynamic range of the method is therefore 3 orders of magnitude. In the embodiment, in the absence of an antibody reaction system, the ultimate sensitivity of the system is verified under the participation of only two reactants with stronger affinities, namely streptavidin and biotin, and the result shows that if the affinity of the antibody is high enough, the method can be explored down to aM, so that a novel technical means is provided for the detection of low-abundance factors.

Example 3 detection of purified novel coronavirus Spike protein

1. Experimental Components

Carboxyl-modified magnetic beads, capture antibodies (SinoBiological 40150-D006), detection antibodies (SinoBiological 40591-MM 43), streptavidin-modified quantum dot microspheres, Spike protein (RBD, SinoBiological), N-hydroxythiosuccinimide (S-NHS), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC), PBS Buffer, Buffer A (0.1% Tween20 in 10mM PBS pH7.4), Buffer B (2% BSA in 10mM pH7.4), Buffer C (0.5% Tween20 in 10mM PBS pH7.4), NaOH, MES-HCl, PBS, EZ-LinkNHS-4-Biotinylation Kit, Desalting Columns (Zeba' S Spainin Desalting Columns).

2. Preparation method

2.1 covalent coupling of magnetic beads to Capture antibodies

Firstly, taking 20uL of magnetic bead suspension liquid into a 1.5mL EP tube, placing the EP tube into a magnetic separation frame, enriching the magnetic beads, and removing supernatant.

Use 0.5mL of H₂And O, swirling for 15s in a centrifugal tube to uniformly mix the magnetic beads, putting the EP tube into a magnetic separation frame, enriching the magnetic beads, and removing supernatant.

Thirdly, adding 0.5mL of NaOH into the centrifugal tube, swirling for 15s to uniformly mix the magnetic beads, placing the EP tube into a magnetic separation frame, enriching the magnetic beads, and removing supernatant. This step was repeated twice.

Fourth add 0.5mL of H₂And O, swirling for 15s in a centrifugal tube to uniformly mix the magnetic beads, putting the EP tube into a magnetic separation frame, enriching the magnetic beads, and removing supernatant. This step is repeated once.

Fifthly, adding 0.5mL of MES (methyl methacrylate) with a pH value of 5.0 into the centrifuge tube, vortexing for 15s to uniformly mix the magnetic beads, placing the EP tube into a magnetic separation frame, enriching the magnetic beads, and removing the supernatant. This step is repeated once.

Sixthly, adding 0.1mL MES with a pH value of 5.0 into a centrifugal tube, vortexing for 15S to uniformly mix magnetic beads, then adding 0.1mL 100mg/mL EDC and 50mg/mL S-NHS, vortexing for 15S, placing an EP tube into a horizontal shaking table, and reacting for 40min at room temperature. The EP tube is placed in a magnetic separation frame, magnetic beads are enriched, and supernatant is removed.

Add 0.5mL MES pH5.0 to centrifuge tubes, vortex for 15s to mix the beads well, place the EP tube in a magnetic separation rack, enrich the beads, and remove the supernatant. This step is repeated once. Add 0.1mL MES pH5.0 heavy suspension beads, vortex for 15 s.

In addition, 88. mu.g of the capture antibody (40150-D006) was diluted with 0.1mL MES pH5.0 and added to the suspension of magnetic beads, vortexed for 15s, and the EP tube was placed on a horizontal shaker and allowed to react at room temperature for 1 h.

The self-lifting is to place the EP tube in a magnetic separation frame, enrich the magnetic beads and remove the supernatant. The beads were resuspended in 0.4mL Tris-HCl pH7.4, vortexed for 15s, and the EP tube was placed on a horizontal shaker and allowed to react at room temperature for 1 h.

The EP tube is placed in a magnetic separation frame to enrich the magnetic beads and remove the supernatant. Adding 0.5mL Buffer A, vortexing for 15s to mix the magnetic beads uniformly, placing the EP tube in a magnetic separation frame, enriching the magnetic beads, and removing the supernatant. This step was repeated twice.

The PBS with 0.5mL is added, the magnetic beads are uniformly mixed by 15s of vortex, the EP tube is placed in a magnetic separation frame, the magnetic beads are enriched, and the supernatant is removed.

Adding 0.15mL Buffer B into the water solution, vortexing for 15s to mix the magnetic beads uniformly, and storing at 4 ℃.

2.2 biotinylation modification of detection antibodies

First, 1mg of detection antibody was diluted with 1mL of 10mM PBS to a concentration of 1mg/mL, and stored at 4 ℃ for later use.

And secondly, taking a tube of NHS-PEG4-Biotin which is subpackaged in the kit, and adding 0.17 mL of ultrapure water for dissolving to obtain a NHS-PEG4-Biotin solution with the concentration of 20 mu M.

And thirdly, adding 6.65 mu L of NHS-PEG4-Biotin solution into the detection antibody solution, and reacting for 1h at room temperature.

The buffer was replaced with a Desalting column (Zeba. RTM. Spin desaling Columns), and the excess NHS-PEG4-Biotin solution was removed from the system.

Fifthly, the concentration of the detection antibody after biotinylation is determined by using the Nanodrop, and the detection antibody is stored at 4 ℃.

3. Experimental methods

First, the concentration of Spike protein was diluted to 0, 0.01, 0.1, 1, 10, 100pg/mL using Buffer B.

Secondly, the magnetic beads marked with the capture antibodies are diluted by 50 times by using Buffer B, the concentration of the biotin-marked detection antibodies is diluted to 4 mu g/mL, and the SA-Qbeads is diluted to 1.67 nM.

And thirdly, respectively taking 25 mu L of diluted Spike protein with different concentrations, magnetic beads marked with capture antibodies, biotin-marked detection antibodies and SA-Qbeads, uniformly mixing in a vortex manner, and reacting for 1h at 37 ℃.

Fourthly, washing is carried out for 6 times by using a Buffer C, and the supernatant is removed.

Fifthly, adding 20 mu L of PBS to resuspend the magnetic beads, taking 5 mu L of the resuspended magnetic beads, transferring the magnetic beads to a cover glass, adsorbing the magnetic beads to the bottom of the glass slide by using a magnet, and carrying out single-molecule imaging by using a fluorescence microscope.

Sixthly, respectively imaging the magnetic beads distributed on the bottom surface of the glass by using a low-power objective lens in a open-cut field imaging mode and a fluorescence imaging mode to obtain two sets of data of the open-cut field and fluorescence. The antigen concentration is obtained by the ratio of the number of magnetic beads containing immune complexes (i.e. the number of active beads) to the total number of magnetic beads.

A series of Spike protein concentrations were performed, 3 replicates per concentration point.

4. Results of the experiment

As shown in FIG. 6, it is understood that in this example, the range of the Spike protein detection is 0.01-100 pg/mL, and the lower limit of the detection is 10 fg/mL.

Comparative example 1: detection of pseudoviruses expressing the S protein of a novel coronavirus on their surface

1. Experimental Components

The test sample was pseudovirus with surface expression of the novel coronavirus S protein, and the rest of the experimental components were identical to those in example 3.

2. Preparation method

2.1 covalent coupling of magnetic beads to Capture antibodies

The procedure was carried out in the same manner as in example 3.

2.2 biotinylation modification of detection antibodies

The procedure was carried out in the same manner as in example 3.

3. Experimental methods

Pseudoviruses were used as test samples, including solutions without the pseudoviruses and serial solutions containing 2, 5, 20, 100 and 200 pseudoviruses per 100. mu.L. The remaining steps were performed in the same manner as the corresponding steps in example 3.

4. Results of the experiment

From the results of FIG. 7, it can be seen that the number of detected pseudoviruses ranged from 2 to 200, which is in the linear range of the detection method. In the case of the pseudovirus numbers of 2 and 5, the ratio of the number of magnetic beads having fluorescence to the total number of magnetic beads was low, but it was still significantly distinguishable from the control group having no virus. The detection sensitivity of the same antibody on a chemiluminescence platform is about 50 pseudoviruses at present, and the result of the embodiment is 25 times higher than that of the chemiluminescence method.

Example 4 magnetic bead assay for detection of IL-6

1. Experimental Components

The capture antibody used in this experiment was 8C9 (hangzhou huakui jinqi biotechnology limited), the detection antibody was 9a2 (hangzhou huakui jinqi biotechnology limited), the detection sample was IL-6, and the other experimental components were identical to those of example 1.

2. Preparation method

2.1 covalent coupling of magnetic beads to Capture antibodies

The procedure was carried out in the same manner as in example 1.

2.2 biotinylation modification of detection antibodies

The procedure was carried out in the same manner as in example 1.

3. Experimental methods

The concentration of IL-6 was diluted to 0, 0.05, 0.2, 0.5, 2, 5, 20pg/mL using Buffer B.

Secondly, the magnetic beads marked with the capture antibodies are diluted by 150 times by using Buffer B, the concentration of the biotin-marked detection antibodies is diluted to 4 mu g/mL, and the SA-Qbeads is diluted to 8 nM. The remaining steps were performed in the same manner as in example 1.

4. Results of the experiment

As shown in FIG. 8, it is understood that in this example, the IL-6 detection range is 0.05-20 pg/mL, which is within the linear range of the detection method, and the lower limit of the detection can reach 50 fg/mL. The detection sensitivity of the same antibody on a chemiluminescence platform is more than 1pg/ml at present, and the result of the embodiment is about 20 times higher than that of the chemiluminescence method.

Claims

1. A method of detecting a signal molecule comprising the steps of:

(3) counting particles in the selected field of view by bright field;

2. A method of detecting one or more target molecules, comprising the steps of:

(3) counting particles in the selected field of view by bright field;

3. The method according to any one of claims 1 or 2, wherein the number of microparticles immobilized to the surface and/or inside of the solid phase carrier is indefinite.

4. The method of claim 2, wherein the target molecule is selected from one or more of a protein, a polypeptide, an amino acid, an antigen, an antibody, a receptor, a ligand, or a nucleic acid.

5. The method of claim 2 or 3, wherein the specific binding reaction is selected from one or more of an immune reaction, a hybridization reaction, a receptor-ligand interaction.

6. The method of any one of claims 1 or 2, wherein the particles are magnetic particles.

7. The method of claim 6, wherein the magnetic particles are magnetic beads.

8. The method of claim 7, wherein the magnetic beads have a particle size of 600nm to 10 μm.

9. The method of claim 8, wherein the magnetic beads have a particle size of 1 μ ι η to 5 μ ι η.

10. The method of claim 1 or 2, wherein the signaling molecule is selected from one or more of a chromophore, a digoxigenin-labeled probe, a metal nanoparticle, or an enzyme.

11. The method of claim 1 or 2, wherein the signal molecule is selected from one or more of an organic small molecule fluorescent probe, a quantum dot pellet, a fluorescent pellet, a three-dimensional DNA nanostructure reporter probe, an upconversion luminescent nanomaterial pellet, a rolling circle amplified fluorescent molecule amplification structure, or an aptamer fluorescent molecule amplification structure.

12. The method of claim 11, wherein the quantum dot globules are within 110nm in size.

13. The method according to any one of claims 1 or 2, wherein in step (2), the microparticles are immobilized on the surface and/or inside a solid support by an applied magnetic and/or electric field and/or a gel.

14. The method of any one of claims 1 or 2, wherein the solid support is selected from a multi-well plate, a flat plate, or a flow channel.

15. The method of any one of claims 1 or 2, wherein the coordinates of the particles in the image are determined by bright field microscopy imaging.

16. The method of claim 15, wherein the coordinates of the particles are determined based on differences in brightness of the particles.

17. The method of claim 15, wherein the counting of step (4) is determined by coordinates of the particles in the image.

18. The method according to claim 1 or 2, wherein the method for determining the concentration of the target molecule in step (5) is:

and (4) determining the concentration of the target molecule by combining a standard curve according to the proportional relation of the number of the particles obtained in the step (3) and the step (4).

19. Use of the method of any one of claims 1 to 18 in the preparation of a diagnostic reagent for the detection of biomolecules.

20. A test device for carrying out the method of any one of claims 1 to 18, comprising:

21. The apparatus of claim 20, wherein the signal acquisition unit comprises a magnification assembly for magnifying particles within the selected field of view.

22. The apparatus of claim 21, wherein the magnifying component is an objective lens.

23. The apparatus of claim 22, wherein the objective lens is a low power objective lens.

24. The device of claim 20, wherein the solid support is at least partially an optically permeable support.

25. The device of claim 24, wherein the solid support is disposed above or below the signal acquisition unit.

26. The device of claim 25, wherein the solid support is configured to be removable from above or below the signal acquisition unit.

27. The device of claim 20, wherein the solid 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.

28. The device of claim 20, wherein the solid support is a multi-well plate or a flat plate.

29. The device of claim 20, further comprising a magnetic field generating device or an electric field generating device for immobilizing and dispersing the microparticles on the surface and/or inside the solid phase carrier.

30. The apparatus of claim 20, wherein the solid support is a carousel rotatable relative to the signal acquisition unit, wherein the carousel comprises at least one optically transparent detection site that is detected by the signal acquisition unit when the detection site is in the optical path of the signal acquisition unit.

31. The apparatus of claim 30, wherein the carousel is configured to sequentially rotate in a stepwise manner between a plurality of stations and to perform the following operations on the solution to be detected at the detection site at the plurality of stations: fixing the microparticles in the solution and rinsing.

32. The apparatus of claim 31, wherein the plurality of stations comprises a detection station located in the optical path of the signal acquisition unit, at least one pre-treatment station located upstream of the detection station, where the operation of fixedly dispersing the particles in the solution is performed, and at least one post-treatment station located downstream of the detection station, where the operation of rinsing is performed.

33. The apparatus of claim 20, wherein the signal acquisition unit comprises at least one camera assembly.

34. The device of claim 20, wherein the device further comprises a displacement mechanism for actuating the signal acquisition unit/solid support.

35. The apparatus of claim 34, wherein the displacement mechanism is 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.

36. A computer-readable storage medium for storing a program for performing the method of any one of claims 1-18 and/or data generated by performing the method.

37. An electronic device comprising the computer-readable storage medium of claim 36.