A single-molecule dynamic detection method and system based on external force regulation

Abstract

The present invention discloses a single-molecule dynamic detection method based on external force regulation. The single-molecule dynamic detection method uses an external force regulation module to adjust the binding and dissociation between a target molecule and a detection probe, a capture probe, and a nanoparticle, and uses a single-molecule imaging system to obtain single-molecule imaging information and analyze the dynamics of the target molecule. The present invention also discloses a system for detecting single-molecule dynamics and its application. The present invention can achieve fast, ultrasensitive, and high-throughput single-molecule immunoassay, with a detection limit up to the fM level or even lower; and solves the problem that the probe is not universal, has a lower device cost, a lower use threshold, and a larger throughput.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical detection, processing and analysis, and relates to a single-molecule dynamic detection method and system based on external force regulation for detecting low-abundance proteins, nucleic acids and small biomolecules. Background Art

[0002] In vitro diagnosis technology (IVD) plays an important role in the early diagnosis, prognosis detection and treatment evaluation of many diseases and cancers. Currently, more than 80% of disease diagnoses in clinical practice rely on in vitro diagnosis technology. The detection limit (LoD) of traditional IVD technologies, such as enzyme-linked immunosorbent assay (ELISA), is at the pM level (10 -12M), which greatly restricts the application fields of IVD technology. It is reported that there are still a large number of low-abundance proteins, nucleic acids and small biological molecules in the human body that cannot be detected by traditional IVD technology (J. Am. Chem. Soc. 2019, 141, 1162 - 1170). Therefore, it is of great significance to develop ultrasensitive biosensors with single-molecule level (<pM). Single-molecule array SiMoA and single-molecule counting SMC are the most commercially successful single-molecule detection technologies at present. The former is based on the ELISA principle and constructs an ultra-low reaction system through nanopore array technology to achieve digital detection of the fluorescence signal of single-molecule enzymatic reactions. Its basic principle is similar to that of digital PCR technology (Nature Biotechnology. 2010, 28, 595 - 599); while the latter realizes the counting measurement of single molecules by focusing the laser on the Airy disk to increase the photon yield of fluorescent molecules, which is equivalent to an ultrasensitive flow-through molecular detector (Anal Chem, 1996, 68(4): 690 - 696; Anal Chem, 1998, 70(3): 431 - 437). However, these single-molecule detection technologies all have certain limitations, resulting in the current single-molecule detection technologies mainly serving laboratories and unable to be popularized to clinical IVD. The operation process of SiMoA technology is complex, the processing accuracy requirements of microfluidic chips are high, and the production cost is high. While SMC has insufficient detection throughput, strict requirements for the optical system and environmental stability, and the detection time of both technologies is generally too long; this is because the current single-molecule detection technologies still cannot break through the theoretical limit of traditional detection. In traditional enzyme-linked immunosorbent assay (ELISA), the detection limit depends on the affinity of antibodies and the inhibition treatment of non-specific adsorption. At the same time, in the endpoint readout assay, this brings two key problems: concentration limit and thermodynamics limit, resulting in its detection sensitivity at the pg / mL level.

[0003] As a new detection strategy, kinetic analysis is expected to break through the thermodynamics limit of single-molecule detection and achieve more sensitive detection. Its principle lies in that at the thermodynamic equilibrium state of molecular binding, the binding and dissociation events of molecules still occur continuously. The continuous recording of the process can achieve signal amplification of the detection, and finally achieve detection at the fM level, which is 1000 times better than ELISA technology.

[0004] Unfortunately, dynamic analysis is not yet universal. This is because in immunoassays, dynamic analysis requires rapid binding and dissociation of antibodies and antigens, and signal amplification is achieved by accumulating repeated binding and dissociation events. Therefore, in immunoassays, a cumbersome process is needed to screen for suitable antibodies (weak affinity, rapid binding and rapid dissociation). Commercial antibody reagents often have a high affinity for the target protein. Although the molecules can bind rapidly, the dissociation events occur slowly, resulting in an overly long time-consuming for dynamic analysis immunoassays. As a dynamic detection technique, the single-molecule recognition technology based on Poisson equilibrium, SiMREPS, has achieved single-molecule detection in 15 - 30 minutes in nucleic acid detection where the affinity is easy to regulate, and the detection limit has reached the fM level (Nature Biotechnology, 2015, 33, 7, 730 - 732). However, the application of this technology in protein detection is limited by the complex antibody screening process and the long time-consuming (>2h) caused by inappropriate antibodies.

[0005] With the improvement of medical standards, society has higher requirements for clinical diagnosis. For example, the detection of virus carriers in the public needs to be as fast as possible. Traditional PCR is difficult to meet the requirements of rapid detection. Currently, the diagnostic kits sold at home and abroad basically rely on the protein recognition of viruses, and the total detection time is within 15 - 30 minutes. The total detection time of current single-molecule detection technologies still cannot meet the minimum requirements of clinical detection applications. For example, the single detection of SiMoA takes 2 hours, and SMC often requires more than 4 hours. There are also inherent time-consuming problems in dynamic detection. Therefore, developing a rapid and ultrasensitive single-molecule detection technology has great research significance and practical value.

[0006] The disadvantages of existing single-molecule detection technologies are high cost, low throughput, time-consuming, and lack of universality. SiMoA and SMC technologies rely on detection chips and laser light sources focused into Airy disks, which are costly and not conducive to the application of clinical IVD. Secondly, SiMoA, SMC, and SiMREPS all have the problem of time-consuming detection. Although the detection efficiency of SiMREPS can be improved by selecting suitable affinity antibodies, the complex antibody screening process makes this method lack universality. Finally, all current single-molecule detection technologies have the problem of too low detection throughput, which limits their popularization in clinical practice. Summary of the Invention

[0007] As described in the background, there are currently two key scientific problems in single-molecule detection that urgently need to be solved, namely concentration limitation and thermodynamics limitation. The concentration limitation is due to the fact that the concentration of the molecules to be detected in single-molecule detection is lower than the pM level, and it takes much longer for the molecules to reach the sensor surface at low concentrations (far greater than 60 minutes, and in ELISA, the lowest possible detection limit generally requires overnight incubation). In addition, there is the detection limit brought about by the thermodynamics limitation. For endpoint detection, the detection result is to read out the target molecules bound at a certain moment (after incubation to reach the thermodynamic equilibrium state). Its detection limit can be defined as the critical concentration: at this concentration, no molecule to be detected can be detected no matter how long the equilibrium lasts. The concentration limitation can be alleviated by microfluidic chip technology to accelerate the diffusion of target molecules to the sensor surface, such as SiMoA, but the high-precision chip processing results in a high cost of the detection chip; the thermodynamics limitation has been partly solved by the SiMREPS technology. Its dynamic (process) detection can truly break through the theoretical limit of thermodynamics detection, and the sensitivity is more than 1000 times higher than that of ELISA. However, it is limited by the lack of universality in antibody screening, and SiMREPS does not solve the problem of too long incubation time caused by the concentration limitation.

[0008] The problem to be solved by the present invention is: to break through the bottlenecks of concentration limitation and antibody screening, and develop a rapid, ultrasensitive and high-throughput single-molecule dynamic detection method based on external force regulation.

[0009] In the first aspect of the present invention, there is provided a single-molecule dynamic detection method, which uses an external force regulation module to regulate the binding and dissociation between a target molecule and a detection probe, a capture probe, and a nanoparticle, and uses a single-molecule imaging system to obtain single-molecule imaging information and analyze the dynamics of the molecule to be detected;

[0010] Wherein, the end of the capture probe is complementary to the head of the target molecule, and the end of the target molecule is complementary to the detection probe; the capture probe or the detection probe forms a nanoparticle complex with the nanoparticle; when the nanoparticle complex binds to the target molecule, it is pulled to the vicinity of the detection probe or the capture probe under the drive of the external force regulation module, specifically binds to the capture probe or the detection probe, and then is lifted away from the detection probe or the capture probe under the drive of the external force regulation module.

[0011] In the present invention, the capture probe, the nanoparticle, the detection probe, and the target molecule form a sandwich system. The positions of the detection probe and the capture probe can be interchanged, which will not have an essential impact on the detection result. For example, when the capture probe forms a nanoparticle complex with the nanoparticle, the detection probe binds to the detection chip; when the detection probe forms a nanoparticle complex with the nanoparticle, the capture probe binds to the detection chip.

[0012] In some preferred embodiments, the external force regulation module includes electric field regulation, magnetic force regulation, and non-contact force generating devices.

[0013] Preferably, the electric field regulation uses a two-electrode or three-electrode electric field generating device such as an electrochemical workstation. The materials that can be selected for the working electrode are selected from glassy carbon electrodes, platinum (Pt), gold (Au), silver (Ag), lead (Pb), conductive glass (ITO), and mercury (Hg). The magnetic force regulation uses an electromagnet or a permanent magnet such as a nickel-chromium magnet or magnetic tweezers. The non-contact force generating devices include optical tweezers, acoustic tweezers, thermophoresis, and atomic force microscopy AFM; and / or,

[0014] The external force applied by the external force regulation module ranges from 0.01 - 1000 pN, such as 20 - 60 pN.

[0015] In addition, in the present invention, the capture probe is also called the capture molecule; the detection probe is also called the detection molecule; the target molecule is also called the molecule to be detected. These terms can be used interchangeably without distinction.

[0016] In some preferred embodiments, the target molecule includes proteins and nucleic acids; and / or, the capture probe and the detection probe include natural or engineered antibodies, nucleic acids, and aptamers, or any chemical substance or metabolite that binds to the target molecule.

[0017] In some preferred embodiments, the single-molecule imaging system includes SPRi, TIRFM, dark-field imaging, and iSCAT; and / or, the particle size of the nanoparticles ranges from 30 nm to 5 μm, and the materials are selected from, including but not limited to, nanosilicon, nanogold, nanosilver, polystyrene, and silica magnetic beads coated with superparamagnetic particles.

[0018] Preferably, the structure of the SPRi includes an objective-coupled SPRi and a prism-coupled SPRi. The objective-coupled SPRi preferably uses an objective-type SPRM, such as an objective-type SPRM reconstructed from a total internal reflection microscope. The prism-coupled SPRi is, for example, a Kretschmann prism-coupled structure; and / or, the light source of the SPRi includes an SLED light source or a laser light source with a wavelength of 600 - 800 nm; and / or, the nanoparticles include nanogold or magnetic nanoparticles. The magnification of the objective-coupled SPRi of the present invention is usually 60 - 100 times.

[0019] More preferably, the structure of the SPRi is a prism-coupled SPRi; and / or, the particle size of the nanoparticles ranges from 50 nm to 300 nm, such as 50 nm, 150 nm, or 300 nm. The magnification of the prism-coupled SPRi of the present invention is usually 2 - 60 times, such as 2 - 12, 10, 40, or 60 times.

[0020] In some preferred embodiments, the capture probe or detection probe is adsorbed on the detection chip.

[0021] Preferably, when the target molecule is nucleic acid: 12-25 nucleotides at the end of the capture probe are complementary to the nucleotides at one end of the target molecule, and the nucleotides at the other end of the target molecule are complementary to the detection probe; or, one of the capture probe and the detection probe is a locked nucleic acid, and the other is a biotin-functionalized target molecule such as a single-stranded nucleic acid ssDNA modified with biotin at one end, and the nanoparticle is a streptavidin-coated nanoparticle. In the present invention, the nucleic acid can be single-stranded nucleic acid, such as single-stranded DNA, RNA including mRNA and miRNA, etc. When the target molecule is double-stranded nucleic acid, the double strand can be opened by methods such as helicase or heating to make it a detectable single-stranded DNA fragment.

[0022] When the target molecule is protein or polypeptide or small molecule: the detection probe is an antibody that binds to the target molecule, and the capture probe is an antibody that binds firmly to the target molecule; preferably, the detection probe is an antibody against the target molecule with biotin modified at the Fc end, and the capture probe is an antibody with biotin-functionalized Fc end. The binding constant k of the capture probe to the target molecule on ≥10 10 M -1 s -1 , the dissociation constant k off ≤0.0002 s -1 , the dissociation equilibrium constant KD ≤ 0.2 fM. The binding constant k of the detection probe to the target molecule on ≤5×10 8 M -1 s -1 , the dissociation constant k off ≥0.05 s -1 ; the dissociation equilibrium constant KD ≥ 1.56 pM; the standard free energy of molecular binding ≥ -12 kcal / mol.

[0023] The principle of chemical probes for small biological molecules is not essentially different from that for nucleic acid-like target molecules such as miRNA. For example, for the detection of allosteric transcription factor (aTF), there are DNA binding domain and small molecule recognition domain on aTF. The binding of small molecule can dissociate aTF from a specific DNA sequence, and the detection of small biological molecules is realized through competitive inhibition interaction.

[0024] In some preferred embodiments, (1) the preparation of the detection chip includes the following steps:

[0025] On a glass slide coated with 2 - 3 nm of chromium and 47 nm of gold, first remove the surface particulate impurities and improve the flatness of the gold film. Then, fix a detection chamber such as a PDMS chamber or a silicon - based cell culture plate on the surface of the gold sheet to form a working electrode. Thereafter, use a reducing solution to reduce the thiol - modified detection probe or capture probe, desalt and remove the reducing solution, add a passivating agent to passivate the surface of the gold sheet, wash it, and then add the reduced detection probe or capture probe and incubate. Finally, wash and add a solution to reduce non - specific adsorption and then wash again to obtain the detection chip.

[0026] In the above steps, the detection chip only adsorbs one of the detection probe and the capture probe. That is, when the detection chip adsorbs the detection probe, the capture probe forms a nanoparticle complex with the nanoparticles; when the detection chip adsorbs the capture probe, the detection probe forms a nanoparticle complex with the nanoparticles.

[0027] (2) The preparation of the nanoparticle complex includes the following steps:

[0028] Prepare nucleic acid - coated gold nanoparticles by the salt - aging method, that is, co - incubate gold nanoparticles with the capture probe or detection probe after thiol reduction, centrifuge and resuspend to obtain. Preferably, the co - incubation is carried out in a gradient sodium chloride solution such as 50 - 300 mM sodium chloride solution; more preferably, the concentration is increased by 50 mM every 4 h until the sodium chloride concentration reaches 300 mM, and then the salt concentration is maintained unchanged for incubation for 24 h; or,

[0029] Prepare nucleic acid - coated nanoparticles such as gold nanoparticles using the biotin - avidin system, that is, co - incubate streptavidin - coated gold nanoparticles with biotin - functionalized detection molecules, centrifuge and resuspend to obtain; the biotin - functionalized detection molecule is, for example, single - stranded nucleic acid ssDNA modified with biotin at one end; preferably, the co - incubation is carried out in a gradient concentration of sodium chloride solution such as 50 - 500 mM; more preferably, the concentration is increased by 50 mM every 4 h until the sodium chloride concentration reaches 500 mM, and then the salt concentration is maintained unchanged for incubation for 24 h; or,

[0030] Prepare antibody - coated nanoparticles such as gold nanoparticles and / or magnetic nanoparticles using the biotin - avidin system, that is, co - incubate streptavidin - coated gold nanoparticles with Fc - end biotin - functionalized detection antibodies, centrifuge and resuspend to obtain.

[0031] There is no sequence priority between (1) and (2).

[0032] In some more preferred embodiments, the single - molecule dynamic detection method includes the following steps:

[0033] (a) Place the prepared detection chip at the observation position of the single-molecule imaging system, and add the nanoparticle complex and the target molecule into the detection chamber; preferably, before adding the nanoparticle complex and the target molecule, first add PBS buffer or SSC buffer or Tris-HCL buffer containing 300 mM NaCl. The SSC buffer is, for example, 1× or 2× SSC buffer.

[0034] (b) Use the external force regulation module to apply an external force, and collect single-molecule imaging data and analyze the dynamics of the target molecule at a frame rate of 15 - 60 FPS, such as 16.7 or 30 FPS: First, apply an attractive force to the nanoparticle complex to accelerate the diffusion of the target molecule and the nanoparticle complex; then, apply a repulsive force to the nanoparticle complex; adjust the binding time between the target molecule and the detection probe or the capture probe, and the binding time is 5 - 500 s, preferably 25 - 250 s.

[0035] Similarly, when adjusting the binding time, only the binding time between the target molecule and the detection probe, or the target molecule and the capture probe needs to be adjusted. The adjustment target depends on whether the detection probe or the capture probe is bound to the nanoparticle complex.

[0036] (c) Collect and record SPRi data, perform differential processing on the data to obtain single-molecule imaging information with background noise removed; count the information on particle binding and dissociation at the same position to obtain the binding time of each molecule; finally, screen out the events within the binding time dominated by specific binding, perform concentration response fitting, and obtain a concentration response curve with higher specificity; preferably, the binding time is the estimated value obtained by performing a single-exponential fit on the overall binding time distribution, and is fitted through the origin-exponential fit-Exp Go1 model, and the iterative algorithm is orthogonal distance regression.

[0037] In some more preferred embodiments, when the external force applied by the external force regulation module is an electric field force, apply a voltage of -0.6 V - +1.0 V, such as -0.4 V - +0.8 V; preferably, first use a positive electric field, such as 0 - 0.8 V (excluding 0), preferably +0.4 V, to apply an attractive force to the nanoparticles for 5 min - 20 min; then use a negative electric field, such as -0.4 - 0 V (excluding 0), preferably -0.2 V, to apply a repulsive force to the nanoparticles for 10 - 45 min.

[0038] In the present invention, through research, it is found that an electrochemical reaction will occur when an excessive voltage is applied to the gold surface, resulting in unexpected experimental results. For example, when the voltage is less than -0.6V, the Au-s bond between the capture probe and the gold surface will be reduced to mercapto -SH. The magnetic force is contributed by an electromagnet or a permanent magnet, the magnetic field strength is constant, and the magnitude of the magnetic force is mainly determined by the distance between the magnet and the particle (1 - 3 cm, preferably 1.5 cm in the present invention).

[0039] When the external force applied by the external force regulation module is a magnetic force, a power supply of 12vDC, a duty cycle of 25%, and 20s is used to drive the magnetic tweezers. First, an attractive force is applied to the nanoparticles for 15 minutes; then a repulsive force is applied to the nanoparticles for 15 minutes.

[0040] In some specific embodiments, (1) the preparation of the detection chip has one or more of the following characteristics: the glass slide is a BK-4 or BK-7 glass slide; the surface particles and impurities are removed and the flatness of the gold film is improved by using a cleaning solution and a hydrogen flame. The cleaning solution is a piranha cleaning solution, or alcohol and pure water; the reducing solution is TCEP or DTT, such as 5μM TCEP or 0.1M DTT; the passivating agent is thiol polyethylene glycol, mercaptoethanol, or thiol polyethylene glycol methoxy and thiol polyethylene glycol biotin, such as 1μM thiol polyethylene glycol; PBS is used for cleaning; thiol ethanol or BSA is used to reduce non-specific adsorption, such as 1μM thiol polyethylene glycol; the ratio of the capture probe or the detection probe to the passivating molecule is 1:100 - 1:1000; and / or,

[0041] The concentration of the capture probe or the detection probe is, for example, 50nM; and / or,

[0042] The capture probe is a locked nucleic acid single strand. For example, the locked nucleic acid molecule accounts for 10 - 50% of the single strand, preferably 20%.

[0043] The cleaning solution of the present invention has strong oxidizing properties and can clean the organic matter on the surface of the glass slide. It can be anhydrous ethanol and pure water, or a piranha cleaning solution. The preparation method of the piranha cleaning solution is as follows: Measure 70mL of concentrated sulfuric acid with a graduated cylinder and pour it into a beaker. Then measure 30mL of hydrogen peroxide with a graduated cylinder and slowly pour it into the beaker containing concentrated sulfuric acid, stirring with a glass rod while pouring. The piranha cleaning solution can effectively clean the surface of the glass slide and is beneficial to subsequent probe modification.

[0044] The detection cavity of the present invention is a perforated mold with antioxidant, water resistance, and good thermal stability, which can be relatively firmly fixed on the surface of the gold-plated chip. If necessary, it can be glued firmly with epoxy resin. The preparation method of the detection cavity, such as a PDMS cavity, is to use Dow Corning SYLGARD DC184, in which the A and B adhesives are mixed at a volume ratio of 10:1, evacuated, and then left standing in an oven at 37 degrees for more than 2 hours. A detection cavity is made on the cured PDMS with a puncher with a diameter of 1mm.

[0045] Since the sulfhydryl groups of substances such as ssDNA-SH and mEPG-SH can react with each other to form disulfide bonds -S-S-, the purpose of the reducing solution of the present invention is to reduce the sulfhydryl group -SH. Considering the influence of the reducing solution on the modification effect, tris-(2-carboxyethyl)phosphine hydrochloride TCEP and dithiothreitol DTT are preferred. Among them, the latter needs to be desalted by a Sephadex gel column (such as NAP-25) during application to remove DTT.

[0046] The cleaning solution of the present invention is a liquid that can clean glass slides, and generally PBS is used.

[0047] The second aspect of the present invention provides a system for detecting single-molecule dynamics, wherein the system includes:

[0048] (i) A nanoparticle complex as defined in the first aspect of the present invention;

[0049] (ii) A detection probe or a capture probe as defined in the first aspect of the present invention.

[0050] Preferably, the system further includes a detection chip as defined in the first aspect of the present invention; and / or, a single-molecule imaging system as defined in the first aspect of the present invention and / or an external force regulation module as defined in the first aspect of the present invention.

[0051] The third aspect of the present invention provides an application of the system as described in the second aspect of the present invention in detecting single-molecule dynamics.

[0052] On the basis of conforming to the common knowledge in the art, the above preferred conditions can be combined arbitrarily to obtain various preferred examples of the present invention.

[0053] The reagents and raw materials used in the present invention are all commercially available.

[0054] The positive and progressive effects of the present invention are as follows:

[0055] 1. It can achieve rapid, ultrasensitive and high-throughput single-molecule immunoassay; the detection limit can reach the fM level or even lower;

[0056] 2. By external force regulation, the problem that target molecules such as antibody molecules in the dynamic detection scheme are not universal is solved;

[0057] 3. Compared with SiMoA and SMC, the device cost is lower, the use threshold is lower, and the throughput is larger;

[0058] 4. The detection method using a prism coupling structure preferably used in the present invention has a large field of view and can record the signal amplification brought by the specific binding and dissociation of target molecules with high throughput, and is more suitable for the throughput requirements of detection technologies in clinical practice. Description of the Drawings

[0059] Figure 1 Schematic diagram of the principle of single-molecule dynamic detection under external force control based on the surface plasmon resonance imaging SPRi platform modified based on the Kretschmann prism coupling structure.

[0060] Figure 2 The probability distribution of binding time under different external forces obtained based on Monte Carlo simulation: a. Potential barrier with external force 0.048×K B T; b. Potential barrier without external force 0.193×K B T.

[0061] Figure 3 Characteristic binding times for different binding behaviors.

[0062] Figure 4 The effect of electric field on intermolecular interactions: a. Binding time changes with applied voltage; b. Binding and dissociation events change with applied voltage.

[0063] Figure 5 The concentration response curves plotted for particles with different binding time ranges are shown: a. Binding time distribution of hsa-miR-29a; b. Concentration standard curve fitted according to the time screening window.

[0064] Figure 6 The workflow of the present invention and the concentration response curve of miRNA are shown: a. Workflow based on detecting fM level of miRNA under 15 min regulation of electric field force; b. Concentration response curves of two miRNAs (hsa-miR-155 and hsa-miR-21).

[0065] Figure 7 Dynamic detection of proteins under different external force regulation: a. Aβ under electric field regulation 1-42 Standard curve of Aβ under magnetic field regulation; b. 1-42 The standard curve of DETAILED DESCRIPTION

[0066] The existing single-molecule dynamic analysis technology is limited by the screening of antibodies and cannot become a universal detection technology to replace endpoint detection. At the same time, the weak affinity of the complexly designed and screened antibodies further exacerbates the concentration limitation problem, making the single-molecule dynamic analysis time-consuming longer than traditional endpoint detection. The present invention uses nanoparticles to replace traditional enzyme-catalyzed luminescent or chemiluminescent molecules, and through the optimization of the optical path design and the deep learning recognition model, it can be used in a large field of view (>mm 2) The prism - type surface plasmon resonance imaging (SPRi) platform dynamically analyzes the scattered light of particles in real - time, records and analyzes the binding and dissociation events of individual particles with high throughput. More importantly, by utilizing the physical and chemical properties of nanoparticles, external forces of different magnitudes and directions can be applied to the detection system. With the regulation of nanoparticles by external forces, the kinetic characteristics of ligand - binding assays can be adjusted, ultimately realizing a fast and ultrasensitive single - molecule dynamic detection technology. The core principle of this technical solution lies in using external forces to regulate the binding and dissociation behavior of target molecules bound to nanoparticles with receptors on the chip surface. When applying an attractive force, the target molecules bind to nanoparticles with higher concentrations and quickly reach the sensor surface with the nanoparticles, changing from passive diffusion to active diffusion, and solving the problem of too long incubation time caused by concentration limitations. In the stage of applying a repulsive force, through the micro - regulation of the nanoparticle - ligand sandwich system, commercial high - affinity reagents also exhibit kinetic characteristics of rapid binding and rapid dissociation with target molecules, achieving a universal single - molecule dynamic detection. Finally, based on the large - field - of - view prism - type SPRi with high - throughput characteristics, after optimizing the optical path, a larger number of target molecules can be detected simultaneously, thus promoting the clinical practice of single - molecule detection.

[0067] The present invention constructs a single - molecule detection system with a sandwich structure, obtains large - field - of - view imaging data in real - time on a prism - type SPRi, manually counts the binding and dissociation events of individual nanoparticles to obtain the binding time of each molecule. On the one hand, the manipulation of nanoparticles is to accelerate the diffusion of molecules by external forces, changing passive diffusion to active diffusion; on the other hand, when the nanoparticles bind to target molecules, a repulsive force is applied to reduce the binding time, enabling fine - tuning of antibody probes with different affinity magnitudes and making dynamic analysis a universal technology. Then, by setting a binding - time window matching the micro - regulation, the influence of non - specific adsorption is excluded to the greatest extent, achieving ultrasensitive detection of low - abundance proteins, nucleic acids, and biological small molecules at the fM level ( Figure 1 ).

[0068] The object of the present invention can be achieved through the following technical solutions:

[0069] 1) To explore the influence of the magnitude and direction of the applied external force on the molecular binding time, this invention patent conducts a one - dimensional estimation through Monte Carlo simulation. According to the Langevin equation:

[0070]

[0071] Since the sampling rate in the experiment is 0.02 s, the quadratic term contributed by it is much smaller than the characteristic time of Brownian motion (10 -6 s), so part of it can be ignored The equation becomes:

[0072]

[0073] z n =z n-1 +dz

[0074] Among them, dz can be divided into two parts. One is caused by the deformation of biomolecules such as nucleic acid strands, aptamers or antibodies. The other is caused by Brownian motion. From the study of Brownian motion, the displacement caused by it will follow a Gaussian distribution. The standard deviation D is the diffusion coefficient.

[0075]

[0076] During the simulation process, if the displacement of the nanoparticle is greater than a certain value, it is determined that the nanoparticle dissociates. Monte Carlo simulation plays an important role in this experiment. We recorded the dissociation times of millions of nanoparticles to obtain their distribution ( Figure 2 ). Under the repulsive force, the binding potential barrier between molecules changes from Figure 2 0.193K of b B T to Figure 2 0.048K of a B T. According to the formula F=-kΔx, it can be known that the magnitude of the external force applied at this time is 20-60 pN. Figure 2 The energy of a is 0.2×10 -21 J, Figure 2 The energy of b is 0.8×10 -21 J, 1K B T = 4.14×10 -21 J.

[0077] The change in the potential barrier of the molecule with and without repulsive force will be intuitively reflected in the binding time distribution, proving the feasibility of external force regulation.

[0078] 2) According to the above simulation results, the present invention uses nanoparticles as reporting agents and manipulating agents to construct a sandwich structure similar to enzyme-linked immunosorbent assay (ELISA), and optimizes the parameters of the applied external force to achieve fine-tuning of the kinetic parameters of the nanoparticles to molecules. Specifically, it includes:

[0079] a) Below is the capture probe modified on the chip surface, which is a receptor probe that can specifically and firmly bind to the target molecule. In addition to the capture probe on the chip surface, there are also passivating molecules used to inhibit non-specific adsorption, and the ratio between the two is between 1:100 and 1:1000;

[0080] b) In the middle is the target molecule (i.e., the molecule to be detected), such as low-abundance proteins, nucleic acids, and small biological molecules. The capture probe and the detection probe bind to different sites of the target molecule respectively. For example, the capture probe and the detection probe bind to the N-terminus and C-terminus of a polypeptide respectively. If the target molecule is a nucleic acid, they bind to the 5' and 3' ends respectively.

[0081] c) Above, a probe that can repeatedly bind and dissociate with the target molecule is used as the detection probe. The detection probe is anchored on nanoparticles serving as reporter molecules and manipulation molecules, amplifying the signal through the reporter molecule, and achieving rapid and ultrasensitive detection of the target molecule with the aid of external force regulation.

[0082] d) In the constructed sandwich system, the positions of the detection probe and the capture probe can be interchanged without having an essential impact on the system.

[0083] 3) The surface plasmon resonance imaging (SPRi) technology proposed in the present invention can be divided into SPRi based on the Kretschmann prism coupling structure and objective lens type SPRM with an oil lens coupling structure. Among them, the prism type SPRi has a large field of view and can record the signal amplification brought by the specific binding and dissociation of target molecules with high throughput, which is more suitable for the throughput requirements of detection techniques in clinical practice. To achieve large-field-of-view and high-throughput single-molecule dynamic detection, the present invention has optimized the optical path and detection system as follows:

[0084] a) A superluminescent light-emitting diode (SLED) is selected as the light source. Compared with a laser light source with extremely high coherence, the SLED light source has fewer coherence fringes, bringing a cleaner background for imaging.

[0085] b) Prism coupling is selected to achieve surface plasmon resonance (SPR), which has a larger field of view than objective lens coupling. However, its disadvantage is insufficient resolution. Therefore, the prism type SPRi is often more commonly used for the detection of a large number of samples (bulk solution sample). The present invention has greatly improved the signal-to-noise ratio (SNR) of the image through light source improvement and differential image processing, and finally enables the prism type SPRi to detect gold nanoparticles (particle size ≥ 30 nm) and magnetic nanoparticles (superparamagnetic particle core, silica shell, particle size about 300 nm) in real time.

[0086] c) The interactions of biomolecules (such as nucleic acid hybridization and antigen-antibody binding) need to be carried out in a salt solution, while gold nanoparticles are extremely unstable in a high-salt solution. This is because in a high-salt solution, the Debye double layer of gold particles will be compressed, and the stability between particles is thus broken, which causes great interference to the dynamic detection based on external forces proposed in the present invention. Therefore, the present invention preferably selects an appropriate salt concentration for the single-molecule dynamic detection of 50-nm and 150-nm gold nanoparticles. At this concentration, the interactions between biomolecules can still proceed, and at the same time, the gold nanoparticles can stably exist.

[0087] d) By performing differential processing (subtracting the previous frame from the subsequent frame) on the data using the open-source software ImageJ, the single-molecule imaging information with background noise removed can be obtained. By manually counting the information on the binding and dissociation of particles at the same position, the binding time of each molecule can be obtained, and further screening the events with binding lifetimes more in line with specific binding for concentration-response fitting.

[0088] 4) The external-force-regulated force field generating device proposed in the present invention is realized by selecting an electrochemical workstation and a magnet to meet the requirements of high-throughput regulation and low cost. According to Monte Carlo simulation estimation, the range of the external force is 20 - 60 pN. Through external-force regulation, the binding time of the originally high-affinity receptor-target molecule will be reduced. Different from the binding time of non-specific binding (<5 s or >300 s), repeated binding and dissociation events can be observed within a certain period of time, realizing universal dynamic detection ( Figure 3 ).

[0089] 5) In order to verify the simulation binding and external-force regulation theory, an external-force-regulated single-molecule dynamic detection was first constructed based on nucleic acid chain probes with easy-to-design affinity. A voltage of -0.4 V - +0.8 V was applied to the nucleic acid target molecule and the detection probe with a base-pairing length of 25. By demonstrating the changes in both the total events of molecular pair binding and dissociation and the molecular binding time, it is proved that external-force regulation can indeed play a role in regulating molecular motion and kinetic behavior ( Figure 4 in a and b).

[0090] 6) The single-molecule dynamic detection based on external-force regulation in the present invention involves the regulation of molecular kinetic parameters. The classical research tool for studying molecular interactions, surface plasmon resonance (SPR), and the single-molecule imaging system surface plasmon resonance imaging (SPRi) developed from SPR can not only discretely observe the signals of individual particles but also excellently reflect the binding and dissociation events of individual molecules. Therefore, the present invention example is constructed based on SPRi.

[0091] 7) In the present invention, in order to better identify the molecular dynamics changes regulated by external forces and reduce the interference caused by non-specific adsorption, a binding time window is set to screen for an appropriate molecular binding time. The concentration response curve plotted for the particles within the specific binding time range proves that the binding time within the range of 5 - 500 s (preferably 25 - 250 s) is more likely to be generated by the molecular specific binding under the regulation of external forces. As the time range is narrowed, the specificity will be further increased, but a part of the detection events will be lost. Within the preferred time range of 25 - 250 s, the goodness of fit of the linear fitting can reach 0.98( Figure 5 a and b).

[0092] 8) A major feature of the present invention different from SiMoA and SMC is to achieve ultrasensitive detection at low cost and with high throughput. The detection chip of SiMoA is sold at a price of $2000, while the total cost of the detection chip used in this technology is within 50 yuan; in addition, the detection time can be controlled within 15 - 30 minutes. Through the ultrasensitive detection of two miRNAs (hsa-miR-155, hsa-miR-21), it is proved that this technology can achieve sub-fM level detection within 15 minutes( Figure 6 a and b).

[0093] 9) The difference between the present invention and SiMREPS is that both the target analyte and the probe of SiMREPS are nucleic acids with easily adjustable affinities, and it can only be well applied to short-chain nucleic acids (such as miRNA). In the detection of proteins, SiMREPS can only meet the requirements of dynamic analysis by screening antibody Fab and FcR. The external force regulation method of the present invention can be compatible with existing commercial reagents. The detection antibody used in the antibody kit, the binding constant k on ≥10 10 M -1 s -1 with the target molecule, and the dissociation constant k off ≤0.0002 s -1 ; the dissociation equilibrium constant KD ≤ 0.2 fM; the binding and dissociation of various antibodies with a standard molecular binding free energy (standard free energy) ≤ -25 kcal / mol are finely adjusted.

[0094] The present invention will be further described below by way of examples, but the present invention is not limited to the scope of the described examples. The experimental methods without specific conditions noted in the following examples are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0095] Example 1:

[0096] Dynamic analysis detection is carried out using a surface plasmon resonance microscope (SPRM), in combination with an electrochemical workstation, and the electrochemical workstation provides an electric field force for the detection system. The method for dynamic analysis of nucleic acid detection with external force regulation for a target nucleic acid with a base complementary strand length of 25 base pairs between the target nucleic acid and the detection probe is as follows:

[0097] (1) Use a commercial total internal reflection microscope (Olympus IX-81) as the detection instrument, select an objective lens with a magnification of 60 times and a numerical aperture N.A = 1.49, the light source is an ultra-wideband light source SLED, the intensity of the light source control current is 140 mA, and the observation field of view is 512×512 pixels (full field of view: 33.28×33.28 μm 2 ). Use micromanager to control the CCD camera (Photometrics) to record the SPRM imaging signal. Cellsens software is used to control the incident angle of the optical path. The electrochemical workstation is CHI660e from Shanghai Chenhua, and the three-electrode method is adopted.

[0098] (2) On a BK-7 glass slide coated with 3 nm of chromium and 47 nm of gold, rinse it successively with absolute ethanol and pure water and then dry it. Then, treat it with a hydrogen flame to remove surface particle impurities and further improve the flatness of the gold film. After that, use epoxy resin glue to fix the self-made polydimethylsiloxane (PDMS) chamber (Dow Corning SYLGARD DC184, the A and B adhesives are mixed at a volume ratio of 10:1, evacuated and left standing in an oven at 37 °C for more than 2 h, and a detection chamber is made on the cured PDMS with a puncher with a diameter of 1 mm) on the gold surface and use conductive silver glue to fix the wire on the gold surface to form a working electrode. Subsequently, reduce the disulfide bond in the capture probe (thiol-modified single-stranded nucleic acid) with a 0.1 M dithiothreitol (DTT) solution (purchased from Shanghai Sangon, product number A100281) for 1 h, and then perform desalting treatment with a Sephadex gel column (such as NAP-25) to remove DTT. Immediately afterwards, add a solution containing 1 μM of thiol polyethylene glycol to the detection chamber formed by the PDMS and the gold chip and incubate for 30 s to passivate the gold surface. After washing three times with 1×PBS, add a solution containing a capture probe (complementary length to the target nucleic acid is 25 bp) with a concentration of 50 nM and incubate for 2 h. After the incubation is completed, remove the solution, wash three times with 1×PBS solution, and then incubate with 1 μM of thiol polyethylene glycol for 30 min to reduce non-specific adsorption; finally, wash with 1×PBS to obtain the detection chip.

[0099] (3) Prepare nucleic acid-coated gold nanoparticles by the salt aging method. Take 10 12Gold nanoparticles at a concentration of NPs / mL were co-incubated with 1 μM of thiol-functionalized detection probes (specifically binding to 25 bp at the other end of the target nucleic acid) in a 50 mM sodium chloride (NaCl) solution environment. The NaCl concentration was increased by 50 mM every 4 h until it reached 300 mM, and then the salt concentration was maintained unchanged and incubated on a shaker at room temperature for 24 h. Finally, the gold nanoparticles coated with the detection nucleic acid molecules were collected by centrifugation and resuspended. The thiol-modified detection probes were reduced with DTT solution for 1 h before use, and then desalted and the reducing solution was removed.

[0100] (4) Place the detection chip at the SPRM observation position and change the incident light angle. As the SPR effect occurs, the light intensity collected by the CCD camera drops sharply, and the light intensity reaches the lowest value near the SPR angle. The detection is carried out near the SPR angle. Add 10 μL of 1×PBS buffer to the detection chamber; then add 10 μL of gold particles with a particle concentration of 10 10 NPs / mL and a particle size of 50 nm. The surface of the particles has been linked with thiol-functionalized 25 bp detection probes and 1 μL of sample solution containing the target nucleic acid through the salt aging method.

[0101] (5) After completing the preparation work for the experiment, conduct the detection. First, apply a downward attractive force to the particles for 5 min to accelerate the diffusion of the target nucleic acid and the nanoparticles, and then apply an upward repulsive force to the particles for 10 min to adjust the binding time of the molecules. Collect and record SPRM data at a frame rate of 16.7 FPS. Then, perform differential processing (subtracting the previous frame from the next frame) on the data using the open-source software ImageJ to obtain single-molecule imaging information with background noise removed. By manually counting the information on the binding and dissociation of particles at the same position, the binding time of each molecule can be obtained. Further screen events with a binding lifetime that better conforms to specific binding for concentration-response fitting to obtain a better curve with a goodness of fit R 2 better curve.

[0102] Example 2:

[0103] In Example 1, a high-resolution surface plasmon resonance microscope (SPRM) was used as a single-molecule imaging platform for single-molecule dynamic detection based on external force regulation. It has extremely high sensitivity to particle size and can observe and distinguish nanoparticles with a particle size of 30 - 160 nm. However, in the single-molecule dynamic detection based on the sandwich structure of the present invention, it is not necessary to use particles with a very small particle size. Therefore, the SPRM with a small field of view cannot well meet the actual application. Therefore, the inventor further optimized the detection system and used a surface plasmon resonance imaging platform (SPRi) with a large field of view for dynamic analysis and detection, and an electrochemical workstation was connected to provide an electric field force for the detection system by the electrochemical workstation. The method for dynamic analysis of nucleic acid detection by external force regulation of the target molecule (the target molecule in this example is nucleic acid, so it is also called target nucleic acid) and the base complementary strand of the detection probe with a length of 25 base pairs (hereinafter referred to as bp, the complementary strand length can be 12 - 50 bp or longer) is as follows:

[0104] (1) A single-molecule dynamic detection platform under external force regulation rebuilt based on the surface plasmon resonance imaging (SPRi) of the Kretschmann prism coupling structure on an optical platform was used as the detection instrument. All the optical devices used in the optical path were purchased from Thorlabs and Daheng Optoelectronics, and the air-bearing optical platform was from LBTEK. A variable lens group with a magnification of 2 - 12 times (preferably used to observe gold particles with a size of 50 nm - 5 μm) and a numerical aperture N.A = 0.06 - 0.40 was selected. The light source was a super-wideband light source SLED (Qphotonics) from Superlum. The intensity of the light source control current was 140 mA and the power was 2 mW. The observation field of view was 2048×2048 pixels (field of view: 1.33×1.33 mm 2 ). The CCD camera (Photometrics Prime) was controlled by micro manager to record the imaging signal of SPRi. The electrochemical workstation was CHI660e from Shanghai Chenhua, and the three-electrode method was used.

[0105] (2) On a BK-7 glass slide coated with 3 nm of chromium and 47 nm of gold (the BK-7 glass slide was purchased from Thermo Fisher Scientific and the coating was subsequently completed at the Suzhou Institute of Nano-Tech and Nano-Bionics, Chinese Academy of Sciences through a magnetron sputtering process). Preferably, first, the glass slide was rinsed and dried with a piranha solution prepared by mixing concentrated sulfuric acid (Sinopharm-Hushi, 53100368) and 30% hydrogen peroxide (Sinopharm-Hushi, 80070961) in a volume ratio of 7:3 (Preparation method: Measure 70 mL of concentrated sulfuric acid with a graduated cylinder and pour it into a beaker. Then measure 30 mL of hydrogen peroxide with a graduated cylinder and slowly pour it into the beaker containing concentrated sulfuric acid while stirring with a glass rod. The piranha solution can effectively clean the surface of the glass slide and is beneficial for subsequent probe modification). Then, it was treated with a hydrogen flame to remove surface particulate impurities and further improve the flatness of the gold film. After that, a self-made polydimethylsiloxane (PDMS) chamber (Dow Corning SYLGARD DC184, the A and B adhesives were mixed in a volume ratio of 10:1, evacuated and left standing in an oven at 37 °C for more than 2 h, and a detection chamber was fabricated on the cured PDMS with a puncher with a diameter of 1 mm) was fixed on the gold surface with epoxy resin glue and a wire was fixed on the gold surface with conductive silver glue to form a working electrode. Subsequently, the disulfide bonds in the capture probe were reduced with a 5 μM solution of TCEP (tricarboxyethylphosphine) for 1 h. Immediately afterwards, 1 μM mercapto-polyethylene glycol was added to the detection chamber formed by the PDMS and the gold chip and incubated for 30 s to passivate the gold surface. After washing three times with 1×PBS, a solution containing a capture probe with a concentration of 50 nM (a locked nucleic acid single strand complementary to the target nucleic acid with a length of 25 bp, and the locked nucleic acid accounts for 10-50% of the overall strand segment, preferably 20%) was added and incubated for 2 h. After the incubation was completed, the solution was removed, washed three times with 1×PBS solution, and then incubated with 1 μM mercapto-polyethylene glycol for 30 min to reduce non-specific adsorption; finally, after washing with 1×PBS, the detection chip was obtained.

[0106] (3) Prepare gold nanoparticles coated with nucleic acid probes. Preferably, use the biotin-streptavidin system to replace the Au-S bond in the classical salt aging method, making the modification process more simple and stable. Mix 10 12 NPs / mL of streptavidin-coated gold nanoparticles (Nanopartz, C11-150-TS-PBS-50-1, particle size 150 nm) with 1 μM of thiol-functionalized detection probe (synthesized by Shanghai Sangon Biotech, a single-stranded nucleic acid ssDNA modified with biotin at one end, which can complementarily pair and bind with the target nucleic acid, and the pairing length is 25 bp) in an environment of 50 mM sodium chloride (NaCl) solution. During this period, the concentration was increased by 50 mM every 4 h until the sodium chloride concentration reached 500 mM, and then the salt concentration was maintained unchanged and incubated on a shaker at room temperature for 24 h. Finally, the gold nanoparticles coated with the detection probe were collected by centrifugation and resuspended. Through the research of the present invention, it was found that due to the coating of the nucleic acid strand, the originally salt-intolerant gold nanoparticles can still maintain stability in a 500 mM - 1 M NaCl buffer solution.

[0107] (4) Place the detection chip at the observation position on the prism, and drop a refractive index matching liquid (Olympus, F30CC, refractive index n = 1.518) between the two. Change the incident light angle to 69 - 72 degrees (preferably 71 degrees), and the SPR effect can be generated. As the SPR effect enhances, the light intensity collected by the CCD camera drops sharply, and the light intensity reaches the lowest value near the SPR angle. The detection is carried out near the SPR angle. First, inject a sodium citrate buffer solution containing 150 mM NaCl, that is, 1×SSC buffer solution, into the detection chamber through the fluid channel, and then slowly introduce a sample solution mixed with nanoparticles and target molecules. Among them, the particle concentration is 10 10 NPs / mL, and the flow rate is 5 μL / min. At the same time, insert the counter electrode and the reference electrode into the detection sample to form a circuit for the electrochemical workstation to apply an electric field force.

[0108] (5) Sample detection. Preferably, first apply a downward attractive force of 15 min (positive electric field, preferably +0.4 V) to the negatively charged gold nanoparticles to accelerate the diffusion of the target nucleic acid and nanoparticles, and then apply an upward repulsive force to the particles for 45 min (negative electric field, preferably -0.2 V) to adjust the binding time of the target molecule and the probe, and collect and record SPRi data at a frame rate of 16.7 FPS.

[0109] (6) Process the collected images, including performing differential processing (subtracting the previous frame from the next frame) on the images through the open-source software imageJ to obtain single-molecule imaging information with background noise removed; by manually counting the information of particle binding and dissociation at the same position, the binding time of each molecule can be obtained, and further screen events with a binding lifetime more in line with specific binding. According to the different times of specific adsorption and non-specific adsorption of the particles, the two can be distinguished for analysis.

[0110] (7) Cumulatively count the binding and dissociation events of specific adsorption to obtain the dynamic analysis results. The results of the total number of binding and dissociation events and the binding time (this binding time is the estimated value obtained by performing single-exponential fitting on the overall binding time distribution, fitted by the origin-exponential fitting-Exp Go1 model, and the iterative algorithm is orthogonal distance regression) changing with the applied voltage prove the effect of external force regulation ( Figure 4 a and b).

[0111] Example 3:

[0112] MicroRNA miRNA has broad application prospects as a biomarker for early cancer screening. In this example, hsa-miR-29a (miR-29a) is selected as the target nucleic acid, and the base complementary strand length with the detection probe is 12bp. The method for rapid detection of nucleic acid by external force-regulated dynamic analysis is as follows:

[0113] (1) A single-molecule dynamic detection platform under external force regulation, which is rebuilt from surface plasmon resonance imaging SPRi based on the Kretschmann prism coupling structure on an optical platform, is used as the detection instrument. All the optical devices used in the optical path are purchased from Thorlabs and Daheng Optoelectronics, and the air-bearing optical platform is from LBTEK (LBTEK). A lens with a magnification of 60 times and a numerical aperture N.A = 1.49 is selected. The light source is the ultra-wideband light source SLED (Qphotonics) of Superlum. The intensity of the light source control current is 140 mA, the power is 2 mW, and the observation field of view is 2048×2048 pixels (field of view: 221.2×221.2 μm 2 ). The micro manager is used to control the CCD camera (Photometrics Prime) to record the imaging signal of SPRi. The electrochemical workstation is CHI660e of Shanghai Chenhua, and the three-electrode method is adopted.

[0114] (2) On a BK-7 glass slide coated with 3 nm of chromium and 47 nm of gold (the BK-7 glass slide was purchased from Thermo Fisher Scientific and then coated with a magnetron sputtering process at the Suzhou Institute of Nano-Tech and Nano-Bionics, Chinese Academy of Sciences), first rinse and dry with a piranha solution prepared by mixing concentrated sulfuric acid (Sinopharm - Shanghai Reagent, 53100368) and 30% hydrogen peroxide (Sinopharm - Shanghai Reagent, 80070961) in a volume ratio of 7:3. Then, treat with a hydrogen flame to remove surface particulate impurities and further improve the flatness of the gold film. After that, use epoxy resin glue to fix the self-made polydimethylsiloxane (PDMS) chamber (Dow Corning, the A and B adhesives are mixed in a volume ratio of 10:1, evacuated and then left to stand in an oven at 37 °C for more than 2 h, and a detection chamber is fabricated on the cured PDMS with a puncher with a diameter of 0.2 mm) on the gold film surface and use conductive silver glue to fix the wire on the gold surface to form a working electrode. Subsequently, reduce the disulfide bond in the capture probe with a 1 μM - 50 μM TCEP (tris(2-carboxyethyl)phosphine) solution for 1 h (it was found through screening that the amount of TCEP needs to be 100 times or more of the amount of DNA. In this example, the preferred concentration is 5 μM). Immediately afterwards, add a 1 μM mercapto-PEG solution to the detection chamber formed by the PDMS and the gold film and incubate for 30 s to passivate the gold surface. After washing three times with 1×PBS, add a solution containing a capture probe with a concentration of 10 - 500 nM (preferably 50 nM) (a locked nucleic acid single strand complementary to the target miRNA with a length of 12 bp, inserting 10 - 50% of the locked nucleic acid molecules into the whole segment, and the preferred locked nucleic acid ratio is 20%) and incubate for 6 h. After the incubation is completed, remove the solution, wash three times with 1×PBS solution, and then incubate with 1 μM mercapto-PEG for 30 min to reduce non-specific adsorption; finally, wash with 1×PBS to obtain the detection chip.

[0115] (3) Prepare nucleic acid-coated gold nanoparticles using the biotin-streptavidin system (BAS). Incubate 10 12 NPs / mL streptavidin-coated gold nanoparticles (Nanopartz, C11-50-TS-PBS-50-1, particle size 50 nm) with 1 μM biotin-functionalized detection molecule (single-stranded nucleic acid ssDNA) in TE buffer for 30 min, and finally centrifuge and resuspend to collect the gold nanoparticles coated with the detection probe.

[0116] (4) Place the detection chip at the observation position on the prism, and drop a refractive index matching liquid (Olympus, F30CC, refractive index n = 1.518) between the two. Adjust the incident light angle to 71 degrees to generate the SPR effect. As the SPR effect enhances, the light intensity collected by the CCD camera drops sharply, and the light intensity reaches the lowest value near the SPR angle. The detection is carried out near the SPR angle. First, inject a sodium citrate buffer solution containing 300 mM NaCl, i.e., 2×SSC buffer solution, into the detection chamber through the fluid channel, and then slowly introduce a sample solution mixed with nanoparticles and target molecules. Among them, the particle concentration is 10 10 NPs / mL, and the flow rate is 5 μL / min. At the same time, insert the counter electrode and reference electrode into the detection sample to form a circuit for the electrochemical workstation to apply an electric field force.

[0117] (5) Sample detection. Preferably, first apply a downward attractive force to the negatively charged gold nanoparticles for 5 min (positive electric field, +0.4 V) to accelerate the diffusion of the target nucleic acid and nanoparticles, and then apply an upward repulsive force to the particles for 15 min (negative electric field, -0.2 V) to adjust the binding time of the target molecule and the probe, and collect and record SPRi data at a frame rate of 16.7 FPS.

[0118] (6) Process the collected images. Perform differential processing (subtracting the previous frame from the next frame) on the images through the open-source software imageJ to obtain single-molecule imaging information with background noise removed; manually count the binding and dissociation events of the particles at the same position, and thus obtain the binding lifetime of each molecule; finally, screen out specific binding events according to the binding time. According to the different adsorption times of specific adsorption and non-specific adsorption of the particles, the two can be distinguished for analysis.

[0119] (7) Cumulatively count the binding and dissociation events of specific adsorption to obtain the dynamic analysis result. The binding time distribution map of miR-29a can be divided into specific adsorption and non-specific adsorption, and can be obtained by further optimizing the parameters. In the time window of 25 - 250 s, the concentration standard curve has a good linear fitting goodness R 2 = 0.98( Figure 5 of a), Figure 5 The dotted line in b represents the particle events detected in the blank group. The detection limit LoD of the sample is obtained through the intersection of the dotted line and the concentration response curve.

[0120] Example 4:

[0121] To prove that the present invention has the ability to rapidly and ultrasensitively detect nucleic acids, two cancer-related miRNAs, hsa-miR-21 (miR-21) and hsa-miR-155 (miR-155), were selected as target nucleic acids in this example, and the base complementary strand lengths with the detection probes were both 12 bp. The specific method is as follows:

[0122] (1) A single-molecule dynamic detection platform under external force regulation, which is reconstructed from a surface plasmon resonance imaging SPRi based on a Kretschmann prism coupling structure on an optical platform, was used as the detection instrument. All the optical devices used in the optical path were purchased from Thorlabs and Daheng Optoelectronics, and the air-bearing optical platform was from LBTEK (LBTEK). A lens with a magnification of 40 times and a numerical aperture N.A = 0.65 was selected. The light source was a super-wideband light source SLED (Qphotonics) from Superlum. The intensity of the light source control current was 140 mA, the power was 2 mW, and the observation field of view was 2048×2048 pixels (field of view: 1106×1106 μm 2 ). The micro manager was used to control the CCD camera (Photometrics Prime) to record the imaging signal of SPRi. The electrochemical workstation was CHI660e from Shanghai Chenhua, and the three-electrode method was adopted.

[0123] (2) On a BK-7 glass slide coated with 3 nm of chromium and 47 nm of gold (the BK-7 glass slide was purchased from Thermo Fisher Scientific and then coated with a magnetron sputtering process at the Suzhou Institute of Nano-Tech and Nano-Bionics, Chinese Academy of Sciences), first rinse and dry with a piranha solution prepared by mixing concentrated sulfuric acid (Sinopharm - Shanghai Reagent, 53100368) and 30% hydrogen peroxide (Sinopharm - Shanghai Reagent, 80070961) in a volume ratio of 7:3. Then, treat with a hydrogen flame to remove surface particulate impurities and further improve the flatness of the gold film. After that, fix a reusable silicon-based cell culture plate (SARSTEDT, flexiPERM) on the surface of the gold film, and at the same time, fix the working electrode on the surface of the gold film with solder. Subsequently, reduce the disulfide bond in the capture probe with a 1 μM - 50 μM TCEP (tris(2-carboxyethyl)phosphine) solution for 1 h (the amount of TCEP needs to be 100 times or more the amount of DNA, and the preferred concentration is 5 μM). Immediately afterwards, add a solution containing 1 μM mercapto-polyethylene glycol to the detection chamber formed by the silicon-based culture plate and the gold film and incubate for 30 s to passivate the gold surface. After washing three times with 1×PBS, add a solution containing a capture probe with a concentration of 10 - 500 nM (preferably 50 nM) (a locked nucleic acid single strand complementary to the target miRNA with a length of 12 bp, inserting 10 - 50% of the locked nucleic acid molecules into the whole segment, and the preferred locked nucleic acid ratio is 20%), and incubate for 6 h. After incubation, remove the solution, wash three times with 1×PBS solution, and then incubate with 1 μM mercapto-polyethylene glycol for 30 min to reduce non-specific adsorption; finally, wash with 1×PBS to obtain the detection chip.

[0124] (3) Prepare nucleic acid-coated gold nanoparticles using the biotin-streptavidin system (BAS). Incubate 10 12 NPs / mL streptavidin-coated gold nanoparticles (Nanopartz, C11-50-TS-PBS-50-1, particle size 50 nm) with 1 μM biotin-functionalized detection molecule (single-stranded nucleic acid ssDNA) in TE buffer for 30 min, and finally centrifuge and resuspend to collect the gold nanoparticles coated with the detection probe.

[0125] (4) Place the detection chip at the observation position on the prism, drop a refractive index matching liquid (Olympus, F30CC, refractive index n = 1.518) between the two, and adjust the incident light angle to 71 degrees to generate the SPR effect. As the SPR effect increases, the light intensity collected by the CCD camera drops sharply, and the light intensity reaches the lowest value near the SPR angle. The detection is carried out near the SPR angle. First, inject a sodium citrate buffer solution containing 300 mM of NaCl, i.e., 2×SSC buffer solution, into the detection chamber through the fluid channel, and then slowly introduce a sample solution mixed with nanoparticles and target molecules. Among them, the particle concentration is 10 10NPs / mL, with a flow rate of 5 μL / min. Meanwhile, insert the counter electrode and reference electrode into the detection sample to form a circuit for the electrochemical workstation to apply an electric field force.

[0126] (5) Sample detection. Preferably, first apply a downward attractive force on the negatively charged gold nanoparticles for 5 min (positive electric field, +0.4 V) to accelerate the diffusion of the target nucleic acid and nanoparticles, and then apply an upward repulsive force on the particles for 15 min (negative electric field, -0.2 V) to adjust the binding time of the target molecule and the probe, and collect and record SPRi data at a frame rate of 30 FPS.

[0127] (6) Process the collected images. Perform differential processing (subtracting the previous frame from the latter frame) on the images through the open-source software ImageJ to obtain single-molecule imaging information with background noise removed; manually count the binding and dissociation events of the particles at the same position, thereby obtaining the binding lifetime of each molecule; finally, screen out the specifically binding events according to the binding time. According to the different times of specific adsorption and non-specific adsorption of the particles, the two can be distinguished for analysis.

[0128] (7) By counting the binding and dissociation events within the specifically binding time, the binding and dissociation event curves of the two miRNAs with respect to concentration changes can be obtained, a standard curve can be fitted, and based on the mean + 3 std (n = 3) of the binding and dissociation events of the blank control group as the baseline, the detection limits (limit of Detection, LoD) of the two target nucleic acids can be calculated. The detection limits of miR-21 and miR-155 are 0.26 and 0.17 fM ( Figure 6 a and b), respectively, where the R2 of miR-21 is 0.96 and the R2 of miR-155 is 0.83.

[0129] Example 5:

[0130] Use the surface plasmon resonance imaging system SPRi to perform high-throughput dynamic analysis detection based on external force regulation, in combination with an electrochemical workstation, and use the electrochemical workstation to provide an electric field force for the detection system. Perform single-molecule detection on the Alzheimer's biomarker Aβ 1-42 :

[0131] (1) The single-molecule dynamic detection platform under external force regulation, which is rebuilt from surface plasmon resonance imaging SPRi based on the Kretschmann prism coupling structure on an optical platform, is used as the detection instrument. All the optical devices used in the optical path are purchased from Thorlabs and Daheng Optoelectronics, and the air-bearing optical platform is from LBTEK. A lens with a magnification of 10 times and a numerical aperture N.A = 0.25 is selected. The light source is the super-wideband light source SLED (Qphotonics) of Superlum. The intensity of the light source control current is 140 mA, the power is 2 mW, and the observation field of view is 2048×2048 pixels (field of view: 1.33×1.33 mm 2 ). The micro manager is used to control the CCD camera (Photometrics Prime) to record the imaging signal of SPRi. The electrochemical workstation is CHI660e of Shanghai Chenhua, and the three-electrode method is adopted.

[0132] (2) On a BK-7 glass slide coated with 3 nm of chromium and 47 nm of gold (the BK-7 glass slide is purchased from Thermo Fisher Scientific and the subsequent coating is completed by magnetron sputtering process at the Suzhou Institute of Nano-tech and Nano-bionics, Chinese Academy of Sciences), first, it is rinsed and dried with the piranha solution mixed by concentrated sulfuric acid (Guoyao-Hushi, 53100368) and 30% hydrogen peroxide (Guoyao-Hushi, 80070961) in a volume ratio of 7:3. Then it is treated with a hydrogen flame to remove surface particle impurities and further improve the flatness of the gold film. After that, the reusable silicon-based cell culture plate (SARSTEDT, flexiPERM) is fixed on the gold surface, and at the same time, the working electrode is fixed on the gold surface with solder. Subsequently, the disulfide bond in the capture probe is reduced with a 1 μM - 50 μM TCEP (tris(2-carboxyethyl)phosphine) solution for 1 h (in the present invention, the amount of substance of TCEP needs to be 100 times or more of the amount of substance of DNA, and the preferred concentration in this example is 5 μM). Immediately afterwards, a mixture of thiol-terminated methoxypolyethylene glycol (HS-mPEG, Mw = 500 Da) and thiol-terminated polyethylene glycol biotin (HS-PEG5K-biotin, Mw = 3400 - 15000 Da, preferably 5000, 7000 Da) with a concentration ratio of 50:1 - 500:1 (preferably 100:1) is added to the detection chamber formed by the silicon-based culture plate and the gold sheet and incubated for 4 h to modify the gold surface. Then a streptavidin solution with a concentration of 1 mg / mL is added and incubated for 1 h. Then a capture antibody with biotin modified at the Fc end (polyclonal antibody clone 12F4, specifically binding to Aβ 1-42Incubate with the solution of the C-terminus, BioLegend, Inc.) at a concentration of 5 - 50 ng / mL (preferably 10 ng / mL) for 2 h. Finally, block with 1% BSA for 30 min to reduce non-specific adsorption. Among them, before each of the above steps, first remove the stock solution of the previous step and wash three times with 1×PBS solution.

[0133] (3) Prepare gold nanoparticles coated with the detection antibody. Mix 10 12 NPs / mL of streptavidin-coated gold nanoparticles (Nanopartz, C11-50-TS-PBS-50-1, particle size 50 nm) with 500 ng / mL - 5 μg / mL (preferably 2 μg / mL) of the Fc-terminus biotinylated detection antibody (polyclonal antibody clone 6E10, specifically binding to the N-terminus of Aβ 1-42 , BioLegend, Inc.) and incubate for 30 min. Finally, centrifuge and resuspend to collect the gold nanoparticles coated with the detection antibody.

[0134] (4) Place the detection chip on the prism at the observation position, drop the refractive index matching solution (Olympus, F30CC, refractive index n = 1.518) between the two, adjust the incident light angle to 71 degrees, and the SPR effect can be generated. As the SPR effect increases, the light intensity collected by the CCD camera drops sharply, and the light intensity reaches the lowest value near the SPR angle. The detection is carried out near the SPR angle. First, inject the Tris-HCL buffer solution containing 300 mM NaCl into the detection chamber through the fluid channel, and then slowly introduce the sample solution mixed with nanoparticles and the target protein. Among them, the particle concentration is 10 10 NPs / mL, and the flow rate is 5 - 15 μL / min, preferably 15 μL / min. At the same time, insert the counter electrode and the reference electrode into the detection sample solution to form a circuit, and apply an electric field force through the electrochemical workstation.

[0135] (5) Sample detection. Preferably, first apply a downward attractive force (positive electric field, +0.4 V) to the negatively charged gold nanoparticles for 5 min to accelerate the diffusion of the target protein and the nanoparticles, and then apply an upward repulsive force (negative electric field, -0.2 V) to the particles for 15 min to adjust the binding time of the target protein and the detection probe, and collect and record the SPRi data at a frame rate of 15 - 60 FPS (preferably 16.7 FPS).

[0136] (6) Process the collected images. Use the open-source software ImageJ to perform differential processing on the images (subtract the previous frame from the subsequent frame) to obtain single-molecule imaging information with background noise removed. Manually count the binding and dissociation events of particles at the same position, and thus obtain the binding lifetime of each molecule. Finally, screen out specific binding events according to the binding time. According to the different times of specific adsorption and non-specific adsorption of particles, the two can be distinguished for analysis.

[0137] (7) Cumulatively count the binding and dissociation events of specific adsorption to obtain the dynamic analysis results ( Figure 7 a). The detection limit of the target polypeptide under electric field regulation is 4.34 fg / mL (~1.08 fM). It can be seen that the regulation range of the electric field force is small, and it is difficult to achieve satisfactory regulation of the binding of antigen-antibody with strong affinity. Subsequently, a larger external force is selected to regulate the particles.

[0138] Example 6:

[0139] Based on Example 5, in this example, the electric field regulation is changed to magnetic field regulation, and a larger external force is used to adjust the binding time of the high-affinity antibody and the target protein. The general dynamic analysis method for external force regulation is as follows:

[0140] (1) Use a single-molecule dynamic detection platform under external force regulation rebuilt from surface plasmon resonance imaging SPRi based on the Kretschmann prism coupling structure on an optical platform as the detection instrument. All the optical devices used in the optical path are purchased from Thorlabs and Daheng Optoelectronics, and the air-bearing optical platform comes from LBTEK. Select a lens with a magnification of 4 times and a numerical aperture N.A = 0.25. The light source is the ultra-wideband light source SLED (Qphotonics) of Superlum. The intensity of the light source control current is 140 mA, the power is 2 mW, and the observation field of view is 2048×2048 pixels (field of view: 3.325×3.325 mm 2 ). Use Micro Manager to control the CCD camera (Photometrics Prime) to record the imaging signal of SPRi. The magnetic force is provided by magnetic tweezers, and the magnetic tweezers are OP-2025 of Magnetech Corporation, which are fixed 1.5 cm above the chip through an optical bracket.

[0141] (2) On a BK-7 glass slide coated with 3 nm of chromium and 47 nm of gold (the BK-7 glass slide was purchased from Thermo Fisher Scientific and the coating was completed by magnetron sputtering process at the Suzhou Institute of Nano-Tech and Nano-Bionics, Chinese Academy of Sciences), first rinse and dry with a piranha solution prepared by mixing concentrated sulfuric acid (Sinopharm-Hushi, 53100368) and 30% hydrogen peroxide (Sinopharm-Hushi, 80070961) at a volume ratio of 7:3. Then treat with a hydrogen flame to remove surface particulate impurities and further improve the flatness of the gold film. After that, fix a reusable silicon-based cell culture plate (SARSTEDT, flexiPERM) on the surface of the gold film. Subsequently, reduce the disulfide bond in the capture probe with a 1 μM - 50 μM TCEP (tris(2-carboxyethyl)phosphine) solution for 1 h (the amount of TCEP needs to be 100 times or more of the amount of DNA, and the preferred concentration is 5 μM). Immediately add a mixture of HS-mPEG (Mw = 500 Da) and HS-PEG5K-biotin (Mw = 3400 - 15000 Da, preferably 5000, 7000 Da) at a concentration ratio of 50:1 - 500:1 (preferably 100:1) to the detection chamber formed by the silicon-based culture plate and the gold film and incubate for 4 h to modify the gold surface. Then add streptavidin at a concentration of 1 mg / mL and incubate for 1 h. Next, add a solution of the capture antibody with biotin modified at the Fc end (polyclonal antibody clone 12F4, specifically binding to the C-terminus of Aβ 1-42 , BioLegend, Inc.) at a concentration of 5 - 50 ng / mL (preferably 10 ng / mL) and incubate for 2 h. Finally, block with 1% BSA for 30 min to reduce non-specific adsorption. Note that before each step, the stock solution of the previous step should be removed first and the sample should be washed three times with 1×PBS solution.

[0142] (3) Prepare magnetic nanoparticles coated with the detection antibody. Incubate 10 12 NPs / mL of streptavidin-coated magnetic nanoparticles (Aladdin, S8040-A300nm-1EA, particle size 300 nm) with 500 ng / mL - 5 μg / mL (preferably 2 μg / mL) of the Fc-end biotin-functionalized detection antibody (polyclonal antibody clone 6E10, specifically binding to the N-terminus of Aβ 1-42 , BioLegend, Inc.) for 30 min. Finally, centrifuge and resuspend to collect the magnetic nanoparticles coated with the detection antibody.

[0143] (4) Place the detection chip at the observation position on the prism, and drop a refractive index matching liquid (Olympus, F30CC, refractive index n = 1.518) between the two. Adjust the incident light angle to 71 degrees to generate the SPR effect. As the SPR effect enhances, the light intensity collected by the CCD camera drops sharply, and the light intensity reaches the lowest value near the SPR angle. The detection is carried out near the SPR angle. First, inject a Tris-HCL buffer solution containing 300 mM NaCl into the detection chamber through the fluid channel, and then slowly introduce a sample solution mixed with nanoparticles and target proteins. Among them, the particle concentration is 10 10 NPs / mL, and the flow rate is 5 - 15 μL / min, preferably 15 μL / min.

[0144] (5) For sample detection, preferably use a 12vDC power supply with a duty cycle of 25% and a duration of 20 s to drive the magnetic tweezers. First, apply a downward attractive force to the magnetic nanoparticles for 5 min (the magnetic tweezers are 1.5 cm below the chip) to accelerate the diffusion of the target protein and nanoparticles. Then, apply an upward repulsive force to the particles for 15 min (the magnetic tweezers are 1.5 cm above the chip) to adjust the binding time of the target protein and the detection probe, and collect and record SPRi data at a frame rate of 15 - 60 FPS (preferably 16.7 FPS).

[0145] (6) Process the collected images. Perform differential processing (subtracting the previous frame from the next frame) on the images through the open-source software imageJ to obtain single-molecule imaging information with background noise removed; manually count the binding and dissociation events of the particles at the same position, and thus obtain the binding lifetime of each molecule; finally, screen out specific binding events according to the binding time. According to the different times of specific adsorption and non-specific adsorption of the particles, the two can be distinguished and analyzed.

[0146] It is found that under magnetic field regulation, due to the greater external force applied, the binding time of molecules can be shorter, which is beneficial to signal amplification for dynamic analysis. Correspondingly, cumulative statistics are performed on the binding and dissociation events of specific adsorption to obtain the dynamic analysis result, proving that appropriate external force regulation can obtain a more sensitive detection limit of 0.21 fg / mL (~0.05 fM) ( Figure 7 of b).

[0147] In the present invention:

[0148] 1. For a single-molecule imaging system, in addition to SPRi in the technical solution, any single-molecule imaging technology can be an alternative, such as total internal reflection fluorescence microscopy TIRFM, dark-field imaging, interference scattering imaging iSCAT, etc.

[0149] 2. For the module regulated by external forces, except for the electrochemical workstation for electric field regulation and the nickel-chromium magnet for magnetic force regulation, numerous non-contact force generating devices can meet the requirements, such as magnetic tweezers, optical tweezers, acoustic tweezers, thermophoresis, and atomic force microscope AFM.

[0150] 3. The nanoparticles serving as reporter molecules and manipulation molecules are nanoparticles or microspheres with a particle size ranging from 30 nm to 5 μm, and the materials include but are not limited to silica (silica spheres), gold (gold particles), silver (silver particles), and polystyrene PS, as well as composite materials such as silica magnetic beads coated with superparamagnetic particles, etc.; their physical and chemical properties are suitable for being regulated by external forces such as electric field force and magnetic field force.

[0151] 4. The capture probes and detection probes, in addition to the antibodies in the technical solution, also include single-stranded nucleic acids, aptamers, engineered antibodies, nucleic acids, aptamers, and any chemical substances, metabolites, etc. that can bind to target molecules.

[0152] 5. For the SPRi system, in addition to the SLED light source, a laser light source with a suitable wavelength band can also be used. For example, a laser light source of 600 - 800 nm is used for SPR excitation of a 50-nm gold film. Or a silver film or a silicon nitride / silicon oxide structure is used to achieve SPR. The structure of SPRi can be an objective lens coupling type or a prism coupling (Kretschmann structure).

[0153] 6. For magnetic field regulation, an electromagnet or a permanent magnet can be used; for electric field regulation, in addition to the electrochemical workstation in the technical solution, any two-electrode or three-electrode electric field generating device can be used instead.

[0154] The above embodiments merely illustrate the principles and effects of the present invention by way of example, and are not intended to limit the present invention. Any person familiar with this technology can modify or change the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or changes made by those with ordinary knowledge in the technical field without departing from the spirit and technical idea disclosed by the present invention should still be covered by the present invention.

Claims

1. A single-molecule dynamic detection method, characterized in that, It uses an external force regulation module to regulate the binding and dissociation between the target molecule and the "detection probe, capture probe, and nanoparticle", and uses a single-molecule imaging system to obtain single-molecule imaging information and analyze the dynamics of the target molecule; Among them, the end of the capture probe is complementary to the head of the target molecule, and the end of the target molecule is complementary to the detection probe; the capture probe or the detection probe forms a nanoparticle complex with the nanoparticle; when the nanoparticle complex binds to the target molecule, it is pulled near the detection probe or the capture probe under the drive of the external force regulation module and specifically binds to the capture probe or the detection probe, and then is lifted away from the detection probe or the capture probe under the drive of the external force regulation module; among them, the external force regulation module includes electric field regulation, magnetic force regulation, and non-contact force generating devices.

2. The single-molecule dynamic detection method according to claim 1, characterized in that, The electric field regulation uses a two-electrode or three-electrode electric field generating device, and the material of the working electrode is selected from glassy carbon electrode, platinum, gold, silver, lead, conductive glass, and mercury; the magnetic force regulation uses an electromagnet or a permanent magnet; the non-contact force generating device includes optical tweezers, acoustic tweezers, thermophoresis, and atomic force microscope AFM; and / or, The external force applied by the external force regulation module ranges from 0.01 - 1000 pN.

3. The single-molecule dynamic detection method according to claim 2, wherein The electric field regulation uses an electrochemical workstation.

4. The single-molecule dynamic detection method according to claim 2, wherein The magnetic force regulation uses a nickel-chromium magnet or a magnetic tweezer.

5. The single-molecule dynamic detection method according to claim 2, wherein The external force applied by the external force regulation module ranges from 20 - 60 pN.

6. The single-molecule dynamic detection method according to claim 1, wherein The target molecule includes proteins and nucleic acids; and / or, the capture probe and the detection probe include any chemical substance that binds to the target molecule.

7. The single-molecule dynamic detection method according to claim 1, wherein The capture probe and the detection probe include any metabolite that binds to the target molecule.

8. The single-molecule dynamic detection method according to claim 1, characterized in that, The capture probe and the detection probe include natural or engineered antibodies, nucleic acids, and aptamers.

9. The single-molecule dynamic detection method according to claim 1, wherein The single-molecule imaging system includes SPRi, TIRFM, dark-field imaging, and iSCAT; and / or, the particle size of the nanoparticle is 30 nm - 5 μm, and the material includes nanosilicon, nanogold, nanosilver, polystyrene, and silica magnetic beads coated with superparamagnetic particles.

10. The single-molecule dynamic detection method according to claim 9, characterized in that The structure of the SPRi includes an objective-coupled SPRi and a prism-coupled SPRi; and / or, the light source of the SPRi includes an SLED light source or a laser light source of 600 - 800 nm; and / or, the nanoparticle includes nanogold or magnetic nanoparticles.

11. The single-molecule dynamic detection method according to claim 10, wherein The objective-coupled SPRi is an objective-type SPRM.

12. The single-molecule dynamic detection method according to claim 11, wherein, The objective-coupled SPRi is an objective-type SPRM rebuilt from a total internal reflection microscope.

13. The single-molecule dynamic detection method according to claim 10, wherein The structure of the SPRi is a prism-coupled SPRi; and / or, the particle size of the nanoparticle is 50 nm - 300 nm.

14. The single-molecule dynamic detection method according to claim 13, wherein The particle size of the nanoparticle is 50 nm, 150 nm, or 300 nm.

15. The single-molecule dynamic detection method according to claim 10, characterized in that, The prism-coupled SPRi is a Kretschmann prism-coupled structure.

16. The single-molecule dynamic detection method according to any one of claims 1 to 15, characterized in that The capture probe or the detection probe is adsorbed on the detection chip.

17. The single-molecule dynamic detection method according to claim 16, wherein When the target molecule is nucleic acid: 12 - 25 nucleotides at the end of the capture probe are complementary to the nucleotides at one end of the target molecule, and the nucleotides at the other end of the target molecule are complementary to the detection probe; or, one of the capture probe and the detection probe is a locked nucleic acid, and the other end is a single-stranded nucleic acid ssDNA modified with biotin, and the nanoparticle is a nanoparticle coated with streptavidin; When the target molecule is a protein or a small molecule: the detection probe is an antibody that binds to the target molecule, and the capture probe is an antibody that binds firmly to the target molecule.

18. The single-molecule dynamic detection method according to claim 17, wherein, When the target molecule is a protein or a small molecule, the detection probe is an antibody against the target molecule with biotin modified at the Fc end, and the capture probe is an antibody with biotin-functionalized Fc end.

19. The single-molecule dynamic detection method according to claim 18, wherein When the target molecule is a protein or a small molecule, the binding constant between the capture probe and the target molecule k on ≥10 10 M -1 s -1 , and the dissociation constant k off ≤0.0002 s -1 , and the dissociation equilibrium constant KD ≤ 0.2 fM; the binding constant k between the detection probe and the target molecule on ≤5×10 8 M -1 s -1 , and the dissociation constant k off ≥0.05 s -1 ; the dissociation equilibrium constant KD ≥ 1.56 pM.

20. The single-molecule dynamic detection method according to claim 16, wherein, (1) The preparation of the detection chip comprises the following steps: On a glass slide coated with 2 - 3 nm of chromium and 47 nm of gold, first remove surface particulate impurities and improve the flatness of the gold film, then fix a detection chamber or a silicon-based cell culture plate on the surface of the gold film to form a working electrode; thereafter, reduce the thiol-modified detection probe or capture probe with a reducing solution, desalt and remove the reducing solution, add a passivating agent to passivate the surface of the gold film, wash and then add the reduced detection probe or capture probe and incubate; finally, wash and add a solution to reduce non-specific adsorption and wash, thus obtaining the detection chip; and / or, (2) The preparation of the nanoparticle complex comprises the following steps: Prepare nucleic acid-coated gold nanoparticles by the salt aging method, that is, co-incubate gold nanoparticles with the capture probe or detection probe after thiol reduction, centrifuge and resuspend to obtain; or, Prepare nucleic acid-coated nanoparticles by the biotin-avidin system, that is, co-incubate gold nanoparticles coated with streptavidin with biotin-functionalized detection molecules, centrifuge and resuspend to obtain; or, Prepare antibody-coated nanoparticles by the biotin-avidin system, that is, co-incubate nanoparticles coated with streptavidin with detection antibodies with biotin-functionalized Fc ends, centrifuge and resuspend to obtain; There is no order of priority between (1) and (2).

21. The single-molecule dynamic detection method according to claim 20, wherein The detection chamber is a PDMS chamber.

22. The single-molecule dynamic detection method according to claim 20, wherein In the step of preparing nucleic acid-coated nanoparticles by the salt aging method, the co-incubation is carried out in a gradient sodium chloride solution.

23. The single-molecule dynamic detection method according to claim 22, wherein The concentration of the gradient sodium chloride solution is 50 - 300 mM.

24. The single-molecule dynamic detection method according to claim 23, wherein The concentration of the gradient sodium chloride solution is increased by 50 mM every 4 h until the sodium chloride concentration reaches 300 mM, and then the salt concentration is maintained unchanged for incubation for 24 h.

25. The single-molecule dynamic detection method according to claim 20, wherein The nucleic acid-coated nanoparticles prepared by the biotin-avidin system are gold nanoparticles.

26. The single-molecule dynamic detection method according to claim 20, wherein In the step of preparing nucleic acid-coated nanoparticles by the biotin-avidin system, the biotin-functionalized detection molecule is a single-stranded nucleic acid ssDNA with one end modified with biotin.

27. The single-molecule dynamic detection method according to claim 20, wherein In the step of preparing nucleic acid-coated nanoparticles by the biotin-avidin system, the co-incubation is carried out in a gradient sodium chloride solution.

28. The single-molecule dynamic detection method according to claim 27, wherein, The concentration of the gradient sodium chloride solution is 50 - 500 mM.

29. The single-molecule dynamic detection method according to claim 28, wherein, The gradient sodium chloride solution increases its concentration by 50 mM every 4 h until the sodium chloride concentration reaches 500 mM, and then the salt concentration is maintained unchanged for incubation for 24 h.

30. The single-molecule dynamic detection method according to claim 20, wherein The nanoparticles coated with antibodies prepared by the biotin-avidin system are gold nanoparticles and magnetic nanoparticles.

31. The single-molecule dynamic detection method according to claim 20, wherein It includes the following steps: (a) Place the detection chip at the observation position of the single-molecule imaging system, and add the nanoparticle complex and the target molecule into the detection chamber; (b) Use the external force regulation module to apply an external force, and collect the imaging data of single molecules at a frame rate of 15 - 60 FPS and analyze the dynamics of the target molecule: First, apply an attractive force to the nanoparticle complex to accelerate the diffusion of the target molecule and the nanoparticle complex; then, apply a repulsive force to the nanoparticle complex; adjust the binding time between the target molecule and the detection probe or capture probe, and the binding time is 5 - 500 s; (c) Collect and record SPRi data, perform differential processing on the data to obtain single-molecule imaging information with background noise removed; count the information of particle binding and dissociation at the same position to obtain the binding time of each molecule; Finally, screen out the events within the binding time dominated by specific binding, perform concentration response fitting, and obtain a concentration response curve with higher specificity.

32. The single-molecule dynamic detection method according to claim 31, characterized in that Before adding the nanoparticle complex and the target molecule, first add PBS buffer or SSC buffer or Tris-HCL buffer containing 300 mM NaCl.

33. The single-molecule dynamic detection method according to claim 32, wherein The SSC buffer is 1× or 2× SSC buffer.

34. The single-molecule dynamic detection method according to claim 31, wherein The frame rate for collecting the imaging data of single molecules and analyzing the dynamics of the target molecule is 16.7 or 30 FPS.

35. The single-molecule dynamic detection method according to claim 31, wherein The binding time is 25 - 250 s.

36. The single-molecule dynamic detection method according to claim 31, wherein The binding time is the estimated value obtained by performing single-exponential fitting on the overall binding time distribution, and is fitted by the origin-exponential fitting-Exp Go1 model, and the iterative algorithm is orthogonal distance regression.

37. The single-molecule dynamic detection method according to claim 31, wherein when the external force applied by the external force regulation module is an electric field force, apply -0.6v - +1.0v; when the external force applied by the external force regulation module is a magnetic field force, use a power supply of 12v DC, duty cycle 25%, and 20 s to drive the magnetic tweezers, first apply an attractive force to the nanoparticles for 15 min; then apply a repulsive force to the nanoparticles for 15 min.

38. The single-molecule dynamic detection method according to claim 37, wherein when the external force applied by the external force regulation module is an electric field force, apply a voltage of 0.4 V - +0.8 V.

39. The single-molecule dynamic detection method according to claim 37, characterized in that, when the external force applied by the external force regulation module is an electric field force, first use a positive electric field to apply an attractive force to the nanoparticles for 5 min - 20 min; then use a negative electric field to apply a repulsive force to the nanoparticles for 10 - 45 min.

40. The single-molecule dynamic detection method according to claim 39, wherein, The positive electric field is 0 - 0.8 V; the negative electric field is -0.4 - 0 V.

41. The single-molecule dynamic detection method according to claim 40, characterized in that, The positive electric field is +0.4 V, and the negative electric field is -0.2 V.

42. The single-molecule dynamic detection method according to claim 20, wherein, (1) The preparation of the detection chip has one or more of the following characteristics: the glass slide is a BK-4 or BK-7 glass slide; the surface particles and impurities are removed and the flatness of the gold film is improved by using a washing solution and a hydrogen flame, and the washing solution is a piranha solution or a combination of alcohol and pure water; the reducing solution is TCEP or DTT; the passivating agent is thiol polyethylene glycol, mercaptoethanol, or a combination of methoxy thiol polyethylene glycol and biotin thiol polyethylene glycol; PBS is used for washing; mercaptoethanol or BSA is used to reduce non-specific adsorption; the ratio of the capture probe or the detection probe to the passivating molecule is 1:100 - 1:1000; and / or, the concentration of the capture probe or the detection probe is 50 nM; and / or, the capture probe is a locked nucleic acid single strand.

43. The single-molecule dynamic detection method according to claim 42, characterized in that, The reducing solution is 5 μM TCEP or 0.1 M DTT.

44. The single-molecule dynamic detection method according to claim 42, characterized in that, The passivating agent is 1 μM thiol polyethylene glycol.

45. The single-molecule dynamic detection method according to claim 42, wherein 1 μM thiol polyethylene glycol is used to reduce non-specific adsorption.

46. The single-molecule dynamic detection method according to claim 42, wherein In the locked nucleic acid single strand, the locked nucleic acid molecules account for 10 - 50% of the single strand.

47. The single-molecule dynamic detection method according to claim 46, wherein In the locked nucleic acid single strand, the locked nucleic acid molecules account for 20% of the single strand.

48. A system for detecting single molecule dynamics, characterized in that, The system includes: (i) The nanoparticle complex defined in the single-molecule dynamic detection method according to any one of claims 1 to 47; (ii) The detection probe or the capture probe defined in the single-molecule dynamic detection method according to any one of claims 1 to 47.

49. The system according to claim 48, wherein The system further includes the detection chip defined in the single-molecule dynamic detection method according to any one of claims 16 to 47; and / or, the single-molecule imaging system and / or the external force regulation module defined in the single-molecule dynamic detection method according to any one of claims 1 to 47.

50. The application of the system according to claim 48 or 49 in the detection of single-molecule dynamics.

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