Method for measuring concentration of analyte on basis of periodically variable external regulation force, and oscillator system

Abstract

A method for measuring the concentration of an analyte on the basis of a periodically variable external regulation force, and an oscillator system, which aim to solve the problem of non-specific adsorption in existing immunoassay techniques leading to reduced sensitivity and specificity in the measurement of analyte concentrations in samples. The measurement method comprises: obtaining a detection system, and setting a periodically variable external regulation force; binding both a capture probe and a detection probe with an analyte in a sample; making labeled particles, which are bound to the detection probe, perform periodic reciprocating motion under the action of the external regulation force; and by means of a monitoring and imaging apparatus, recording motion imaging signals of the labeled particles to select specifically bound molecules, and compiling statistics to calculate the proportion of the specifically bound molecules so as to obtain the analyte concentration. A specifically bound analyte can be accurately selected, thereby enhancing the capability and detection sensitivity of specific detection of analytes. The method has no complex elution processes, has a short detection response time, and exhibits high-sensitivity detection capability in complex environments.

Description

Method for detecting concentration of to-be-detected substance based on periodical change regulation of external force and oscillator system TECHNICAL FIELD

[0001] The present application belongs to the field of chemical and biological detection technology, and particularly relates to a method for detecting concentration of to-be-detected substance based on periodical change regulation of external force and an oscillator system. BACKGROUND

[0002] Although due to the availability of polymerase chain reaction (PCR) and related technologies, the detection of many nucleic acid molecular markers can easily reach the sub-molecular detection limit (LOD), it is difficult to perform such similar amplification for protein analytes. The most sensitive and specific immunoassay often adopts a "sandwich" assay form, in which enzyme-linked immunosorbent assay (ELISA) requires the analyte to be captured on the chip surface by one antibody and detected by the binding of a second antibody, which is usually coupled with an enzymatic developing reagent for signal amplification. Electrochemical chemiluminescence technology (ECL) requires the analyte to be captured by an antibody that can be coupled with magnetic particles and adsorbed to the surface by a magnetic field, and detected by the binding of a second antibody, which is usually coupled with luminol for luminescence by oxidation reaction in a chemiluminescent substrate solution for specific signal amplification.

[0003] However, even with strict washing and high-quality antibodies, non-specific binding of probes to the assay surface and / or free molecules in the solution will produce a non-negligible background signal level. Therefore, conventional technologies often cannot reliably achieve an LOD below 1 pM (10 -12 M), resulting in low accuracy in detecting the concentration of to-be-detected substances, which may lead to many diseases not being detected at an early stage. SUMMARY

[0004] The present application provides a method for detecting concentration of to-be-detected substance based on periodical change regulation of external force and an oscillator system, aiming to solve the problem of decreased sensitivity and specificity in detecting the concentration of to-be-detected substances in samples due to non-specific adsorption in existing immunoassay technologies. TECHNICAL SOLUTION

[0005] To solve the above technical problems, the technical scheme adopted by the present application is as follows:

[0006] In a first aspect, the present application provides a method for detecting concentration of to-be-detected substance based on periodical change regulation of external force, comprising the following steps:

[0007] S100, one end of a long molecular tether is bound to the surface of a chip, and the other end is bound to a capture probe; a detection probe is combined with a labeled particle; the capture probe and the detection probe are both combined with a to-be-detected substance in a sample to obtain a detection system;

[0008] S200, setting a controllable external force which can periodically change, and the labeled particles make periodic reciprocating motion under the action of the controllable external force;

[0009] S300, using a monitoring imaging device to record the motion imaging signal of the labeled particles;

[0010] S400, screening specific signals according to the motion imaging signal of the labeled particles, and obtaining the number of labeled particles specifically combined with the to-be-detected substance in the sample;

[0011] S500, statistics the proportion of the number of specifically combined labeled particles in the number of all labeled particles, and obtains the concentration of the to-be-detected substance in the sample.

[0012] Further scheme: in the step S200, the controllable external force which can periodically change is provided by a physical field which can periodically change.

[0013] Further scheme: the physical field which can periodically change is a magnetic field which can periodically change, the magnetic field which can periodically change is obtained by an electromagnet or a permanent magnet, the size range of the magnetic field which can periodically change is 0.01-1000pN, and the labeled particles are magnetic particles.

[0014] Based on the above scheme, the magnetic field which can periodically change can be obtained by the electromagnet and the permanent magnet, which is simple and convenient. In addition, the size range of the magnetic field which can periodically change is 0.01-1000pN, which is the most appropriate range for driving the labeled particles to drive the to-be-detected substance in the sample to make reciprocating motion, which can avoid that the to-be-detected substance specifically combined with the labeled particles is separated from the labeled particles due to the too large regulation force of the magnetic field.

[0015] Further scheme: each of the labeled particles is specifically combined with the to-be-detected substance in the sample one by one;

[0016] In the step S400, the method for obtaining the number of labeled particles specifically combined with the to-be-detected substance in the sample is:

[0017] By digitally demodulating the motion imaging signals of all the collected labeled particles, the amplitude spectrum and the frequency spectrum of the motion of the labeled particles are obtained; and according to the amplitude spectrum and the frequency spectrum, the number of specifically combined labeled particles whose motion amplitude conforms to the length of the long molecular belt and whose motion frequency corresponds to the frequency of the controllable external force is obtained.

[0018] Based on the above scheme, the labeled particles have the function of reporter molecules, and since it is difficult to directly detect the concentration of the to-be-detected substance in the sample, the concentration of the to-be-detected substance is expressed by the motion imaging signal of the labeled particles specifically combined with the to-be-detected substance in the sample; the motion imaging signal of the labeled particles is digitally demodulated, so that the motion imaging signal of the labeled particles is converted into the number of labeled particles, and the specific combination of the labeled particles is more clear, and the concentration of the to-be-detected substance in the sample is easier to detect.

[0019] Further scheme: in the step S100, the step of binding one end of the long molecular tether on the chip surface and the other end of the capture probe includes:

[0020] S110, adding a long molecular tether to the chip, and after incubation for 12 hours, binding one end of the long molecular tether on the chip surface;

[0021] S111, adding a capture probe solution modified with biotin for incubation, and binding the capture probe on the other end of the long molecular tether.

[0022] Further scheme: the capture probe is an antibody, a peptide nucleic acid, or a single-stranded nucleic acid.

[0023] Based on the above scheme, in order to make the binding effect between the capture probe and the to-be-detected substance best, therefore, the antibody, the peptide nucleic acid, or the single-stranded nucleic acid is selected as the capture probe as the optimal scheme.

[0024] Further scheme: in the step S100, the step of binding the detection probe with the labeled particles includes:

[0025] S120, adding a detection probe solution modified with biotin to a labeled particle solution coated with streptavidin for incubation;

[0026] S121, after incubation, collecting the labeled particles modified with the detection probe by separation, and resuspending the labeled particles to a set concentration.

[0027] Further scheme: the detection probe is an antibody, a peptide nucleic acid, or a single-stranded nucleic acid.

[0028] Based on the above scheme, in order to make the binding effect between the detection probe and the to-be-detected substance best, therefore, the antibody, the peptide nucleic acid, or the single-stranded nucleic acid is selected as the detection probe as the optimal scheme.

[0029] Further scheme: the particle size of the labeled particles is 100 nm to 10 µm; the labeled particles have physical response characteristics and are regulated under the periodically changeable regulation external force.

[0030] Based on the above scheme, the labeled particles are regulated by the periodically changeable regulating external force, so that the labeled particles can reciprocate.

[0031] In a second aspect, the present application provides a vibrator system, comprising:

[0032] a detection system; the detection system comprises capture probes bound on the chip through long molecular lacing, detection probes modified with labeled particles, and a sample; the capture probes and the detection probes are both combined with the detection object in the sample;

[0033] a periodically changeable regulating external force; the labeled particles reciprocate under the action of the regulating external force. Advantages

[0034] 1. In the present application, by applying a periodically changeable regulating external force, the labeled particles reciprocate under the action of the periodically changeable regulating external force, and then the detection probes drive the detection object specifically combined with the labeled particles to reciprocate; substances non-specifically combined with the detection probes cannot make the labeled particles produce periodic motion under the action of the periodically changeable regulating external force, so that the specifically combined detection object is screened out in the sample, greatly improving the detection specificity, and then the concentration of the detection object in the sample can be accurately obtained.

[0035] 2. The present application records the motion imaging signal of the labeled particles under the action of the periodically changeable regulating external force by the monitoring imaging device, so as to obtain the concentration of the detection object in the sample, without complex elution process, and the process is simpler, and the shortest response time of detection is less than 1 minute, which not only can realize rapid and high-sensitivity detection of the concentration of the detection object in a complex environment, but also has the potential to develop into a portable automatic instrument.

[0036] 3. The scattering light signal intensity of the labeled particles used in the present application is higher than that of traditional electrochemical luminescence and fluorescence signal, and excellent signal-to-noise ratio makes the motion imaging signal of the labeled particles easier to be monitored, so as to more accurately obtain the detection object specifically combined with the detection probes, and improve the accuracy of the detection of the concentration of the detection object. BRIEF DESCRIPTION OF DRAWINGS

[0037] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following will briefly introduce the drawings needed to be used in the embodiments. It should be understood that the following drawings only show some of the embodiments of the present application, and therefore should not be regarded as a limitation to the scope. For those skilled in the art, other related drawings can also be obtained without creative labor.

[0038] Fig. 1 is a flowchart of the detection method of the concentration of the to-be-detected substance based on the periodically changing regulation external force in the present application;

[0039] Fig. 2 is a structural diagram of the chip in the present application;

[0040] Fig. 3 is a flowchart of the process of the capture probe being modified on the chip by the long molecular tie in the present application;

[0041] Fig. 4 is a flowchart of the process of the detection probe being combined with the labeled particle in the present application;

[0042] Fig. 5 is a flowchart of the process of screening the specifically combined labeled particle in the present application;

[0043] Fig. 6 is a structural diagram of the detection system in the present application;

[0044] Fig. 7 is a comparison chart of the results of whether the detection system in the present application contains the periodically changing regulation external force. DETAILED DESCRIPTION

[0045] The technical solutions of the embodiments of the present application will be described in detail below with reference to the drawings of the embodiments of the present application. It should be understood that the specific embodiments described herein are only used to explain the present application, and are not used to limit the present application. Based on the embodiments of the present application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of the present application.

[0046] Embodiment one:

[0047] As shown in Fig. 1, the present embodiment provides a detection method of the concentration of the to-be-detected substance based on the periodically changing regulation external force, which comprises the following steps:

[0048] S100, one end of the long molecular tie is combined on the surface of the chip, and the other end is combined with the capture probe; the detection probe is combined with the labeled particle; the capture probe and the detection probe are both combined with the to-be-detected substance in the sample, and a detection system is obtained;

[0049] S200, a regulation external force which can periodically change is set, and the labeled particle makes periodic reciprocating motion under the action of the regulation external force;

[0050] S300, a monitoring imaging device is used to record the motion imaging signal of the labeled particle;

[0051] S400, screening specific signals according to the motion imaging signals of the labeled particles, and obtaining the number of labeled particles specifically combined with the to-be-detected object in the sample;

[0052] S500, counting the proportion of the number of specifically combined labeled particles in the number of all labeled particles, and obtaining the concentration of the to-be-detected object in the sample.

[0053] More specifically, the step S100 includes:

[0054] In the step S100, the chip includes a BK-7 glass sheet and a gold sheet, and a detection cavity is formed between the BK-7 glass sheet and the gold sheet, as shown in FIG. 2, the step includes: cleaning the BK-7 glass sheet plated with 2 nm chromium and 47 nm gold with alcohol and pure water, and treating the BK-7 glass sheet with a hydrogen flame to remove particle impurities on the surface of the BK-7 glass sheet and further improve the flatness of the gold film; and then fixing the BK-7 glass sheet on the surface of the gold sheet by heating the sealing film to form the detection cavity.

[0055] In the step S100, the capture probe can be an antibody, a peptide nucleic acid, or a single-stranded nucleic acid, etc. In addition, the capture probe can also be any chemical substance or metabolic product capable of being combined with the to-be-detected object.

[0056] It should be noted that: the binding rate constant kon of the capture probe and the to-be-detected object is ≤5×10 8 M -1 s -1 , the dissociation rate constant koff is ≥0.05 s -1 , and the dissociation equilibrium constant KD is ≥1.56 pM; after the capture probe is combined with the to-be-detected object, the standard free energy of the to-be-detected object is greater than or equal to -12 kcal / mol.

[0057] As a further solution, as shown in FIG. 3, one end of the long molecular tether is combined on the surface of the chip, and the other end is combined with the capture probe, and the step includes:

[0058] S110, adding a long molecular tether to the chip, and after incubation for 12 hours, combining one end of the long molecular tether on the surface of the chip;

[0059] S111, adding a capture probe solution modified with biotin for incubation, and combining the capture probe on the other end of the long molecular tether.

[0060] In the step S110, the long molecular tether can be a biomacromolecule (e.g., a polypeptide chain, double-stranded DNA modified with biotin and thiol at two ends, etc.), a polymer molecule (e.g., polyethylene glycol (PEG), polylysine (PLL), etc.), or a compound (e.g., a straight-chain alkane, etc.), etc.

[0061] In addition, the method for binding one end of the long molecular tether to the chip surface includes a self-assembly method and a covalent binding method, etc. Specifically, when the chip surface is plated with a gold film, the long molecular tether (one end modified with a thiol group -SH) is combined with the chip by using the self-assembly method. The thiol group (-SH) at the end of the long molecular tether directly fixes the long molecular tether to the electrode surface of the chip by forming an Au-S bond through the covalent binding of the thiol group and the gold (Au) surface. When the chip surface is plated with a silicon film, the long molecular tether (one end modified with an amino group -NH2) is combined with the chip by using the covalent binding method. The amino group (-NH2) at the end of the long molecular tether dehydrates and condenses with silicon on the chip to fix the long molecular tether to the electrode surface of the chip.

[0062] It should be noted that, when the long molecular tether is combined with the chip, an anti-non-specific adsorption molecule can be added to the chip to inhibit non-specific adsorption on the chip. The anti-non-specific adsorption molecule can be a polymer molecule (e.g., polyethylene glycol (PEG), etc.), a natural macromolecule (e.g., chitosan, etc.), or a biomolecule (e.g., bovine serum albumin (BSA), etc.), etc.

[0063] In the step S110 of the embodiment, the long molecular tether is selected as a double-stranded DNA (HS-dsDNA-biotin) solution, and the anti-non-specific adsorption molecule is selected as a thiol polyethylene glycol methoxy (HS-mPEG) solution. The molar concentration ratio of the thiol polyethylene glycol methoxy solution and the double-stranded DNA solution is 1000:1, the molecular weight Mw of the thiol polyethylene glycol methoxy solution is 5000 Da, and the length of the double-stranded DNA is 721 bp. After incubation overnight, the original solution on the chip is removed, and the chip is washed three times with a 1×PBS solution (a phosphate buffered saline solution with a concentration of 0.01 M).

[0064] In the step S111, the capture probe solution can be a solution of a polyclonal antibody clone R4-6A2 with a concentration of 10 ng / mL, which specifically binds to the C-terminus of IFN-γ, BioLegend, Inc. The incubation time is 2 hours. After the incubation is completed, the original solution of the polyclonal antibody clone R4-6A2 is removed, and the chip is washed three times with a 1×PBS solution (a phosphate buffered saline solution with a concentration of 0.01 M).

[0065] It should be noted that in addition to the capture probe, there is a passivation molecule on the chip to inhibit non-specific adsorption of the capture probe and the to-be-detected substance. The ratio of the capture probe to the passivation molecule ranges from 1:00 to 1:1000.

[0066] On the basis of the above scheme, the particle size of the labeling particle in the step S100 is 100 nm to 10 µm; the labeling particle has a physical response characteristic and is regulated under the periodically changeable regulation external force. For example, the labeling particle is regulated by a magnetic field in a periodically changeable magnetic field, the labeling particle is regulated by an electric field in a periodically changeable electric field, or the labeling particle is regulated by a capture force in an optical potential well formed by laser, and the like.

[0067] The detection probe in the step S100 is an antibody, a peptide nucleic acid, or a single-stranded nucleic acid, etc. In addition, the capture probe can also be any chemical substance or metabolite capable of combining with the to-be-detected substance.

[0068] It should be noted that the binding rate constant kon of the detection probe and the to-be-detected substance is ≥10 10 M -1 s -1 The dissociation rate constant koff is ≤0.0002 s -1 , and the dissociation equilibrium constant KD is ≤0.2 aM; after the detection probe combines with the to-be-detected substance, the standard free energy of the to-be-detected substance is less than or equal to -25 kcal / mol.

[0069] As a further scheme, as shown in FIG. 4, the step of combining the detection probe with the labeling particle includes:

[0070] S120, adding the detection probe solution modified with biotin to the labeling particle solution coated with streptavidin for incubation;

[0071] S121, after the incubation is completed, the labeling particle modified with the detection probe is collected by separation, and the labeling particle is resuspended to a set concentration; wherein the concentration of the resuspended labeling particle is set by an operator according to specific conditions.

[0072] Specifically, before the step S120, the labeling particle is added to the streptavidin solution for incubation to obtain a labeling particle solution coated with the streptavidin, wherein the concentration of the streptavidin solution is 10 12The particle size of the labeled particles can be 2.8 pm. In the step S120, the detection probe solution can be a solution of polyclonal antibody clone XMG1.2, specific to the N-terminus of IFN-gamma, BioLegend, Inc., with a concentration of 500 ng / mL, and the incubation time is 30 minutes.

[0073] As a further solution, to enable the capture probe and the detection probe to bind to the to-be-detected substance in the sample, 40 pL of a sample solution is added to the chip modified with the capture probe and incubated for 2 hours, and then 40 pL of the labeled particles coated with the detection probe with a particle concentration of 10 5 NP / mL is added to complete the binding of the capture probe and the detection probe to the to-be-detected substance in the sample.

[0074] The sample solution contains to-be-detected substances and non-detected substances. The to-be-detected substance in the sample can be a low-abundance protein, nucleic acid, or biological small molecule (for example, a polypeptide). The capture probe and the detection probe are respectively bound to different sites of the to-be-detected substance. For example, when the to-be-detected substance is a nucleic acid, the capture probe is bound to the 5' end of the nucleic acid, and the detection probe is bound to the 3' end of the nucleic acid. When the to-be-detected substance is a polypeptide, the capture probe is bound to the N-terminus of the polypeptide, and the detection probe is bound to the C-terminus of the polypeptide.

[0075] On the basis of the above solution, a more specific case of the step S200 is:

[0076] In the step S200, the periodically changeable external control force is provided by a periodically changeable physical field.

[0077] As a preferred solution of the periodically changeable physical field, the periodically changeable physical field can be a periodically changeable magnetic field. The periodically changeable magnetic field can be arranged above the detection system. The periodically changeable magnetic field is obtained by an electromagnet or a permanent magnet, and is driven by an electric motor, so that the electromagnet or the permanent magnet can move periodically, thereby generating a periodically changeable magnetic field.

[0078] The magnitude of the periodically changeable magnetic field ranges from 0.01 pN to 1000 pN. For example, the periodically changeable magnetic field can be set to 20 pN-500 pN-800 pN-500 pN-20 pN. It should be noted that the periodically changeable range of the magnetic field is not limited to the above solution, but can also be other range change solutions, as long as the magnitude of the magnetic field ranges from 0.01 pN to 1000 pN.

[0079] When the periodically-varying physical field can be a periodically-varying magnetic field, the corresponding marking particles can be magnetic particles, which are regulated by the magnetic field in the periodically-varying magnetic field. The magnetic particles include magnetic particles (e.g., Fe, Ni, Co, Mn, etc.) at the core site, and a hydrophilic polymer coating, a hydrophobic polymer coating, or a silica coating outside the coating. Various types of functional groups are linked outside the coating by chemical synthesis to achieve different purposes.

[0080] As another solution of the periodically-varying physical field, the periodically-varying physical field can also be a periodically-varying electric field, and the corresponding marking particles are charged particles, which are regulated by the electric field in the periodically-varying electric field; or the periodically-varying physical field can also be a laser-formed optical potential well, and the corresponding marking particles are transparent particles, which are captured by the trapping force in the laser-formed optical potential well.

[0081] On the basis of the above solutions, more specific cases of the step S300 are as follows:

[0082] The monitoring imaging device can use, but is not limited to, a surface plasmon resonance microscope (SPRM) (modified based on a commercial total internal reflection microscope (Olympus IX-81)) for analysis and monitoring. An oil lens with a magnification of 60 times and a numerical aperture N.A = 1.49 is used; the light source is an ultra-wideband light source SLED, and the intensity of the light source control current is 150 mA; the observation field is 1024x1024 pixels (full field of view: 110.4x110.4 μm 2 ); the movement imaging signal of the marking particles is recorded by a micro manager software CCD camera (Photometrics); and the Cell lens software (a flow cytometry acquisition and analysis software) is used to control the incident angle of the light path.

[0083] The detection system is placed on the monitoring imaging device, and the periodically changeable physical field is arranged above the detection system. The incident light angle is changed, and as the SPR effect occurs, the light intensity collected by the CCD camera sharply decreases. The resonance angle is the reflection angle corresponding to the dark band when the reflection intensity is the lowest. The detection is performed near the resonance angle. In the first 5 minutes, the labeled particles are attracted to the chip surface by the gravitational magnetic field; in the last 15 minutes, the periodically changeable physical field is started to make the labeled particles reciprocate under the action of the periodically changeable physical field, and the CCD camera records the motion imaging signal of the labeled particles at a frame rate of 16.7 FPS.

[0084] Since SPR is very sensitive to the change of the refractive index of the medium on the surface of the metal film, when the properties of the medium change or the molecular weight changes, the resonance angle will change. Therefore, by detecting the change of the resonance angle over time, the change of the medium on the surface of the metal film can be reflected. When a molecule (called Ligand) is fixed on the surface of the chip, if another molecule (called Analyte) can be combined, a corresponding change in the resonance angle will be detected, and if it cannot be combined, the resonance angle will not change.

[0085] In further schemes, the surface plasmon resonance microscope (SPRM) mechanism can be an objective coupling type or a prism coupling type.

[0086] As another scheme, for the surface plasmon resonance microscope (SPRM), in addition to using an SLED light source, a laser light source with a suitable waveband can also be used; for example, a laser light source with a waveband of 600-800 nm. In addition to the surface plasmon resonance microscope (SPRM), total internal reflection fluorescence microscope (TIRFM), dark field imaging technology, or interference imaging iSCAT technology can also be used to record the motion imaging signal of the labeled particles.

[0087] On the basis of the above scheme, a single labeled particle corresponds to the combination of the to-be-detected substance in the sample;

[0088] In the step S400, the method for obtaining the number of labeled particles specifically combined with the to-be-detected substance in the sample is:

[0089] The motion imaging signals of all the collected labeled particles are digitally demodulated to obtain the amplitude spectrum (f in FIG. 5) and the frequency domain spectrum (g in FIG. 5) of the motion of the labeled particles; and the number of specifically combined labeled particles with a motion amplitude conforming to the length of the long molecular belt and a motion frequency corresponding to the frequency of the regulated external force is obtained according to the amplitude spectrum and the frequency domain spectrum.

[0090] As shown in FIG. 5, a specific case is that after applying a periodically changing control external force to make the labeled particles reciprocate under the action of the control external force, signal acquisition and processing are needed. The monitoring imaging device can simultaneously obtain a bright-field image (a) and a surface plasmon resonance image (d) of the labeled particles.

[0091] The bright-field image is used for screening (c) of individual labeled particles, aiming to exclude non-specific interference caused by aggregation of the labeled particles. As shown in (b), due to the inevitable spontaneous aggregation of the labeled particles, aggregates not forming the detection system may produce similar signals to the detection system (specifically bound labeled particles) under the action of the periodically changing control external force, thus causing greater background interference and being not conducive to screening of specific signals.

[0092] The surface plasmon resonance image is used for motion analysis of the labeled particles. First, the parabolic pattern (e) corresponding to each labeled particle needs to be located to obtain an amplitude spectrum (f) (extracting the brightness information of each labeled particle pattern over time). Since the surface plasmon resonance microscope is sensitive to the surface state, the brightness change of the labeled particle pattern represents the change in the distance from the surface, i.e., reflects the amplitude of the oscillation of the labeled particle under the action of the periodically changing external force. Then, the brightness information collected for each labeled particle is subjected to Fourier processing for frequency spectrum analysis to obtain a frequency domain spectrum (g). At this time, the dominant frequency component in the specific signal is presented in the form of a peak. Only the labeled particles forming the oscillator system will produce a corresponding peak at the frequency of the applied external force. Statistical analysis of the oscillator amplitude at this frequency can screen out those specifically bound labeled particles with extremely high specificity.

[0093] In the step S500, according to the number of specifically bound labeled particles screened out in the step S400, the proportion of the number of specifically bound labeled particles to the number of all labeled particles is calculated (h). Then, the detection substance in a series of standard samples is detected to obtain the corresponding number of specifically bound labeled particles and draw a standard curve (i). According to the standard curve, the concentration of the detection substance in the sample is detected in the subsequent detection, realizing high-sensitivity and high-specificity detection of the detection substance in the sample and improving the detection accuracy of the concentration of the detection substance.

[0094] The following further illustrates the present application by another comparative experiment, i.e., whether to adopt the step S200 of "setting a periodically changing control external force, and making the labeled particles reciprocate periodically under the action of the control external force":

[0095] In the step S400, the method for obtaining the number of labeled particles specifically combined with the to-be-detected substance in the sample is:

[0096] As shown in FIG. 7, a specific case is that after the periodic change of the control external force is applied to make the labeled particles reciprocate under the action of the control external force, signal acquisition and processing are needed. The monitoring imaging device can obtain the surface plasmon resonance image (A) of the labeled particles, and locate the parabolic pattern (B) corresponding to each labeled particle.

[0097] As a control, the surface plasmon resonance image recorded under the condition of no periodic change of the control external force (i.e., the image is directly recorded without the step S200) is located, and the specific binding screening process consistent with the experimental group is adopted to prove the effectiveness of the signal modulation method of the present application.

[0098] The surface plasmon resonance image is used for motion analysis of the labeled particles; first, the parabolic pattern (B) corresponding to each labeled particle needs to be located, and the brightness information (C) of each labeled particle pattern over time is extracted. Since the surface plasmon resonance microscope is sensitive to the surface state, the brightness change of the labeled particle pattern represents the change in the distance from the surface, i.e., reflects the amplitude of the oscillation of the labeled particle under the action of the periodic change of the external force.

[0099] The brightness change amplitude of the particle without external force control is close to the background, on the contrary, the brightness of the particle under the control of the periodic external force changes periodically, in short, there is a significant difference in the amplitude of the brightness of the particle with or without periodic external force control (D).

[0100] Then, the brightness information collected for each labeled particle is subjected to Fourier processing for frequency spectrum analysis, and the amplitude (E) and probability density (F) change spectrum with frequency are obtained. At this time, the dominant frequency component in the specific signal is in the form of a peak. Only the labeled particles forming the oscillator system will produce a corresponding peak at the frequency of the applied external force. Statistical analysis of the amplitude of the oscillator at this frequency can screen out those specifically combined labeled particles with extremely high specific discrimination ability.

[0101] In the step S500, according to the number of specifically combined labeled particles screened out in the step S400, the proportion of the number of specifically combined labeled particles to the number of all labeled particles is calculated (G). Then, the to-be-detected substance in a series of standard samples is detected to obtain the corresponding number of specifically combined labeled particles and a standard curve (H) is drawn.

[0102] Therefore, the periodic external force regulation method used in the present application can significantly improve the specificity and sensitivity (smaller error and lower detection limit) of the analysis. The concentration of the to-be-detected substance in the sample is obtained in the subsequent detection according to the standard curve, so that the high-sensitivity and high-specificity detection of the to-be-detected substance in the sample is realized, and the detection accuracy of the concentration of the to-be-detected substance is improved.

[0103] Embodiment two:

[0104] The present embodiment provides a vibrator system, which comprises:

[0105] A detection system, which comprises a capture probe combined on the chip by a long molecular tether, a detection probe modified with a label particle, and a sample; the capture probe and the detection probe are both combined with the to-be-detected substance in the sample;

[0106] A periodically changeable regulation external force; the label particle makes periodic reciprocating motion under the action of the regulation external force.

[0107] The present application will be further described in combination with experiments:

[0108] Test principle: a periodically changeable magnetic field is applied to the detection system to form the vibrator system, and the magnetic particle (label particle) makes periodic reciprocating motion under the action of the periodically changeable magnetic field; the motion imaging signal of the magnetic particle is recorded by the monitoring imaging device, and then the specific signal is screened out through the motion imaging signal, so that the number of magnetic particles specifically combined with the to-be-detected substance in the sample is obtained; the proportion of the number of specifically combined magnetic particles in the number of all magnetic particles is counted, and the concentration of the to-be-detected substance in the sample is obtained.

[0109] Step one: establish a detection system as shown in FIG. 6, the step comprising:

[0110] A1, the capture probe (polyclonal antibody clone R4-6A2) is modified on the chip by the long molecular tether. Specifically, add a molar concentration ratio of 1000:1 of a mercapto polyethylene glycol methoxy solution and a long molecular tether (double-stranded DNA solution) to the chip, incubate overnight, remove the original solution on the chip and wash with 1xPBS solution with a concentration of 0.01M for three times; add streptavidin with a concentration of 1mg / mL for incubation for 1 hour, remove the streptavidin original solution and wash with 1xPBS solution with a concentration of 0.01M for three times; add a polyclonal antibody clone R4-6A2 (capture probe) solution modified with biotin and with a concentration of 10ng / mL for incubation for 2 hours, remove the polyclonal antibody clone R4-6A2 original solution and wash with 1xPBS solution with a concentration of 0.01M for three times, and modify the capture probe on the long molecular tether.

[0111] A2, the detection probe (polyclonal antibody clone XMG1.2) is combined with the magnetic particles. Specifically, add magnetic particles with a particle size of 2.8µm to a streptavidin solution with a concentration of 10 12 NPs / mL, incubate to obtain a magnetic particle solution coated with the streptavidin; add a polyclonal antibody clone XMG1.2 (detection probe) solution modified with biotin and with a concentration of 500ng / mL to the magnetic particle solution coated with streptavidin for incubation for 30 minutes; after incubation, collect the magnetic particles modified with the detection probe by separation, and resuspend the magnetic particles to a set concentration.

[0112] A3, the capture probe and the detection probe are both combined with the to-be-detected substance in the sample. Specifically, add 40µL of a sample solution to the chip modified with the capture probe and incubate for 2 hours, and then add 40µL of the detection probe coated magnetic particles with a particle concentration of 10 5 NPs / mL, complete the combination of the capture probe and the detection probe with the to-be-detected substance in the sample.

[0113] Step two: establish an oscillator system, which includes the following steps:

[0114] B1, set a periodically changing magnetic field, and the magnetic particles make periodic reciprocating motion under the action of the magnetic field.

[0115] Step three: record the motion imaging signal of the magnetic particles, which includes the following steps:

[0116] C1, monitor the oscillator system by the monitoring imaging device (surface plasmon resonance microscope) and record the motion imaging signal of the magnetic particles.

[0117] Step four: screening specific signals according to the motion imaging signals of the magnetic particles, and obtaining the number of magnetic particles specifically combined with the to-be-detected substance in the sample; specifically, the amplitude and frequency spectrum of the motion of the magnetic particles are obtained by digitally demodulating all the motion imaging signals of the magnetic particles collected; and the number of the specifically combined magnetic particles with the motion amplitude conforming to the length of the long molecular belt and the motion frequency corresponding to the frequency of the external force is obtained according to the amplitude and frequency spectrum.

[0118] Step five: calculating the proportion of the number of the specifically combined magnetic particles in the number of all the magnetic particles, and obtaining the concentration of the to-be-detected substance in the sample.

[0119] The present application is not limited to the above-mentioned optional embodiments, and anyone can derive other various forms of products under the inspiration of the present application, but regardless of any changes in shape or structure, any technical solutions falling within the scope defined by the claims of the present application fall within the protection scope of the present application.

Claims

1. A method for detecting the concentration of a test substance based on the periodic variation of an external force, characterized by, The method comprises the following steps: S100, one end of a long molecular tether is combined on a chip surface, and the other end is combined with a capture probe; A detection probe is combined with a labeled particle; the capture probe and the detection probe are both combined with a to-be-detected substance in a sample to obtain a detection system; S200, a controllable external force capable of periodic change is set, and the labeled particle makes periodic reciprocating motion under the action of the controllable external force; S300, a monitoring imaging device is used to record a motion imaging signal of the labeled particle; S400, specific signals are screened out according to the motion imaging signal of the labeled particle, and the number of labeled particles specifically combined with the to-be-detected substance in the sample is obtained; S500, the proportion of the number of specifically combined labeled particles in the number of all labeled particles is counted, and the concentration of the to-be-detected substance in the sample is obtained.

2. The method according to claim 1, wherein In the step S200, the controllable external force capable of periodic change is provided by a physical field capable of periodic change.

3. The method according to claim 2, wherein the method is characterized by, The physical field capable of periodic change is a magnetic field capable of periodic change, the magnetic field capable of periodic change is obtained by an electromagnet or a permanent magnet, the size of the magnetic field capable of periodic change ranges from 0.01 to 1000 pN, and the labeled particle is a magnetic particle.

4. The method according to claim 1, wherein Each of the labeled particles is correspondingly combined with the to-be-detected substance in the sample; In the step S400, the number of labeled particles specifically combined with the to-be-detected substance in the sample is obtained by: Carrying out digital demodulation on the motion imaging signals of all the collected labeled particles to obtain an amplitude spectrum and a frequency spectrum of the motion of the labeled particles; and according to the amplitude spectrum and the frequency spectrum, the number of specifically combined labeled particles, whose motion amplitude conforms to the length of the long molecular tether and whose motion frequency corresponds to the frequency of the controllable external force, is obtained.

5. The method according to claim 1, wherein the method is characterized by, In the step S100, the step of combining one end of the long molecular tether on the chip surface and the other end with the capture probe comprises: S110, long molecular tethers are added to the chip, and after incubation for 12 hours, one end of the long molecular tether is combined on the chip surface; S111, the capture probe solution modified by biotin is added for incubation, and the capture probe is combined on the other end of the long molecular tether.

6. The method according to claim 1 or 5, wherein The capture probe is an antibody, a peptide nucleic acid or a single-stranded nucleic acid.

7. The method according to claim 1, wherein the method is characterized by, In the step S100, the step of combining the detection probe with the labeled particle comprises: S120, the detection probe solution modified by biotin is added to the labeled particle solution coated with streptavidin for incubation; S121, after incubation, the labeled particle modified by the detection probe is collected by separation, and the labeled particle is resuspended to a set concentration.

8. The method according to claim 1 or 7, wherein the method is characterized by, The detection probe is an antibody, a peptide nucleic acid or a single-stranded nucleic acid.

9. The method according to claim 1, wherein the method is characterized by, The particle size of the labeled particle ranges from 100 nm to 10 µm; the labeled particle has physical response characteristics and is regulated under the controllable external force capable of periodic change.

10. A system of transducers, characterized in that The method comprises: a detection system; The detection system comprises a capture probe combined on the chip through a long molecular tether, a detection probe modified with a labeled particle, and a sample; The capture probe and the detection probe are both combined with the to-be-detected substance in the sample; The periodically changeable regulation external force; the mark particle makes periodic reciprocating motion under the action of the regulation external force.

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