Method for detecting concentration of object to be detected by regulating external force based on periodic change and vibrator system

Abstract

The invention relates to the technical field of chemical and biological detection, particularly provides a method for detecting the concentration of a to-be-detected object based on periodic change regulation and control of external force and an oscillator system, and aims to solve the problem that the sensitivity and specificity of concentration detection of the to-be-detected object in a sample are reduced due to non-specific adsorption in an existing immunodetection technology. The system comprises a detection system and periodically changing regulation and control external force; the capture probe and the detection probe are both combined with an object to be detected in a sample; the labeled particles combined with the detection probe do periodic reciprocating motion under the action of regulation and control external force; recording motion imaging signals of the labeled particles through a monitoring imaging device, screening the specific binding molecules, and counting the proportion of the specific binding molecules to obtain the concentration of the to-be-detected object. According to the invention, the specifically-bound to-be-detected object can be accurately screened out, and the capability of specifically detecting the to-be-detected object and the detection sensitivity are improved. The method has the advantages of no complex elution process, short detection response time, and high-sensitivity detection capability in a complex environment.

Description

Technical Field

[0001] The present invention belongs to the technical field of chemical and biological detection, and in particular relates to a detection method and an oscillator system for detecting the concentration of an object to be detected based on periodic variation regulation of external force. Background Art

[0002] While many nucleic acid markers are readily detectable to submolecular limits of detection (LOD) due to the availability of polymerase chain reaction (PCR) and related technologies, protein analytes are difficult to amplify in this manner. The most sensitive and specific immunoassays tend to be in the form of a "sandwich" assay, where the enzyme-linked immunosorbent assay (ELISA) requires the analyte to be captured by an antibody on a chip surface and detected by binding of a second antibody, which is often coupled to an enzymatic developer for signal amplification. Electrochemiluminescence (ECL) requires the analyte to be captured by an antibody that can be coupled to magnetic particles and adsorbed to the surface by a magnetic field, and detected by binding of a second antibody, which is often coupled to luminol for specific signal amplification.

[0003] However, even with stringent washing and high-quality antibodies, nonspecific binding of the probe to the assay surface and / or free molecules in the assay solution can produce non-negligible background signal levels. As a result, conventional techniques are often unable to reliably achieve levels below 1 pM (10 -12 The LOD of the target substance is low, which leads to low detection accuracy of the concentration of the target substance, and may cause many diseases to fail to be detected in the early stage. Summary of the invention

[0004] The present invention provides a detection method and an oscillator system for detecting the concentration of an object to be detected based on periodic variation regulation of external force, aiming to solve the problem of decreased sensitivity and specificity of detection of the concentration of an object to be detected in a sample due to nonspecific adsorption in existing immunoassay technology.

[0005] In order to solve the above technical problems, the technical solution adopted by the present invention is:

[0006] In a first aspect, the present invention provides a method for detecting the concentration of an object to be detected based on periodic variation regulation of external force, comprising the following steps:

[0007] S100, combining one end of the long molecular tether on the chip surface and the other end thereof with a capture probe; combining the detection probe with the labeled particle; combining the capture probe and the detection probe with the object to be detected in the sample to obtain a detection system;

[0008] S200, setting a periodically variable regulating external force, wherein the labeled particles perform periodic reciprocating motion under the action of the regulating external force;

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

[0010] S400, screening out specific signals according to the motion imaging signals of the labeled particles, and obtaining the number of labeled particles specifically bound to the object to be detected in the sample;

[0011] S500, calculating the ratio of the number of specifically bound labeled particles to the number of all labeled particles, and obtaining the concentration of the object to be detected in the sample.

[0012] A further solution: in the step S200, the periodically variable regulating external force is provided by a periodically variable physical field.

[0013] A further solution: the periodically variable physical field is a periodically variable magnetic field, the periodically variable magnetic field is obtained by an electromagnet or a permanent magnet, and the magnitude of the periodically variable magnetic field ranges from 0.01 to 1000 pN; the labeling particles are magnetic particles.

[0014] Based on the above scheme, a periodically variable magnetic field can be obtained by the electromagnet and the permanent magnet, which is simple and convenient. In addition, the magnitude range of the periodically variable magnetic field is 0.01 to 1000 pN, which is the most suitable range for driving the labeled particles to drive the objects to be detected in the sample to reciprocate, and can avoid the excessive regulation force of the magnetic field, which causes the objects to be detected that specifically bind to the labeled particles to separate from the labeled particles.

[0015] A further solution is that a single labeled particle is combined with an object to be detected in the sample in a one-to-one correspondence;

[0016] In step S400, the method for obtaining the number of labeled particles specifically bound to the object to be detected in the sample is:

[0017] By digitally demodulating the collected motion imaging signals of all the labeled particles, the amplitude spectrum and frequency spectrum of the motion of the labeled particles are obtained; based on the amplitude spectrum and frequency spectrum, the number of specifically bound labeled particles whose motion amplitude matches the length of the long molecular tether and whose motion frequency corresponds to the frequency of the regulated external force is obtained.

[0018] Based on the above scheme, the labeled particles have the function of reporter molecules. Since it is difficult to directly detect the concentration of the object to be detected in the sample, the concentration of the object to be detected is expressed by the motion imaging signal of the labeled particles that specifically bind to the object to be detected in the sample; the motion imaging signal of the labeled particles is digitally demodulated to convert the motion imaging signal of the labeled particles into the number of labeled particles, thereby making the specific binding of the labeled particles clearer and the concentration of the object to be detected in the sample easier to detect.

[0019] Further solution: In step S100, the step of binding one end of the long molecular tether to the chip surface and the other end to the capture probe comprises:

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

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

[0022] A further solution: 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 achieve the best binding effect between the capture probe and the object to be detected, it is optimal to select antibodies, peptide nucleic acids or single-stranded nucleic acids as the capture probe.

[0024] Further solution: In step S100, the step of combining the detection probe with the labeling particle includes:

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

[0026] S121. After the incubation is completed, the labeled particles modified with the detection probe are collected by separation, and the labeled particles are resuspended to a set concentration.

[0027] A further solution: 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 achieve the best binding effect between the detection probe and the object to be detected, it is the best solution to select antibodies, peptide nucleic acids or single-stranded nucleic acids as the detection probe.

[0029] A further solution: the particle size of the labeling particle is 100 nm to 10 μm; the labeling particle has both physical response characteristics and is regulated under the periodically variable regulating external force.

[0030] Based on the above solution, the labeling particles are regulated by the regulating external force that can change periodically, so that the labeling particles can perform reciprocating motion.

[0031] In a second aspect, the present invention provides an oscillator system, comprising:

[0032] Detection system; the detection system comprises a capture probe bound to the chip via a long molecular tether, a detection probe modified with a labeled particle, and a sample; the capture probe and the detection probe are both bound to an object to be detected in the sample;

[0033] A regulating external force that can be changed periodically; the labeled particles perform periodic reciprocating motion under the action of the regulating external force.

[0034] The beneficial effects of the present invention are:

[0035] 1. In the present invention, by applying a periodically changeable regulating external force, the labeled particles are made to reciprocate under the action of the periodically changeable regulating external force, and then the detection probe drives the object to be detected that specifically binds to the labeled particles to reciprocate; while the substance that non-specifically binds to the detection probe cannot cause the labeled particles to produce periodic movement under the action of the periodically changeable regulating external force, thereby screening out the specifically bound detection object in the sample, greatly improving the detection specificity, and then being able to accurately obtain the concentration of the object to be detected in the sample.

[0036] 2. The present invention records the motion imaging signals of the labeled particles under the action of a periodically variable regulated external force through the monitoring imaging device, thereby obtaining the concentration of the object to be detected in the sample. No complicated elution process is required, the process is simpler, and the shortest response time of the detection is less than 1 minute. It can not only realize rapid and highly sensitive detection of the concentration of the object to be detected in a complex environment, but also has the potential to be further developed into a portable automated instrument.

[0037] 3. The scattered light signal intensity of the labeled particles used in the present invention is higher than that of traditional electrochemiluminescence and fluorescence signals. The excellent signal-to-noise ratio makes it easier to monitor the motion imaging signal of the labeled particles, thereby more accurately obtaining the object to be detected that specifically binds to the detection probe, thereby improving the accuracy of the concentration detection of the object to be detected. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying creative work.

[0039] Figure 1 It is a schematic flow chart of a method for detecting the concentration of an object to be detected based on periodic variation and regulation of external force in the present invention;

[0040] Figure 2 It is a schematic diagram of the structure of the chip in the present invention;

[0041] Figure 3 It is a schematic diagram of the process of modifying the capture probe on the chip through the long molecular tether in the present invention;

[0042] Figure 4 It is a schematic diagram of the process of combining the detection probe and the labeling particle in the present invention;

[0043] Figure 5 It is a schematic diagram of the process of screening specifically bound labeled particles in the present invention;

[0044] Figure 6 It is a schematic diagram of the structure of the detection system in the present invention. DETAILED DESCRIPTION

[0045] The following will be combined with the accompanying drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work belong to the protection scope of the present invention.

[0046] Embodiment 1:

[0047] like Figure 1 As shown, this embodiment provides a method for detecting the concentration of an object to be detected based on periodic variation regulation of external force, comprising the following steps:

[0048] S100, combining one end of the long molecular tether on the chip surface and the other end thereof with a capture probe; combining the detection probe with the labeled particle; combining the capture probe and the detection probe with the object to be detected in the sample to obtain a detection system;

[0049] S200, setting a periodically variable regulating external force, wherein the labeled particles perform periodic reciprocating motion under the action of the regulating external force;

[0050] S300, using a monitoring imaging device to record the motion imaging signal of the labeled particles;

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

[0052] S500, calculating the ratio of the number of specifically bound labeled particles to the number of all labeled particles, and obtaining the concentration of the object to be detected in the sample.

[0053] A more specific example of step S100 is as follows:

[0054] In step S100, the chip includes a BK-7 glass slide and a gold sheet, and a detection cavity is formed between the BK-7 glass slide and the gold sheet. Figure 2 As shown, the steps include: first rinsing the BK-7 glass slide plated with 2nm chromium and 47nm gold with alcohol and pure water, and treating it with hydrogen flame to remove particulate impurities on the surface of the BK-7 glass slide and further improve the flatness of the gold film; then fixing the BK-7 glass slide on the surface of the gold sheet by heating with a sealing film to form the detection cavity.

[0055] In step S100, the capture probe may be an antibody, a peptide nucleic acid or a single-stranded nucleic acid, etc. In addition, the capture probe may also be any chemical substance or metabolite that can bind to the object to be detected.

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

[0057] As a further solution: Figure 3 As shown, the step of binding one end of the long molecular tether to the chip surface and the other end to the capture probe comprises:

[0058] S110, adding a long molecular tether to the chip, incubating for 12 hours, and then binding one end of the long molecular tether to the surface of the chip;

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

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

[0061] In addition, the method of combining one end of the long molecular tether on the surface of the chip includes a self-assembly method and a covalent bonding method. Specifically, when the surface of the chip is plated with a gold film, the combination of the long molecular tether (one end of which is modified with a thiol group -SH) and the chip adopts a 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 on the chip through the Au-S bond formed by the covalent bonding of the thiol group and the gold (Au) surface. When the surface of the chip is plated with a silicon film, the combination of the long molecular tether (one end of which is modified with an amino group -NH2) and the chip adopts a covalent bonding method. The amino group (-NH2) at the end of the long molecular tether undergoes a dehydration condensation reaction with the silicon on the chip to fix the long molecular tether to the electrode surface on the chip.

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

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

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

[0065] It should be noted that: in addition to the capture probes, the chip also has passivation molecules to inhibit non-specific adsorption of the capture probes and the object to be detected. The ratio of the capture probes to the passivation molecules is between 1:00 and 1:1000.

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

[0067] The detection probe in 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 that can bind to the object to be detected.

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

[0069] As a further solution: Figure 4 As shown, the step of combining the detection probe with the labeling particle comprises:

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

[0071] S121. After the incubation is completed, the labeled particles modified with the detection probe are collected by separation, and the labeled particles are resuspended to a set concentration; wherein the concentration of the resuspended labeled particles is set by the operator according to specific circumstances.

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

[0073] As a further solution, in order to make the capture probe and the detection probe bind to the object to be detected in the sample, 40 μL of the sample solution was added to the chip modified with the capture probe and incubated for 2 hours, and then 40 μL of a particle concentration of 10 5 The labeled particles coated with the detection probe at NP / mL complete the binding of the capture probe and the detection probe to the object to be detected in the sample.

[0074] The sample solution contains objects to be detected and non-detectable objects. The objects to be detected in the sample can be low-abundance proteins, nucleic acids or small biological molecules (e.g., polypeptides), etc. The capture probe and the detection probe are respectively bound to different sites of the object to be detected, for example, when the object to be detected 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 object to be detected 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] Based on the above solution, a more specific example of step S200 is as follows:

[0076] In the step S200, the periodically variable regulating external force is provided by a periodically variable 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 driven by a motor, so that the electromagnet or the permanent magnet can perform periodic motion, thereby generating a periodically changeable magnetic field.

[0078] The magnitude range of the periodically variable magnetic field is 0.01 to 1000 pN. For example, the periodically variable magnetic field can be set to 20 pN-500 pN-800 pN-500 pN-20 pN. It should be noted that the periodic variation range of the magnetic field is not the above-mentioned scheme, but can also be other range variation schemes, as long as the magnitude range of the magnetic field is within 0.01 to 1000 pN.

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

[0080] Another scheme of the periodically changeable physical field is: the periodically changeable physical field can also be a periodically changeable electric field, and the corresponding marking particles are charged particles, and the charged particles will be regulated by the electric field in the periodically changeable electric field; or, the periodically changeable physical field can also be an optical potential well formed by laser, and the corresponding marking particles are transparent particles, and the transparent particles will be subject to capture force in the optical potential well formed by laser.

[0081] Based on the above solution, a more specific example of step S300 is as follows:

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

[0083] Wherein, the detection system is placed on the monitoring imaging device, and the periodically variable physical field is set above the detection system. The incident light angle is changed. As the SPR effect occurs, the light intensity collected by the CCD camera drops sharply. The reflection angle corresponding to the dark band when the reflected light intensity is the lowest is the resonance angle (Resonance Angle), and the detection is performed near the resonance angle. In the first 5 minutes, the labeled particles are pulled to the surface of the chip by the gravitational magnetic field; in the next 15 minutes, the periodically variable physical field is started, so that the labeled particles reciprocate under the action of the periodically variable physical field, and the CCD camera records the motion imaging signal of the labeled particles at a frame rate of 16.7FPS.

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

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

[0086] As another solution, for the surface plasmon resonance microscope SPRM, in addition to using the SLED light source, a laser light source with a suitable wavelength band can also be used; for example: a laser light source with a wavelength band of 600 to 800 nm. In addition, in addition to the surface plasmon resonance microscope SPRM, total internal reflection fluorescence microscopy 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 is combined with the object to be detected in the sample in a one-to-one correspondence;

[0088] In step S400, the method for obtaining the number of labeled particles specifically bound to the object to be detected in the sample is:

[0089] By digitally demodulating the motion imaging signals of all the collected labeled particles, the amplitude spectrum of the motion of the labeled particles is obtained ( Figure 5 f graph in) and frequency spectrum ( Figure 5 g in the figure); according to the amplitude spectrum and the frequency spectrum, the number of specifically bound labeled particles whose movement amplitude conforms to the length of the long molecular tether and whose movement frequency corresponds to the frequency of the regulated external force is obtained.

[0090] like Figure 5 As shown in the figure, a specific case is: after applying a periodically changing control external force so that the labeled particle reciprocates under the control external force, signal acquisition and processing are required. The monitoring imaging device can simultaneously obtain a bright field image (a) and a surface plasmon resonance image (d) of the labeled particle.

[0091] Among them, the bright field image is used for screening of a single labeled particle (c), with the purpose of eliminating non-specific interference caused by aggregation of the labeled particles: as shown in Figure (b), since the labeled particles will inevitably aggregate spontaneously, those aggregates that have not formed the detection system may produce signals similar to the detection system (specifically bound labeled particles) under the action of periodically variable regulated external forces, thereby causing greater background interference, which is not conducive to the screening of specific signals.

[0092] The surface plasmon resonance image is used for the 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) (the brightness information of all the labeled particle patterns 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 its distance from the surface, that is, it reflects the amplitude of the oscillation of the labeled particle under the action of the periodically variable external force. After that, the brightness information collected from each labeled particle is subjected to Fourier processing for spectrum analysis to obtain a frequency 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 that form the oscillator system will produce a corresponding peak at the frequency of the applied external force. The oscillator amplitude corresponding to the frequency is statistically analyzed, that is, the specifically bound labeled particles can be screened out with extremely high specific discrimination ability.

[0093] In step S500, according to the number of specifically bound labeled particles screened in step S400, the ratio of the number of specifically bound labeled particles to the number of all labeled particles is calculated (h). Then, the corresponding number of specifically bound labeled particles is obtained by detecting the detection object in a series of concentrations of standard samples and a standard curve (i) is drawn. According to the standard curve, the concentration of the object to be detected in the sample is detected in the subsequent detection, so as to achieve high sensitivity and high specificity detection of the object to be detected in the sample and improve the detection accuracy of the concentration of the object to be detected.

[0094] Embodiment 2:

[0095] This embodiment provides an oscillator system, including:

[0096] Detection system; the detection system comprises a capture probe bound to the chip via a long molecular tether, a detection probe modified with a labeled particle, and a sample; the capture probe and the detection probe are both bound to an object to be detected in the sample;

[0097] A regulating external force that can be changed periodically; the labeled particles perform periodic reciprocating motion under the action of the regulating external force.

[0098] The present invention will be further described below in conjunction with experiments:

[0099] Experimental principle: A periodically changing magnetic field is applied to the detection system to form the oscillator system, and the magnetic particles (labeled particles) perform periodic reciprocating motion under the action of the periodically changing magnetic field; the motion imaging signals of the magnetic particles are recorded by the monitoring imaging device, and then the specific signals are screened out by the motion imaging signals to obtain the number of magnetic particles specifically bound to the object to be detected in the sample; the ratio of the number of specifically bound magnetic particles to the number of all magnetic particles is counted to obtain the concentration of the object to be detected in the sample.

[0100] Step 1: Create Figure 6 The detection system shown includes the following steps:

[0101] A1. The capture probe (polyclonal antibody clone R4-6A2) is modified on the chip through the long molecular tether. Specifically, add a thiol polyethylene glycol methoxy solution and a long molecular tether (double-stranded DNA solution) with a molar concentration ratio of 1000:1 to the chip, remove the stock solution on the chip after incubation overnight, and wash it three times with a 1×PBS solution with a concentration of 0.01M; add streptavidin with a concentration of 1mg / mL for incubation for 1 hour, remove the streptavidin stock solution and wash it three times with a 1×PBS solution with a concentration of 0.01M; 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 stock solution and wash it three times with a 1×PBS solution with a concentration of 0.01M, and modify the capture probe on the long molecular tether.

[0102] A2, combining the detection probe (polyclonal antibody clone XMG1.2) with the magnetic particles. 12 Magnetic particles with a particle size of 2.8 μm are added to a streptavidin solution of 10.5 NPs / mL, and the mixture is incubated to obtain a magnetic particle solution coated with the streptavidin; a polyclonal antibody clone XMG1.2 (detection probe) solution modified with biotin and having a concentration of 500 ng / mL is added to the magnetic particle solution coated with streptavidin and the mixture is incubated for 30 minutes; after the incubation, the magnetic particles modified with the detection probe are collected by separation, and the magnetic particles are resuspended to a set concentration.

[0103] A3. Bind the capture probe and the detection probe to the object to be detected in the sample. Specifically, add 40 μL of sample solution to the chip modified with the capture probe and incubate for 2 hours, then add 40 μL of particles with a concentration of 10 5The magnetic particles coated with the detection probe at NP / mL complete the binding of both the capture probe and the detection probe with the object to be detected in the sample.

[0104] Step 2: Establish an oscillator system, including the following steps:

[0105] B1. A periodically variable magnetic field is provided, and the magnetic particles perform periodic reciprocating motion under the action of the magnetic field.

[0106] Step 3: Recording the motion imaging signal of the magnetic particles, the steps include:

[0107] C1. The oscillator system is monitored by the monitoring imaging device (surface plasmon resonance microscope) to record the motion imaging signals of the magnetic particles.

[0108] Step 4: Screen out specific signals based on the motion imaging signals of the magnetic particles to obtain the number of magnetic particles specifically bound to the object to be detected in the sample; specifically, obtain the amplitude and frequency spectrum of the movement of the magnetic particles by digitally demodulating the motion imaging signals of all the collected magnetic particles; and obtain the number of specifically bound magnetic particles whose movement amplitude matches the length of the long molecular band and whose movement frequency corresponds to the frequency of the external force based on the amplitude and frequency spectrum.

[0109] Step 5: Count the ratio of the number of specifically bound magnetic particles to the number of all magnetic particles to obtain the concentration of the object to be detected in the sample.

[0110] The present invention is not limited to the above-mentioned optional implementation modes. Anyone can derive other various forms of products under the inspiration of the present invention. However, no matter what changes are made in the shape or structure, all technical solutions that fall within the scope defined by the claims of the present invention fall within the protection scope of the present invention.

Claims

1. A method for detecting the concentration of an object to be detected based on periodic variation and regulation of external force, characterized in that: The following steps are involved: S100, binding one end of the long molecular tether to the chip surface and the other end to the capture probe; Combining the detection probe with the labeling particle; combining the capture probe and the detection probe with the object to be detected in the sample to obtain a detection system; S200, setting a periodically variable regulating external force, wherein the labeled particles perform periodic reciprocating motion under the action of the regulating external force; S300, using a monitoring imaging device to record the motion imaging signal of the labeled particles; S400, screening out specific signals according to the motion imaging signals of the labeled particles, and obtaining the number of labeled particles specifically bound to the object to be detected in the sample; S500, calculating the ratio of the number of specifically bound labeled particles to the number of all labeled particles, and obtaining the concentration of the object to be detected in the sample.

2. The method for detecting the concentration of an object to be detected based on periodic variation and regulation of external force according to claim 1, characterized in that: In the step S200, the periodically variable regulating external force is provided by a periodically variable physical field.

3. The method for detecting the concentration of an object to be detected based on periodic variation and regulation of external force according to claim 2, characterized in that: The periodically variable physical field is a periodically variable magnetic field, the periodically variable magnetic field is obtained by an electromagnet or a permanent magnet, and the magnitude of the periodically variable magnetic field ranges from 0.01 to 1000 pN; the labeling particles are magnetic particles.

4. The method for detecting the concentration of an object to be detected based on periodic variation and regulation of external force according to claim 1, characterized in that: The single labeled particle is combined with the object to be detected in the sample in a one-to-one correspondence; In step S400, the method for obtaining the number of labeled particles specifically bound to the object to be detected in the sample is: By digitally demodulating the collected motion imaging signals of all the labeled particles, the amplitude spectrum and frequency spectrum of the motion of the labeled particles are obtained; based on the amplitude spectrum and frequency spectrum, the number of specifically bound labeled particles whose motion amplitude matches the length of the long molecular tether and whose motion frequency corresponds to the frequency of the regulated external force is obtained.

5. The method for detecting the concentration of an object to be detected based on periodic variation and regulation of external force according to claim 1, characterized in that: In step S100, the step of binding one end of the long molecular tether to the chip surface and the other end to the capture probe comprises: S110, adding a long molecular tether to the chip, incubating for 12 hours, and then binding one end of the long molecular tether to the surface of the chip; S111, adding the capture probe solution modified with biotin for incubation, and binding the capture probe to the other end of the long molecular tether.

6. The method for detecting the concentration of an object to be detected based on periodic variation and regulation of external force according to claim 1 or 5, characterized in that: The capture probe is an antibody, a peptide nucleic acid or a single-stranded nucleic acid.

7. The method for detecting the concentration of an object to be detected based on periodic variation and regulation of external force according to claim 1, characterized in that: In step S100, the step of combining the detection probe with the labeling particle includes: S120, adding the detection probe solution modified with biotin to the labeled particle solution coated with streptavidin for incubation; S121. After the incubation is completed, the labeled particles modified with the detection probe are collected by separation, and the labeled particles are resuspended to a set concentration.

8. The method for detecting the concentration of an object to be detected based on periodic variation and regulation of external force according to claim 1 or 7, characterized in that: The detection probe is an antibody, a peptide nucleic acid or a single-stranded nucleic acid.

9. The method for detecting the concentration of an object to be detected based on periodic variation and regulation of external force according to claim 1, characterized in that: The particle size of the labeling particle is 100nm-10μm; the labeling particle has a physical response characteristic and is regulated under the periodically changeable regulating external force.

10. An oscillator system, characterized in that include: Testing system; The detection system includes a capture probe bound to the chip via a long molecular tether, a detection probe modified with a labeled particle, and a sample; The capture probe and the detection probe both bind to the object to be detected in the sample; A regulating external force that can be changed periodically; the labeled particles perform periodic reciprocating motion under the action of the regulating external force.

Images