Detection method and oscillator system for detecting concentration of an object to be detected based on periodic variation and regulation of external force

Abstract

The present invention relates to the field of chemical and biological detection technology, and specifically provides a detection method and an oscillator system for the concentration of an object to be detected based on periodic variation and regulation of an external force, aiming to solve the problem in existing immunoassay technology that the sensitivity and specificity of the detection of the concentration of an object to be detected in a sample are reduced due to nonspecific adsorption. The present invention includes a detection system and a periodic variation and regulation external force; both the capture probe and the detection probe are bound to the object to be detected in the sample; the labeled particles bound to the detection probe perform periodic reciprocating motion under the action of the regulation external force; the motion imaging signal of the labeled particles is recorded by a monitoring imaging device to screen specific binding molecules, and the proportion of the specific binding molecules is counted to obtain the concentration of the object to be detected. The present invention can accurately screen out specifically bound objects to be detected, thereby improving the specific detection capability and detection sensitivity of the object to be detected. The present invention does not require a complex elution process, has a short detection response time, and has a highly sensitive detection capability in a complex environment.

Description

Technical Field

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

[0002] While the availability of polymerase chain reaction (PCR) and related technologies makes it easy to detect many nucleic acid markers with submolecular limits of detection (LOD), protein analytes face challenges with similar amplification. The most sensitive and specific immunoassays often utilize a "sandwich" assay format, where enzyme-linked immunosorbent assays (ELISAs) require the analyte to be captured by an antibody on a chip surface and detected by binding of a second antibody, which is typically coupled to an enzymatic developer for signal amplification. Electrochemiluminescence (ECL) techniques require the analyte to be captured by an antibody that can be coupled to magnetic particles and adsorbed to a surface by a magnetic field, and detected by binding of a second antibody, which is typically coupled to luminol and used to oxidize in a chemiluminescent substrate solution to produce luminescence, providing 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 solution can produce non-negligible background signal levels. Consequently, conventional techniques are often unable to reliably achieve levels below 1 pM (10 -12 The low LOD of the detected substance (M) results in a low detection accuracy, which may lead to the failure to detect many diseases in their early stages. 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 and regulation of external force, aiming to solve the problem in existing immunoassay technology of nonspecific adsorption leading to decreased sensitivity and specificity in detecting the concentration of an object to be detected in a sample.

[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 an external force, comprising the following steps:

[0007] S100, binding one end of the long molecular tether to the chip surface and the other end to the capture probe; binding the detection probe to the labeled particle; binding the capture probe and the detection probe to 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 based on the motion imaging signals of the labeled particles to obtain the number of labeled particles that specifically bind 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 to obtain the concentration of the substance to be detected in the sample.

[0012] Further solution: In 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 using the electromagnet and the permanent magnet, which is simple and convenient. Furthermore, the magnitude of the periodically variable magnetic field, ranging from 0.01 to 1000 pN, is optimal for driving the labeled particles and the analytes in the sample to reciprocate. This prevents excessive magnetic field regulation, which could cause the analytes specifically bound to the labeled particles to separate from the labeled particles.

[0015] A further solution is that a single labeled particle is combined with a substance 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 motion imaging signals of all the collected labeled particles, the amplitude spectrum and frequency spectrum of the labeled particle motion are obtained; based on the amplitude spectrum and frequency spectrum, the number of specifically bound labeled particles whose motion amplitude conforms to 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 so that the motion imaging signal of the labeled particles is converted into the number of labeled particles, thereby making the specific binding of the labeled particles clearer and 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 chip surface;

[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, peptide nucleic acid or 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 and incubating;

[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, peptide nucleic acid or 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 optimal 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 particles is 100 nm to 10 μm; the labeling particles have both physical response characteristics and are regulated under the periodically variable regulating external force.

[0030] Based on the above solution, the labeling particles are regulated by the periodically changing regulating external force, 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 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 substance to be detected in the sample;

[0033] A regulating external force 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 changing regulating external force, the labeled particles are caused to reciprocate under the action of the periodically changing 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 changing regulating external force, thereby screening out the specifically bound detection object in the sample, greatly improving the ability of detection specificity, and thus 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. This eliminates the need for a complex elution process, simplifies the process, and achieves a minimum response time of less than 1 minute. This not only enables rapid and highly sensitive detection of the concentration of the object to be detected in complex environments, but also has the potential to be further developed into portable automated instruments and equipment.

[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 the motion imaging signal of the labeled particles easier to monitor, 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 following is a brief introduction to the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate 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 any creative work.

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

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

[0041] Figure 3 Schematic diagram of the process of modifying the capture probe on the chip through long molecular tethers in the present invention;

[0042] Figure 4 Schematic diagram of the process of combining the detection probe and the labeling particle in the present invention;

[0043] Figure 5 Schematic diagram of the process of screening specifically bound labeled particles in the present invention;

[0044] Figure 6 It is a schematic structural diagram of the detection system of 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 intended 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 are within the scope of protection of the present invention.

[0046] Example 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 an external force, comprising the following steps:

[0048] S100, binding one end of the long molecular tether to the chip surface and the other end to the capture probe; binding the detection probe to the labeled particle; binding the capture probe and the detection probe to 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 based on the motion imaging signals of the labeled particles to obtain the number of labeled particles that specifically bind 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 to obtain the concentration of the substance to be detected in the sample.

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

[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 coated with 2nm chromium and 47nm gold with alcohol and pure water, and treating it with a 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 slide by heating with a sealing film to form the detection cavity.

[0055] In step S100, the capture probe may be an antibody, peptide nucleic acid, or 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 , the 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 steps of binding one end of the long molecular tether to the chip surface and binding the other end to the capture probe include:

[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 chip surface;

[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 for binding one end of the long molecular tether to the chip surface includes self-assembly and covalent bonding. Specifically, when the chip surface is coated with a gold film, the long molecular tether (one end of which is modified with a thiol group -SH) is bound to the chip by 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 between the thiol group and the gold (Au) surface. When the chip surface is coated with a silicon film, the long molecular tether (one end of which is modified with an amino group -NH2) is bound to the chip by 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, fixing the long molecular tether to the electrode surface on the chip.

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

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

[0064] In step S111, the capture probe solution can use 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., 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 contains passivation molecules to inhibit nonspecific adsorption of the capture probes to the analytes. The ratio of the capture probes to the passivation molecules is between 1:00 and 1:1000.

[0066] Based on the above solution, the particle size of the labeled particles in step S100 is 100 nm to 10 μm; the labeled particles have physical response characteristics and are regulated by the periodically variable external regulating force. For example, the labeled particles are regulated by the magnetic field in a periodically variable magnetic field, by the electric field in a periodically variable electric field, or by a trapping force in an optical potential well formed by a laser.

[0067] The detection probe in step S100 is an antibody, peptide nucleic acid, or 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 includes:

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

[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 the specific situation.

[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 substance to be detected in the sample, 40 μL of 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 a concentration of NP / mL complete the binding of both 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 (for example, 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] A preferred embodiment of the periodically variable physical field is as follows: the periodically variable physical field can be a periodically variable magnetic field; the periodically variable magnetic field can be disposed above the detection system. The periodically variable magnetic field is generated by an electromagnet or a permanent magnet, which is driven by a motor to cause the electromagnet or permanent magnet to perform periodic motion, thereby generating a periodically varying magnetic field.

[0078] The magnitude of the periodically variable magnetic field ranges from 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 periodically variable range of the magnetic field is not limited to the above scheme, and can also be other range variation schemes, as long as the magnitude of the magnetic field ranges from 0.01 to 1000 pN.

[0079] When the cyclically variable physical field can be a cyclically variable magnetic field, the corresponding labeling particles can be magnetic particles, which are regulated by the magnetic field in the cyclically 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 around the outside. Various types of functional groups are linked to the outside of the coating through chemical synthesis to achieve different uses.

[0080] Another solution for the periodically variable physical field is: the periodically variable physical field can also be a periodically variable electric field, and the corresponding marking particles are charged particles, and the charged particles will be regulated by the electric field in the periodically variable electric field; or, the periodically variable 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 a capturing 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, but is not limited to, a surface plasmon resonance microscope (SPRM) (modified from a commercial total internal reflection microscope (Olympus IX-81)) for analysis and monitoring. An oil immersion objective with a magnification of 60x and a numerical aperture (NA) of 1.49 is used; the light source is an ultra-broadband light source (SLED), with a current intensity of 150 mA; and the observation field of view is 1024×1024 pixels (full field of view: 110.4×110.4 μm). 2 ); a CCD camera (Photometrics) with micro manager software was used to record the motion imaging signal of the labeled particles; and the incident angle of the light path was controlled using Cell lens software (a flow cytometer acquisition and analysis software).

[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] Because SPR is very sensitive to changes in 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 in the resonance angle over time, it is possible to reflect the changes in the medium on the surface of the metal film. When a molecule is fixed to the surface of the chip (called a ligand molecule), if it can bind to another molecule (called an analyte molecule), a corresponding change in 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] Alternatively, in addition to using an SLED light source, the surface plasmon resonance microscope (SPRM) can also utilize a laser light source with a suitable wavelength band, for example, a laser light source with a wavelength band of 600-800 nm. Furthermore, in addition to the surface plasmon resonance microscope (SPRM), total internal reflection fluorescence microscopy (TIRFM), dark-field imaging, or interferometric imaging (iSCAT) can also be used to record the motion imaging signals 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 labeled particle motion 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 example is: after applying a periodically changing control external force to cause the labeled particles to reciprocate 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 particles.

[0091] Among them, the bright field image is used for screening of a single labeled particle (c), with the purpose of eliminating nonspecific 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 a periodically changing regulated external force, thereby causing greater background interference, which is not conducive to the screening of specific signals.

[0092] The surface plasmon resonance image is used to analyze the motion of the labeled particles. First, the parabolic pattern (e) corresponding to each labeled particle must be located to obtain an amplitude spectrum (f) (extracting the brightness information of each labeled particle pattern over time). Because surface plasmon resonance microscopy is sensitive to surface conditions, changes in the brightness of the labeled particle pattern represent changes in its distance from the surface, reflecting the amplitude of the labeled particle's oscillation under the influence of a periodically varying external force. The brightness information collected from each labeled particle is then Fourier-processed for spectral analysis to obtain a frequency spectrum (g). The dominant frequency component in the specific signal appears as a peak. Only labeled particles that form an oscillator system will produce a peak corresponding to the frequency of the applied external force. Statistical analysis of the oscillator amplitude corresponding to this frequency allows for the highly specific discrimination of specifically bound labeled particles.

[0093] In step S500, based on the number of specifically bound labeled particles screened in step S400, the ratio of the number of specifically bound labeled particles to the total number of labeled particles is calculated (h). The number of specifically bound labeled particles corresponding to the concentration of the test substance in a standard sample with a series of concentrations is then determined, and a standard curve (i) is plotted. Based on the standard curve, the concentration of the test substance in the sample is determined in subsequent tests, achieving highly sensitive and specific detection of the test substance in the sample, thereby improving the accuracy of the detection of the test substance concentration.

[0094] Example 2:

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

[0096] Detection 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 substance to be detected in the sample;

[0097] A regulating external force 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 total number of 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, a thiol polyethylene glycol methoxyl solution and a long molecular tether (double-stranded DNA solution) with a molar concentration ratio of 1000:1 are added to the chip. After incubation overnight, the original solution on the chip is removed and the chip is washed three times with a 1×PBS solution with a concentration of 0.01M; streptavidin with a concentration of 1mg / mL is added and incubated for 1 hour, the streptavidin stock solution is removed and the chip is washed three times with a 1×PBS solution with a concentration of 0.01M; a polyclonal antibody clone R4-6A2 (capture probe) solution modified with biotin and with a concentration of 10ng / mL is added and incubated for 2 hours, the polyclonal antibody clone R4-6A2 stock solution is removed and the chip is washed three times with a 1×PBS solution with a concentration of 0.01M. The capture probe is modified 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 were added to a streptavidin solution of 100 NPs / mL, and the mixture was 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 was added to the magnetic particle solution coated with streptavidin and incubated for 30 minutes; after the incubation was completed, the magnetic particles modified with the detection probe were collected by separation and the magnetic particles were resuspended to the set concentration.

[0103] A3. Bind the capture probe and the detection probe to the substance 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 a concentration of NP / mL complete the binding of both the capture probe and the detection probe to 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. Monitor the oscillator system through the monitoring imaging device (surface plasmon resonance microscope) and record the motion imaging signals of the magnetic particles.

[0108] Step 4: Filter out specific signals based on the motion imaging signals of the magnetic particles to obtain the number of magnetic particles that are 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 motion amplitude is consistent with the length of the long molecular band and whose motion 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 substance to be detected in the sample.

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

Claims

1. A method for detecting the concentration of an object to be detected based on periodic 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 both 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 based on the motion imaging signals of the labeled particles to obtain the number of labeled particles that specifically bind 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 to obtain the concentration of the substance 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 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, which is obtained by an electromagnet or a permanent magnet. 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 motion imaging signals of all the collected labeled particles, the amplitude spectrum and frequency spectrum of the labeled particle motion are obtained; based on the amplitude spectrum and frequency spectrum, the number of specifically bound labeled particles whose motion amplitude conforms to 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 includes: 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 chip surface; 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, peptide nucleic acid or 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 and incubating; 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, peptide nucleic acid or 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 particles is 100 nm to 10 μm; the labeling particles have physical response characteristics and are regulated under the periodically changeable regulating external force.

10. An oscillator system for executing the method for detecting the concentration of an object to be detected based on periodic variation and regulation of external force according to any one of claims 1 to 9, 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 substance to be detected in the sample; A regulating external force can be changed periodically; the labeled particles perform periodic reciprocating motion under the action of the regulating external force.

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