WO2021120943A1 - 一种检测样品体系中微量蛋白的方法 - Google Patents

一种检测样品体系中微量蛋白的方法 Download PDF

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WO2021120943A1
WO2021120943A1 PCT/CN2020/128990 CN2020128990W WO2021120943A1 WO 2021120943 A1 WO2021120943 A1 WO 2021120943A1 CN 2020128990 W CN2020128990 W CN 2020128990W WO 2021120943 A1 WO2021120943 A1 WO 2021120943A1
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complex
antibody
electrical pulse
pulse signal
immunomagnetic beads
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PCT/CN2020/128990
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French (fr)
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柳可
熊贵
王哲
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瑞芯智造(深圳)科技有限公司
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Priority to EP20903562.5A priority Critical patent/EP4075134A4/en
Priority to US17/787,573 priority patent/US20220404348A1/en
Priority to JP2022538402A priority patent/JP7401136B2/ja
Publication of WO2021120943A1 publication Critical patent/WO2021120943A1/zh

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles
    • G01N33/54333—Modification of conditions of immunological binding reaction, e.g. use of more than one type of particle, use of chemical agents to improve binding, choice of incubation time or application of magnetic field during binding reaction
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483—Physical analysis of biological material
    • G01N33/487—Physical analysis of biological material of liquid biological material
    • G01N33/48707—Physical analysis of biological material of liquid biological material by electrical means
    • G01N33/48721—Investigating individual macromolecules, e.g. by translocation through nanopores
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2470/00—Immunochemical assays or immunoassays characterised by the reaction format or reaction type
    • G01N2470/04—Sandwich assay format

Definitions

  • the invention relates to the technical field of protein detection, in particular to a method for detecting trace proteins in a sample system.
  • enzyme-linked immunoassay is currently the most widely used immunoassay method.
  • the method is to label the secondary antibody with the enzyme, the specificity of the antigen-antibody reaction is combined with the action of the enzyme-catalyzed substrate, and the test result is judged according to the color change after the enzyme acts on the substrate, and its sensitivity can reach the nanogram level.
  • Commonly used enzymes for labeling include horseradish peroxidase (HRP), alkaline phosphatase (AP) and so on.
  • enzyme-linked immunoassay does not require special equipment and is simple to detect, it is widely used in disease detection.
  • Commonly used methods include indirect method, sandwich method and BAS-ELISA method.
  • the indirect method is to first coat the protein to be tested in the well plate, then add the primary antibody, the enzyme-labeled secondary antibody, and the substrate to develop color, and then quantitatively detect the antigen through an instrument (such as a microplate reader).
  • This method is simple to operate, but due to its high background and poor specificity, it has been gradually replaced by the sandwich method.
  • the sandwich method uses two kinds of antibodies to capture and fix the target antigen, which greatly improves the specificity of the reaction while ensuring sensitivity.
  • the immunomagnetic bead capture method is a new type of immunological technology that combines the magnetic field response capability of magnetic beads with immune specificity.
  • Immunomagnetic beads have the advantages of immobilized reagent characteristics, high sensitivity and specificity of immunological reaction.
  • Nanopore DNA sequencing technology is mainly used for nucleic acid analysis. Both of these techniques have not been used in the detection of antigens and other proteins.
  • the present invention provides a method for detecting trace proteins in a sample system.
  • the charge state of a single magnetic bead complex is detected by immunospecific binding to magnetic beads, thereby calculating the absolute number of trace proteins, and the detection limit is as low as a single protein Molecules can detect trace proteins below the detection limit of conventional immunoassays, and can be widely used in immunological detection, microbial detection, cell separation and other fields.
  • a method for detecting trace proteins in a sample system including:
  • the composite is passed through the micro-nanopore device in the form of a single particle and an electrical pulse signal is generated, the electrical pulse signal is analyzed to obtain the charge state or the volume/mass state of the composite, and the sample system is calculated based on the charge state or volume/mass state The number of proteins to be tested in.
  • the above method further includes: in a solution system suitable for antibody modification reactions, covalently binding the above-mentioned primary antibody to the functional group on the surface of the immunomagnetic bead to obtain the above-mentioned primary antibody-modified immunomagnetic bead;
  • the degree of modification of the primary antibody on the surface of the immunomagnetic bead is adjusted by adjusting the concentration ratio of the primary antibody to the immunomagnetic bead in the solution system.
  • the particle size of the immunomagnetic beads is at least 1-1000 times that of the nanoparticles.
  • the particle size of the immunomagnetic beads is generally 100 nanometers to 10 microns, and the particle size of the nanoparticles is generally less than 1 micron.
  • the magnetic particles are separated from the unreacted excess nanoparticles by magnetic sedimentation.
  • the above-mentioned micro-nanopore device is a micro-nanopore single particle counting device based on Coulter's principle.
  • the device includes two chambers filled with electrolyte, and a device communicating with the two chambers filled with electrolyte.
  • Micro-nanopores when the composite body passes through the micro-nanopores in the form of single particles, it temporarily blocks the flow of ions in the micro-nanopores to form an electric pulse signal.
  • the electrical pulse signal reflects the charge state of the complex
  • the state and/or quantity of the nanoparticles bound in the complex is obtained by analyzing the peak fluctuations of the electrical pulse signal, according to the state and/or
  • the quantity and the principle of double antibody sandwich structure reaction are used to calculate the quantity of the protein to be tested in the above sample system.
  • the above-mentioned micro-nanoporous device is a device with a double-layer membrane structure with micropores.
  • the device includes two layers of nano-films with micro-pores with a set spacing distance, and the micro-pores on the two nano-films are opposite to each other. Connect the cavities on both sides of the nano-film, the cavities are filled with electrolyte, and the cavities on both sides are respectively arranged with electrodes for maintaining ion transmission; when the composite body continuously passes through two micropores in the form of single particles Generate a pair of electrical pulse signals with time intervals.
  • the above method further includes: controlling the electrical pulse signal interval between different complexes to be far greater than the time interval in the electrical pulse signal pair by adjusting the concentration of the above-mentioned complex in the sample, and by adjusting the pulse of the electrical pulse signal Intensity threshold analysis, filters out the signal generated by the immunomagnetic beads that are not bound to the nanoparticles.
  • the particle surface potential of the composite body is obtained by calculating the electric mobility.
  • the above-mentioned electrical mobility is calculated by the following formula:
  • ⁇ represents the electric mobility
  • v(x) represents the speed of the composite particles continuously passing through the two micropores
  • E(x) represents the electric field distribution
  • the particle surface potential ⁇ and the electric mobility ⁇ have the following corresponding relationship:
  • ⁇ represents the viscosity of the electrolyte
  • t represents the residence time of the particles in the micropores
  • A represents the correction coefficient of each micropore
  • V represents the potential difference
  • the volume/mass state is positively correlated with the integrated area of the electrical pulse signal, and the number of captured proteins on the complex is proportional to the volume/mass increase of the complex relative to the immunomagnetic bead.
  • the standard curve relationship between the signal integration area and the number of captured proteins on the complex is used to calculate the number of captured proteins on the complex, and then the number of proteins to be tested in the above-mentioned sample system is calculated.
  • the present invention uses immunomagnetic beads to capture the trace protein to be tested.
  • the immunomagnetic beads can be attracted by a magnetic field and have specific functional groups on the surface to covalently bind to proteins with biological activity.
  • the immunomagnetic beads are used as the test Carrier of protein.
  • the immunomagnetic bead-coated antibody binds to the antigen (protein to be tested) with the determinants of fit, and is efficiently separated from complex sample systems such as plasma, and is recognized by the antibody labeled with nanoparticles to realize the detection of the protein to be tested. Quantitative testing.
  • one or more antigen proteins can be captured.
  • the sandwich structure of the double antibody is beneficial to improve the capture specificity, and the second antibody is connected to the nanometer
  • the particles can adjust the total charge of the double-antibody sandwich structure complex. Therefore, the method of the present invention can control the amount of protein captured, which can be as low as less than 10 proteins/magnetic beads, and use nanoparticles with a small particle size to accurately count the captured antigen proteins to be tested.
  • Figure 1 is a schematic diagram of the principle of the double-antibody sandwich structure-single-protein complex in an embodiment of the present invention
  • Fig. 2 is a schematic diagram of the principle of the double-antibody sandwich structure-multi-protein complex in an embodiment of the present invention
  • FIG. 3 is a schematic diagram of a micro-nanopore single particle counting device based on Coulter's principle in an embodiment of the present invention
  • FIG. 4 is a schematic diagram of the principle of judging the charge state of the composite particles by analyzing the subtle fluctuations of the electric pulse signal peaks in the embodiment of the present invention
  • FIG. 5 is a schematic diagram of a double-layer membrane structure device with micropores in an embodiment of the present invention.
  • FIG. 6 is a schematic diagram of the principle of preparing a double-layer membrane structure device by micro-nano processing and integral molding in an embodiment of the present invention
  • FIG. 7 is a schematic diagram of a pair of electric pulse signals generated during the process of the composite body continuously passing through two micropores in the form of a single particle in an embodiment of the present invention
  • FIG. 8 is a graph of real electrical pulse signals versus pulses generated during the process of continuous passage through two micropores in the form of single particles of a composite in an embodiment of the present invention
  • Fig. 9 is a graph showing the linear correspondence between the number of captured proteins and the electric mobility (migration speed) in the embodiment of the present invention.
  • Fig. 10 is an electrical pulse signal diagram (A) and a standard curve diagram (B) of the relationship between the electrical pulse signal integrated area (volume/mass of the complex) and the number of captured proteins on the complex in the embodiment of the present invention.
  • the present invention provides a method for detecting trace protein in a sample system
  • the sample system can be a simple system containing a single protein component, or a complex system containing one or more proteins and other components, including blood, plasma, serum, tissue fluid, urine, Various samples such as cerebrospinal fluid.
  • the method of the present invention can detect trace proteins in samples, such as micrograms, nanograms, picograms, etc., with the lowest detection limit as low as a single protein molecule, and can detect trace proteins below the detection limit of conventional immunoassays. It is widely used in immunological detection, microbial detection, cell separation and other fields.
  • the method for detecting trace protein in a sample system of the present invention includes the following steps:
  • the double-antibody sandwich structure complex is passed through the micro-nanopore device in the form of a single particle and an electric pulse signal is triggered.
  • the electric pulse signal is analyzed to obtain the charge state or volume/mass state of the complex, and the sample system is calculated according to the charge state or volume/mass state The number of proteins to be tested.
  • the primary antibody and the secondary antibody are respectively combined with different antigenic determinants on the protein to be tested to form a double antibody sandwich structure, as shown in Figure 1, where the Y-shaped structure represents the antibody, and the larger sphere represents the immunomagnetic field. Beads, the surface of which is modified with a primary antibody, the smaller spheres represent nanoparticles, and the surface is modified with a secondary antibody.
  • the rhombus structure represents the protein to be tested.
  • the structure of the formed double antibody sandwich structure complex can be expressed as immunomagnetic beads-one Anti-test protein-secondary antibody-nanoparticle.
  • the double antibody sandwich structure complex shown in Figure 1 has only one protein to be tested (diamond-shaped structure in the figure), so it can also be called a "single protein complex".
  • the sandwich structure of the double antibody of the present invention is beneficial to improve the capture specificity.
  • the source and type of the primary antibody and the secondary antibody are not particularly limited, such as various animal-derived polyclonal antibodies or recombinantly expressed monoclonal antibodies, such as IgG antibodies from mice, sheep, rabbits, and horses.
  • the primary antibody is mouse IgG and the secondary antibody is goat anti-mouse antibody.
  • the primary antibody is covalently combined with the functional groups on the surface of the immunomagnetic beads to obtain the primary antibody-modified immunomagnetic beads.
  • the functional groups on the surface of the immunomagnetic beads can be amino and carboxyl groups.
  • Such immunomagnetic beads become amino magnetic beads and carboxyl magnetic beads, respectively.
  • the functional groups on the surface of the immunomagnetic beads are separated from the corresponding groups on the primary antibody.
  • the primary antibody is modified to the surface of the immunomagnetic beads by forming a covalent bond.
  • a typical reaction of primary antibody modified immunomagnetic beads is in EDC(1-Ethyl-3-(3'-dimethylaminopropyl)carbodiimide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide Hydrochloride) and NHS (N-Hydroxysuccinimide, N-hydroxysuccinimide).
  • EDC 1-Ethyl-3-(3'-dimethylaminopropyl)carbodiimide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide Hydrochloride) and NHS (N-Hydroxysuccinimide, N-hydroxysuccinimide).
  • the immunomagnetic beads (Ademtech) are replaced with MES buffer (2-(N-morpholine ethanesulfonic acid) three times, and the solution is balanced to a pH of 4-5 and an ionic strength of 0.1M.
  • the concentration ratio of the primary antibody to the immunomagnetic beads in the solution system by adjusting the concentration ratio of the primary antibody to the immunomagnetic beads in the solution system, the degree of modification of the primary antibody on the surface of the immunomagnetic beads is adjusted, from single modification to multiple modification, so as to adjust the binding secondary antibody
  • the number of modified nanoparticles to form a special complex as shown in Figure 2.
  • This double-antibody sandwich structure complex has multiple proteins to be tested (diamond-shaped structure in the figure), so it can also be called "polyprotein" Complex".
  • the concentration of the primary antibody is less than 1 microgram per milligram of immunomagnetic beads, so that the degree of modification of the primary antibody on the surface of the immunomagnetic beads is less than 50 antibodies per immunomagnetic bead; -5 micrograms per milligram of immunomagnetic beads, so that the degree of modification of the primary antibody on the surface of the immunomagnetic beads is less than 300 antibodies per immunomagnetic bead; the concentration of the primary antibody is higher than 50 micrograms per milligram of immunomagnetic beads, making the primary antibody immune Saturation on the magnetic beads.
  • This method can control the amount of primary antibody modification on the immunomagnetic beads. The degree of modification was further confirmed by electron microscopy.
  • the nano-particles can be nano-particles of various materials and various suitable particle sizes and charging conditions, such as polyethylene nano-microspheres, nano-magnetic beads, nano-silicon balls, nano-emulsions, etc.
  • the size of the nano-particles For example, it can be substantially smaller than 1 micron, and generally speaking, the particle size of the corresponding immunomagnetic beads is at least larger than that of nanoparticles, which can achieve better results and facilitate the formation of a special composite structure.
  • the preparation principle and method of the nanoparticles modified by the secondary antibody are similar to those of the immunomagnetic beads modified by the primary antibody.
  • the secondary antibody on the nanoparticle there are many ways to modify the secondary antibody on the nanoparticle, and the present invention is not limited.
  • the secondary antibody in a solution system suitable for the antibody modification reaction, is covalently combined with the functional group on the surface of the nanoparticle to obtain the secondary antibody-modified nanoparticle. Since the nanoparticles have a certain amount of electricity, the number of nanoparticles in the double-antibody sandwich structure composite formed by the present invention will affect the charge state of the entire composite.
  • the present invention obtains the quantity of nanoparticles on a single complex by analyzing the charge state of the complex, and then analyzes the quantity of the protein to be tested.
  • the reaction of the secondary antibody modified nanoparticles is realized under the action of EDC and NHS.
  • the secondary antibody is a goat anti-mouse antibody
  • the preparation method of goat anti-mouse antibody-labeled nanoparticles includes the following steps: 1 mg nanoparticle balance pH is 8.0, concentration 0.1M PBS solution to the final concentration of the nanoparticles 0.1mg/mL; then add EDC and NHS respectively, until the final concentration of EDC and NHS in the solution are both 0.2mg/mL, after activation for 30 minutes, centrifuge and discard. clear.
  • the precipitate after discarding the supernatant was reconstituted with PBS solution, goat anti-mouse antibody (Cat.: 4T20, HyteSt) was added, labeled for 25 minutes, and BSA was added to the concentration of 0.5% to stop the reaction.
  • goat anti-mouse antibody Cat.: 4T20, HyteSt
  • the protein detection method of the present invention is based on electric pulse signals.
  • the double-antibody sandwich structure complex formed by the immune reaction will cause electric pulse signals when it is electrophoresed in the electrolyte and passes through the micropores, and the complexes can be obtained by analyzing the electric pulse signals.
  • the charge state the quantity of the protein to be tested in the sample system can be calculated based on the charge state.
  • the double-antibody sandwich structure complex passes through the micro-nanopore device in the form of a single particle and triggers an electrical pulse signal.
  • the electrical pulse signal is analyzed to obtain the charge state of the complex, and the charge state of the sample system is calculated based on the charge state.
  • the amount of protein is based on electric pulse signals.
  • micro-nanopore single particle counting device based on the Coulter principle (for example, US Patent 2,656,508,1953) to detect the signals of different complexes in the micro-nanopore detection device.
  • the micro-nano hole single particle counting device includes two chambers filled with electrolyte, namely chamber 31 and chamber 32 in the figure, and the only micro-nano hole communicating with the two chambers filled with electrolyte. 33.
  • the flow of ions in the micro-nanopore 33 is temporarily blocked to form an electric pulse signal.
  • the electrical pulse signal can reflect the size, binding state, charge form and even geometric form of ions.
  • the electrical pulse signal intensity threshold is analyzed to determine the charge state of the composite particles, and the subtle fluctuations of the electrical pulse signal peaks can be analyzed to determine whether the composite particles are Binding of nanoparticles, and the number of bindings. For example, in Figure 4, when two nanoparticles are bound to the composite particle, two peaks are reflected on the electrical pulse signal; when the composite particle is not bound with nanoparticles, no peak is reflected on the electrical pulse signal; When three nanoparticles are bound to the composite particles, three peaks are reflected in the electrical pulse signal.
  • the micro-nanoporous device is a device with a microporous double-layer membrane structure, as shown in FIG. 1 and micropore 2) of the nano film, the micropores on the two layers of nano film are opposite to connect the cavities on both sides of the nano film (cavity 1 and cavity 2 in the figure), the cavity is filled with electrolyte, and both sides
  • the cavities of the cavities are respectively arranged with electrodes for maintaining ion transmission; when the protein magnetic bead complex continuously passes through the two micropores in the form of single particles, a pair of electrical pulse signals with a time interval are generated.
  • the pore size of the double-layer membrane structure device can range from 1 nanometer to 10 micrometers, which can be specifically set according to the requirement of allowing the protein magnetic bead complex to continuously pass in the form of single particles.
  • Nano film can be selected from silicon nitride, silicon dioxide, silicon and other semiconductor film-forming materials or films made of high molecular polymers.
  • the double-layer membrane structure device is equipped with two electrodes, which can be silver/silver chloride or platinum electrodes. By applying an external electric field, a balanced ion transmission is maintained, which is reflected in a stable opening current.
  • the protein magnetic bead complex generates a pair of electrical pulse signals with a time interval during the process of continuously passing through two micropores under the action of driving force.
  • the double-layer membrane structure device of the present invention can be obtained by various suitable methods.
  • the double-layer membrane structure device is prepared by controlling the spacing of the double-layer membrane by micro-nano processing and integral molding.
  • the specific principles and steps are shown in Figure 6.
  • Step 1 Prepare a double-sided polished matrix material (silicon or quartz, etc.) as a support layer, and prepare 1 nanometer to 1 nanometer on the upper and lower surfaces by thermal oxidation or vapor deposition. 10 micron nano film (film-forming layer in the picture).
  • Step 2 Use the pre-prepared photolithography template to define the size and shape of the micropores and the position on the film. Coating photoresist on the upper and lower surfaces, using a template to expose and develop.
  • the upper and lower templates can be precisely aligned to ensure that the two micro-holes maintain a micron alignment accuracy in space.
  • gas etching is used to etch micropores on the film using unexposed photoresist as a sacrificial layer.
  • Step 3 Using a wet etching method, the etching solution etches the support layer through the micropores to connect the two micropores.
  • the complex 1, unbound magnetic beads (i.e., immunomagnetic beads that are not bound to nanoparticles) and complex 2 generate a pair of electrical pulse signals in the process of passing through two micropores continuously in the form of single particles.
  • the electrical pulse signals of composite 1 and composite 2 exceeded the set threshold, while the electrical pulse signals of unbound magnetic beads did not exceed the set threshold.
  • Figure 8 shows the true pair of electrical pulse signals generated during the process of the composite passing through two micropores in the form of a single particle. It can be seen that each composite particle can generate a pair of electrical pulse signals when passing through the micropores. And the two electric pulse signals in the pair of electric pulse signals have a certain time interval.
  • the electrical pulse signal interval between different complexes is controlled to be much larger than the time interval in the electrical pulse signal pair, and
  • the signal generated by the immunomagnetic beads that are not bound to the nanoparticles is filtered out.
  • the number of proteins captured on the immunomagnetic beads determines the number of secondary antibody-modified nanoparticles bound to it, thereby determining the charge amount of the complex, and the charge amount and electromigration of the complex Rate related. Therefore, as shown in Fig. 9, the number of capture proteins has a linear relationship with the electrical mobility (migration speed).
  • the electrical mobility By calculating the electrical mobility, the particle surface potential can be obtained, and then the amount of the protein to be measured can be calculated.
  • the electrical mobility is calculated by the following formula:
  • ⁇ represents the electric mobility
  • v(x) represents the speed of the composite particles continuously passing through the two micropores
  • E(x) represents the electric field distribution.
  • the distance between two micropores is 1000 nanometers
  • the time interval for a single composite particle to pass through the two micropores is 1 millisecond
  • the potential difference is 100 millivolts.
  • the electric mobility can be calculated as 10 -8 m 2 V -1 s -1 .
  • the particle surface potential ⁇ and the electric mobility ⁇ have the following correspondence:
  • ⁇ represents the viscosity of the electrolyte.
  • t represents the residence time of the particles in the micropores, which can be obtained from experiments
  • A represents the correction coefficient of each micropore, which can be obtained from the standard particles for each hole
  • V represents the potential difference.
  • the correction coefficient A is 6 ⁇ 10 -6 V 2 s, and the particle surface potential ⁇ can be obtained as 60 millivolts.
  • the quantity of the protein to be tested in the sample system is calculated according to the volume/mass state of the complex.
  • the volume/mass state of the complex is positively correlated with the integrated area of the electrical pulse signal, and the number of captured proteins on the complex is proportional to the volume/mass increase of the complex relative to the immunomagnetic beads.
  • the standard curve relationship between the integrated area of the electrical pulse signal (which characterizes the volume/mass state of the complex) and the number of captured proteins on the complex the number of captured proteins on the complex is calculated, and then the number of proteins to be tested in the sample system is calculated.
  • the volume/mass of the complex represents the volume, mass, or a combination of the two, because according to the reaction principle of the double-antibody sandwich structure in the present invention, the number of captured proteins on the immunomagnetic beads determines the two bound to it.
  • the number of anti-modification nanoparticles, and the number of nanoparticles determines the increase in the volume or mass of the composite.

Abstract

一种检测样品体系中微量蛋白的方法，包括：提供待测蛋白的一抗修饰的免疫磁珠和二抗修饰的纳米颗粒；将一抗修饰的免疫磁珠和二抗修饰的纳米颗粒与含有待测蛋白的样品体系混合孵育，形成双抗夹心结构复合体；使复合体以单颗粒形式通过微纳孔装置并引发电脉冲信号，分析电脉冲信号得到复合体的电荷状态或者体积/质量状态，根据电荷状态或者体积/质量状态计算样品体系中待测蛋白的数量。本发明通过免疫特异性结合磁珠的方式，检测单个磁珠复合体的电荷状态，从而计算出微量蛋白的绝对数量，检出限低至单个蛋白分子，可以检出常规免疫检测检出下限以下的微量蛋白，可广泛应用于免疫学检测、微生物检测、细胞分离等领域。

Description

一种检测样品体系中微量蛋白的方法 技术领域
本发明涉及蛋白质检测技术领域，尤其涉及一种检测样品体系中微量蛋白的方法。
背景技术
在几种常用的免疫学检测技术中，酶联免疫检测是目前应用最广泛的免疫检测方法。该方法是将二抗标记上酶，抗原抗体反应的特异性与酶催化底物的作用结合起来，根据酶作用底物后的显色颜色变化来判断试验结果，其敏感度可达纳克水平。常见用于标记的酶有辣根过氧化物酶(HRP)、碱性磷酸酶(AP)等。
由于酶联免疫法无需特殊的仪器，检测简单，因此被广泛应用于疾病检测。常用的方法包括间接法、夹心法以及BAS－ELISA法。间接法是先将待测的蛋白包被在孔板内，然后依次加入一抗、标记了酶的二抗和底物显色，通过仪器(例如酶标仪)定量检测抗原。这种方法操作简单，但由于高背景而特异性较差，目前已逐渐被夹心法取代。夹心法利用两种抗体对目标抗原进行捕获和固定，在确保灵敏度的同时大大提高了反应的特异性。免疫磁珠捕获法是一种将磁珠磁场响应能力与免疫特异性相结合的新型免疫学技术。免疫磁珠具有固相化试剂特质及免疫学反应高灵敏度、高 专一性等优点。
此处，还有基于库尔特原理的系列细胞计数分选技术，该技术对微米尺寸的细胞进行分选。纳米孔DNA测序技术，主要用于核酸物质分析。这两种技术均未用于抗原等蛋白质的检测中。
发明内容
本发明提供一种检测样品体系中微量蛋白的方法，通过免疫特异性结合磁珠的方式，检测单个磁珠复合体的电荷状态，从而计算出微量蛋白的绝对数量，检出限低至单个蛋白分子，可以检出常规免疫检测检出下限以下的微量蛋白，可广泛应用于免疫学检测、微生物检测、细胞分离等领域。
本发明通过如下技术方案实现：
一种检测样品体系中微量蛋白的方法，包括：
提供待测蛋白的一抗修饰的免疫磁珠和二抗修饰的纳米颗粒；
将上述一抗修饰的免疫磁珠和二抗修饰的纳米颗粒与含有上述待测蛋白的样品体系混合孵育，形成双抗夹心结构复合体；
使上述复合体以单颗粒形式通过微纳孔装置并引发电脉冲信号，分析上述电脉冲信号得到上述复合体的电荷状态或体积/质量状态，根据上述电荷状态或体积/质量状态计算上述样品体系中待测蛋白的数量。
在优选实施例中，上述方法还包括：在适于抗体修饰反应的溶液体系中，使上述一抗与免疫磁珠表面的功能基团共价结合得到上述一抗修饰的 免疫磁珠；
在优选实施例中，通过调节上述溶液体系中上述一抗与上述免疫磁珠的浓度比例，来调节上述一抗在上述免疫磁珠表面的修饰程度。
在优选实施例中，上述免疫磁珠的粒径至少是上述纳米颗粒的1-1000倍。
在优选实施例中，上述免疫磁珠的粒径大体上为100纳米到10微米，上述纳米颗粒的粒径大体上小于1微米。
在优选实施例中，通过磁力沉降将磁性颗粒和未反应过量的纳米颗粒分离。
在优选实施例中，上述微纳孔装置是基于库尔特原理的微纳孔单颗粒计数装置，该装置包括两个充满电解液的腔室，以及联通上述两个充满电解液的腔室的微纳孔，当上述复合体以单颗粒形式通过上述微纳孔时，短暂堵塞离子在上述微纳孔中的流动，形成电脉冲信号。
在优选实施例中，上述电脉冲信号反映上述复合体的电荷状态，通过分析上述电脉冲信号的峰波动得到上述复合体中结合的上述纳米颗粒的状态和/或数量，根据上述状态和/或数量以及双抗夹心结构反应原理计算上述样品体系中待测蛋白的数量。
在优选实施例中，上述微纳孔装置是带微孔的双层膜结构装置，该装置包括两层具有设定间隔距离的带微孔的纳米薄膜，两层纳米薄膜上的微孔相对而将上述纳米薄膜两侧的腔体连通，上述腔体内充满电解液，并且 两侧的腔体分别布置有用于维持离子传输的电极；当上述复合体以单颗粒形式连续通过两个微孔的过程中产生具有时间间隔的一对电脉冲信号。
在优选实施例中，上述方法还包括：通过调节样品中上述复合体的浓度控制不同复合体间的电脉冲信号间隔远大于电脉冲信号对内的时间间隔，并通过对上述电脉冲信号的脉冲强度阈值分析，滤掉未结合纳米颗粒的免疫磁珠产生的信号。
在优选实施例中，对上述复合体的电脉冲信号，通过计算电迁移率得到复合体的粒子表面电位。
在优选实施例中，上述电迁移率通过如下公式计算：
Figure PCTCN2020128990-appb-000001
其中，μ表示电迁移率，v(x)表示复合体粒子连续通过两个微孔的速度，E(x)表示电场分布；
粒子表面电位ξ与电迁移率μ存在如下对应关系：
Figure PCTCN2020128990-appb-000002
其中，η表示电解液的粘度；
以及，粒子表面电位ξ公式如下：
Figure PCTCN2020128990-appb-000003
其中，t表示粒子在微孔中停留时间，A表示每个微孔的纠正系数，V 表示电势差。
在优选实施例中，上述体积/质量状态与电脉冲信号积分面积正相关，上述复合体上的捕获蛋白数与复合体相对于上述免疫磁珠的体积/质量增量成正比，根据上述电脉冲信号积分面积与复合体上的捕获蛋白数的标准曲线关系推算复合体上的捕获蛋白数，进而计算上述样品体系中待测蛋白的数量。
本发明采用免疫磁珠捕获待测微量蛋白，利用免疫磁珠可被磁场吸引及其表面具有特异性功能基团的特性，与具有生物活性的蛋白进行共价结合，免疫磁珠用作待测蛋白的承载体。免疫反应中免疫磁珠包被抗体与具有契合决定簇的抗原(待测蛋白)结合，从血浆等复杂样品体系中高效地分离，并且被标记有纳米颗粒的抗体所识别，实现待测蛋白的定量检测。
此外，在优选实施例中，通过调节免疫磁珠表面的抗体修饰程度，可实现一个或多个抗原蛋白的捕获，所形成的双抗夹心结构有利于提高捕获特异性，同时二抗连接的纳米颗粒能够调节双抗夹心结构复合体的总电荷。因此，本发明的方法能够控制蛋白捕获量，可低至小于10个蛋白/磁珠，结合使用粒径较小的纳米颗粒精确计数捕获的待测抗原蛋白。
附图说明
图1为本发明实施例中双抗夹心结构——单蛋白复合体的原理示意图；
图2为本发明实施例中双抗夹心结构——多蛋白复合体的原理示意图；
图3为本发明实施例中基于库尔特原理的微纳孔单颗粒计数装置示意图；
图4为本发明实施例中通过对电脉冲信号峰的细微波动进行分析判断复合体颗粒的电荷状态的原理示意图；
图5为本发明实施例中带微孔的双层膜结构装置示意图；
图6为本发明实施例中通过微纳加工一体成型的方式制备双层膜结构装置的原理示意图；
图7为本发明实施例中复合体以单颗粒形式连续通过两个微孔的过程中产生一对电脉冲信号的示意图；
图8为本发明实施例中复合体以单颗粒形式连续通过两个微孔的过程中产生的真实电脉冲信号对脉冲图；
图9为本发明实施例中捕获蛋白数与电迁移率(迁移速度)呈线性对应关系图。
图10为本发明实施例中电脉冲信号图(A)以及电脉冲信号积分面积(复合体的体积/质量)与复合体上的捕获蛋白数的标准曲线关系图(B)。
具体实施方式
下面通过具体实施方式结合附图对本发明作进一步详细说明。在以下的实施方式中，很多细节描述是为了使得本发明能被更好的理解。然而，本领域技术人员可以毫不费力的认识到，其中部分特征在不同情况下是可 以省略的，或者可以由其他材料、方法所替代。
另外，说明书中所描述的特点、操作或者特征可以以任意适当的方式结合形成各种实施方式。同时，方法描述中的各步骤或者动作也可以按照本领域技术人员所能显而易见的方式进行顺序调换或调整。因此，说明书和附图中的各种顺序只是为了清楚描述某一个实施例，并不意味着是必须的顺序，除非另有说明其中某个顺序是必须遵循的。
本发明提供了一种检测样品体系中微量蛋白的方法，
本发明实施例中，样品体系可以是各种含有蛋白单一成分的简单体系，也可以是含有一种或多种蛋白同时含有其他成分的复杂体系，包括血液、血浆、血清、组织液、尿液、脑脊液等各种样本。
本发明的方法能够检测样本中的微量蛋白，例如微克、纳克、皮克等级别的蛋白，最低检出限低至单个蛋白分子，可以检出常规免疫检测检出下限以下的微量蛋白，可广泛应用于免疫学检测、微生物检测、细胞分离等领域。
本发明的检测样品体系中微量蛋白的方法包括如下步骤：
提供待测蛋白的一抗修饰的免疫磁珠和二抗修饰的纳米颗粒；
将一抗修饰的免疫磁珠和二抗修饰的纳米颗粒与含有待测蛋白的样品体系混合孵育，形成双抗夹心结构复合体；
使双抗夹心结构复合体以单颗粒形式通过微纳孔装置并引发电脉冲信号，分析电脉冲信号得到复合体的电荷状态或体积/质量状态，根据电 荷状态或体积/质量状态计算样品体系中待测蛋白的数量。
本发明实施例中，一抗和二抗分别与待测蛋白上的不同抗原决定簇结合形成双抗夹心结构，如图1所示，其中Y形结构表示抗体，较大的圆球表示免疫磁珠，其表面修饰有一抗，较小的圆球表示纳米颗粒，其表面修饰有二抗，菱形结构表示待测蛋白，所形成的双抗夹心结构复合体的结构可表示为免疫磁珠-一抗-待测蛋白-二抗-纳米颗粒。图1所示的双抗夹心结构复合体中只有一个待测蛋白(图中菱形结构)，因此也可被称为“单蛋白复合体”。本发明的双抗夹心结构有利于提高捕获特异性。
本发明实施例中，一抗和二抗的来源和种类没有特别限定，例如各种动物来源的多抗或重组表达的单抗等，例如鼠、羊、兔、马等的IgG抗体。在本发明的一个实施例中，一抗是鼠IgG，二抗是羊抗鼠抗体。
本发明实施例中，将一抗修饰到免疫磁珠上的方法有多种，本发明不做限制。在一个优选实施例中，在适于抗体修饰反应的溶液体系中，使一抗与免疫磁珠表面的功能基团共价结合得到一抗修饰的免疫磁珠。其中，免疫磁珠表面的功能基团可以是氨基和羧基等，这样的免疫磁珠分别成为氨基磁珠和羧基磁珠，免疫磁珠表面的功能基团与一抗上的对应基团之间通过形成共价结合而将一抗修饰到免疫磁珠表面。一种典型的一抗修饰免疫磁珠的反应是在EDC(1-Ethyl-3-(3’-dimethylaminopropyl)carbodiimide，1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐)与NHS(N-Hydroxysuccinimide，N-羟基丁二酰亚胺)的作用下实现的。例如，在一个优选实施例中，免疫磁珠 (Ademtech)经MES缓冲液(2-(N-吗啡啉乙磺酸)置换三次，平衡至溶液pH为4-5、离子强度0.1M。平衡后的溶液中分别加入DMSO溶解的EDC与NHS各1mg，反应20分钟后离心弃上清；然后以MES缓冲液复溶弃上清后的沉淀物，加入一抗鼠IgG(Cat.:ab151276,Abcam)0.1mg；混合包被1小时，然后加入终止剂BSA(Cat.:10735108001，Roche)至BSA的浓度为1％，终止反应。
在优选实施例中，通过调节溶液体系中一抗与免疫磁珠的浓度比例，来调节一抗在免疫磁珠表面的修饰程度，从单修饰到多修饰，以此来调节可结合的二抗修饰的纳米颗粒的数目，从而形成特殊复合体，如图2所示，这种双抗夹心结构复合体中有多个待测蛋白(图中菱形结构)，因此也可被称为“多蛋白复合体”。例如，在一个优选实施例中，一抗浓度低于1微克每毫克免疫磁珠，使得一抗在免疫磁珠表面上的修饰程度低于50个抗体每个免疫磁珠；一抗浓度在1-5微克每毫克免疫磁珠，使得一抗在免疫磁珠表面上的修饰程度低于300个抗体每个免疫磁珠；一抗浓度高于50微克每毫克免疫磁珠，使得一抗在免疫磁珠上饱和。通过此方法可以控制免疫磁珠上的一抗修饰量。通过电镜进一步确认修饰程度。
本发明实施例中，纳米颗粒可以是各种材料和各种合适粒径和带电情况的纳米颗粒，例如聚乙烯纳米微球、纳米磁珠、纳米硅球、纳米乳胶等，纳米颗粒的粒径例如可以是大体上小于1微米，同时一般而言相对应的免疫磁珠的粒径至少是大于纳米颗粒的，能够取得较好的效果，利于形成特殊的复合结构。
本发明实施例中，二抗修饰的纳米颗粒的制备原理和方法与一抗修饰的免疫磁珠类似。将二抗修饰到纳米颗粒上的方法有多种，本发明不做限制。在一个优选实施例中，在适于抗体修饰反应的溶液体系中，使二抗与纳米颗粒表面的功能基团共价结合得到二抗修饰的纳米颗粒。由于纳米颗粒上带有一定电量，本发明形成的双抗夹心结构复合体中纳米颗粒的数量就会影响整个复合体的电荷状态。本发明正是通过对复合体的电荷状态进行分析得到单个复合体上纳米颗粒的数量，并进而分析得到待测蛋白的数量。
在本发明的一个典型的实施例中，二抗修饰纳米颗粒的反应是在EDC与NHS的作用下实现的。例如，在一个优选实施例中，二抗是羊抗鼠抗体，羊抗鼠抗体标记的纳米颗粒(Cat.:F122270，aladdin)的制备方法包括如下步骤：l mg纳米颗粒平衡pH为8.0、浓度为0.1M的PBS溶液至纳米颗粒的终浓度为0.1mg/mL；然后分别加入EDC与NHS，至EDC与NHS在溶液中的终浓度均为0.2mg/mL，活化30分钟后，离心弃上清。将弃上清之后的沉淀物以PBS溶液复溶，加入羊抗鼠抗体(Cat.:4T20，HyteSt)，标记25分钟，加入BSA至其浓度为0.5％终止反应。
本发明的蛋白检测方法是基于电脉冲信号进行的，免疫反应所形成的双抗夹心结构复合体在电解液中电泳并通过微孔时会引发电脉冲信号，分析电脉冲信号就能得到复合体的电荷状态，根据电荷状态即可计算样品体系中待测蛋白的数量。在本发明的一个实施例中，双抗夹心结构复合体以单颗粒形式通过微纳孔装置并引发电脉冲信号，分析电脉冲信号得到复合体的电荷状态，根据电荷状态计算样品体系中待测蛋白的数量。
有多种方法可以实现本发明中基于电脉冲信号的蛋白检测方法。其中一种方法是基于库尔特原理(例如美国专利2,656,508，1953)的微纳孔单颗粒计数装置检测不同复合体在微纳孔检测装置中的信号。如图3所示，微纳孔单颗粒计数装置包括两个充满电解液的腔室，即图中腔室31和腔室32，以及联通两个充满电解液的腔室的唯一的微纳孔33，当复合体以单颗粒形式在驱动力(例如，电场、压力或电渗流等)的作用下通过微纳孔33时，短暂堵塞离子在微纳孔33中的流动，形成电脉冲信号。该电脉冲信号可反映离子的大小、结合状态以及电荷形态甚至几何形态等。通过分析电脉冲信号的峰波动得到复合体中纳米颗粒的结合状态(是否结合)以及纳米颗粒的结合数量，根据纳米颗粒的结合状态和/或数量以及双抗夹心结构反应原理计算样品体系中待测蛋白的数量。如图4所示，在本发明的一个实施例中，通过分析电脉冲信号强度的阈值，判断复合体颗粒的电荷状态，对电脉冲信号峰的细微波动进行分析，可以得出复合体颗粒是否结合纳米颗粒，以及结合数量。例如，在图4中，当复合体颗粒上结合有两个纳米颗粒，在电脉冲信号上反映出两个峰；当复合体颗粒上未结合纳米颗粒，在电脉冲信号上未反映出峰；当复合体颗粒上结合有三个纳米颗粒，在电脉冲信号上反映出三个峰。
在本发明的另一个实施例中，微纳孔装置是带微孔的双层膜结构装置，如图5所示，该装置包括两层具有设定间隔距离的带微孔(图中微孔1和微孔2)的纳米薄膜，两层纳米薄膜上的微孔相对而将纳米薄膜两侧的腔体(图中腔体1和腔体2)连通，腔体内充满电解液，并且两侧的腔体分别布置有用于维持离子传输的电极；当蛋白磁珠复合体以单颗粒形式连续 通过两个微孔的过程中产生具有时间间隔的一对电脉冲信号。
上述实施例中，双层膜结构装置的微孔尺寸范围可以是1纳米到10微米，具体可以根据允许蛋白磁珠复合体以单颗粒形式连续通过的要求设定。纳米薄膜可以选择氮化硅、二氧化硅、硅等任何半导体成膜材料或高分子聚合物制成的薄膜。两层纳米薄膜之间具有设定间隔距离，该设定间隔距离可以是1纳米到1微米。双层膜结构装置装配有两个电极，可以是银/氯化银或铂电极。通过施加外加电场，维持平衡的离子传输，体现在稳定的开孔电流。蛋白磁珠复合体在驱动力作用下连续通过两个微孔的过程中产生具有时间间隔的一对电脉冲信号。
本发明的双层膜结构装置可以通过各种合适的方法得到。例如，在本发明的一个实施例中，通过微纳加工一体成型的方式控制双层膜的间距而制备双层膜结构装置。具体原理和步骤如图6所示，步骤一：准备双面抛光的基质材料(硅或者石英等)作为支撑层，在其上表面和下表面上通过热氧化或者气相沉积的方法制备1纳米到10微米的纳米薄膜(图中成膜层)。步骤二：通过预先制备的光刻模板来限定微孔的尺寸和形状以及在膜上的位置。在上下表面涂覆光刻胶，用模板曝光并显影。上下模板可以精确对齐保证两个微孔在空间上保持微米的对准精度。然后用气体刻蚀以未曝光的光刻胶为牺牲层在膜上刻蚀出微孔。步骤三：用湿刻蚀的方法，刻蚀液通过微孔将支撑层刻蚀从而将两个微孔连通。
如图7所示，复合体1、未结合磁珠(即未结合纳米颗粒的免疫磁珠)和复合体2以单颗粒形式连续通过两个微孔的过程中产生一对电脉冲信号， 它们具有一定的时间间隔，该时间间隔反映的是通过微孔的时间，与电迁移率有关，而电迁移率又与电荷状态有关。复合体1和复合体2的电脉冲信号超过了设定的阈值，而未结合磁珠的电脉冲信号未超过设定的阈值。图8示出了复合体以单颗粒形式连续通过两个微孔的过程中产生的真实的电脉冲信号对，可以看出，每个复合体颗粒通过微孔均能产生一对电脉冲信号，并且该对电脉冲信号中的两个电脉冲信号具有一定的时间间隔。
在本发明的一个优选实施例中，通过调节样品中复合体的浓度(例如，小于10皮克每毫升)控制不同复合体间的电脉冲信号间隔远大于电脉冲信号对内的时间间隔，并通过对电脉冲信号的脉冲强度阈值分析，滤掉未结合纳米颗粒的免疫磁珠产生的信号。
根据本发明的双抗夹心结构反应原理，免疫磁珠上捕获的蛋白数决定了与其结合的二抗修饰的纳米颗粒的数量，从而决定复合体的带电量，而复合体的带电量与电迁移率相关。因此，如图9所示，捕获蛋白数与电迁移率(迁移速度)呈线性对应关系。通过计算电迁移率可以得到粒子表面电位，进而计算待测蛋白的数量。
在本发明的一个实施例中，电迁移率通过如下公式计算：
Figure PCTCN2020128990-appb-000004
其中，μ表示电迁移率，v(x)表示复合体粒子连续通过两个微孔的速度，E(x)表示电场分布。
例如，两个微孔之间距离1000纳米，单个复合体颗粒通过两个微孔 的时间间隔为1毫秒，电势差为100毫伏。从而可以计算出电迁移率为10 -8m 2V -1s -1。
同时，粒子表面电位ξ与电迁移率μ存在如下对应关系：
Figure PCTCN2020128990-appb-000005
其中，η表示电解液的粘度。
以及，粒子表面电位ξ公式如下：
Figure PCTCN2020128990-appb-000006
其中，t表示粒子在微孔中停留时间，可由实验得到，A表示每个微孔的纠正系数，可由标准粒子针对每个孔得到，V表示电势差。
例如，在本发明的一个实施例中，纠正系数A为6×10 -6V 2s，可得粒子表面电位ξ为60毫伏。
在本发明的另一个实施例中，根据复合体的体积/质量状态计算样品体系中待测蛋白的数量。具体而言，如图10所示，复合体的体积/质量状态与电脉冲信号积分面积正相关，复合体上的捕获蛋白数与复合体相对于免疫磁珠的体积/质量增量成正比，根据电脉冲信号积分面积(其表征复合体的体积/质量状态)与复合体上的捕获蛋白数的标准曲线关系推算复合体上的捕获蛋白数，进而计算样品体系中待测蛋白的数量。本实施例中，复合体的体积/质量表示复合体的体积、质量或二者的组合，因为根据本发明中的双抗夹心结构反应原理，免疫磁珠上捕获蛋白数决定了与其结合 的二抗修饰的纳米颗粒的数量，而纳米颗粒的数量决定了复合体的体积或质量的增加。
以上应用了具体个例对本发明进行阐述，只是用于帮助理解本发明，并不用以限制本发明。对于本发明所属技术领域的技术人员，依据本发明的思想，还可以做出若干简单推演、变形或替换。

Claims (10)

  1. 一种检测样品体系中微量蛋白的方法，其特征在于，所述方法包括：
    提供待测蛋白的一抗修饰的免疫磁珠和二抗修饰的纳米颗粒；
    将所述一抗修饰的免疫磁珠和二抗修饰的纳米颗粒与含有所述待测蛋白的样品体系混合孵育，形成双抗夹心结构复合体；
    使所述复合体以单颗粒形式通过微纳孔装置并引发电脉冲信号，分析所述电脉冲信号得到所述复合体的电荷状态或体积/质量状态，根据所述电荷状态或体积/质量状态计算所述样品体系中待测蛋白的数量。
  2. 根据权利要求1所述的检测方法，其特征在于，所述方法还包括：在适于抗体修饰反应的溶液体系中，使所述一抗与免疫磁珠表面的功能基团共价结合得到所述一抗修饰的免疫磁珠。
  3. 根据权利要求2所述的检测方法，其特征在于，通过调节所述溶液体系中所述一抗与所述免疫磁珠的浓度比例，来调节所述一抗在所述免疫磁珠表面的修饰程度。
  4. 根据权利要求1所述的检测方法，其特征在于，所述免疫磁珠的粒径至少是所述纳米颗粒的1-1000倍；
    优选地，所述免疫磁珠的粒径大体上为100纳米到10微米，所述纳米颗粒的粒径大体上小于1微米。
  5. 根据权利要求1所述的检测方法，其特征在于，所述微纳孔装置是基于库尔特原理的微纳孔单颗粒计数装置，该装置包括两个充满电解液的 腔室，以及联通所述两个充满电解液的腔室的微纳孔，当所述复合体以单颗粒形式通过所述微纳孔时，短暂堵塞离子在所述微纳孔中的流动，形成电脉冲信号。
  6. 根据权利要求5所述的检测方法，其特征在于，所述电脉冲信号反映所述复合体的电荷状态，通过分析所述电脉冲信号的峰波动得到所述复合体中结合的所述纳米颗粒的状态和/或数量，根据所述状态和/或数量以及双抗夹心结构反应原理计算所述样品体系中待测蛋白的数量。
  7. 根据权利要求1所述的检测方法，其特征在于，所述微纳孔装置是带微孔的双层膜结构装置，该装置包括两层具有设定间隔距离的带微孔的纳米薄膜，两层纳米薄膜上的微孔相对而将所述纳米薄膜两侧的腔体连通，所述腔体内充满电解液，并且两侧的腔体分别布置有用于维持离子传输的电极；当所述复合体以单颗粒形式连续通过两个微孔的过程中产生具有时间间隔的一对电脉冲信号。
  8. 根据权利要求7所述的检测方法，其特征在于，所述方法还包括：通过调节样品中所述复合体的浓度控制不同复合体间的电脉冲信号间隔远大于电脉冲信号对内的时间间隔，并通过对所述电脉冲信号的脉冲强度阈值分析，滤掉未结合纳米颗粒的免疫磁珠产生的信号。
  9. 根据权利要求8所述的检测方法，其特征在于，对所述复合体的电脉冲信号，通过计算电迁移率得到复合体的粒子表面电位；
    优选地，所述电迁移率通过如下公式计算：
    Figure PCTCN2020128990-appb-100001
    其中，μ表示电迁移率，v(x)表示复合体粒子连续通过两个微孔的速度，E(x)表示电场分布；
    粒子表面电位ξ与电迁移率μ存在如下对应关系：
    Figure PCTCN2020128990-appb-100002
    其中，η表示电解液的粘度；
    以及，粒子表面电位ξ公式如下：
    Figure PCTCN2020128990-appb-100003
    其中，t表示粒子在微孔中停留时间，A表示每个微孔的纠正系数，V表示电势差。
  10. 根据权利要求1所述的检测方法，其特征在于，所述体积/质量状态与所述电脉冲信号积分面积正相关，所述复合体上的捕获蛋白数与所述复合体相对于所述免疫磁珠的体积/质量增量成正比，根据所述电脉冲信号积分面积与所述复合体上的捕获蛋白数的标准曲线关系推算所述复合体上的捕获蛋白数，进而计算所述样品体系中待测蛋白的数量。
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