WO2015106588A1 - 纳米模拟酶免疫层析检测方法 - Google Patents

纳米模拟酶免疫层析检测方法 Download PDF

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WO2015106588A1
WO2015106588A1 PCT/CN2014/088540 CN2014088540W WO2015106588A1 WO 2015106588 A1 WO2015106588 A1 WO 2015106588A1 CN 2014088540 W CN2014088540 W CN 2014088540W WO 2015106588 A1 WO2015106588 A1 WO 2015106588A1
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magnetic
molecule
detection
analyte
magnetic nanoparticles
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French (fr)
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阎锡蕴
段德民
张德玺
冯静
宋丽娜
杨东玲
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中国科学院生物物理研究所
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Priority to JP2016547006A priority patent/JP6449309B2/ja
Priority to EP14879160.1A priority patent/EP3096140A4/en
Publication of WO2015106588A1 publication Critical patent/WO2015106588A1/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
    • 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
    • G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
    • 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
    • G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
    • 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
    • G01N33/54346—Nanoparticles
    • 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
    • G01N33/54386—Analytical elements
    • G01N33/54387—Immunochromatographic test strips
    • G01N33/54388—Immunochromatographic test strips based on lateral flow
    • 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
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • 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
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/11—Orthomyxoviridae, e.g. influenza virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/415—Assays involving biological materials from specific organisms or of a specific nature from plants
    • G01N2333/42—Lectins, e.g. concanavalin, phytohaemagglutinin

Definitions

  • the invention belongs to the field of nano materials and biomedical nanotechnology.
  • the present invention relates to magnetic nanoparticle mimetic enzymes and provides a method thereof for use in immunochromatographic biomolecule detection.
  • Colloidal gold immunochromatography is a method of detecting colloidal gold as a marker developed in the early 1990s. It combines immunoaffinity, blotting and spot thin layer chromatography. Due to the continuous reaction of the narrower cellulose membrane, all the samples are concentrated and aggregated, which improves the sensitivity of the reaction and accelerates the reaction speed. The operation time is only 3 to 15 minutes. The principle is to fix a specific antibody (or antigen) to a certain zone of the nitrocellulose membrane.
  • Magnetic nanoparticles have good biocompatibility, which has the unique properties of nanomaterials, such as small particle size, large specific surface area, high coupling capacity, magnetic responsiveness and superparamagnetism, and can be under constant magnetic field. Aggregation and localization, absorption of electromagnetic wave heat in an alternating magnetic field, using these characteristics, magnetic nanoparticles are widely used in magnetic resonance contrast agents, magnetic targeting drug carriers, cell and biomolecule separation, biosensing and detection, and magnetic induction tumors. Hyperthermia and other biological fields.
  • Magnetic Immunochromatography has evolved as a new generation of single-part rapid quantitative detection technology. It replaces traditional markers with superparamagnetic nanoparticles (colloidal gold, latex particles). Etc. to perform immunochromatography, and finally read the magnetic field intensity of the magnetic particles combined at the detection line by a magnetic signal reader to qualitatively and quantitatively judge the sample to be tested.
  • a magnetic signal reader to qualitatively and quantitatively judge the sample to be tested.
  • domestically formed magnetic signal readers have not yet been listed, and only a few foreign companies (such as MagnaBioSciences in the United States) have this technology. However, its price is expensive, thus limiting the development and promotion of magnetic immunochromatography.
  • magnetic nanoparticles can catalyze the substrate of horseradish peroxidase, such as catalyzing 3,3,5,5-tetramethyl Aniline (TMB) produces a blue product that catalyzes the formation of a brown precipitate of diaminobenzidine (DAB), which catalyzes the formation of an orange-red product from o-phenylenediamine (OPD).
  • TMB horseradish peroxidase
  • DAB diaminobenzidine
  • OPD o-phenylenediamine
  • the catalytic activity depends on pH, temperature and hydrogen peroxide concentration. The catalytic mechanism is consistent with the ping-pong mechanism.
  • the catalytic activity of the magnetic nanoparticles is enhanced as the particle size of the particles is decreased, and the smaller the particle size of the particles, the higher the catalytic activity. After the magnetic particles have a particle size on the order of micrometers, the catalytic activity is reduced to near zero.
  • the mimetic enzyme activity of magnetic nanoparticles has more advantages than the horseradish peroxidase (HRP) of protein preparations: (1) proteases are easily denatured at extreme pH and temperature, and are also easily degraded by proteases.
  • the magnetic nanoparticles are stable under extreme conditions; (2) the production cost of the protease is high, and the preparation of the magnetic nanoparticles is simple and inexpensive; (3) since the magnetic nanoparticles have superparamagnetism, the magnets can be recycled and reused; At the same time, based on the magnetic controllability of magnetic nanoparticles, it has expanded its application as a mimic enzyme.
  • the object of the present invention is to overcome the deficiencies of the existing immunochromatography technology, and provide a nano-simulative enzyme immunochromatographic detection method, the basic principle of which is the same as the colloidal gold test strip, firstly, a specific antigen-specific antibody A and magnetic The nanoparticle is coupled to prepare a magnetic particle pad, and then another antibody B against the antigen is immobilized on a specific zone of the nitrocellulose membrane to form a detection line (T line), and the antibody against the antibody A (secondary antibody) is also A specific zone fixed to the nitrocellulose membrane forms a quality control line (C line), parallel to the T line, and a magnetic immunochromatographic test paper is assembled and prepared.
  • T line detection line
  • C line quality control line
  • the sample When one end of the dried nitrocellulose membrane is immersed in the sample, the sample will move forward along the membrane due to capillary action.
  • the antibody A on the magnetic particle When moving to the magnetic particle pad, the antibody A on the magnetic particle will react with the antigen to form an antigen-antibody.
  • the complex of A-magnetic particles continues to move by capillary action to the T-line region where another antibody B is immobilized, the antigen in the sample will react with this antibody B, and finally the antibody B-antigen-antibody A- Magnetic particle
  • the granule complex; the magnetic particle antibody probe without antigen binding continues to move forward, and binds to the secondary antibody at the C line to form a secondary antibody-antibody A-magnetic particle complex, and the magnetic particles are at the T line and the C line. Gather.
  • the concentration of the antigen in the sample is high, then there are many magnetic particles accumulated at the T line, which will show the color of the magnetic particles; if the concentration of the antigen in the sample is very low, the magnetic particles accumulated at the T line are few. Not enough to show the color of the magnetic particles.
  • peroxide and hydrogen-donating substrates such as TMB, DAB, etc. are added, and a large amount of precipitate is generated by the peroxidase catalysis of the magnetic particles to enhance the strong detection signal to detect the low concentration. Antigenic substance.
  • the technology integrates the catalytic activity and magnetic separation characteristics of the magnetic nanoparticle peroxidase, and is subjected to enzymatic action by magnetic particles by adding a peroxide and a hydrogen supply substrate such as o-phenylenediamine (DAB) after chromatography.
  • a peroxide and a hydrogen supply substrate such as o-phenylenediamine (DAB) after chromatography.
  • DAB o-phenylenediamine
  • a method for detecting a nano-mimetic enzyme immunochromatography comprising: 1) preparing a magnetic nanoparticle probe by coupling a biomolecule that specifically binds the antigen to be tested to a surface of the magnetic nanoparticle with a suitable size to prepare a magnetic sample Particle pad; 2) Assembly and preparation of magnetic immunochromatographic test paper; 3) Chromatographic reaction, the antigen to be detected reacts with magnetic nanoprobe, detection line antibody, and quality control line antibody to form a sandwich complex, and the positive sample will be at the T line.
  • Forming magnetic particle aggregation 4) color reaction, adding peroxide and hydrogen supply substrate such as TMB, DAB, etc., utilizing the enzymatic action of magnetic particles to generate a large amount of precipitate to enhance the detection signal; 5) according to the experimental results, achieving the target Qualitative and semi-quantitative detection of molecules.
  • the present invention provides a nano-synthesis enzyme immunochromatographic detection method, wherein the assembly and preparation of a magnetic immunochromatographic test strip is a magnetic particle pad, a sample pad, and a water-absorbing pad that binds an antibody corresponding to the antigen to be detected.
  • the mats are sequentially attached to the bottom plate in a staggered manner, and then assembled on the upper layer covered with a transparent plastic sealing film, wherein the coated film is pre-coated with a detection line and a quality control line for the antigen to be tested.
  • the nano-mimetic enzyme immunochromatographic detection method described above is characterized in that the magnetic nanoparticles may be any one of a spherical shape, a rod shape, a cuboid shape, a triangular shape, a polygonal shape, and the like; Nano to 500 nanometers; it may be bare magnetic particles, or may be a protein shell such as a virus coat, a transferrin coat, a ferritin-coated magnetic particle; it may be Fe 3 O 4 magnetic particles, or It is an Fe 2 O 3 magnetic particle whose outer layer is modified with a divalent iron ion reagent.
  • the above nano-mimetic enzyme immunochromatography detection method is characterized in that the hydrogen supply substrate comprises tetramethylbenzidine TMB, tetramethylbenzidine sulfate TMBS, o-phenylenediamine OPD, diamino group.
  • the hydrogen supply substrate comprises tetramethylbenzidine TMB, tetramethylbenzidine sulfate TMBS, o-phenylenediamine OPD, diamino group.
  • Benzidine DAB diaminobenzidine tetrahydrochloride DAB-4HCl, 5-aminosalicylic acid 5-AS, o-toluidine OT or diazonium diamine salt ABTS;
  • the peroxide comprises hydrogen peroxide or urea peroxide Wait.
  • the nano-mimetic enzyme immunochromatography detection method described above is characterized in that the specific biomolecule comprises a protein, a nucleic acid, a polypeptide; and the target molecule is present in a solution or a body fluid.
  • the invention aims at the current lack of signal amplification function of colloidal gold and relatively low sensitivity, and combines the latest scientific findings to provide a nano-simulative enzyme immunochromatographic detection method, which integrates magnetic nanoparticle peroxidase catalytic activity and magnetic
  • the separation property is integrated, and after chromatography, a large amount of brown precipitate is generated by using the enzyme activity of the magnetic particles by adding a peroxide and a hydrogen supply substrate such as o-phenylenediamine (DAB), and the detection signal is amplified 10-100 times, so that the detection result is obtained.
  • DAB o-phenylenediamine
  • the present invention provides the following:
  • a method for detecting a nano-mimetic enzyme immunochromatography for detecting a sample in a liquid sample comprising the steps of:
  • a color reaction is carried out by adding a hydrogen donor substrate and a peroxide to the capture probe passed through step 4).
  • analyte is a protein, a polypeptide or a nucleic acid.
  • the hydrogen donor substrate comprises tetramethylbenzidine (TMB), tetramethylbenzidine sulfate (TMBS), o-phenylenediamine (OPD), diaminobenzidine ( DAB), diaminobenzidine tetrahydrochloride (DAB-4HCl), 5-aminosalicylic acid (5-AS), o-toluidine (OT) or diazonium diammonium salt (ABTS).
  • TMB tetramethylbenzidine
  • TMBS tetramethylbenzidine sulfate
  • OPD o-phenylenediamine
  • DAB diaminobenzidine
  • DAB-4HCl diaminobenzidine tetrahydrochloride
  • 5-aminosalicylic acid 5-AS
  • OT o-toluidine
  • ABTS diazonium diammonium salt
  • a nano-mimetic enzyme immunochromatographic detection device for detecting a test substance in a liquid sample, the device comprising the following items arranged in sequence on a bottom plate:
  • a sample pad for receiving the liquid sample and filtering impurities in the sample
  • a magnetic nanoparticle pad comprising magnetic nanoparticles coupled to a first molecule capable of specifically binding to the analyte
  • a detection line comprising a second molecule capable of specifically binding to the analyte
  • An absorbent pad generally made of a thicker filter paper or similar absorbent material, for providing motive power
  • the particle diameter of the magnetic nanoparticles is preferably in the range of 10 nm to 500 nm
  • the magnetic nanoparticles are preferably Fe 3 O 4 magnetic nanoparticles
  • the analyte is preferably a protein, a polypeptide or a nucleic acid, and more preferably, the analyte is a protein and the first molecule and the second molecule are specific antibodies to the protein, preferably Monoclonal antibodies,
  • first molecule and the magnetic nanoparticle are preferably coupled by an EDC-NHS method
  • the hydrogen donor substrate preferably comprises tetramethylbenzidine (TMB), tetramethylbenzidine sulfate (TMBS), o-phenylenediamine (OPD), diaminobenzidine (DAB), diaminobenzidine IV Hydrochloric acid (DAB-4HCl), 5-aminosalicylic acid (5-AS), o-toluidine (OT) or diazonium diammonium salt (ABTS), and
  • the peroxide preferably comprises hydrogen peroxide and urea peroxide.
  • a quality control line is further disposed after the detection line, the quality control line being fixed with a third molecule capable of specifically binding to the first molecule.
  • FIG. 1 Schematic diagram of colloidal gold immunochromatography
  • Figure 2 Schematic representation of one embodiment of a nano-mimetic enzyme immunochromatography according to the present invention
  • FIG. 3 Screening of high affinity Acacia toxin monoclonal antibodies by enzyme-linked immunosorbent assay (ELISA);
  • Figure 4 Subtype identification of acacia toxin monoclonal antibody
  • Figure 5 Western blotting method to identify the antigenic epitope of the antibody recognizing Acacia toxin
  • Figure 6 Screening of paired antibodies against acacia toxin by double-antibody sandwich enzyme-linked immunosorbent assay (ELISA);
  • Figure 8 Preparation of magnetic particle antibody probe and dot blot test
  • Figure 9 Comparison of the sensitivity of magnetic particle nanosynthesis enzyme immunochromatography and colloidal gold immunochromatography to detect the sensitivity of acacia.
  • Figure 10 Comparison of the sensitivity of nano-mimetic enzyme immunochromatography with traditional colloidal gold assay for influenza virus detection.
  • Example 1 Nano-mimetic enzyme immunochromatographic assay for the detection of acacia toxin
  • Acacia toxin is a component of the seeds of the legume Acacia. It is one of the most toxic plant toxins found so far. It is very toxic to humans, animals and insects. Chew a Acacia seed. In order to cause death, the present invention detects acacia toxin (Acacia toxin and below)
  • the mentioned ricin is provided by the Academy of Military Medical Sciences as an example to illustrate the sensitivity and practicability of the nano-analog enzyme immunochromatographic assay.
  • Hybridoma cells methods for the preparation of monoclonal antibodies are known in the art and can be found, for example, in Kohler and Milstein, Nature 256:495, 1975; Yeh et al, Proc. Natl. Acad. Sci. USA, 1979; Yeh et al. , Int. J.
  • Splenocytes were fused with SP2/0-Agl4 murine myeloma cells in the presence of polyethylene glycol (PEG) and HAT selective medium (containing hypoxan, hypopterin, aminopterin and thymidine) Hybridomas were screened for thymidin medium to obtain hybridoma cells.
  • PEG polyethylene glycol
  • HAT selective medium containing hypoxan, hypopterin, aminopterin and thymidine
  • Hybridomas were screened for thymidin medium to obtain hybridoma cells.
  • the antibody which has strong binding ability to the natural acacia toxin was screened by ELISA, and four antibodies were obtained, which were named Abrin-1, Abrin-2, Abrin-3, Abrin-4, and hybridoma cells secreting these antibodies were simultaneously obtained. , followed by Abrin-1, Abrin-2, Abrin-3, Abrin-4.
  • the specific method is as follows: firstly, 50 ⁇ l of 2 ⁇ g/ml of acacia toxin protein was coated overnight in a 96-well ELISA plate; washed three times with PBST, and 5% BSA-PBS was added for 1 hour; hybridoma cell culture with monoclonal antibody was separately added.
  • the supernatant was incubated at 37 ° C for 1 h; washed three times with PBST, incubated with HRP-labeled goat anti-mouse antibody for 1 h; washed three times with PBST, and added TMB chromogenic substrate (200 ng/ml TMB, 0.03% H 2 O 2 , pH 4.5) Color development, 50 ⁇ l / well, reaction at 37 ° C for 15 min, 50 ⁇ l / well 2M sulfuric acid solution was added to terminate the reaction, and the plate reader was read at 450 nm. As can be seen from the ELISA results, the affinity of Abrin-3 and Abrin-4 can reach 1:5000, while the affinity of Abrin-1 and Abrin-2 is as high as 1:50,000 (Fig. 3).
  • the specific methods are as follows: Abrin-1, Abrin-2, Abrin-3, and Abrin-4 were cultured in large quantities to prepare cell suspensions, and BALB/C mice were injected intraperitoneally with 6-week-old BALB/C mice. (Sigma-Aldrich) 0.5 ml/only, after about ten days, ascites was collected and the supernatant was centrifuged. Monoclonal antibodies were purified from ascites by protein G affinity chromatography (Roche). Purified monoclonal antibodies are sterile filtered, refrigerated or cryopreserved.
  • the mouse antibody subtype identification kit (BD Pharmingen) was used, and according to the instruction, the antibody Abrin-1 was identified as belonging to IgG2a, and Abrin-2, Abrin-3 and Abrin-4 belonged to the IgGl subtype (Fig. 4).
  • the peroxidase HR is first labeled to the antibody Abrin-1 by a glutaraldehyde two-step method or a sodium periodate method, and then the Abrin-2, Abrin-3, and Abrin-4 antibodies are at 0.02M.
  • PBS (pH 7.2) was diluted to 2 ⁇ g / ml, then added to a 96-well plate in an amount of 50 ⁇ l / well, coated at 4 ° C overnight; washed three times with PBST, added 5% BSA-PBS for 1 h; 100, 10, 1, 0.1 ng/ml of acacia toxin and 10 ng/ml of ricin as a negative control (Ctrl), incubated at 37 ° C for 1 h; washed three times with PBST, and added HRP-labeled Abrin at a concentration of 1 ⁇ g/ml -1 antibody was incubated for 1 h; washed three times with PBST, added with TMB chromogenic substrate (200 ng/ml TMB, 0.03% H 2 O 2 , pH 4.5), 50 ⁇ l/well, reacted at 37 ° C for 15 min, added The reaction was stopped with 50 ⁇ l/well 2M sulfuric acid solution and
  • EDC-NHS carbodiimide hydrochloride
  • NHS hydroxysuccinimide
  • MNPs@Abrin-1 is formed on the three ferromagnetic particles.
  • the specific steps are as follows: Weigh an appropriate amount of ferroferric oxide magnetic particles, add 50 mg/ml of NHS, 50 ⁇ l of EDC, incubate for 30 minutes at room temperature, and wash with deionized water to remove excess NHS/EDC.
  • a. Preparation of coating film The antibody Abrin-2 of Acacia toxin and the goat anti-mouse antibody (secondary antibody) purchased by the company were diluted to 0.5 mg//, respectively, with a coating buffer (0.02 M phosphate buffer, pH 7.2). Ml and 1 mg/ml were uniformly sprayed on a 3.5 cm wide nitrocellulose membrane at a spacing of 0.8 cm in a sequence of 1 ⁇ l/cm using a quantitative spray device to form a detection line (T line). Antibody band and quality control line (C line) antibody band. After drying at room temperature for 30 min, it was immersed in a blocking solution (0.02 M PBS containing 0.5% BSA, pH 7.2) for 10 min, dried at 25-35 ° C for 8 hours, and sealed with a desiccant for use.
  • a coating buffer 0.02 M phosphate buffer, pH 7.2
  • Preparation of magnetic particle probe pad uniformly spray the processed magnetic particle probe (as prepared in 8) with a special nozzle of a film sprayer at a thickness of 50 ⁇ l/cm on a 0.8 cm wide fiberglass mat. Freeze-dried overnight, sealed with desiccant for later use.
  • sample pad Dip a 1.8 cm wide sample pad (hydrophilic glass fiber) into the sample pad treatment solution (1-5% Casein, 0.1-1% PVA (Polyvinyl Alcohol), 0.01 -0.2% Tween 20, 0.02 M PBS, pH 7.2) was treated for 1 hour, taken out and dried at 25-35 ° C for 8 hours.
  • PVA Polyvinyl Alcohol
  • test strip 3.5cm coated film, 0.8cm magnetic particle probe pad, 1.8cm sample pad and 2.5cm absorbent pad are sequentially pasted on the backing (base plate) in the form of 2mm intertwined (the stacking sequence is shown in Figure 2), covered with a transparent plastic sealing film, assembled into test paper; The machine will cut the assembled test paper into 0.5cm wide finished test strips; place the cut test strips in the card slot on the plastic low card, cover the upper cover, and use the press card to put the upper and lower plastics The card is pressed tightly, and the desiccant is added to the room temperature for storage (the above-mentioned coating film, magnetic particle probe pad, sample pad, absorbent pad and the interdigitated width can be appropriately adjusted by those skilled in the art according to actual needs).
  • Micropipette Take 50 ⁇ l of gradient aconite sample solution (concentration 100ng/ml, 10ng/ml, 1ng/ml, 0ng/ml) onto the sample pad on the test card, then add 50 ⁇ l chromatography buffer (1% Tween 20,0.5% Triton X -100,1% NP-40,0.05% NaN 3, 20mM PBS, pH 7.2), waiting for the reaction 15min.
  • gradient aconite sample solution concentration 100ng/ml, 10ng/ml, 1ng/ml, 0ng/ml
  • 50 ⁇ l chromatography buffer 1% Tween 20,0.5% Triton X -100,1% NP-40,0.05% NaN 3, 20mM PBS, pH 7.2
  • the monoclonal antibody Abrin-1 conjugated to the azithroxin on the magnetic particle the complex formed by the combination with the acacia toxin in the sample solution continues to move forward, and the acacia toxin at the detection line (T line)
  • Another antibody, Abrin-2 binds to form agglomeration of magnetic particles, while the magnetic particle Abrin-1 antibody probe that does not bind to the acacia toxin continues to move to the quality control line (line C) to form magnetic particles by interacting with its secondary antibody. Gather.
  • Example 2 Nano-mimetic enzyme immunochromatographic detection method for detecting influenza virus
  • Antibody source purchased by the company, numbered FluA-1 and FluA-2 (manufacturer: Medix Biochemica, article number: FluA-1: 100081, FluA-2: 100083).
  • the micropipette takes 50 ⁇ l of the influenza virus sample solution containing the gradient concentration (the virus titer is 1.25 ⁇ 10 4 , 6.25 ⁇ 10 3 , 3.1 ⁇ 10 3 , 1.56 ⁇ 10 3 , 7.8 ⁇ 10 2 , 3.9 ⁇ in terms of PFU. 10 2 , 1.95 ⁇ 10 2 )
  • Add to the sample pad on the test card then add 50 ⁇ l of chromatography buffer (1% Tween 20, 0.5% Triton X-100, 1% NP-40, 0.05% NaN 3 , 20 mM PBS, pH 7.2), waiting for the reaction to proceed for 15 min.
  • the magnetic particle Since the magnetic particle is conjugated with the influenza virus monoclonal antibody FluA-1, the complex formed after binding to the influenza virus in the sample solution continues to move forward, and the other line of the influenza virus at the detection line (T line) An antibody FluA-2 binds to form agglomeration of magnetic particles, while a magnetic particle FluA-1 antibody probe that does not bind to influenza virus continues to move to the quality control line (line C) to form agglomeration of magnetic particles by its secondary antibody.
  • the present invention is a nano-simulative enzyme immunochromatographic detection method, which integrates the catalytic activity and magnetic separation characteristics of magnetic nanoparticle peroxidase, and adds peroxide and hydrogen supply after immunochromatography.
  • the substrate such as o-phenylenediamine (DAB)
  • DAB o-phenylenediamine
  • the method has the advantages of simple, rapid and high sensitivity, and is very suitable for on-site detection.
  • the function of the granule peroxidase has a broad application prospect and is a novel, novel and practical new technology.

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Abstract

一种用于检测液体样品中的待测物的纳米模拟酶免疫层析检测方法及检测装置。检测方法包括以下步骤：1）提供检测探针，将磁性纳米颗粒与能够与待测物特异性结合的第一分子偶联制备检测探针；2）提供固定化的能够与待测物特异性结合的第二分子作为捕获探针；3）液体样品与检测探针接触；4）将与检测探针接触过的液体样品与捕获探针接触；以及5）向经过步骤4）的捕获探针加入供氢底物和过氧化物进行显色反应。

Description

纳米模拟酶免疫层析检测方法 技术领域
本发明属于纳米材料及生物医学纳米技术领域。本发明涉及磁性纳米颗粒模拟酶，并提供了其应用于免疫层析生物分子检测的方法。
背景技术
胶体金免疫层析法是20世纪90年代初发展起来的以胶体金为标记物的检测方法，它把免疫亲和技术、印渍技术和斑点薄层层析技术组合在一起。由于用该层析条检测的时候，所有样品均经过较窄的纤维素膜的持续性反应，实际上对被测物质起到了浓缩、聚集作用，提高了反应的灵敏度，加快了反应速度，整个操作时间仅需3～15分钟。其原理是将特异的抗体(或抗原)先固定于硝酸纤维素膜的某一区带，当干燥的硝酸纤维素膜一端浸入样品后，由于毛细管作用，样品将沿着该膜向前移动(层析)，当移动至固定有抗体、抗原的区域时，样品中相应的抗原即与该抗体发生特异性结合，若用免疫胶体金作标记物可使该区域显示一定的颜色，从而实现特异性的免疫诊断。传统的胶体金免疫层析技术具有操作简便、经济、快速等特点，但由于灵敏度相对较低，严重制约了其在生物分子检测方面的广泛应用，信号放大是解决免疫层析技术灵敏度低的关键所在。
磁性纳米颗粒具有良好的生物相容性，其既具有纳米材料所特有的性质，如粒径小、比表面积大、偶联容量高，又具有磁响应性及超顺磁性，可以在恒定磁场下聚集和定位、在交变磁场下吸收电磁波产热，利用这些特性，磁性纳米颗粒被广泛应用于磁共振对比剂、磁靶向药物载体、细胞与生物分子分离、生物传感与检测以及磁感应肿瘤热疗等生物学领域。
最近几年，磁性免疫层析(Magnetic ImmunoChromatographic Test，MICT)作为一种新一代单人份快速定量检测技术逐渐发展起来，它是以超顺磁性纳米颗粒代替传统的标记物(胶体金，乳胶颗粒等)来进行免疫层析，最后通过磁信号阅读仪读取结合在检测线处磁颗粒的磁场强度，从而对所检样品进行定性定量判断。目前国内成型的磁信号阅读仪还没有上市，国际上也只有少数外国公司(如美国MagnaBioSciences公司)具有该项技术， 但其价格昂贵，因而限制了磁性免疫层析的发展和推广。
近年来，我们课题组研究发现磁纳米颗粒具有内在的模拟酶活性，可替代过氧化物酶进行免疫检测(阎锡蕴等，Nature Nanotechnology.2007)。最近，我们课题组又研制了三功能(识别、催化、磁性)于一体的新型免疫组化检测试剂，发现磁颗粒表面包裹蛋白分子后仍然具有酶活性(阎锡蕴等，Nature Nanotechnology.2012)。这种催化活性与辣根过氧化物酶相似，在过氧化氢存在下，磁性纳米颗粒可以催化辣根过氧化物酶的底物，如可以催化3，3，5，5-四甲基联苯胺(TMB)生成蓝色的产物，催化二氨基联苯胺(DAB)生成棕色沉淀，催化邻苯二胺(OPD)生成橘红色产物，催化活性依赖于pH值、温度和过氧化氢浓度，其催化机理符合乒乓机制。同时还发现磁性纳米颗粒的催化活性随着颗粒粒径的减小而增强，颗粒的粒径越小，其催化活性越高。磁性颗粒的粒径达微米量级后，催化活性降低至接近零值。磁性纳米颗粒的模拟酶活性，相比蛋白制剂的辣根过氧化物酶(horseradishperoxidase，HRP)，具有更多的优势：(1)蛋白酶在极端pH和温度下容易变性，同时也容易被蛋白酶降解，而磁性纳米颗粒在极端条件下很稳定；(2)蛋白酶的生产成本很高，而磁性纳米颗粒制备简单、廉价；(3)由于磁性纳米颗粒具有超顺磁性，用磁铁可以回收反复利用；同时基于磁性纳米颗粒的磁可控性，拓展了其作为模拟酶的应用领域。
发明内容
本发明的目的是克服现有免疫层析技术存在的不足，提供一种纳米模拟酶免疫层析检测方法，其基本原理同胶体金试纸条，首先将某一特定抗原特异的抗体A与磁性纳米颗粒偶联，制备磁颗粒垫，然后将另一种针对此抗原的抗体B固定于硝酸纤维素膜的特定区带形成检测线(T线)，将抗抗体A的抗体(二抗)也固定于硝酸纤维素膜的特定区带形成质控线(C线)，与T线平行，组装和制备磁性免疫层析试纸。当干燥的硝酸纤维素膜一端浸入样品后，由于毛细管作用，样品将沿着该膜向前移动，当移动至磁颗粒垫时，磁颗粒上的抗体A就会与抗原反应，生成抗原-抗体A-磁颗粒的复合物，继续经毛细管作用移动到固定有另一抗体B的T线区域时，样品中的抗原就会与这一抗体B发生反应，最后生成抗体B-抗原-抗体A-磁颗 粒复合物；而没有结合抗原的磁颗粒抗体探针继续向前移动，在C线处与二抗结合形成二抗-抗体A-磁颗粒复合物，磁颗粒就会在T线和C线处聚集。如果样品中的抗原浓度较高，那么在T线处聚集的磁颗粒也多，会显示出磁颗粒的颜色；如果样品中的抗原浓度很低时，则T线处聚集的磁颗粒很少，不足以显示出磁颗粒的颜色，这时加入过氧化物和供氢底物如TMB、DAB等，利用磁颗粒的过氧化物酶催化作用产生大量沉淀而增强强检测信号，以检测出低浓度的抗原物质。该技术集磁性纳米颗粒过氧化物酶催化活性及磁分离特性于一体，层析后通过加入过氧化物和供氢底物如邻苯二胺(DAB)等，利用磁颗粒的酶催化作用产生棕色沉淀，从而增强检测信号，提高灵敏度，使得检测结果通过肉眼观察即可以判定，免除了对仪器的依赖性，基于此新技术的生物样本检测，简便快速、非常适合现场使用。
本发明通过以下技术方案来实现：
纳米模拟酶免疫层析检测方法，所述方法包括：1)采用合适尺寸的磁性纳米颗粒，将特异性结合待测抗原的生物分子偶联于其表面制备特异性纳米颗粒探针，制备出磁颗粒垫；2)组装和制备磁性免疫层析试纸；3)层析反应，待检抗原与磁纳米探针、检测线抗体、质控线抗体反应形成夹心复合物，阳性样品会在T线处形成磁颗粒聚集；4)显色反应，加入过氧化物和供氢底物如TMB、DAB等，利用磁颗粒的酶催化作用产生大量沉淀而增强检测信号；5)根据实验结果，实现对靶标分子的定性和半定量检测。
进一步地，本发明提供纳米模拟酶免疫层析检测方法，其中所述组装和制备磁性免疫层析试纸条是将包被膜、结合了待检抗原所对应抗体的磁颗粒垫、样品垫、吸水垫以相互交错的形式依次粘贴在底板上，然后在上层覆盖透明塑料密封膜组装而成，其中所述的包被膜上预包被有待检抗原的检测线和质控线。
更进一步地，上述的纳米模拟酶免疫层析检测方法，其特征在于：所述磁性纳米颗粒可以是球形、棒形、立方形、三角形、多角形等形状的任意一种；其粒径在10纳米至500纳米范围内；其可以是裸露的磁颗粒、也可以是蛋白外壳如病毒外壳、转铁蛋白外壳、铁蛋白外壳包被的磁颗粒；其可以是Fe3O4磁颗粒，也可以是外层修饰有二价铁离子试剂的Fe2O3磁 颗粒。
更进一步地，上述的纳米模拟酶免疫层析检测方法，其特征在于：所述供氢底物包括四甲基联苯胺TMB、四甲基联苯胺硫酸盐TMBS、邻苯二胺OPD、二氨基联苯胺DAB、二氨基联苯胺四盐酸DAB-4HCl、5-氨基水杨酸5-AS、邻联甲苯胺OT或连氮二铵盐ABTS；所述过氧化物包括过氧化氢或过氧化脲等。
更进一步地，上述的纳米模拟酶免疫层析检测方法，其特征在于：所述特异性生物分子包括蛋白、核酸、多肽；所述的靶标分子存在于溶液或体液中。
本发明技术方案突出的实质性特点和显著的进步主要体现在：
本发明针对目前胶体金无信号放大功能、灵敏度相对较低的不足，结合最新的科学发现，提供一种纳米模拟酶免疫层析检测方法，该技术集磁性纳米颗粒过氧化物酶催化活性及磁分离特性于一体，层析后通过加入过氧化物和供氢底物如邻苯二胺(DAB)等，利用磁颗粒的酶活性产生大量棕色沉淀，放大检测信号10-100倍，使得检测结果通过肉眼观察即可以判定，免除了对仪器的依赖性，基于此新技术的生物样本检测，简便快速、灵敏度高、非常适合现场使用，是一项具有新颖性、创造性、实用性的新技术。
更具体地，本发明提供以下各项：
1.一种用于检测液体样品中的待测物的纳米模拟酶免疫层析检测方法，所述方法依次包括以下步骤：
1)提供检测探针，所述检测探针通过将磁性纳米颗粒与能够与所述待测物特异性结合的第一分子偶联制备；
2)提供捕获探针，所述捕获探针是固定化的能够与所述待测物特异性结合的第二分子；
3)使所述液体样品与所述检测探针接触；
4)使与所述检测探针接触过的所述液体样品与所述捕获探针接触；以及
5)向经过步骤4)的所述捕获探针中加入供氢底物和过氧化物进行显色反应。
2.根据1所述的方法，其中所述磁性纳米颗粒的粒径在10纳米至500 纳米范围内。
3.根据1所述的方法，其中所述磁性纳米颗粒是Fe3O4磁性纳米颗粒。
4.根据1所述的方法，其中所述待测物是蛋白质、多肽或核酸。
5.根据1所述的方法，其中所述待测物是蛋白质，并且所述第一分子和所述第二分子是针对所述蛋白质的特异性抗体，优选是单克隆抗体。
6.根据5所述的方法，其中所述第一分子与所述磁性纳米颗粒通过EDC-NHS法偶联。
7.根据1所述的方法，其中所述供氢底物包括四甲基联苯胺(TMB)、四甲基联苯胺硫酸盐(TMBS)、邻苯二胺(OPD)、二氨基联苯胺(DAB)、二氨基联苯胺四盐酸(DAB-4HCl)、5-氨基水杨酸(5-AS)、邻联甲苯胺(OT)或连氮二铵盐(ABTS)。
8.根据1所述的方法，其中所述过氧化物包括过氧化氢和过氧化脲。
9.一种用于检测液体样品中的待测物的纳米模拟酶免疫层析检测装置，所述装置包括依次设置在底板上的以下各项：
样品垫，所述样品垫用于承接所述液体样品并滤过所述样品中的杂质；
磁性纳米颗粒垫，所述磁性纳米颗粒垫包含与能够与所述待测物特异性结合的第一分子偶联的磁性纳米颗粒；
检测线，所述检测线包含能够与所述待测物特异性结合的第二分子；
吸收垫，所述吸收垫一般由较厚的滤纸或类似的吸水材料制成，用于提供层析的动力，
其中所述磁性纳米颗粒的粒径优选在10纳米至500纳米范围内，
其中所述磁性纳米颗粒优选是Fe3O4磁性纳米颗粒，
其中所述待测物优选是蛋白质、多肽或核酸，并且更优选地，所述待测物是蛋白质而所述第一分子和所述第二分子是针对所述蛋白质的特异性抗体，优选是单克隆抗体，
其中所述第一分子与所述磁性纳米颗粒优选通过EDC-NHS法偶联，
其中所述供氢底物优选包括四甲基联苯胺(TMB)、四甲基联苯胺硫酸盐(TMBS)、邻苯二胺(OPD)、二氨基联苯胺(DAB)、二氨基联苯胺四盐酸(DAB-4HCl)、5-氨基水杨酸(5-AS)、邻联甲苯胺(OT)或连氮二铵盐(ABTS)， 并且
其中所述过氧化物优选包括过氧化氢和过氧化脲。
10.根据9所述的检测装置，其中在所述检测线之后还设置有质控线，所述质控线固定有能够与所述第一分子特异性结合的第三分子。
附图说明
下面结合附图对本发明技术方案作进一步说明：
图1：胶体金免疫层析法示意图
图2：根据本发明纳米模拟酶免疫层析法的一个实施方案的示意图；
图3：酶联免疫(ELISA)方法筛选高亲和力的相思子毒素单克隆抗体；
图4：相思子毒素单克隆抗体的亚型鉴定；
图5：免疫印迹(Western blotting)方法鉴定抗体识别相思子毒素的抗原表位；
图6：双抗夹心酶联免疫(ELISA)方法筛选相思子毒素的配对抗体；
图7：磁性纳米颗粒的制备；
图8：磁颗粒抗体探针的制备及点印迹(Dot blot)检验；
图9：磁颗粒纳米模拟酶免疫层析方法与胶体金免疫层析方法检测相思子毒素灵敏度的比较。
图10：纳米模拟酶免疫层析法与传统胶体金法对流感病毒检测的灵敏度比较。
具体实施方式
以下结合具体实施例来描述本发明，要理解本发明的范围不受限于具体实施例。
实施例1：纳米模拟酶免疫层析检测方法检测相思子毒素
1)杂交瘤细胞的制备
相思子毒素是豆科植物相思子种子中的成分，是迄今为止所发现的毒性最强的植物毒素之一，对人、动物和昆虫都有很大的毒性，嚼服一粒相思子种子足以致人死亡，本发明以检测相思子毒素(相思子毒素以及下文中 提到的蓖麻毒素均由军事医学科学院提供)为例来说明纳米模拟酶免疫层析检测方法的灵敏度和实用性。实验中使用的抗相思子毒素的单克隆抗体Abrin-1、Abrin-2、Abrin-3、Abrin-4，分别来自分泌单克隆抗体Abrin-1、Abrin-2、Abrin-3、Abrin-4的杂交瘤细胞(单克隆抗体的制备方法是本领域中已知的，并且可以参见例如Kohler和Milstein，Nature 256：495，1975；Yeh等，Proc.Natl.Acad.Sci.USA，1979；Yeh等，Int.J.Cancer，1982)，具体为：采用硫酸铵沉淀法获得相思子种子中的粗毒素，再通过分子筛柱层析、离子交换柱层析、冷冻干燥等步骤获得高纯度的毒素；经甲醛灭活后的相思子毒素全毒素作为免疫原对BALB/C小鼠进行免疫接种，每次皮下注射20μg蛋白/每只鼠，每两星期一次，共三次。取脾细胞之前加强免疫一次。加强免疫之后三天，取脾脏，并将脾细胞悬浮于RPMI培养基中。在聚乙二醇(PEG)存在下，将脾细胞和SP2/0-Agl4鼠骨髓瘤细胞进行融合，并用HAT选择性培养基(含次黄嘌呤hypoxantin、氨基蝶呤aminopterin和胸腺嘧啶脱氧核苷thymidin的培养基)对杂交瘤进行筛选，获得杂交瘤细胞。用ELISA的方法筛选与天然相思子毒素有强结合能力的抗体，获得四株抗体，分别命名为Abrin-1、Abrin-2、Abrin-3、Abrin-4，同时获得分泌这些抗体的杂交瘤细胞，依次是Abrin-1、Abrin-2、Abrin-3、Abrin-4。
2)ELISA方法鉴定单克隆抗体Abrin-1、Abrin-2、Abrin-3、Abrin-4对相思子毒素的亲和力
具体方法如下：首先在96孔ELISA板中过夜包被50μl的2μg/ml的相思子毒素蛋白；用PBST洗三遍，加入5％BSA-PBS封闭1h；分别加入单克隆抗体的杂交瘤细胞培养上清，37℃孵育1h；用PBST洗三遍，加入HRP标记的山羊抗小鼠抗体孵育1h；用PBST洗三遍，加入TMB显色底物(200ng/ml的TMB，0.03％的H2O2，pH4.5)显色，50μl/孔，37℃反应15min，加入50μl/孔2M的硫酸溶液终止反应，酶标仪450nm读数。从ELISA结果可以看出，Abrin-3、Abrin-4的亲和力可以达到1∶5000，而Abrin-1、Abrin-2的亲和力高达1∶50000(如图3)。
3)亲和层析纯化单克隆抗体
具体方法如下：大量培养扩增杂交瘤细胞Abrin-1、Abrin-2、Abrin-3、Abrin-4，分别制成细胞悬液，取六周龄BALB/C小鼠腹腔注射降植烷 (Sigma-Aldrich)0.5ml/只，约十天后，收集腹水，离心取上清液。通过蛋白G亲和层析(Roche)，从腹水中纯化单克隆抗体。纯化好的单克隆抗体无菌过滤，冷藏或冷冻保存。
4)单克隆抗体亚型鉴定
采用鼠抗体亚型鉴定试剂盒(BD Pharmingen)，按照说明书操作，鉴定出抗体Abrin-1属于IgG2a，Abrin-2、Abrin-3、Abrin-4属于IgGl亚型(如图4)。
5)免疫印迹(Western blotting)方法对抗体识别相思子毒素抗原表位的鉴定
具体方法是：用0.1M的二硫苏糖醇(DTT)将相思子毒素A、B链的二硫键还原打开，利用Western blotting方法鉴定抗体识别相思子毒素的表位。结果发现，Abrin-1、Abrin-2、Abrin-3、Abrin-4均在26kD的位置出现条带，而在34kD位置没有条带如图5)，这说明这四种抗体均识别相思子毒素的A链(分子量大小约为30kD)。
6)双抗夹心ELISA方法筛选配对抗体
具体方法是：首先将过氧化物酶HR通过戊二醛二步法或过碘酸钠法标记到抗体Abrin-1上，然后将Abrin-2、Abrin-3、Abrin-4抗体以0.02M的PBS(pH7.2)稀释至2μg/ml，然后以50μl/孔的量加入到到96孔板中，4℃包被过夜；用PBST洗三遍，加入5％BSA-PBS封闭1h；分别加入100、10、1、0.1ng/ml的相思子毒素以及10ng/ml的蓖麻毒素作为阴性对照(Ctrl)，37℃孵育1h；用PBST洗三遍，加入1μg/ml浓度的HRP标记的Abrin-1抗体孵育1h；用PBST洗三遍，加入TMB显色底物(200ng/ml的TMB，0.03％的H2O2，pH4.5)显色，50μl/孔，37℃反应15min，加入50μl/孔2M的硫酸溶液终止反应，酶标仪450nm读数。从ELISA结果可以看出(如图6)，Abrin-1与Abrin-2配对后效果最好。
7)Fe3O4磁性纳米颗粒的制备
在王水浸泡过的烧杯中，分别加入0.3g的CoCl2-6H2O、0.675g的FeCl3-6H2O和20mL的乙二醇，搅拌至完全溶解；加入1.5g无水NaAc，0.15g PAA，继续搅拌30min；置于反应釜中200℃反应14h；降温，将其中溶液倒入离心管中，磁分离并去除上清，加入乙醇超声洗4次，50-60℃ 干燥，制备的磁颗粒MNPs粒径约350nm左右(如图7)。
8)磁颗粒探针的制备
首先采用EDC-NHS(EDC：碳化二亚胺盐酸盐；NHS：羟基丁二酰亚胺)将磁颗粒表面的羧基活化，然后将相思子毒素单克隆抗体Abrin-1偶联于350nm四氧化三铁磁性颗粒上形成MNPs@Abrin-1。具体步骤如下：称取适量四氧化三铁磁性颗粒，加入50mg/ml的NHS，EDC各50μl，室温孵育30分钟，用去离子水清洗，除去多余的NHS/EDC。加入1ml、pH6.0的乙酸钠溶液，加入100μg的Abrin-1抗体，混匀，4℃孵育2小时，PBS洗涤，加入50mM pH 7.4Tris-C1封闭活化的羧基，PBS重悬，4℃保存；偶联效果采用点印迹(Dot blot)方法检测，即在硝酸纤维素膜上分别点上1mg/ml的羊抗鼠抗体、相思子毒素Abrin、蓖麻毒素各1μl，待干燥后加入MNPs@Abrin-1溶液，反应后的实验结果显示这种磁颗粒探针只与羊抗鼠抗体、相思子毒素Abrin结合，而不与蓖麻毒素Ricin结合，这说明偶联的MNPs@Abrin-1抗体探针特异性很好(如图8)。
9)纳米模拟酶免疫层析法试纸条的组装(如图2)
a.包被膜的制备：用包被缓冲液(0.02M磷酸盐缓冲液，pH 7.2)将相思子毒素的抗体Abrin-2、公司购买的羊抗鼠抗体(二抗)分别稀释为0.5mg/ml与1mg/ml，使用定量喷膜装置以1μl/cm的量按前后顺序将二者以0.8cm的间隔均匀喷印于3.5cm宽度的硝酸纤维素膜上，分别形成检测线(T线)抗体带与质控线(C线)抗体带。室温晾干30min后于封闭液(含有0.5％BSA的0.02M PBS，pH 7.2)中浸泡10min后于25-35℃烘干8小时，加入干燥剂封存备用。
b.磁颗粒探针垫的制备：使用喷膜仪的专用喷头将处理好的磁颗粒探针(如8)中所制备的)以50μl/cm的量均匀喷涂于0.8cm宽度的玻璃纤维垫上，过夜冷冻干燥，加入干燥剂封存备用。
c.样品垫的处理：将1.8cm宽度的样品垫(亲水性的玻璃纤维)浸入样品垫处理液(1-5％Casein(酪蛋白)，0.1-1％PVA(聚乙烯醇)，0.01-0.2％Tween 20，0.02M PBS，pH 7.2)处理1小时，取出后于25-35℃烘干8小时。
d.试纸条的组装及切割：将3.5cm包被膜、0.8cm磁颗粒探针垫、 1.8cm样品垫、2.5cm吸水垫以相互交错2mm的形式依次粘贴在背衬(底板)上(层叠顺序如图2所示)，覆盖一层透明塑料密封膜，组装成试纸板；使用切条机将组装好的试纸板切成0.5cm宽的成品试纸条；将切割好的试纸条置于塑料低卡上的卡槽内，盖上上盖，使用压卡机将上下两片塑料卡压紧，加入干燥剂室温封存备用(上述包被膜、磁颗粒探针垫、样品垫、吸水垫的尺寸以及相互交错的宽度可以根据实际需要由本领域技术人员适当调整)。
10)加样检测
微量移液器取50μl含有梯度浓度的相思子毒素样品溶液(浓度依次是100ng/ml、10ng/ml、1ng/ml、0ng/ml)加入检测卡上的样品垫上，再加入50μl层析缓冲液(1％Tween 20，0.5％Triton X-100，1％NP-40，0.05％NaN3，20mM PBS，pH 7.2)，等待反应进行15min。由于磁颗粒上偶联有相思子毒素的单克隆抗体Abrin-1，它与样品溶液中的相思子毒素结合后形成的复合物继续先前移动时，会与检测线(T线)处相思子毒素的另一抗体Abrin-2结合形成磁颗粒的聚集，而没有结合相思子毒素的磁颗粒Abrin-1抗体探针会继续移动到质控线(C线)处通过与其二抗作用形成磁颗粒的聚集。
11)酶催化放大检测信号
在检测线T线(Abrin-2抗体带)和质控线C线(羊抗鼠抗体带)处加入50μl的显色溶液(过氧化氢H2O2浓度为530mM；过氧化物酶的生色底物二氨基联苯胺(3，3’-diaminobenzidine，DAB)浓度为816mM)，反应5min；由于磁颗粒的过氧化物模拟酶活性，会在磁颗粒聚集的T线和C线处生成大量棕褐色的不溶性沉淀物，从而将检测信号放大，其检测灵敏度是传统胶体金试纸条检测法的100倍(如图9所示)。
实施例2：纳米模拟酶免疫层析检测方法检测流感病毒
1)抗体来源：公司购买，编号为FluA-1和FluA-2(生产商：Medix Biochemica，货号：FluA-1：100081，FluA-2：100083)。
2)Fe3O4磁性纳米颗粒的制备同实施例1。
3)磁颗粒探针的制备同实施例1，但需要将偶联的抗体替换为流感病 毒的抗体FluA-1。
4)纳米模拟酶免疫层析法试纸条的组装同实施例1，但需要将检测线(T线)处的抗体替换为流感病毒的抗体FluA-2。
5)加样检测
微量移液器取50μl含有梯度浓度的流感病毒样品溶液(病毒滴度以PFU计依次是1.25×104、6.25×103、3.1×103、1.56×103、7.8×102、3.9×102、1.95×102)加入检测卡上的样品垫上，再加入50μl层析缓冲液(1％Tween 20，0.5％Triton X-100，1％NP-40，0.05％NaN3，20mM PBS，pH 7.2)，等待反应进行15min。由于磁颗粒上偶联有流感病毒的单克隆抗体FluA-1，它与样品溶液中的流感病毒结合后形成的复合物继续先前移动时，会与检测线(T线)处流感病毒的的另一抗体FluA-2结合形成磁颗粒的聚集，而没有结合流感病毒的磁颗粒FluA-1抗体探针会继续移动到质控线(C线)处通过与其二抗作用形成磁颗粒的聚集。
6)酶催化放大检测信号
在检测线T线(FluA-2抗体带)和质控线C线(羊抗鼠抗体带)处加入50μl的显色溶液(过氧化氢H2O2浓度为530mM；过氧化物酶的生色底物二氨基联苯胺(3，3’-diaminobenzidine，DAB)浓度为816mM)，反应5min；由于磁颗粒的过氧化物模拟酶活性，会在磁颗粒聚集的T线和C线处生成大量棕褐色的不溶性沉淀物，从而将检测信号放大，其检测灵敏度是传统胶体金试纸条检测法的8倍(如图10所示)。
综上所述，本发明为一种纳米模拟酶免疫层析检测方法，该技术集磁性纳米颗粒过氧化物酶催化活性及磁分离特性于一体，免疫层析后通过加入过氧化物和供氢底物如邻苯二胺(DAB)等，利用磁颗粒的酶活性产生大量棕色沉淀，放大检测信号10-100倍，使得检测结果通过肉眼观察即可以判定，免除了对仪器的依赖性。该方法操作步骤简单、快捷、灵敏度高、非常适合现场检测使用，粒过氧化物酶的功能，具有十分广阔的应用前景，是一项具有新颖性、创造性、实用性的新技术。
以上仅是本发明的具体应用范例。应理解，这些实施例仅用于说明本专利而不用于限制本发明的范围。

Claims (10)

  1. 一种用于检测液体样品中的待测物的纳米模拟酶免疫层析检测方法，所述方法依次包括以下步骤：
    1)提供检测探针，所述检测探针通过将磁性纳米颗粒与能够与所述待测物特异性结合的第一分子偶联制备；
    2)提供捕获探针，所述捕获探针是固定化的能够与所述待测物特异性结合的第二分子；
    3)使所述液体样品与所述检测探针接触；
    4)使与所述检测探针接触过的所述液体样品与所述捕获探针接触；以及
    5)向经过步骤4)的所述捕获探针中加入供氢底物和过氧化物进行显色反应。
  2. 根据权利要求1所述的方法，其中所述磁性纳米颗粒的粒径在10纳米至500纳米范围内。
  3. 根据权利要求1所述的方法，其中所述磁性纳米颗粒是Fe3O4磁性纳米颗粒。
  4. 根据权利要求1所述的方法，其中所述待测物是蛋白质、多肽或核酸。
  5. 根据权利要求1所述的方法，其中所述待测物是蛋白质，并且所述第一分子和所述第二分子是针对所述蛋白质的特异性抗体，优选是单克隆抗体。
  6. 根据权利要求5所述的方法，其中所述第一分子与所述磁性纳米颗粒通过EDC-NHS法偶联。
  7. 根据权利要求1所述的方法，其中所述供氢底物包括四甲基联苯胺(TMB)、四甲基联苯胺硫酸盐(TMBS)、邻苯二胺(OPD)、二氨基联苯胺(DAB)、二氨基联苯胺四盐酸(DAB-4HCl)、5-氨基水杨酸(5-AS)、邻联甲苯胺(OT)或连氮二铵盐(ABTS)。
  8. 根据权利要求1所述的方法，其中所述过氧化物包括过氧化氢和过氧化脲。
  9. 一种用于检测液体样品中的待测物的纳米模拟酶免疫层析检测装置，所述装置包括依次设置在底板上的以下各项：
    样品垫，所述样品垫用于承接所述液体样品并滤过所述样品中的杂质；
    磁性纳米颗粒垫，所述磁性纳米颗粒垫包含与能够与所述待测物特异性结合的第一分子偶联的磁性纳米颗粒；
    检测线，所述检测线包含能够与所述待测物特异性结合的第二分子；
    吸收垫，所述吸收垫由吸水材料制成，用于提供层析的动力，
    其中所述磁性纳米颗粒的粒径优选在10纳米至500纳米范围内，
    其中所述磁性纳米颗粒优选是Fe3O4磁性纳米颗粒，
    其中所述待测物优选是蛋白质、多肽或核酸，并且更优选地，所述待测物是蛋白质而所述第一分子和所述第二分子是针对所述蛋白质的特异性抗体，优选是单克隆抗体，
    其中所述第一分子与所述磁性纳米颗粒优选通过EDC-NHS法偶联，
    其中所述供氢底物优选包括四甲基联苯胺(TMB)、四甲基联苯胺硫酸盐(TMBS)、邻苯二胺(OPD)、二氨基联苯胺(DAB)、二氨基联苯胺四盐酸(DAB-4HCl)、5-氨基水杨酸(5-AS)、邻联甲苯胺(OT)或连氮二铵盐(ABTS)，并且
    其中所述过氧化物优选包括过氧化氢和过氧化脲。
  10. 根据权利要求9所述的检测装置，其中在所述检测线之后还设置有质控线，所述质控线固定有能够与所述第一分子特异性结合的第三分子。
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