WO2019096325A1 - 一种双酰胺复合物及其制备方法和用途 - Google Patents

一种双酰胺复合物及其制备方法和用途 Download PDF

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WO2019096325A1
WO2019096325A1 PCT/CN2018/116375 CN2018116375W WO2019096325A1 WO 2019096325 A1 WO2019096325 A1 WO 2019096325A1 CN 2018116375 W CN2018116375 W CN 2018116375W WO 2019096325 A1 WO2019096325 A1 WO 2019096325A1
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compound
solution
antibody
cholinesterase
complex
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PCT/CN2018/116375
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French (fr)
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李谷丰
王贻杰
夏丽
邓双胜
刘勇
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江苏达骏生物科技有限公司
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Priority to CN201880072542.6A priority Critical patent/CN111566081B/zh
Publication of WO2019096325A1 publication Critical patent/WO2019096325A1/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
    • G01N33/54366—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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C231/00—Preparation of carboxylic acid amides
    • C07C231/02—Preparation of carboxylic acid amides from carboxylic acids or from esters, anhydrides, or halides thereof by reaction with ammonia or amines
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C233/00—Carboxylic acid amides
    • C07C233/64—Carboxylic acid amides having carbon atoms of carboxamide groups bound to carbon atoms of six-membered aromatic rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C319/00—Preparation of thiols, sulfides, hydropolysulfides or polysulfides
    • C07C319/22—Preparation of thiols, sulfides, hydropolysulfides or polysulfides of hydropolysulfides or polysulfides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C323/00—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups
    • C07C323/23—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and nitrogen atoms, not being part of nitro or nitroso groups, bound to the same carbon skeleton
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C323/00—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups
    • C07C323/23—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and nitrogen atoms, not being part of nitro or nitroso groups, bound to the same carbon skeleton
    • C07C323/24—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and nitrogen atoms, not being part of nitro or nitroso groups, bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C323/00—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups
    • C07C323/23—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and nitrogen atoms, not being part of nitro or nitroso groups, bound to the same carbon skeleton
    • C07C323/24—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and nitrogen atoms, not being part of nitro or nitroso groups, bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton
    • C07C323/29—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and nitrogen atoms, not being part of nitro or nitroso groups, bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton containing six-membered aromatic rings
    • 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
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances

Definitions

  • the present invention relates to a method for rapidly detecting cholinesterase activity, in particular to a bisamide complex capable of detecting organic phosphorus and carbamate substances.
  • Carbamate pesticides are synthetic pesticides developed after organic phosphates. Carbamate pesticides generally have no special odor, are stable under acidic conditions, and are decomposed by alkali. Most species are less toxic than organic phosphates. However, since most organophosphorus pesticides are highly toxic compounds, they not only pollute natural resources such as soil and water sources, but also accumulate in animals and plants, and become a hidden danger to people's lives through the food chain.
  • carbamate pesticides are used, Not a highly toxic compound, but carcinogenic, the International Agency for Research on Cancer listed carbamates as Class 2A carcinogens in 2007. After the occurrence of food poisoning, rapid screening to detect whether it is caused by organophosphorus or carbamate pesticides or rodenticides is of great significance for timely rescue of the injured.
  • organophosphorus or carbamate pesticides The focus of pesticide testing is on organophosphorus or carbamate pesticides. In order to prevent and control the impact of organophosphorus pesticide residues on human health, it is necessary to strengthen the detection of organophosphorus pesticide residues in food. At present, the country has developed a national standard for rapid detection of organophosphorus and carbamate pesticides.
  • the present invention provides a rapid and capable method for detecting trace amounts of organophosphorus and carbamate pesticides, and has the ability to achieve trace organic phosphorus and The rapid, high sensitivity and selective detection of carbamate pesticides, and the advantages of low detection cost and simple operation.
  • Cholinesterase is a type of glycoprotein that exists in the body in the form of multiple isoenzymes. Generally divided into true cholinesterase and pseudocholinesterase. In the clinic, measuring serum cholinesterase activity is an important means to assist in the diagnosis of organophosphate poisoning and to assess liver parenchymal damage.
  • the existing methods for detecting cholinesterase mainly include colorimetric method and continuous monitoring rate method.
  • the colorimetric method is unstable in color development, especially when the room temperature exceeds 20 °C, the influence is large, and the error is large.
  • the analyzer is not suitable for rapid detection and analysis on site.
  • the technique of the present invention can be implemented, and only a small amount of sample is needed for on-site fast analysis.
  • An object of the present invention is to disclose a bisamide complex, a process for the preparation thereof, and a method for detecting an organophosphorus and a carbamate substance using the complex, and a method for detecting a cholinesterase using the complex.
  • the present invention discloses, in a first aspect, a bisamide complex composed of a compound of the following structure: A-[B] n -[C] m , wherein N ⁇ M ⁇ 1, A, B and C Connected by an amide bond, wherein A is a polymer, and B and C are compounds;
  • Polymer A is a high molecular polymer whose surface is activated by an amino group or a carboxyl group;
  • Compound B is specifically a compound of the formula:
  • R1, R2 and R3 are amino groups or a carboxyl group, and the remaining group is H, an alkane group, an aryl group, a nitro group, a halogen group, a hydroxyl group or a derivative thereof.
  • Compound C is a compound containing a free primary amino group or a carboxyl group, which may be a small molecule compound having a molecular weight of less than 5,000 Daltons, or a nucleic acid, a polypeptide or a proteinaceous macromolecular compound having a molecular weight of more than 5,000 Daltons.
  • the molar ratio of polymer A, compound B and compound C is 1:N:M, wherein N ⁇ M ⁇ 1, and the bisamide compound can react with a thiol compound such as thiocholine to release a derivative of Compound C.
  • the compound A may be selected from amino microspheres or carboxyl microspheres, and the preferred amino microspheres have a particle diameter of from 0.5 to 30 ⁇ m, more preferably from 5 to 15 ⁇ m, still more preferably from 6 ⁇ m, 8 ⁇ m, or 10 ⁇ m.
  • Compound A can also select magnetic nanopolystyrene particles having a particle diameter of 1-100 nm.
  • compound B may be selected from any compound conforming to the above structural formula, for example, 5,5'-dithiobis(2-nitrobenzoic acid CAS number: 69-78-3, or 4,4'-dithiodiphenylamine CAS No.: 722-27-0, or 4,4'-dithiobisbenzoic acid CAS No.: 1155-51-7, or 2,2'-dithiobis(3-methylbenzoic acid) CAS No.: 13363-59-2.
  • Compound C is a compound containing a free primary amino group or a carboxyl group, which may be a small molecule compound having a molecular weight of less than 5,000 Daltons, or a nucleic acid, a polypeptide or a proteinaceous macromolecular compound having a molecular weight of more than 5,000 Daltons.
  • the second aspect of the invention discloses a method for preparing a composite, comprising the following steps:
  • the step (1) means that the EDC solution and the NHS solution are added to the solution of the compound B, and reacted at room temperature to obtain a solution.
  • the amount ratio of EDC to NHS species is 2:1.
  • the step (2) means that the compound C is added to the solution obtained in the step (1).
  • step (3) refers to adding a solution of the polymer A to the solution of the compound B-compound C conjugate such that the compound B-compound C conjugate is covalently bonded to the polymer A. Forming a derivative of polymer A-compound B-compound C.
  • the compound A may be selected from amino microspheres or carboxyl microspheres having a particle diameter of from 0.5 to 30 ⁇ m, and preferably having an amino microsphere having a particle diameter of from 5 to 15 ⁇ m, more preferably 6 ⁇ m, 8 ⁇ m, or 10 ⁇ m.
  • polymer A is a polystyrene amino microsphere having a particle size of 10 ⁇ m.
  • Compound B is 5,5'-dithiobis(2-nitrobenzoic acid) (abbreviated as DTNB).
  • Compound C is procalcitonin (PCT for short).
  • the molar ratio of DTNB to PCT is 5-20:1.
  • the polymer A may be selected from amino microspheres having a particle size of from 0.5 to 30 [mu]m, preferably having an amino microsphere size of from 5 to 15 [mu]m, more preferably from 6 [mu]m, 8 [mu]m, or 10 [mu]m.
  • polymer A may select magnetic nanopolystyrene particles having a spherical particle size of from 1 to 100 nm.
  • the mass molar ratio (g: mol) of the amino microspheres to DTNB is: 300-900:1, preferably 330:1, 600:1 or 700:1.
  • compound C is sulfadiazine
  • compound A is an amino microsphere
  • compound B is DTNB
  • the molar ratio of DTNB to sulfadiazine is 1:0.5 to 3, and the preferred molar ratio of DTNB to sulfadiazine is 1:1.5.
  • the complex of the present invention is capable of reacting with thiocholine while releasing Compound C, thereby detecting the content of Compound C by an immunological method, thereby indirectly reflecting the concentration of the organophosphorus and carbamate pesticide.
  • a third aspect of the invention discloses a method for detecting organophosphorus and carbamate substances, which mainly comprises the following steps:
  • test solution (sample solution) is sequentially reacted with cholinesterase, acylated thiocholine and bisamide complex to release compound C, and then compound C is detected by antigen antibody or ligand receptor immunological analysis method.
  • the content indirectly, reflects the concentration of organophosphorus and carbamate pesticides.
  • the acylated thiocholine is poorly water-soluble and unstable, and a halogen salt of a well-soluble acylated thiocholine such as thioacetylcholine iodide or thiobutyrylcholine iodide can be generally selected.
  • Immunological detection methods and techniques mainly include: enzyme immunoassay technology, radioimmunoassay technology, immunofluorescence technology, immunogold gold labeling technology.
  • fluorescent immunochromatography is used.
  • Polymer A is an amino microsphere, polymer A has a particle size of 0.5-30 ⁇ m, and compound B is 5,5'-dithiobis (2-nitro Benzoic acid) (DTNB for short);
  • Compound C is procalcitonin (PCT).
  • the detection method of the fluorescent immunochromatographic analysis is as follows: 1. The sample is dropped on the binding pad embedded with cholinesterase, and the aqueous solution in the sample dissolves the cholinesterase, and the migration occurs toward the end of the absorbent paper; During the migration, the migrated liquid reacts with a binding pad containing thioacetylcholine iodide. Under certain conditions, cholinesterase can catalyze the formation of thiocholine and acetic acid by iodinated thioacetylcholine; 3.
  • the base further migrates forward with the liquid, and further reacts with the amino microsphere-DTNB-compound C complex binding pad, and the thiocholine can catalyze the amino microsphere-DTNB-compound C complex (hereinafter referred to as the complex).
  • a compound C-TNB or a compound C-thiocholine-NTB soluble derivative (hereinafter referred to as a compound C derivative) is produced; since the microspheres in the complex are large in diameter and cannot pass through the nitrocellulose membrane (NC membrane), they are trapped At the bottom of the NC membrane, however, the formation of the compound C derivative can continue to move on the NC membrane as the liquid continues, thereby separating the unreacted complex and the released compound C derivative; 4.
  • the released compound C is derived. Further moving forward with the liquid, further reacting with the fluorescent microsphere-labeled antibody 1 (hereinafter referred to as fluorescent microsphere antibody I) to form a compound C-fluorescent microsphere antibody I complex; 5. Forming a compound C-fluorescent micro The balloon antibody I complex migrates forward with the liquid on the NC membrane, reacts with the antibody 11 or the compound C-hapten coated on the NC membrane, so that the fluorescent microspheres are trapped due to the fluorescent microspheres. Under a certain wavelength of light excitation, a special wavelength signal can be emitted, and the content of the compound C can be analyzed by collecting the intensity of the special wavelength signal by the instrument; 6. The content level of the compound C directly reflects the content of the thiocholine.
  • the level of cholinesterase activity indirectly reflects the level of organophosphorus or carbamate pesticides (hereinafter referred to as pesticides) in the sample.
  • pesticides organophosphorus or carbamate pesticides
  • the magnetic nanoparticle separation-enzyme-labeled immunochemiluminescence analysis technique is used to detect that the polymer A is an amino-activated magnetic nano-polystyrene particle, and the polymer A has a magnetic nanoparticle with a particle diameter of 1-100 nm.
  • Polystyrene particles, magnetic nanoparticles can be achieved by uniformly coating nano-sized polystyrene particles through ferroferric oxide, or by encapsulating nano-sized particles by any other magnetic and adhesive substance.
  • Compound B is 5,5'-dithiobis(2-nitrobenzoic acid) (abbreviated as DTNB);
  • Compound C is procalcitonin (PCT for short).
  • the detection method is: 1.
  • the sample is added dropwise to the lyophilized porcine lipase, and the aqueous solution in the sample dissolves the cholinesterase to form a uniform stable liquid system;
  • the substrate iodide thioacetylcholine, magnetic nanoparticle-DTNB-compound C complex (hereinafter referred to as complex) is sequentially added and mixed.
  • complex The substrate iodide thioacetylcholine, magnetic nanoparticle-DTNB-compound C complex
  • complex is sequentially added and mixed.
  • cholinesterase can catalyze the formation of thiocholine and acetonitrile by iodinated thioacetylcholine; the resulting thiocholine further reacts with the complex to form compound C-TNB or compound C-thiocholine- NTB soluble complex (hereinafter referred to as compound C); 3.
  • the magnetic substance is used to adsorb the unreacted complex: since the complex can be adsorbed by the magnetic substance, the unreacted complex and The released compound C is separated; 4.
  • the antigen (hereinafter referred to as: labeled antigen) reacts to form a coating antibody 1-compound C-labeled antibody II or a compound C-labeled antigen complex, and then removes unreacted labeled antibody II or labeled antigen by washing;
  • Horseradish peroxidase, alkaline phosphatase can catalyze luminescent substrates such as luminol or its derivatives, emit special wavelength signals, and collect the intensity of special wavelength signals by instrument.
  • the content level of compound C directly reflects the level of thiocholine, while the level of thiocholine directly reflects the activity of cholinesterase; 7, choline
  • the level of enzyme activity indirectly reflects the content of organophosphorus or carbamate pesticides (hereinafter referred to as pesticides) in the sample.
  • pesticides organophosphorus or carbamate pesticides
  • a fourth aspect of the invention discloses a test strip for detecting an organophosphorus and a urethane substance, the test strip comprising the following components:
  • substrate binding pad containing acylated thiocholine
  • labeled antibody pad a monoclonal antibody-label conjugate coated with a compound C;
  • detection line and quality control line on the reaction membrane the detection line is coated with another monoclonal antibody of compound C-hapten or compound C; the quality control line is coated with anti-antibody against the antibody in step 4);
  • Absorbent paper An absorbent paper for providing a capillary force required for detection.
  • test strip as described above, characterized in that the test strip further comprises a bottom plate, wherein the bottom plate is pasted with an enzyme bonding pad, a substrate bonding pad, a composite bonding pad, a fluorescent microsphere pad, which are sequentially overlapped, Reaction film and absorbent paper.
  • the enzyme binding pad contains two forms, one is embedded in a special material and is present on the bottom plate together with the test strip; the other form is liquid or lyophilized powder is stored in a special container. , and the bottom plate portion is independent of each other.
  • the affixed enzyme binding pad, substrate binding pad, complex binding pad, fluorescent microsphere pad, reaction film, and absorbent paper are sequentially lapped.
  • the cholinesterase is commercially available or synthetic acetylcholinesterase (AChE) or butyrylcholinesterase (BChE), and the cholinesterase can be purified by plasma or by recombinant DNA technology. preparation.
  • AChE acetylcholinesterase
  • BChE butyrylcholinesterase
  • the acylated thiocholine comprises acetylthiocholine, propionylthiocholine, butyrylthiocholine, benzoylthiocholine, and the like.
  • the acylated thiocholine has poor water solubility and is unstable.
  • a halogen salt of a water-soluble acylated thiocholine can be selected as a substrate, for example, thioacetylcholine iodide, thiobutyroylcholine iodide, etc. .
  • the amino microsphere-DTNB-Compound C complex is a complex capable of reacting with thiocholine to release Compound C, which is composed of amino microspheres, DTNB, and Compound C. The three are coupled together.
  • the microsphere composite has microspheres of carboxyl microspheres.
  • reaction membrane is an NC membrane.
  • the content level in the sample should be inversely proportional to the ratio of the T value/C value.
  • the content level in the sample should be proportional to the ratio of T value/C value.
  • a method for detecting cholinesterase activity which utilizes cholinesterase to catalyze the reaction of an acylated thiocholine with the aforementioned bisamide complex of the invention to form a compound C derivative Then, the content of the compound C is detected by an antigen antibody or a ligand receptor immunological analysis method, thereby indirectly reflecting the activity of the cholinesterase.
  • the reaction of releasing the compound C derivative with the aforementioned bisamide complex of the present invention may cause a large interference to the detection, thereby detecting the cholinesterase activity of the sample.
  • the influence of the compound C derivative produced by the sulfhydryl compound contained in the sample on the detection result is excluded.
  • the enzyme activity value of the enzyme-catalyzed reaction can be calculated by subtracting the enzyme activity value corresponding to the background in the enzyme activity.
  • the conversion of the compound C derivative content to the cholinesterase activity was calculated based on the standard curve of the compound C and cholinesterase activity, which was obtained by multiple experiments using the cholinesterase standard.
  • the preferred method for detecting cholinesterase activity is to first detect the cholinesterase activity corresponding to the compound C derivative produced in the sample without the addition of acylated thiocholine, and then detect the addition of the acyl group.
  • the method for detecting cholinesterase activity comprises the following steps:
  • test solution (sample solution) reacts with the complex described above to release a derivative of compound C, and then the compound C derivative is detected by antigen antibody or ligand receptor immunological analysis method.
  • Content according to the content, the enzyme activity 1 is calculated;
  • sample detection the analyte solution (sample solution) is sequentially reacted with the acylated thiocholine and the bisamide complex according to claim 1, releasing a complex of compound C, and then passing the antigen antibody or ligand.
  • the receptor immunological assay method detects the content of the compound C derivative, and calculates the enzyme activity 2 based on the content;
  • a background test strip for detecting cholinesterase activity is disclosed, characterized in that the test strip comprises the following components:
  • labeled antibody pad a monoclonal antibody-label conjugate coated with a compound C;
  • Detection line and quality control line on the reaction membrane the detection line is coated with another monoclonal antibody of compound C-hapten or compound C; the quality control line is coated with anti-antibody against the antibody in step (2) ;
  • Absorbent paper used to provide absorbent paper for detecting the required capillary force.
  • test strip for detecting cholinesterase activity, characterized in that the test strip comprises the following components:
  • substrate binding pad containing acylated thiocholine
  • labeled antibody pad a monoclonal antibody-label conjugate coated with anti-C;
  • detection line and quality control line on the reaction membrane the detection line is coated with another monoclonal antibody of compound C-hapten or compound C; the quality control line is coated with anti-antibody against the antibody in step 3);
  • Absorbent paper An absorbent paper for providing the capillary force required for the detection.
  • Acylated thiocholine refers to acylated thiocholine and its halogen salts, including but not limited to iodinated acetylthiocholine, acetylcholine chloride, acetylthiocholine bromide, and propidium iodide Acylthiocholine, propionylthiocholine chloride, propionylthiocholine, iodinated butyrylthiocholine, butyrylthiocholine chloride, butyrylthiocholine bromide , benzoylthiocholine iodide, benzoylthiocholine chloride, benzoylthiocholine bromide, and the like.
  • Compound C refers to a class of polypeptide, nucleic acid or protein macromolecules having a molecular weight greater than 5 KD, for example: bovine serum albumin, human serum albumin, chicken ovalbumin, hemocyanin, procalcitonin, alpha-fetoprotein Carcinoembryonic antigen, human chorionic gonadotropin, follicle stimulating hormone, and the like.
  • Compound C may also be a compound containing a primary amino group having a molecular weight of less than 5 KD. For example: melamine, aniline, sulfonamide, sulfadiazine, sulfamethazine, sulfamethoxazole, clenbuterol, dopamine, and the like.
  • Amino microspheres refer to microspheres whose surface is aminated.
  • the materials of the microspheres are mainly: resin, ethyl cellulose, polypropylene, polystyrene, polyacrylic acid, latex styrene butadiene rubber, polyvinyl acetate and the like.
  • Carboxy microspheres refer to microspheres whose surface is carboxylated.
  • the materials of the microspheres are mainly: resin, ethyl cellulose, polypropylene, polystyrene, polyacrylic acid, latex styrene butadiene rubber, polyvinyl acetate and the like.
  • the microspheres mainly include: fluorescent microspheres, colored microspheres, and the like.
  • DTNB refers to 5,5'-dithiobis(2-nitrobenzoic acid), CAS number: 69-78-3.
  • Coupling means that the coupling is a phenomenon in which other reactions are carried out in a stoichiometric relationship when a chemical reaction occurs.
  • the coupling reaction also as a coupling reaction, a coupling reaction, and an oxidative coupling, is performed by two organic chemical units. A process in which a chemical reaction is carried out to obtain an organic molecule.
  • a conjugate refers to the formation of a corresponding substance by coupling reactions of two organic chemicals.
  • Immunological detection refers to a method for determining the content of a substance to be tested in a sample by using the principle of immunology as an antigen or an antibody (receptor or ligand).
  • the content refers to the amount of a certain component contained in the substance.
  • EDC means 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride), CAS No.: 25952-53-8.
  • NHS means N-hydroxysuccinimide, CAS No.: 6066-82-6.
  • the carbodiimide method refers to the activation of a carboxyl group by a carbodiimide to promote the formation of an amide and an ester.
  • the NC membrane refers to a nitrocellulose membrane, and the pore diameter of each 4 cm of membrane water is defined by the number of seconds. According to the speed of the flow rate, there are 90, 120, 135, 180, 240 and the like.
  • Cholinesterase is a class of glycoproteins that exist in a variety of isoenzymes including, but not limited to, acetylcholinesterase and butyrylcholinesterase.
  • the label refers to fluorescein, radioactive metals, microspheres, biological enzymes and the like.
  • antigenic antibodies in the following examples are commercially available, but these materials can also be self-made, and the source of the antigen-antibody is not critical to the present invention.
  • amino microsphere solution amino microspheres (remark: solid content of 0.01 g / ml) (hereinafter referred to as amino microspheres) diluted 1:10 with PBS;
  • PCT procalcitonin
  • DTNB solution DTNB was dissolved in DMF and formulated into a concentration of 10 mmoL / L solution;
  • EDC solution 1-ethyl-(3-dimethylaminopropyl)carbodiimide (hereinafter referred to as: EDC) is dissolved in water and formulated into a solution having a concentration of 100 mmoL/L;
  • NHS solution N-hydroxysuccinimide (hereinafter referred to as: NHS) is dissolved in water and formulated into a solution having a concentration of 100 mmoL/L;
  • the reacted liquid was transferred to a centrifuge tube, and centrifuged at 10,000 r/min for 4 min at 4 ° C; the supernatant was discarded, and washed with PBS solution 5 times.
  • the precipitate was obtained after centrifugation, dried at a low temperature and vacuum dried, and stored at 4 ° C in a sealed and protected from light.
  • amino microsphere solution amino microspheres (remark: solid content of 0.01 g / ml) (hereinafter referred to as amino microspheres) diluted 1:10 with PBS;
  • Antigen solution sulfamethazine (SD) was prepared into a 10 mmoL/L solution with PBS solution;
  • DTNB solution DTNB was dissolved in DMF and formulated into a concentration of 10 mmoL / L solution;
  • EDC solution 1-ethyl-(3-dimethylaminopropyl)carbodiimide (hereinafter referred to as: EDC) is dissolved in water and formulated into a solution having a concentration of 100 mmoL/L;
  • NHS solution N-hydroxysuccinimide (hereinafter referred to as: NHS) is dissolved in water and formulated into a solution having a concentration of 100 mmoL/L;
  • SD sulfamethazine
  • the reacted liquid was transferred to a centrifuge tube, and centrifuged at 10,000 r/min for 4 min at 4 ° C; the supernatant was discarded, and washed with PBS solution 5 times.
  • the precipitate was obtained after centrifugation, dried at a low temperature and vacuum dried, and stored at 4 ° C in a sealed and protected from light.
  • the enzyme dilution buffer (hereinafter referred to as: enzyme diluted solution) was prepared as follows: 3 g of Na2HPO4 ⁇ 12H2O; 0.25 g of NaH2PO4 ⁇ 2H2O; 8.7 g of NaCl; 10 g of bovine serum albumin; 5 g of trehalose; 1 g of sodium azide; 10 g of hydroxypropyl-beta-cyclodextrin was first dissolved in 800 mL of deionized water, adjusted to pH 8.0, and finally made up to 1 L with deionized water.
  • the substrate dilution buffer (hereinafter referred to as: substrate liquid) was prepared as follows: 5 g of hydroxyethylidene diphosphate; 30 g of isopropanol; 2 g of trehalose; 3 g of sodium sulfite; 0.5 g of benzoic acid; First, dissolve thoroughly with 800mL of deionized water, adjust the pH to 6.0, and finally dilute to 1L with deionized water.
  • the first form diluted with enzyme diluted solution to 0.1 ⁇ 3U / mL of acetylcholinesterase (purchased from Beijing Suobao company), and then pipetted 1 ⁇ 2mL tiled 5mm * 300mm glass fiber paper Top, placed at room temperature (25 ° C ⁇ 5 ° C), humidity ⁇ 30%, ventilated and dried for 24 hours, dried and stored at 4 ° C for use.
  • the second form diluted with enzyme diluted solution to 0.1 ⁇ 3U / mL of acetylcholinesterase (purchased from Beijing Suobao company), directly stored in a liquid state in 0.5 ⁇ 100mL plastic bottles or glass bottles, or frozen
  • the state of the dry powder is stored in a 0.1 to 100 mL plastic bottle (hole) or a glass bottle (hole).
  • Substrate binding pad preparation dilute thioacetylcholine iodide with substrate solution to 1-5 mg / mL, and then pipette 1 to 2 m L to 5 mm * 300 mm glass fiber paper, placed Room temperature (25 ° C ⁇ 5 ° C), humidity ⁇ 30%, ventilated and dried for 24 hours, dried and stored at 4 ° C for use.
  • the amino microsphere-DTNB-antigen complex of Example 1 was diluted with an enzyme diluted solution at a ratio of 1:10, and then pipetted by 1-2 m to a glass fiber paper of 5 mm*300 mm. Placed at room temperature (25 ° C ⁇ 5 ° C), humidity ⁇ 30%, ventilated and dried for 24 hours, after drying, the amino microsphere-DTNB- PCT complex binding pad was stored at 4 ° C for use.
  • microsphere dilution buffer (hereinafter referred to as microsphere dilution solution) was prepared as follows: 3 g Na2HPO4 ⁇ 12H2O; 0.25 g NaH2PO4 ⁇ 2H2O; 5 g hydroxyethylidene diphosphate; 30 g isopropanol; 5 g sucrose; Sodium sulfite; 1 g of sodium azide, first dissolved in 800 mL of deionized water, then adjusted to pH 6, 5, and finally made up to 1 L with deionized water.
  • the amino microsphere-DTNB-antigen complex of Example 2 was supplemented with a microsphere dilution, diluted 1:10, and then pipetted with 1 to 2 m L of glass fiber paper of 5 mm*300 mm. Above, placed at room temperature (25 ° C ⁇ 5 ° C), humidity ⁇ 30%, ventilated and dried for 24 hours, after drying, the amino microsphere-DTNB- SD composite bond pad was stored at 4 ° C for use.
  • PCT anti-procalcitonin
  • monoclonal antibody I available from sea peptide Biotechnology (Shanghai) Co., Ltd.
  • pH7.4 0.05M PBS solution was dialyzed 3 times the volume of The replacement ratio was 1:100, and after completion of dialysis, PCT monoclonal antibody I and monoclonal antibody II were adjusted to a concentration of 1 mg/mL with pH 7.4 0.05 M PBS.
  • SD anti-sulfadiazine
  • the liquid is shaken by ultrasonic for 30 seconds, and then 200 ⁇ L of 1.0 M ethanolamine solution (prepared with ultrapure water) and 100 ⁇ L of 2.0 M glycine solution (prepared with ultrapure water) are added, room temperature (25 ° C ⁇ 5 ° C).
  • the reaction was carried out for 1 hour, and the shaker was inverted and mixed at a speed of 60 rpm.
  • Example 8 Coating of test lines and quality control lines on a reaction membrane (PCT antibody coating)
  • Anti-PCT monoclonal antibody II and goat anti-mouse IgG secondary antibody (hereinafter referred to as secondary antibody, purchased from Haipept Biotechnology (Shanghai) Co., Ltd.) coated onto NC membrane
  • the anti-PCT monoclonal antibody II-adjusted coating concentration was 0.5 mg/mL and the secondary antibody-adjusted coating concentration was 0.5 mg/mL, and the sprayed film amount was 0.8 ⁇ L/cm, using pH 7.4 0.05 M PBS;
  • the detection line is coated with the monoclonal antibody II of the compound C, the quality control line is coated with the secondary antibody, the two regions are separated by 6 mm, the quality control line is 10 mm from the top of the NC membrane, and the detection line is 9 mm from the bottom end of the NC membrane;
  • Example 9 Coating of test lines and quality control lines on a reaction membrane (SD-BSA antigen coating)
  • the secondary antibody of SD-BSA conjugate and goat anti-SD monoclonal antibody (hereinafter referred to as secondary antibody, purchased from Haipept Biotechnology (Shanghai) Co., Ltd.) was coated onto the NC membrane.
  • the SD-BSA conjugate was adjusted to a coating concentration of 0.5 mg/mL with a pH of 7.4 0.05 M PBS and the secondary antibody was adjusted to a coating concentration of 0.5 mg/mL, and the sprayed film amount was 0.8 ⁇ L/cm;
  • the detection line is coated with SD-BSA conjugate, the quality control line is coated with the secondary antibody, the two regions are separated by 6Mm, the quality control line is 10mm from the top of the NC membrane, and the detection line is 9mm from the bottom end of the NC membrane;
  • Example 10 Assembly of cholinesterase fluorescent microsphere immunochromatography background card and detection card
  • Background card a blank pad, a composite bond pad obtained in Example 4 or Example 5, a fluorescent microsphere pad obtained in Example 6, an NC film obtained in Example 8 or Example 9, and a paste were sequentially laminated on a substrate.
  • Absorbent paper the bonded test strip is cut into 4mm test strips by a cutting machine, assembled into the prepared test card holder, filled into the aluminum foil bag, added with desiccant, sealed and stored in a dry environment at room temperature. It can be kept for at least one year.
  • the specific structure of the background card is shown in Figure 1.
  • Example 3 The substrate bond pad obtained in Example 3, the composite bond pad obtained in Example 4 or Example 5, the fluorescent microsphere pad obtained in Example 6, and Example 8 or Example 9 were successively pasted on the substrate.
  • the NC film and the absorbent paper, the bonded test strip is cut into 4mm test strips by a cutting machine, assembled into the prepared test card shell, filled into the aluminum foil bag, and after the desiccant is added, the seal is stored. Store in a dry room at room temperature.
  • the specific structure of the test card is shown in Figure 2.
  • Example 11 Test of cholinesterase fluorescent microsphere immunochromatographic assay card
  • the cholinesterase is prepared into distilled water or a buffer solution of pH 6.0 to pH 8.0 to an active concentration of 0 U/L, 15 U/L, 45 U/L, 135 U/L, 405 U/L, and 1215 U/L. Mix and reserve;
  • the cholinesterase fluorescent microsphere immunochromatographic test card is taken out from the aluminum foil bag and placed on the table top;
  • the above sample liquid is extracted by pipetting 100 ⁇ L sample drop into the sample hole of the background card (S);
  • the reaction card completed in the reaction is placed in a dry fluorescence detector for detection, irradiated with a 365 nm fluorescence excitation source, and a signal is acquired at 615 nm to obtain signal values of the detection line (T line) and the control line (C line).
  • the ratio of the detected line value (T value) to the control line value (C value) was compared, and data analysis was performed.
  • the level of the compound C was released from the analysis, and the level of the enzyme activity in the background was calculated from the compound C and the enzyme activity standard curve.
  • Example 10 (1) the cholinesterase fluorescent microsphere immunochromatographic assay obtained in Example 10 (1) was used to detect the sum of the enzymatic activity of the background and the enzyme-catalyzed reaction.
  • Calculating the difference between the first card and the second card is the enzyme activity of the enzyme catalyzed reaction.
  • Compound C is procalcitonin (PCT).
  • cholinesterase fluorescent microsphere immunochromatographic test card detects cholinesterase, which has good linearity between 45 and 1215 U/L, and can identify cholinesterase activity.
  • Example 12 Assembly of an organophosphorus and carbamate fluorescent microsphere immunochromatographic test card
  • the enzyme binding pad obtained in Example 3, the substrate binding pad obtained in Example 3, the composite binding pad obtained in Example 4, the fluorescent microsphere pad obtained in Example 5, and Example 8 were successively pasted on the bottom plate.
  • the obtained NC film and absorbent paper, the bonded test strip is cut into 4 mm test strips by a cutting machine, assembled into a prepared test card case, filled into an aluminum foil bag, and after adding a desiccant, the seal is stored. It can be stored for at least one year in a dry environment at room temperature.
  • the composite bond pad of Example 4 can also be replaced with the composite bond pad of Example 5, and the NC film of Example 8 can be replaced with the NC film of Example 9, and the rest of the preparation methods are the same.
  • the assembled reagent card is shown in Figure 3.
  • the cholinesterase is attached to one end of the sample pad in solid phase.
  • cholinesterase can also be present in the form of a lyophilized powder outside the test card, as shown in Figure 4, at which point the enzyme binding pad is omitted and the rest of the preparation process is the same.
  • Example 13 Test method for fluorescent microsphere immunochromatographic detection card of organophosphorus and carbamate drugs
  • organophosphorus and carbamate pesticide residual fluorescent microsphere immunochromatographic test card and dry fluorescence detector were opened and preheated for 5 minutes.
  • the organic phosphorus and carbamate pesticide residual fluorescent microsphere immunochromatographic test card is taken out from the aluminum foil bag and placed on the table top;
  • the reaction card completed in the reaction is placed in a dry fluorescence detector for detection, irradiated with a 365 nm fluorescence excitation source, and a signal is acquired at 615 nm to obtain signal values of the detection line (T line) and the control line (C line).
  • the ratio of the detected line value (T value) to the control line value (C value) was compared, and data analysis was performed.
  • the compound C is sulfadiazine (SD), and the detection result of the card fluorescent microsphere immunochromatographic test card is as follows: determination:
  • the ratio of the blank control T value / C value should be ⁇ 0.2;
  • the concentration of carbofuran in different vegetables was 100-200 ng/ml, which showed a positive result.
  • the ratio of the blank control T value / C value should be ⁇ 1.1;
  • the concentration of carbofuran in different vegetables was 20 ⁇ 50ng/ml, which showed a positive result.
  • the compound C is sulfadiazine (SD)
  • the detection result of the card fluorescent microsphere immunochromatographic test card is determined according to the concentration of each drug. Calculate the ratio of T value/C value of the instrument test at each concentration. The content level in the sample should be inversely proportional to the ratio of T value/C value. Draw a standard curve and calculate the measured result:
  • the compound C is procalcitonin (PCT) and the detection result of the detection card fluorescent microsphere immunochromatographic test card is based on each drug.
  • concentration of the instrument is calculated as the ratio of the T value/C value of the instrument under each concentration.
  • the content level in the sample should be proportional to the ratio of the T value/C value.
  • the standard curve is drawn and the measured result is calculated:
  • Compound C is procalcitonin (PCT).
  • the drug dichlorvos (concentration 1 mg/mL) was diluted to 20 ug/ml with pH 7.4 0.05 M PBS, and diluted to a concentration of 0 ng/ml, 8.8 ng/ml, 26.4 ng/ml, 79.2 ng/ml, 237.6, respectively.
  • Ng/ml, 712.8 ng/ml 100 ul of the diluted solution was added to the S hole of the test card, reacted at room temperature for 5 min, analyzed by a dry fluorescence analyzer, irradiated with a 365 nm fluorescence excitation source, and collected at 615 nm. , obtain the signal values of the detection line (T line) and the control line (C line).
  • the sensitivity for dichlorvos is up to 9 ng/mL, linear regression is R>0.99, and the linearity is good. It can be seen that the method can perform quantitative detection.
  • the method of the present invention can not only achieve qualitative detection of organophosphorus and carbamate substances, but also perform quantitative detection of a small amount, and the detection sensitivity is high, and can reach less than 10 ppb.

Abstract

本发明涉及一种双酰胺复合物及其快速检测有机磷和氨基甲酸酯类农药的方法。本发明制备出的复合物能有效检测微量的有机磷和氨基甲酸酯类农药，而且使用方便、检测时间短、灵敏性高、稳定性好。故本发明可广泛应用于蔬菜、水果等农产品及环境中的微痕量有机磷和氨基甲酸酯类农药的残留检测，也可用于对食品和环境安全评价、监控其有机磷和氨基甲酸酯类农药的污染情况等。

Description

一种双酰胺复合物及其制备方法和用途 技术领域
本发明涉及一种快速检测胆碱脂酶活性的方法，具体是涉及一种双酰胺复合物，其能够实现对有机磷和氨基甲酸酯类物质的检测。
背景技术
有机磷农药因其具有防治对象多、应用范围广、价格低等特点，已成为目前世界农药三大支柱之一，在农业稳产、高产中发挥着巨大作用。氨基甲酸酯类农药，是在有机磷酸酯之后发展起来的合成农药，氨基甲酸酯类农药一般无特殊气味，在酸性环境下稳定，遇碱分解，大多数品种毒性较有机磷酸酯类低。但是，由于大多数有机磷农药都属于高毒化合物，不仅污染土壤、水源等自然资源，且在动、植物体内产生蓄积，通过食物链，成为危害人们生命安全的隐患，然而氨基甲酸酯类农药虽然不是剧毒化合物，但具有致癌性，国际癌症研究机构在2007年把氨基甲酸酯类列为2A类致癌物。食物中毒发生后，快速筛查出是否是由有机磷或氨基甲酸酯类的农药或鼠药所致，对于及时抢救伤者具有重要意义。
农药的检测重点是有机磷或氨基甲酸酯类农药。为预防和控制有机磷农药残留对人体健康的影响，就需要加强对食品中有机磷农药残留检测的力度，目前关于有机磷和氨基甲酸酯类农药的快速检测方法,国家已经制定出国家标准快速检验方法GB/T5009.199-2003，但是很多农药的检出限已经不能满足《GB2763-2016食品安全国家标准食品中最大农药残留限量》中规定，如何实现微量有机磷农药残留快速、高灵敏性的检测方法成为人们迫切需要解决的问题，对食品安全评价、增强我国农产品国际竞争力等方面具有重要意义。
现有检测有机磷和氨基甲酸酯类农药的方法主要有气相色谱、质谱、气相色谱-质谱联用、液相色谱-质谱联用、高效液相色谱、拉曼光谱等。这些方法虽然具备定量准确、灵敏度高等优点，但多为大型仪器，检测周期长，不适合现场检测和大批量样品的筛选，难于满足预防和控制突发事 件。针对现有的有机磷和氨基甲酸酯类等食品农残检测的不足之处，本发明提供一种快速且能够检测微量有机磷和氨基甲酸酯类农药的方法，具有能实现对微量有机磷和氨基甲酸酯类农药的快速、高灵敏性、选择性检测，且检测成本低，操作简单等优点。
胆碱酯酶(cholinesterase)是一类糖蛋白，以多种同工酶形式存在于体内。一般可分为真性胆碱酯酶和假性胆碱脂酶。在临床中,测定血清胆碱酯酶活性是协助诊断有机磷中毒和评估肝实质细胞损害的重要手段。
现有检测胆碱脂酶的方法主要有比色法和连续监测速率法，比色法显色不稳定，特别是室温超过20℃时影响明显，误差较大；另外连续监测速率法需要依赖生化分析仪，不适合于现场快速检测分析。然而本发明技术可以实现，仅需微量样本即可现场快分析。
发明内容
本发明的目的是公开一种双酰胺复合物，其制备方法和使用该复合物检测有机磷和氨基甲酸酯类物质的方法，同时使用该复合物检测胆碱酯酶的方法。
本发明在第一方面公开一种双酰胺复合物，该复合物由如下结构的化合物组成:A-[B] n-[C] m，其中N≥M≥1，A、B和C三者通过酰氨键连接而成，其中A为聚合物，B和C为化合物；
聚合物A为表面被氨基或羧基活化的高分子聚合物；
化合物B具体为下式结构式的化合物，
Figure PCTCN2018116375-appb-000001
R1、R2、R3中只有一个基团为氨基或羧基，剩余基团为H、烷烃基、芳香基、硝基、卤素、羟基或其衍生物。
化合物C为含有游离伯氨基或羧基的化合物，该化合物可以为分子量 小于5000道尔顿的小分子化合物，或者是分子量大于5000道尔顿的核酸、多肽、蛋白质类大分子化合物。
在一个优选的实施例中聚合物A、化合物B和化合物C的摩尔比为1：N：M，其中N≥M≥1,双酰胺化合物能够与硫代胆碱等巯基类化合物反应，释放出化合物C的衍生物。
其中化合物A可选氨基微球或羧基微球，优选的氨基微球粒径为0.5-30μm，更优选为5-15μm，更优选6μm、8μm、或10μm。化合物A也可选择粒径为1-100nm的磁性纳米聚苯乙烯颗粒。
其中化合物B可选任何符合上述结构式的化合物，例如5,5'-二硫代双(2-硝基苯甲酸CAS号：69-78-3，或是4,4'-二硫代二苯胺CAS号：722-27-0，或是4,4'-二硫代双苯甲酸CAS号：1155-51-7，或是2,2'-二硫双(3-甲基苯甲酸)CAS号：13363-59-2。
化合物C为含有游离伯氨基或羧基的化合物，该化合物可以为分子量小于5000道尔顿的小分子化合物，或者是分子量大于5000道尔顿的核酸、多肽、蛋白质类大分子化合物。
本发明第二方面公开复合物的制备方法，包括如下步骤：
(1)通过碳二亚胺法活化化合物B溶液；
(2)加入化合物C溶液，进行反应，得到化合物B与化合物C的偶联物；
(3)再加入聚合物A溶液，使得化合物B-化合物C偶联物偶联至聚合物A上，得到聚合物A-化合物B-化合物C的衍生物。
在一个具体实施方式中，步骤(1)是指在化合物B溶液中，加入EDC溶液和NHS溶液，室温反应，得到溶液。
在一个具体的实施例中，EDC和NHS物质的量比为2:1。
在一个具体实施方式中，步骤(2)是指在步骤(1)所得溶液中加入化合物C。
在一个具体实施方式中，步骤(3)是指在化合物B-化合物C偶联物的溶液中加入聚合物A溶液，使得化合物B-化合物C偶联物以共价键结合在聚合物A上，形成聚合物A-化合物B-化合物C的衍生物。
在一个具体实施方式中，化合物A可选氨基微球或羧基微球，粒径为 0.5-30μm，优选的氨基微球粒径为5-15μm，更优选6μm、8μm、或10μm。
在一个具体实施例中聚合物A为聚苯乙烯氨基微球，聚合物A球粒径为10μm。
在一个具体实施例中化合物B为5,5'-二硫代双(2-硝基苯甲酸)(简称DTNB)。
在一个具体实施例中化合物C为降钙素原(简称PCT)。
在一个具体实施例中双酰胺复合物为以下的结构(其中微球为聚苯乙烯氨基微球)：
Figure PCTCN2018116375-appb-000002
在一个实施方式中DTNB和PCT的摩尔比为5-20：1。
在一个实施方式中聚合物A可选择氨基微球，粒径为0.5-30μm，优选的氨基微球粒径为5-15μm，更优选6μm、8μm、或10μm。
在另一个具体实施方式中，聚合物A可选择球粒径为1-100nm的磁性纳米聚苯乙烯颗粒。
在一个实施方式中，氨基微球与DTNB的质量摩尔比(g:mol)为：300-900:1，优选的330:1、600:1或700:1。
在一个具体实施例中化合物C为磺胺嘧啶，化合物A为氨基微球，化合物B为DTNB。
在一个实施方式中DTNB和磺胺嘧啶的摩尔比例是1：0.5～3，DTNB和磺胺嘧啶的优选摩尔比例为1:1.5。
在一个具体实施例中双酰胺复合物为以下的结构：
Figure PCTCN2018116375-appb-000003
本发明的复合物能够实现与硫代胆碱反应，同时释放化合物C，从而通过免疫学方法检测化合物C的含量，从而间接反映出有机磷和氨基甲酸酯类农药的浓度。
本发明第三方面公开一种检测有机磷和氨基甲酸酯类物质的方法，主要包括如下步骤:
待测物溶液(样本液)依次与胆碱酯酶，酰化硫代胆碱和双酰胺复合物反应，释放出化合物C，再通过抗原抗体或者配体受体免疫学分析方法检测化合物C的含量，从而间接反映出有机磷和氨基甲酸酯类农药的浓度。
其中，酰化硫代胆碱水溶性差且不稳定，一般可选水溶性好的酰化硫代胆碱的卤素盐，例如碘化硫代乙酰胆碱，碘化硫代丁酰胆碱等。免疫学检测的方法及技术主要有：酶免疫测定技术、放射免疫测定技术、免疫荧光技术、免疫胶体金标记技术等。
在一个具体实施例中使用荧光免疫层析分析技术，聚合物A为氨基微球，聚合物A球粒径为0.5-30μm，化合物B为5,5'-二硫代双(2-硝基苯甲酸)(简称DTNB)；化合物C为降钙素原(简称PCT)。
荧光免疫层析分析的检测方法是：1、将样本滴加在包埋有胆碱酯酶的结合垫上面，样本中水溶液使胆碱脂酶溶解，而向着吸水纸一端发生泳动迁移；2、泳动过程中，迁移的液体与含有碘化硫代乙酰胆碱的结合垫反应， 在一定条件下胆碱酯酶能够催化碘化硫代乙酰胆碱生成硫代胆碱和乙酸；3、生成硫代胆碱随着液体进一步向前泳动，进一步与含有氨基微球-DTNB-化合物C复合物结合垫反应，硫代胆碱能够催化氨基微球-DTNB-化合物C复合物(下面简称复合物)，生成化合物C-TNB或者化合物C-硫代胆碱-NTB可溶性衍生物(下面简称化合物C衍生物)；由于复合物中微球直径较大不能通过硝酸纤维素膜(NC膜)，而被截留在NC膜底端，然而生成化合物C衍生物能够随着液体继续在NC膜上泳动，从而使未反应的复合物和释放出的化合物C衍生物发生分离；4、释放出的化合物C衍生物随着液体进一步向前泳动，进一步与荧光微球标记的抗体1(下面简称荧光微球抗体I)反应，形成化合物C-荧光微球抗体I复合物；5、形成化合物C-荧光微球抗体I复合物随着液体继续在NC膜上向前层析迁移，与包被在NC膜上抗体11或者化合物C-半抗原发生反应，从而使荧光微球被截留下来，由于荧光微球在一定波长光激发下，能发射出特殊波长信号，通过仪器收集特殊波长信号的强弱而分析出化合物C的含量水平；6、化合物C的含量水平，直接反映出了硫代胆碱的含量水平，而硫代胆碱的含量水平直接体现出胆碱酯酶的活性；7、胆碱脂酶活性高低间接反映出样本中有机磷或者氨基甲酸酯类农药(以下简称农药)的含量水平，胆碱酯酶的活性越低，反映出农药含量水平越高；胆碱酯酶的活性越高，反映出农药含量水平越低。
在另一个具体实施例中以磁性纳米颗粒分离-酶标记免疫化学发光分析技术进行检测，聚合物A为氨基活化的磁性纳米聚苯乙烯颗粒，聚合物A球粒径为1-100nm的磁性纳米聚苯乙烯颗粒，磁性纳米颗粒可以通过四氧化三铁均匀包裹纳米级聚苯乙烯颗粒实现，也可以通过其他任何磁性的且具有粘附性质的物质包裹纳米级颗粒实现。化合物B为5,5'-二硫代双(2-硝基苯甲酸)(简称DTNB)；化合物C为降钙素原(简称PCT)。在一个具体实施例中，检测方法是：1、将样本滴加在冻干有胆碱酯酶的微孔中，样本中水溶液使胆碱脂酶溶解，而形成均一稳定液态体系；2、再依次加入底物碘化硫代乙酰胆碱、磁性纳米颗粒-DTNB-化合物C复合物(下面简称复合物)，混匀。在一定条件下胆碱酯酶能够催化碘化硫代乙酰胆碱生成硫代胆碱和乙酸；生成的硫代胆碱，进一步与复合物反应，生成化合物C-TNB或者化合物C-硫代胆碱-NTB可溶性复合物(下面简称化合物C)； 3、反应一段时间后，再采用含磁性的物质吸附未反应完全的复合物：由于复合物能够被磁性物质所吸附，从而使未反应的复合物和释放出的化合物C发生分离；4、释放的化合物C与微孔包被抗体1(下面简称包被抗体I)和酶标记的抗体II(以下简称标记抗体II)或者酶标记的化合物C-半抗原(以下简称：标记抗原)反应，形成包被抗体1-化合物C-标记抗体II或者化合物C-标记抗原复合物，再通过洗涤去除未反应的标记抗体II或者标记抗原；5、酶(可选辣根过氧化物酶，碱性磷酸酶)能够催化鲁米诺或其衍生物等发光底物，发射出特殊波长信号，通过仪器收集特殊波长信号的强弱而分析出化合物C的含量水平；6、化合物C的含量水平，直接反映出了硫代胆碱的含量水平，而硫代胆碱的含量水平直接体现出胆碱酯酶的活性；7、胆碱脂酶活性高低间接反映出样本中有机磷或者氨基甲酸酯类农药(以下简称农药)的含量水平，胆碱酯酶的活性越低，反映出农药含量水平越高；胆碱酯酶的活性越高，反映出农药含量水平越低。
本发明第四方面公开一种检测有机磷和氨基甲酸酯类物质的试纸条，所述试纸条包括以下部件：
1)酶结合垫:含有胆碱酯酶；
2)底物结合垫:含有酰化硫代胆碱；
3)复合物结合垫：包被有氨基微球-DTNB-化合物C复合物；
4)标记抗体垫：包被有抗化合物C的单克隆抗体-标记物偶联物；
5)反应膜上检测线和质控线：检测线包被有化合物C-半抗原或化合物C的另一种单克隆抗体；质控线包被有抗步骤4)中抗体的抗抗体；
6)吸水纸：用于提供检测所需的毛细作用力的吸水纸。
如上所述的试纸条，其特征在于所述试纸条进一步包括底板，其中所述底板上粘贴有依次搭接的酶结合垫、底物结合垫、复合物结合垫、荧光微球垫、反应膜和吸水纸。
特殊注明：其中酶结合垫包含两种形式，一种形式为包埋在特殊材料上，与检测试纸条同时存在于底板上；另一种形式为液体或者冻干粉保存在特殊容器中，与底板部分各自独立存在。
在一个具体实施方式中，依次搭接粘贴的酶结合垫、底物结合垫、复合物结合垫、荧光微球垫、反应膜和吸水纸。
在一个具体实施方式中，所述胆碱酯酶为市售的或合成的乙酰胆碱酯酶(AChE)或丁酰胆碱酯酶(BChE)，胆碱酯酶可以由血浆纯化或通过重组DNA技术制备。
在一个具体实施方式中，所述酰化硫代胆碱包含乙酰硫代胆碱、丙酰硫代胆碱、丁酰硫代胆碱、苯甲酰硫代胆碱等。酰化硫代胆碱的水溶性差且不稳定，一般可选水溶性好的酰化硫代胆碱的卤素盐作为底物，例如碘化硫代乙酰胆碱等，碘化硫代丁酰胆碱等。
在一个具体实施方式中，所述氨基微球-DTNB-化合物C复合物，是一种能够与硫代胆碱反应，释放化合物C的复合物，该复合物由氨基微球、DTNB和化合物C三者偶联而成。
在一个具体实施方式中，所述微球复合物，其微球为羧基微球。
在一个具体实施方式中，所述反应膜为NC膜。
在检测时，如果化合物C的类型为伯氨基化合物类小分子，例如化合物C为磺胺嘧啶(SD)，样本中含量水平应与T值/C值的比值，呈反比例关系。
如果化合物C的类型为多肽、核酸或者蛋白质类大分子，例如化合物C为降钙素原(PCT)，样本中含量水平应与T值/C值的比值，呈正比例关系。
本发明的第五方面，公开了一种检测胆碱酯酶活性的方法，该方法利用胆碱酯酶能够催化酰化硫代胆碱与本发明的前述双酰胺复合物反应生成化合物C衍生物，再通过抗原抗体或者配体受体免疫学分析方法检测化合物C的含量，从而间接反映出胆碱酯酶的活性。
由于主要检测样品例如血液中存在其他巯基类物质，也会与本发明前述双酰胺复合物发生释放化合物C衍生物的反应，这样会对检测形成较大干扰，因此检测样品的胆碱酯酶活性时，要排除掉样品中自带的巯基类化合物所产生的化合物C衍生物对检测结果的影响。这时我们引入背景检测卡，计算出背景所产生的化合物C衍生物，这里为了方便计算，我们也将该背景的干扰换算为胆碱酯酶活性值，这样直接从检测卡所测得的总酶活中减掉背景所对应的酶活值，即可计算出酶催化反应的酶活值。将化合物C衍生物含量换算为胆碱酯酶活性，均依据化合物C与胆碱酯酶活性的标 准曲线计算得到，该标准曲线为利用胆碱酯酶标准品多次实验获得。由此优选的检测胆碱酯酶活性的方法为：先检测不加酰化硫代胆碱的情况下，样品中产生的化合物C衍生物所对应的胆碱酯酶活性1，再检测添加酰化硫代胆碱的情况下，样品中的背景以及酶催化反应的总胆碱酯酶活性2，活性2与活性1的值相减即除掉样品中自带的巯基类化合物对检测结果的影响，获得了酶催化反应的胆碱酯酶活性。
该检测胆碱酯酶活性方法包括如下步骤:
(1)背景检测：待测物溶液(样本液)与前面所述的复合物反应，释放出化合物C的衍生物，再通过抗原抗体或者配体受体免疫学分析方法检测化合物C衍生物的含量，根据该含量计算出酶活性1；
(2)样品检测：待测物溶液(样本液)依次与酰化硫代胆碱和权利要求1所述的双酰胺复合物反应，释放出化合物C的复合物，再通过抗原抗体或者配体受体免疫学分析方法检测化合物C衍生物的含量，根据该含量计算出酶活性2；
(3)计算样品中胆碱酯酶活性：计算活性1和活性2差值，计算出酶催化反应的酶活性水平。
本发明的第六方面，公开了一种检测胆碱酯酶活性的背景试纸条，其特征在于所述试纸条包括以下部件：
1)复合物结合垫：包被有氨基微球-DTNB-化合物C复合物；
2)标记抗体垫：包被有抗化合物C的单克隆抗体-标记物偶联物；
3)反应膜上检测线和质控线：检测线包被有化合物C-半抗原或化合物C的另一种单克隆抗体；质控线包被有抗步骤(2)中的抗体的抗抗体；
4)吸水纸：用于提供检测所需的毛细作用力的吸水纸。
还公开了一种检测胆碱酯酶活性的样品试纸条，其特征在于所述试纸条包括以下部件：
1)底物结合垫:含有酰化硫代胆碱；
2)复合物结合垫：包被有氨基微球-DTNB-化合物C复合物；
3)标记抗体垫：包被有抗化合物C的单克隆抗体-标记物偶联物；
4)反应膜上检测线和质控线：检测线包被有化合物C-半抗原或化合物C的另一种单克隆抗体；质控线包被有抗步骤3)中抗体的抗抗体；
5)吸水纸：用于提供检测所需的毛细作用力的吸水纸。
该发明中的术语解释：
酰化硫代胆碱是指酰化硫代胆碱及其卤素盐，包括但不限于碘化乙酰硫代胆碱、氯化乙酰硫代胆碱、溴化乙酰硫代胆碱、碘化丙酰硫代胆碱、氯化丙酰硫代胆碱、溴化丙酰硫代胆碱、碘化丁酰硫代胆碱、氯化丁酰硫代胆碱、溴化丁酰硫代胆碱、碘化苯甲酰硫代胆碱、氯化苯甲酰硫代胆碱、溴化苯甲酰硫代胆碱等。
化合物C是指一类为多肽、核酸或者蛋白质类大分子，其分子量大于5KD，例如：牛血清白蛋白、人血清白蛋白、鸡卵清蛋白、血蓝蛋白、降钙素原、甲胎蛋白、癌胚抗原、促人绒毛膜性腺激素、卵泡刺激素等。化合物C也可以是一类为含有伯氨基的化合物，其分子量小于5KD。例如：三聚氰胺、苯胺、磺胺、磺胺嘧啶、磺胺甲基嘧啶、磺胺甲恶唑、克伦特罗、多巴胺等。
氨基微球是指表面被氨基化的微球。微球的材质主要有：树脂、乙基纤维素、聚丙乙烯、聚苯乙烯、聚丙烯酸、乳聚丁苯橡胶、聚乙酸乙烯脂等。
羧基微球是指表面被羧基化的微球。微球的材质主要有：树脂、乙基纤维素、聚丙乙烯、聚苯乙烯、聚丙烯酸、乳聚丁苯橡胶、聚乙酸乙烯脂等。微球主要包括：荧光微球、彩色微球等。
DTNB是指5,5'-二硫代双(2-硝基苯甲酸)，CAS号：69-78-3。
偶联是指偶联是一个化学反应发生时其它反应以化学计量学的关系相伴进行的现象，偶联反应，也作偶连反应、耦联反应、氧化偶联，是由两个有机化学单位进行某种化学反应而得到一个有机分子的过程。
偶联物是指将两个有机化学物质通过偶联反应形成相应的物质。
免疫学方法检测是指利用免疫学原理，以待测物作为抗原或抗体(受体或配体)从而测定样品中待测物质含量的方法。
含量是指物质中所包含的某种成分的量。
EDC是指1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐)，CAS号：25952-53-8。
NHS是指N-羟基琥珀酰亚胺，CAS号：6066-82-6。碳二亚胺， (Carbodiimide)，含有N＝C＝N官能团，是一类常用的失水剂。一般由硫脲失硫化氢或脲失水制备，水解得到脲衍生物。主要用于活化羧基，促使酰胺和酯的生成。碳二亚胺法是指利用碳二亚胺活化羧基，促使酰胺和酯的生成。
NC膜是指硝酸纤维素膜，每4cm膜水的层析流速多少秒来定义孔径的，根据流速的快慢常用的有90，120，135，180，240等规格。
胆碱酯酶是一类糖蛋白，以多种同工酶形式存在，胆碱酯酶包括但不限于乙酰胆碱酯酶和丁酰胆碱酯酶。
标记物是指荧光素，放射物性金属，微球，生物酶等。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚明白，以下结合具体实施例，并参照附图，对本发明作进一步的详细说明。
以下实施例中的抗原抗体均市售可得，但这些原料也可以自制，抗原抗体的来源并非本发明的关键。
实施例1：氨基微球-DTNB-PCT(降钙素原)复合物制备
氨基微球溶液：将氨基微球(备注：固体含量为0.01g/ml)(以下简称氨基微球)用PBS按照1:10稀释；
抗原溶液：用PBS溶液将PCT(降钙素原)配制10mmoL/L溶液；
DTNB溶液：将DTNB用DMF溶解，并配制成浓度为10mmoL/L溶液；
EDC溶液：将1-乙基-(3-二甲基氨基丙基)碳酰二亚胺(以下简称：EDC)用水溶解，并配制成浓度为100mmoL/L溶液；
NHS溶液：将N-羟基琥珀酰亚胺(以下简称：NHS)用水溶解，并配制成浓度为100mmoL/L溶液；
DTNB活化：在4mL的棕色玻璃瓶中加入300μL DTNB溶液，再加入840μL的EDC溶液和420μL的NHS溶液，室温振荡反应10～16小时。(投入物质的量比约为DTNB:EDC:NHS＝25：700：350)。
在10mL棕色瓶中加入30μLPCT溶液，将活化好的DTNB溶液全部缓慢加入到PCT溶液，继续室温振荡反应0.5小时(物质的量比约为 DTNB:EDC:NHS:PCT＝25：700：350：2.5)。
反应两小时后再向上述溶液中加入1.0mL稀释好的氨基微球(微球为1mg)，继续室温振荡反应2小时。
将反应后的液体转移到离心管中，以4℃10000r/min的速度离心20min；弃去上清液，再用PBS溶液离心洗涤5次。得离心后沉淀物，在低温真空干燥后得干粉，置于4℃密封避光保存。
实施例2：氨基微球-DTNB-磺氨嘧啶(SD)复合物制备
氨基微球溶液：将氨基微球(备注：固体含量为0.01g/ml)(以下简称氨基微球)用PBS按照1:10稀释；
抗原溶液：用PBS溶液将磺氨嘧啶(SD)配制10mmoL/L溶液；
DTNB溶液：将DTNB用DMF溶解，并配制成浓度为10mmoL/L溶液；
EDC溶液：将1-乙基-(3-二甲基氨基丙基)碳酰二亚胺(以下简称：EDC)用水溶解，并配制成浓度为100mmoL/L溶液；
NHS溶液：将N-羟基琥珀酰亚胺(以下简称：NHS)用水溶解，并配制成浓度为100mmoL/L溶液；
DTNB活化：在4mL的棕色玻璃瓶中加入300μL DTNB溶液，再加入840μL的EDC溶液和420μL的NHS溶液，室温振荡反应10～16小时。(投入物质的量比约为DTNB:EDC:NHS＝25：700：350)。
在10mL棕色瓶中加入450μL磺氨嘧啶(SD)溶液，将活化好的DTNB溶液全部缓慢加入到SD溶液，继续室温振荡反应0.5小时(物质的量比约为DTNB:EDC:NHS:SD＝25：700：350：37.5)。
反应两小时后再向上述溶液中加入1.0mL稀释好的氨基微球(微球为1mg)，继续室温振荡反应2小时。
将反应后的液体转移到离心管中，以4℃10000r/min的速度离心20min；弃去上清液，再用PBS溶液离心洗涤5次。得离心后沉淀物，在低温真空干燥后得干粉，置于4℃密封避光保存。
实施例3：检测试剂卡酶结合垫与底物结合垫制备
按以下方法配制酶稀释缓冲液(以下简称：酶稀液)：分别称取3g  Na2HPO4·12H2O；0.25g NaH2PO4·2H2O；8.7g NaCl；10g牛血清白蛋白；5g海藻糖；1g叠氮钠；10g羟丙基-beta-环糊精，先用800mL去离子水充分溶解，再调节pH值至8.0，最后用去离子水定容至1L。
按以下方法配制底物稀释缓冲液(以下简称：底物液)：分别称取5g羟基亚乙基二磷酸；30g异丙醇；2g海藻糖；3g亚硫酸钠；0.5g苯甲酸；1g叠氮钠，先用800mL去离子水充分溶解，再调节pH值至6.0，最后用去离子水定容至1L。
酶结合垫制备：
第一种形式：将用酶稀液稀释成0.1～3U/mL的乙酰胆碱酯酶(购自北京索来宝公司)，再用移液器取1～2mL平铺在5mm*300mm的玻璃纤维纸上，置于室温(25℃±5℃)，湿度≤30％，通风干燥24小时，干燥后于4℃保存备用。
第二种形式：将用酶稀液稀释成0.1～3U/mL的乙酰胆碱酯酶(购自北京索来宝公司)，直接以液体状态保存在0.5～100mL塑料瓶或者玻璃瓶中，或者采用冻干粉的状态保存在0.1～100mL塑料瓶(孔)或者玻璃瓶(孔)中。
底物结合垫制备：将碘化硫代乙酰胆碱用底物液稀释成1～5 mg/mL，再用移液器量取1～2 mL平铺在5mm*300mm的玻璃纤维纸上，置于室温(25℃±5℃)，湿度≤30％，通风干燥24小时，干燥后于4℃保存备用。
实施例4：复合物氨基微球-DTNB- PCT结合垫制备
将实施例1的氨基微球-DTNB-抗原复合物用酶稀液，按照1:10的比例稀释，再用移液器量取1～2 mL平铺在5mm*300mm的玻璃纤维纸上，置于室温(25℃±5℃)，湿度≤30％，通风干燥24小时，干燥后于4℃保存得氨基微球-DTNB- PCT复合物结合垫备用。
实施例5： 氨基微球-DTNB-SD复合物结合垫制备
按以下方法配制微球稀释缓冲液(以下简称：微球稀释液)：分别称取3g Na2HPO4·12H2O；0.25g NaH2PO4·2H2O；5g羟基亚乙基二磷酸；30g异丙醇；5g蔗糖；3g亚硫酸钠；1g叠氮钠，先用800mL去离子水充分 溶解，再调节pH值至6,5，最后用去离子水定容至1L。
补充将实施例2的氨基微球-DTNB-抗原复合物用微球稀释液，按照1:10的比例稀释，再用移液器量取1～2 mL平铺在5mm*300mm的玻璃纤维纸上，置于室温(25℃±5℃)，湿度≤30％，通风干燥24小时，干燥后于4℃保存得氨基微球-DTNB- SD复合物结合垫备用。
实施例6：降钙素原(PCT)抗体荧光微球垫制备
1、将抗降钙素原(PCT)(以下简称PCT)的单克隆抗体I( 购自 海肽生物科技(上海)有限公司)用pH7.4 0.05M PBS溶液，透析3次，每次体积置换比例为1:100，透析完成后用pH7.4 0.05M PBS将PCT的单克隆抗体I和单克隆抗体II调节成浓度为1mg/mL。
2、在1mL羧基微球(时间分辨荧光微球(铕)：水＝1g：1000mL，Bangs Lab)中，加入0.4mg NHS(用pH5.5 50mM MES缓冲液配制成浓度为1mg/mL的溶液)；再加入0.6mg EDC(用pH5.5 50mM MES缓冲液配制成浓度为1mg/mL的溶液)，室温(25℃±5℃)下反应30分钟，摇床翻转混匀。
3、反应完成后，以4℃10000r/min的速度离心20min，离心完成弃去上清液，得沉淀物；再用pH5.5 50mM MES缓冲液洗涤沉淀物3次，每次洗涤液体为2mL。
4、将上述洗涤好的沉淀物，用0.75mLpH7.4 0.05M PBS重悬，再加入0.5mg透析好的抗PCT的单克隆抗体I，室温(25℃±5℃)下反应2小时，摇床翻转混匀，速度60转/分钟。
5、将上述反应完成后液体用超声波震荡30秒，再加入200μL 1.0M乙醇胺溶液(用超纯水配制)和100μL 2.0M甘氨酸溶液(用超纯水配制)，室温(25℃±5℃)下反应1小时，摇床翻转混匀，速度60转/分钟。(终止反应)
6、将上述反应完成后用液体超声波震荡30秒，以4℃10000r/min的速度离心20min，离心完成弃去上清液，得沉淀物；再用0.1％吐温-20PBS洗涤沉淀物3次，每次洗涤液体为2mL。(去除杂质)
7、将上述洗涤好的沉淀物用5mL酶稀液重悬，再用移液器量取1 mL平铺在5mm*300mm的玻璃纤维纸上，置于室温(25℃±5℃)，湿度 ≤30％，通风干燥24小时，干燥后于4℃保存备用。
实施例7：抗磺胺嘧啶(SD)抗体荧光微球垫制备
1、将抗磺胺嘧啶(SD)(以下简称SD)的单克隆抗体( 购自深 圳市宝安康生物技术有限公司)用pH7.4 0.05M PBS溶液，透析3次，每次体积置换比例为1:100，透析完成后用pH7.4 0.05M PBS将抗SD的单克隆抗体调节成浓度为1mg/mL。
2、在1mL羧基微球((时间分辨荧光微球(铕)：水＝1g：1000mL，Bangs Lab)，加入0.4mg NHS(用pH5.5 50mM MES缓冲液配制成浓度为1mg/mL的溶液)；再加入0.6mg EDC(用pH5.5 50mM MES缓冲液配制成浓度为1mg/mL的溶液)，室温(25℃±5℃)下反应30分钟，摇床翻转混匀。
3、反应完成后，以4℃10000r/min的速度离心20min，离心完成弃去上清液，得沉淀物；再用pH5.5 50mM MES缓冲液洗涤沉淀物3次，每次洗涤液体为2mL。
4、将上述洗涤好的沉淀物，用0.5mL PBS重悬，再加入0.5mg透析好的抗SD的单克隆抗体，室温(25℃±5℃)下反应2小时，摇床翻转混匀，速度60转/分钟。
5、将上述反应完成后液体用超声波震荡30秒，再加入200μL 1.0M乙醇胺溶液(用超纯水配制)和100μL 2.0M甘氨酸溶液(用超纯水配制)，室温(25℃±5℃)下反应1小时，摇床翻转混匀，速度60转/分钟。
6、将上述反应完成后用液体超声波震荡30秒，以4℃10000r/min的速度离心20min，离心完成弃去上清液，得沉淀物；再用0.1％吐温-20PBS洗涤沉淀物3次，每次洗涤液体为2mL。
7、将上述洗涤好的沉淀物用5mL酶稀液重悬，再用移液器量取1 mL平铺在5mm*300mm的玻璃纤维纸上，置于室温(25℃±5℃)，湿度≤30％，通风干燥24小时，干燥后于4℃保存备用。
实施例8：反应膜上检测线和质控线的包被(PCT抗体包被)
抗PCT的单克隆抗体II和羊抗鼠IgG的二抗(以下简称二抗，购自海肽生物科技(上海)有限公司)包被到NC膜上
用pH7.4 0.05M PBS将抗PCT的单克隆抗体II调节包被物浓度为0.5mg/mL和将二抗调节包被物浓度为0.5mg/mL,喷膜量为0.8μL/cm；
检测线包被抗化合物C的单克隆抗体II，质控线包被二抗，两区位置相隔6mm，质控线距NC膜顶端10mm，检测线距NC膜底端9mm；
37℃烘干处理过夜后，于室温干燥的环境下保存备用
实施例9：反应膜上检测线和质控线的包被(SD-BSA抗原包被)
SD-BSA偶联物和羊抗抗SD的单克隆抗体的二抗(以下简称二抗，购自海肽生物科技(上海)有限公司)包被到NC膜上
用pH7.4 0.05M PBS将SD-BSA偶联物调节包被物浓度为0.5mg/mL和将二抗调节包被物浓度为0.5mg/mL,喷膜量为0.8μL/cm；
检测线包被SD-BSA偶联物，质控线包被二抗，两区位置相隔6Mm，质控线距NC膜顶端10mm，检测线距NC膜底端9mm；
37℃烘干处理过夜后，于室温干燥的环境下保存备用。
实施例10：胆碱酯酶荧光微球免疫层析背景卡和检测卡的装配
(1)基于PCT的胆碱酯酶背景卡和检测卡
背景卡：在底板上依次搭连地粘贴空白垫、实施例4或实施例5获得的复合物结合垫、实施例6获得的荧光微球垫、实施例8或实施例9获得的NC膜和吸水纸，粘贴好的试纸条板用切割机裁切成4mm试纸条，并组装到准备好的检测卡卡壳中，装入铝箔袋，加入干燥剂后，封口保存，于室温干燥的环境下至少可保存一年。背景卡具体构造如图1所示。
检测卡：
在底板上依次搭连地粘贴实施例3获得的底物结合垫、实施例4或实施例5获得的复合物结合垫、实施例6获得的荧光微球垫、实施例8或实施例9获得的NC膜和吸水纸，粘贴好的试纸条板用切割机裁切成4mm试纸条，并组装到准备好的检测卡卡壳中，装入铝箔袋，加入干燥剂后，封口保存，于室温干燥的环境下保存。检测卡具体构造如图2所示。
实施例11：胆碱酯酶荧光微球免疫层析检测卡的测试
检测前，先打开干式荧光检测仪，预热5分钟。
首先，由于样品中会存在一些干扰物质，它们与硫代胆碱类似，因此先使用实施例10(1)获得的胆碱酯酶荧光微球免疫层析背景卡检测样品背景干扰：
1、将胆碱酯酶用蒸馏水或者pH6.0～pH8.0的缓冲溶液，配置成0U/L、15U/L、45U/L、135U/L、405U/L、1215U/L的活性浓度，混匀后备用；
2、每批次检测时，以蒸馏水或者pH6.0～pH8.0的缓冲溶液作为空白对照；
3、将胆碱酯酶荧光微球免疫层析测试卡的从铝箔袋中取出，平整放在桌面上；
4、再将上述提取好样本液体用吸管吸取100μL样本液滴加在背景卡的样本孔中(S)；
5、37±2℃反应10分钟；
6、将反应完成的检测卡放入干式荧光检测仪中进行检测，照射365nm荧光激发光源，并在615nm处采集信号，获得检测线(T线)和对照线(C线)的信号值，比较检测线值(T值)与对照线值(C值)的比值，进行数据分析。分析释放出化合物C的含量水平，再通过化合物C和酶活标准曲线计算出背景的酶活性水平。
然后，依据同样的方法，使用实施例10(1)获得的胆碱酯酶荧光微球免疫层析检测卡检测背景和酶催化反应的酶活总和。
计算第一卡和第二卡之间的差值即是酶催化反应的酶活。
备注：该实施例中，化合物C为降钙素原(PCT)。
Figure PCTCN2018116375-appb-000004
上述结果显示胆碱酯酶荧光微球免疫层析测试卡检测胆碱酯酶，在 45～1215U/L之间具有良好的线性，可以进行胆碱酯酶活性鉴定。
实施例12：有机磷和氨基甲酸酯类药物荧光微球免疫层析检测卡的装配
(1)基于PCT的有机磷和氨基甲酸酯类药物检测卡
在底板上依次搭连地粘贴实施例3获得的酶结合垫、实施例3获得的底物结合垫、实施例4获得的复合物结合垫、实施例5获得的荧光微球垫、实施例8获得的NC膜和吸水纸，粘贴好的试纸条板用切割机裁切成4mm试纸条，并组装到准备好的检测卡卡壳中，装入铝箔袋，加入干燥剂后，封口保存，于室温干燥的环境下至少可保存一年。
(2)基于SD的有机磷和氨基甲酸酯类药物检测卡
也可以将实施例4中的复合物结合垫替换为实施例5的复合物结合垫，实施例8中的NC膜替换为实施例9中的NC膜，其余制备方法相同。
装配完的试剂卡如图3所示，胆碱酯酶以固相形式附着于样本垫的一端。
当然，胆碱酯酶还可以以冻干粉形式存在于检测卡之外，如图4所示，这时即省略掉酶结合垫，其余制备过程一样。
实施例13：有机磷和氨基甲酸酯类药物荧光微球免疫层析检测卡的测试方法
检测前，先把有机磷和氨基甲酸酯类农药残留荧光微球免疫层析测试卡和干式荧光检测仪打开，预热5分钟。
1、选取代表性的蔬菜或者水果样本，去除表面泥土，剪成1cm见方的碎片，取5g样本放入50mL塑料离心管中，再加入15mL蒸馏水或者pH6.0～pH8.0的缓冲溶液，上下颠倒50次左右，再静置3～5分钟；
2、每批次检测时，以蒸馏水或者pH6.0～pH8.0的缓冲溶液作为空白对照；
3、将有机磷和氨基甲酸酯类农药残留荧光微球免疫层析测试卡的从铝箔袋中取出，平整放在桌面上；
4、再将上述提取好样本液体用吸管吸取100μL样本液滴加在检测卡的样本孔中(S)；
5、37±2℃反应10分钟；
6、将反应完成的检测卡放入干式荧光检测仪中进行检测，照射365nm荧光激发光源，并在615nm处采集信号，获得检测线(T线)和对照线(C线)的信号值，比较检测线值(T值)与对照线值(C值)的比值，进行数据分析。
实施例14：定性及定量检测实验结果
一、定性判读
1、使用实施例12(2)装配的有机磷及氨基甲酸酯类药物检测卡进行检测，化合物C为磺胺嘧啶(SD)，其检测卡荧光微球免疫层析测试卡的检测结果按照如下方式判定：
空白对照T值/C值的比值应≤0.2；
如果T值/C值的比值<0.3为阴性结果；T值/C值的比值≥0.3为阳性结果。
具体结果如表1所示：
Figure PCTCN2018116375-appb-000005
表1
依据数据显示不同蔬菜添加克百威药物浓度为100～200ng/ml时，表现为阳性结果。
2、使用实施例12(1)中装配的有机磷及氨基甲酸酯类药物检测卡进行检测，化合物C为降钙素原(PCT)，其检测卡荧光微球免疫层析测试卡的检测结果按照如下方式判定：
空白对照T值/C值的比值应≥1.1；
如果T值/C值的比值>0.9为阴性结果；T值/C值的比值≤0.9为阳性 结果。
具体结果如表2所示：
Figure PCTCN2018116375-appb-000006
表2
依据数据显示不同蔬菜添加克百威药物浓度为20～50ng/ml时，表现为阳性结果。
二、定量检测
1、如果化合物C的类型为伯氨基化合物类小分子，该实施例中，化合物C为磺胺嘧啶(SD)，其检测卡荧光微球免疫层析测试卡的检测结果依据每个药物的浓度，计算出每个浓度下仪器测试T值/C值的比值，样本中含量水平应与T值/C值的比值，呈反比例关系，绘制标准曲线，再对测定的结果进行计算：
2、如果化合物C的类型为多肽、核酸或者蛋白质类大分子，该实施例中，化合物C为降钙素原(PCT)其检测卡荧光微球免疫层析测试卡的 检测结果依据每个药物的浓度，计算出每个浓度下仪器测试T值/C值的比值，样本中含量水平应与T值/C值的比值，呈正比例关系，绘制标准曲线，再对测定的结果进行计算：
该实施例中，化合物C为降钙素原(PCT)。
将药物敌敌畏(浓度为1mg/mL)用pH7.4 0.05M PBS，稀释为20ug/ml，再分别稀释成浓度为0ng/ml、8.8ng/ml、26.4ng/ml、79.2ng/ml、237.6ng/ml、712.8ng/ml，将稀释好的溶液吸取100ul滴加到检测卡的S孔中，室温反应5min，用干式荧光分析仪分析，照射365nm荧光激发光源，并在615nm处采集信号，获得检测线(T线)和对照线(C线)的信号值。
浓度 T值/C值
0ppb 2.276631
8.8ppb 2.072447
26.4ppb 1.901383
79.2ppb 1.63292
237.6ppb 1.547983
712.8ppb 1.266685
针对敌敌畏药物的灵敏度最高可达9ng/mL，线性回归R>0.99，线性良好。可见，该方法可以进行定量检测。
通过上述定性及定量检测实验，可以看出，本发明的方法不仅能够对有机磷及氨基甲酸酯类物质实现定性检测，还可以进行微量的定量检测，检测灵敏度很高，能够达到小于10ppb。
尽管已用于解释说明的目的公开了本发明的优选实施方式，但本领域技术人员可理解，可做出各种改变、增添和替换，而不脱离如后附权利要求所公开的本发明的范围和精神。

Claims (24)

  1. 一种双酰胺复合物，其特征在于该复合物由如下结构的化合物组成:A-[B] n-[C] m，其中N≥M≥1，A、B和C三者通过酰胺键连接而成，其中A为聚合物，B和C为化合物；
    聚合物A为表面被氨基或羧基活化的高分子聚合物；
    化合物B具体为下式结构式的化合物，
    Figure PCTCN2018116375-appb-100001
    R1、R2、R3中只有一个基团为氨基或羧基，剩余基团为H、烷烃基、芳香基、硝基、卤素、羟基或其衍生物；
    化合物C为含有游离伯氨基或羧基的化合物，该化合物可以为分子量小于5000道尔顿的小分子化合物，或者是分子量大于5000道尔顿的核酸、多肽、蛋白质类大分子化合物。
  2. 如权利要求1所述的复合物，其特征在于该复合物的化合物A为直径为0.5-30μm的氨基微球或羧基微球，优选6μm，8μm，10μm；
    化合物B为5,5'-二硫代双(2-硝基苯甲酸)(简称DTNB)；
    和/或化合物C为降钙素原(简称PCT)或磺胺嘧啶。
  3. 如权利要求1所述的复合物，其特征在于该复合物的化合物A为粒径1-100nm的磁性氨基微球或羧基微球。
  4. 如权利要求1所述的复合物，其特征在于该复合物的化合物A为被氨基活化的聚苯乙烯微球。
  5. 如权利要求1所述的复合物，其特征在于该复合物与硫代胆碱等巯基类化合物反应，释放出化合物C的衍生物。
  6. 如权利要求1-5任一项所述的复合物的制备方法，其特征在于该方法包括如下步骤：
    A、通过碳二亚胺法活化化合物B溶液；
    B、加入化合物C溶液，进行反应，得到化合物B与化合物C的偶联物；
    C、再加入聚合物A溶液，使得化合物B-化合物C偶联物偶联至聚合物A上，得到聚合物A-化合物B-化合物C的复合物。
  7. 如权利要求6所述的制备方法，步骤(1)是指在化合物B溶液中，加入EDC溶液和NHS溶液，室温反应，得到溶液，其中EDC和NHS物质的量比优选2:1。
  8. 如权利要求6所述的制备方法，其特征在于步骤(2)是指在步骤(1)所得溶液中加入化合物C。
  9. 如权利要求6所述的制备方法，其特征在于步骤(3)是指在化合物B-化合物C偶联物的溶液中加入聚合物A溶液，使得化合物B-化合物C偶联物以共价键结合在聚合物A上，形成聚合物A-化合物B-化合物C的衍生物。
  10. 如权利要求6所述的制备方法，其特征在于化合物A与化合物B的质量摩尔比(g:mol)为：300-900:1，优选330:1。
  11. 如权利要求6所述的制备方法，化合物B为DTNB，化合物C为PCT，DTNB和PCT的摩尔比为5-20：1。
  12. 如权利要求6所述的制备方法，化合物B为DTNB，化合物C为磺胺嘧啶，DTNB和磺胺嘧啶的摩尔比例是1：0.5～3，DTNB和磺胺嘧啶的优选摩尔比例为1:1.5。
  13. 一种检测有机磷和氨基甲酸酯类物质的方法，其特征在于该方法包括如下步骤:
    待测物溶液(样本液)依次与胆碱酯酶，酰化硫代胆碱和权利要求1-5任一项所述的双酰胺复合物反应，释放出化合物C的衍生物，再通过抗原抗体或者配体受体免疫学分析方法检测化合物C衍生物的含量，从而间接反映出有机磷和氨基甲酸酯类农药的浓度。
  14. 如权利要求13所述的检测方法，其特征在于免疫学分析方法是指酶免疫测定技术、放射免疫测定技术、免疫荧光技术或免疫胶体金标记技术。
  15. 如权利要求13所述的检测方法，其特征在于所述酰化硫代胆碱包含乙酰硫代胆碱、丙酰硫代胆碱、丁酰硫代胆碱、苯甲酰硫代胆碱等或其 卤素盐。
  16. 一种检测有机磷和氨基甲酸酯类物质的试纸条，其特征在于所述试纸条包括以下部件：
    1)胆碱酯酶：含有胆碱酯酶的酶结合垫，或者以干粉形式存在于检测卡外的微型容器中的胆碱酯酶；
    2)底物结合垫:含有酰化硫代胆碱；
    3)复合物结合垫：包被有权利要求1-5任一项所述的双酰胺复合物；
    4)标记抗体垫：包被有抗化合物C的单克隆抗体-标记物偶联物；
    5)反应膜上检测线和质控线：检测线包被有化合物C-半抗原或化合物C的另一种单克隆抗体；质控线包被有抗步骤4)中抗体的抗抗体；
    6)吸水纸：用于提供检测所需的毛细作用力的吸水纸。
  17. 如权利要求16所述的试纸条，其特征在于所述试纸条进一步包括底板，其中所述底板上粘贴有依次搭接的酶结合垫、底物结合垫、复合物结合垫、荧光微球垫、反应膜和吸水纸。
  18. 如权利要求16的试纸条，其特征在于所述胆碱酯酶为乙酰胆碱酯酶(AChE)或丁酰胆碱酯酶(BChE)。
  19. 如权利要求16的试纸条，其特征在于所述酰化硫代胆碱包含乙酰硫代胆碱、丙酰硫代胆碱、丁酰硫代胆碱、苯甲酰硫代胆碱等或其卤素盐。
  20. 如权利要求16的试纸条，其特征在于所述反应膜为NC膜。
  21. 一种检测胆碱酯酶活性的方法，其特征在于该方法包括如下步骤:
    该方法利用胆碱酯酶能够催化酰化硫代胆碱与本发明的前述双酰胺复合物反应生成化合物C衍生物，再通过抗原抗体或者配体受体免疫学分析方法检测化合物C的含量，从而间接反映出胆碱酯酶的活性。
  22. 一种如权利要求21所述的检测方法，其特征在于该方法包括如下步骤:
    (1)背景检测：待测物溶液(样本液)与权利要求1-5任一项所述的双酰胺复合物反应，释放出化合物C的衍生物，再通过抗原抗体或者配体受体免疫学分析方法检测化合物C衍生物的含量，间接计算出背景对应的酶活；
    (2)样品检测：待测物溶液(样本液)依次与酰化硫代胆碱和权利要求1-5任一项所述的双酰胺复合物反应，释放出化合物C的衍生物，再通 过抗原抗体或者配体受体免疫学分析方法检测化合物C衍生物的含量，间接计算出背景和酶催化反应的酶活总和；
    (3)计算样品中胆碱酯酶活性：计算样品检测和背景检测的差值，得到酶催化反应的酶活性水平。
  23. 一种检测胆碱酯酶活性的试纸条，其特征在于所述试纸条包括以下部件：
    1)底物结合垫:含有酰化硫代胆碱；
    2)复合物结合垫：包被有权利要求1-5任一项所述的双酰胺复合物；
    3)标记抗体垫：包被有抗化合物C的单克隆抗体-标记物偶联物；
    4)反应膜上检测线和质控线：检测线包被有化合物C-半抗原或化合物C的另一种单克隆抗体；质控线包被有抗步骤3)抗体的抗抗体；
    5)吸水纸：用于提供检测所需的毛细作用力的吸水纸。
  24. 一种检测胆碱酯酶活性的背景试纸条，其特征在于所述试纸条包括以下部件：
    1)复合物结合垫：包被有权利要求1-5任一项所述的双酰胺复合物；
    2)标记抗体垫：包被有抗化合物C的单克隆抗体-标记物偶联物；
    3)反应膜上检测线和质控线：检测线包被有化合物C-半抗原或化合物C的另一种单克隆抗体；质控线包被有抗步骤3)抗体的抗抗体；
    4)吸水纸：用于提供检测所需的毛细作用力的吸水纸。
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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH10124844A (ja) * 1996-10-23 1998-05-15 Hitachi Ltd 磁気記録媒体及びそれを用いた磁気記憶装置
JP2003000298A (ja) * 2001-06-21 2003-01-07 Satake Corp 残留農薬検出方法
CN103323415A (zh) * 2013-07-01 2013-09-25 广州市酒类检测中心 一种检测氨基甲酸酯类的酶抑制法
CN105785021A (zh) * 2016-04-08 2016-07-20 广州万联生物科技有限公司 一种有机磷及氨基甲酸酯类农药多残留胆碱酯酶免疫层析速测卡
CN107305212A (zh) * 2016-04-25 2017-10-31 赵芳 一种有机磷类和氨基甲酸酯类农药的免疫学检测方法及试剂盒

Family Cites Families (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7803782B2 (en) * 2003-05-28 2010-09-28 Roche Madison Inc. Intravenous delivery of polynucleotides to cells in mammalian limb
BR122018071808B8 (pt) * 2003-11-06 2020-06-30 Seattle Genetics Inc conjugado
DE602005012551D1 (de) * 2004-12-22 2009-03-12 California Inst Of Techn Abbaubare polymere und herstellungsverfahren dafür
JP5061435B2 (ja) * 2005-08-01 2012-10-31 東レ株式会社 耐熱樹脂前駆体組成物およびそれを用いた半導体装置
DE602007001103D1 (de) * 2006-03-14 2009-06-25 Cellzome Ag Verfahren zur identifizierung von lrrk2 interagierenden molekülen und zur reinigung von lrrk2
US9139614B2 (en) * 2007-04-25 2015-09-22 The United States Of America, As Represented By The Secretary Of The Navy Modular linkers for conjugation of organic substances to substantially inorganic substances and methods of manufacture and use thereof
EP2040075A1 (en) * 2007-09-24 2009-03-25 Julius-Maximilians-Universität Würzburg Compounds and markers for surface-enhanced raman scattering
CN108191846B (zh) * 2008-12-04 2021-08-20 于崇曦 高穿透性组合物及其应用
US20150322120A1 (en) * 2012-04-17 2015-11-12 University Of Utah Research Foundation Sodium channel sensitive conopeptides and analogs, including compositions and methods thereof
JP2016079305A (ja) * 2014-10-17 2016-05-16 住友精化株式会社 ポリアリーレンスルフィド系樹脂組成物

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH10124844A (ja) * 1996-10-23 1998-05-15 Hitachi Ltd 磁気記録媒体及びそれを用いた磁気記憶装置
JP2003000298A (ja) * 2001-06-21 2003-01-07 Satake Corp 残留農薬検出方法
CN103323415A (zh) * 2013-07-01 2013-09-25 广州市酒类检测中心 一种检测氨基甲酸酯类的酶抑制法
CN105785021A (zh) * 2016-04-08 2016-07-20 广州万联生物科技有限公司 一种有机磷及氨基甲酸酯类农药多残留胆碱酯酶免疫层析速测卡
CN107305212A (zh) * 2016-04-25 2017-10-31 赵芳 一种有机磷类和氨基甲酸酯类农药的免疫学检测方法及试剂盒

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
KYUNG, W.J. ET AL.: "Development of New Linkers for the Formation of Aliphatic C-H Bonds on Polymeric Supports", TETRAHEDRON, vol. 35, no. 19, 31 December 1997 (1997-12-31), pages 6645 - 6652, XP004105656, ISSN: 0040-4020, DOI: doi:10.1016/S0040-4020(97)00222-6 *

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