CN115248243B - Method for detecting AFP (alpha-fetoprotein) based on sandwich electrochemical immunoassay mode of hyperbranched polyethyleneimine - Google Patents
Method for detecting AFP (alpha-fetoprotein) based on sandwich electrochemical immunoassay mode of hyperbranched polyethyleneimine Download PDFInfo
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- 102000013529 alpha-Fetoproteins Human genes 0.000 title claims abstract description 46
- 108010026331 alpha-Fetoproteins Proteins 0.000 title claims abstract description 46
- 238000003018 immunoassay Methods 0.000 title claims abstract description 17
- 229920002873 Polyethylenimine Polymers 0.000 title claims abstract description 12
- 238000000034 method Methods 0.000 title claims description 10
- 239000000243 solution Substances 0.000 claims abstract description 22
- 239000000523 sample Substances 0.000 claims abstract description 19
- SQGYOTSLMSWVJD-UHFFFAOYSA-N silver(1+) nitrate Chemical compound [Ag+].[O-]N(=O)=O SQGYOTSLMSWVJD-UHFFFAOYSA-N 0.000 claims abstract description 12
- 239000000090 biomarker Substances 0.000 claims abstract description 6
- 229910001961 silver nitrate Inorganic materials 0.000 claims abstract description 6
- 239000007864 aqueous solution Substances 0.000 claims abstract description 5
- 229910021397 glassy carbon Inorganic materials 0.000 claims abstract description 4
- 102100023635 Alpha-fetoprotein Human genes 0.000 claims abstract 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 10
- 238000000151 deposition Methods 0.000 claims description 6
- 229920002246 poly[2-(dimethylamino)ethyl methacrylate] polymer Polymers 0.000 claims description 6
- 239000008367 deionised water Substances 0.000 claims description 5
- 229910021641 deionized water Inorganic materials 0.000 claims description 5
- 238000005406 washing Methods 0.000 claims description 5
- 239000004642 Polyimide Substances 0.000 claims description 4
- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 claims description 4
- 229920001721 polyimide Polymers 0.000 claims description 4
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 3
- 238000004108 freeze drying Methods 0.000 claims description 3
- 238000003756 stirring Methods 0.000 claims description 3
- -1 N, N-dimethylamino ethyl Chemical group 0.000 abstract description 3
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- 238000001514 detection method Methods 0.000 description 7
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- 238000004458 analytical method Methods 0.000 description 3
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- JKNCOURZONDCGV-UHFFFAOYSA-N 2-(dimethylamino)ethyl 2-methylprop-2-enoate Chemical compound CN(C)CCOC(=O)C(C)=C JKNCOURZONDCGV-UHFFFAOYSA-N 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
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- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
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- G01N27/416—Systems
- G01N27/48—Systems using polarography, i.e. measuring changes in current under a slowly-varying voltage
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
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- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/308—Electrodes, e.g. test electrodes; Half-cells at least partially made of carbon
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
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- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54366—Apparatus specially adapted for solid-phase testing
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- G01N33/5438—Electrodes
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- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
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- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
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Abstract
The invention discloses a sandwich electrochemical immunoassay method for detecting AFP based on hyperbranched polyethyleneimine, which comprises the steps of putting a glassy carbon electrode into an aqueous solution of the hyperbranched polyethyleneimine, sequentially adding the aqueous solution into a silver nitrate and alpha fetoprotein antibody solution, and incubating to obtain an anti-AFP/HPEI-AgNR/GCE sensor; mixing polymethyl methacrylate N, N-dimethylamino ethyl ester with HAuCl 4 Reacting to obtain PDM@Au, and adding alpha fetoprotein antibody to react to obtain biomarker probe bio-PDM@Au; incubating anti-AFP/HPEI-AgNR/GCE and a alpha fetoprotein standard sample with gradient concentration with a bovine serum sample to form AFP/anti-AFP/HPEI-AgNR/GCE, and then incubating with bio-PDM@Au to form a sandwich immune complex; and (5) measuring electrochemical response signals of the alpha fetoprotein standard sample with gradient concentration to obtain a working curve.
Description
Technical Field
The invention belongs to the field of immunosensors, and relates to a method for detecting AFP (alpha-fetoprotein) by using a sandwich electrochemical immunoassay mode based on hyperbranched polyethyleneimine.
Background
During tumorigenesis and proliferation, a class of substances that are biosynthesized, released, or host-reactive to a tumor by tumor cells are called tumor markers. Such substances may be circulating substances, which may be present in cells, tissues or body fluids. Tumor markers are usually present in the form of metabolites such as antigens, enzymes or hormones, either in the tumor cells or in the body fluids of the host, and in medicine can recognize or diagnose tumors based on their biochemical or immunological properties. Alpha Fetoprotein (AFP) is a glycoprotein, which belongs to the albumin family and is mainly synthesized by fetal liver cells and yolk sac. Alpha fetoprotein has higher concentration in fetal blood circulation, and is reduced after birth until 2-3 months after birth, and alpha fetoprotein is basically replaced by albumin, so that alpha fetoprotein is difficult to detect in blood, and the alpha fetoprotein has extremely low content in serum of an adult. Alpha fetoprotein has many important physiological functions including transport function, bi-directional regulation function as growth regulator, immunosuppression, T lymphocyte induced apoptosis, etc. Alpha fetoprotein is closely related to the occurrence and development of liver cancer and various tumors, can show higher concentration in various tumors, and can be used as a positive detection index of various tumors. The serum marker is mainly used as a serum marker of primary liver cancer in clinic at present and is used for diagnosing and monitoring curative effect of primary liver cancer.
Immunoassay methods are analytical methods that utilize the specific binding of antibodies or antigens to assay for the antigen or antibodies and hapten. Electrochemical immunosensors are an analytical method that combines electrochemical immunoassays with sensing technology. According to the type of the detection signal of the electrochemical immunosensor, it is classified into a potentiometric immunosensor, a capacitive immunosensor, an impedance-type immunosensor, a conductivity-type immunosensor, and a amperometric immunosensor. Among them, the amperometric immunosensor has high sensitivity, is easy for signal amplification, and is widely studied and applied. High sensitivity is achieved by using labels (enzymes or nanocatalysts etc.) as signal amplification indicators. Therefore, the nano material for constructing the electrochemical immunosensor has higher stability, good biocompatibility, electrochemical activity and the like. Such as nano gold, nano silver, carbon nanotubes, conductive polymers, etc., have been widely studied and advanced. However, the nanocomposite prepared from inorganic metal nanoparticles and organic polymers is used for electrochemical immunoassay to further improve the accuracy and electrochemical performance of the immunoassay sensor.
Disclosure of Invention
In view of this, the present invention provides a method for detecting AFP based on a sandwich electrochemical immunoassay format of hyperbranched polyethyleneimine.
The invention specifically provides the following technical scheme:
a sandwich electrochemical immunoassay method for detecting AFP based on hyperbranched polyethyleneimine comprises the following steps:
1) Placing a Glassy Carbon Electrode (GCE) into an aqueous solution of Hyperbranched Polyimide (HPEI), depositing for 240 seconds at a potential of-2.0V, taking out, washing with water, immersing into a silver nitrate solution, circularly scanning for 20 circles at a speed of 100mV/s at a potential of-1.0V, depositing nano silver, immersing into an alpha fetoprotein antibody (anti-AFP) solution, and incubating for 12 hours at 4 ℃ to prepare the sensor anti-AFP/HPEI-AgNR/GCE;
2) Dissolving poly (N, N-dimethylaminoethyl methacrylate) (PDM) in deionized water, adding HAuCl 4 And NaBH 4 Reacting for 24 hours at room temperature, dialyzing, and freeze-drying to obtain a product PDM@Au;
3) Dissolving PDM@Au into a Tris-HCl solution, adding alpha fetoprotein antibody (anti-AFP), stirring at room temperature, placing into a refrigerator for reacting for 12 hours, centrifuging after the reaction is finished, and washing to obtain a biomarker probe bio-PDM@Au;
4) Incubating the sensor anti-AFP/HPEI-AgNR/GCE and a serial gradient concentration alpha fetoprotein standard sample for 50 minutes to form an antigen-antibody complex sensor AFP/anti-AFP/HPEI-AgNR/GCE;
5) Incubating the antigen-antibody complex sensor AFP/anti-AFP/HPEI-AgNR/GCE obtained in the step 4) and the probe bio-PDM@Au obtained in the step 3) for 50 minutes to form a sandwich immune complex;
6) To contain H 2 O 2 And (2) taking PBS as base solution, performing DVP scanning in a potential range of-200V to-800V, and measuring electrochemical response signals of alpha-fetoprotein standard samples with serial gradient concentrations to obtain a working curve.
Further, the number average molecular weight of the hyperbranched polyimide HPEI in the step 1) is 10000, the concentration of the HPEI solution is 1mmol/L, and the concentration of the silver nitrate solution is 5mmol/L.
Advancing oneStep 2) in particular, 0.2g of N, N-dimethylaminoethyl polymethacrylate are dissolved in 50mL of deionized water and 1.0mL of HAuCl are added 4 ，1.0mL NaBH 4 The number average molecular weight of the poly (N, N-dimethylaminoethyl methacrylate) is 2000-5000.
Further, the concentration of the alpha fetoprotein antibody in the step 3) is 0.5mg/L, and the addition amount is 500 mu L.
Further, H as described in step 6) 2 O 2 The concentration of the solution is 3mol/L, and the pH value of the solution is 5.5.
The invention has the beneficial effects that: the sandwich electrochemical immunoassay method is constructed and used for detecting tumor marker alpha fetoprotein AFP, anti-AFP/HPEI-AgNR/GCE is used as an immunosensor interface, anti-AFP marked polymethyl methacrylate N, N-dimethylaminoethyl ester/nano gold compound is used as a biomarker pointer, and the sandwich electrochemical immunoassay method has the function of enhancing signals.
Drawings
In order to make the objects, technical solutions and advantageous effects of the present invention clearer, the present invention provides the following drawings:
FIG. 1 is a graph of current response versus AFP concentration.
Detailed Description
Preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings.
Example 1
A sandwich electrochemical immunoassay method for detecting AFP based on hyperbranched polyethyleneimine comprises the following detection steps:
1. preparation of electrochemical immunosensors
The Glassy Carbon Electrode (GCE) was treated with 0.3 μm and 0.05 μm Al, respectively 2 O 3 Polishing powder to mirror surface, sequentially adding ethanol, acetone and distilled water, ultrasonic cleaning, naturally drying, placing the electrode in 1mmol/L hyperbranched polyethyleneimine HPEI (Mn=10000) water solution, depositing at-2.0V potential for 240 seconds, taking out, cleaning with water, soaking in 5mmol/L silver nitrate solution, depositing nano silver, and placing at-1.0V potential for 100mV/sAnd (3) carrying out speed circulation scanning for 20 circles, immersing in an anti-AFP antibody solution, and incubating for 12 hours at 4 ℃ to prepare the sensor anti-AFP/HPEI-AgNR/GCE.
1. Preparation of biomarker probes
1) 0.2g of N, N-dimethylaminoethyl polymethacrylate (PDMAEMA, mn=2000-5000) was dissolved in 50mL of deionized water, and 1.0mL of HAuCl was added 4 ，1.0mL NaBH 4 Reacting for 24 hours at room temperature, dialyzing, freeze-drying to obtain nano-gold modified PDMEMA, which is marked as PDM@Au;
2) Dissolving PDM@Au into 2mL Tris-HCl solution, adding 500 mu L alpha fetoprotein antibody (anti-AFP, 0.5 mg/L), stirring at room temperature, placing into a refrigerator for reacting for 12 hours, centrifuging after the reaction is finished, and washing to obtain biomarker probe bio-PDM@Au.
2. Electrochemical immunoassay
1) Incubating the sensor anti-AFP/HPEI-AgNR/GCE and AFP standard samples and bovine serum samples with different concentrations for 50 minutes to form an antigen-antibody complex sensor AFP/anti-AFP/HPEI-AgNR/GCE;
2) AFP/anti-AFP/HPEI-AgNR/GCE and probe bio-17-cell of antigen-antibody complex sensor obtained in step 1)
Incubating PDM@Au for 50 minutes to form a sandwich immune complex;
3) To contain 3mol/L H 2 O 2 And (2) using PBS (pH=5.5) as a base solution, performing DVP scanning in a potential range of-200V to-800V, measuring an electrochemical response signal of an AFP standard sample, and making a standard curve graph.
4) Incubating a sensor anti-AFP/AgNR@QDs/GCE and a serum sample to be detected for 30 minutes to form an antigen-antibody complex sensor AFP to be detected/anti-AFP/AgNR@QDs/GCE, continuously incubating with a probe bio-PEI-Ag for 30 minutes to form a sandwich immune complex, measuring an electrochemical response signal in the serum sample to be detected according to the method of the step 3), and obtaining a concentration value of the AFP in the serum according to a standard curve obtained in the step 3), thus obtaining the concentration of the AFP in the serum sample to be detected.
Example 2 analysis of test results
The bio-PDM@Au is used as a capture probe, the anti-AFP/HPEI-AgNR/GCE is used as an immunosensor of a beacon to detect standard samples of AFP with different concentrations, the detection method is differential pulse voltammetry, experimental results are shown in figure 1, the DPV response current of the immunosensor is increased along with the increase of the AFP concentration, the detection range of the specific antigen of alpha fetoprotein is 0.01-40ng/mL, the detection limit is 0.001ng/mL, and the experimental results show that the prepared immunosensor has high sensitivity and high response speed, and the response linear range of the electrochemical immunosensor can meet the detection of serum samples.
Finally, it is noted that the above-mentioned preferred embodiments are only intended to illustrate rather than limit the invention, and that, although the invention has been described in detail by means of the above-mentioned preferred embodiments, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention as defined by the appended claims.
Claims (5)
1. A method for detecting AFP based on a sandwich electrochemical immunoassay mode of hyperbranched polyethyleneimine is characterized by comprising the following steps:
1) Placing a glassy carbon electrode into an aqueous solution of hyperbranched polyimide, depositing for 240 seconds at a potential of-2.0V, taking out, washing with water, immersing into a silver nitrate solution, circularly scanning for 20 circles at a speed of 100mV/s at a potential of-1.0V, depositing nano silver, immersing into a alpha fetoprotein antibody solution, and incubating at 4 ℃ for 12 hours to prepare an anti-AFP/HPEI-AgNR/GCE sensor;
2) Dissolving poly (N, N-dimethylaminoethyl methacrylate) in deionized water, adding HAuCl 4 And NaBH 4 Reacting for 24 hours at room temperature, dialyzing, and freeze-drying to obtain a product PDM@Au;
3) Dissolving PDM@Au into a Tris-HCl solution, adding alpha fetoprotein antibody, stirring at room temperature, placing into a refrigerator for reacting for 12 hours, centrifuging and washing after the reaction is finished to obtain a biomarker probe bio-PDM@Au;
4) Incubating the sensor anti-AFP/HPEI-AgNR/GCE and a serial gradient concentration alpha fetoprotein standard sample for 50 minutes to form an antigen-antibody complex sensor AFP/anti-AFP/HPEI-AgNR/GCE;
5) Incubating the antigen-antibody complex sensor AFP/anti-AFP/HPEI-AgNR/GCE obtained in the step 4) and the probe bio-PDM@Au obtained in the step 3) for 50 minutes to form a sandwich immune complex;
6) To contain H 2 O 2 And (2) taking PBS as base solution, performing DVP scanning in a potential range of-200V to-800V, and measuring electrochemical response signals of alpha-fetoprotein standard samples with serial gradient concentrations to obtain a working curve.
2. The method for detecting AFP by using a sandwich electrochemical immunoassay method based on hyperbranched polyethyleneimine according to claim 1, wherein the hyperbranched polyimide in the step 1) has a number average molecular weight of 10000, the concentration of the aqueous solution is 1mmol/L, and the concentration of the silver nitrate solution is 5mmol/L.
3. The method for detecting AFP according to claim 1, wherein the step 2) is specifically to dissolve 0.2g of poly (N, N-dimethylaminoethyl methacrylate) in 50mL of deionized water and add 1.0mL of HAuCl 4 ，1.0mL NaBH 4 The number average molecular weight of the poly (N, N-dimethylaminoethyl methacrylate) is 2000-5000.
4. The method for detecting AFP according to claim 1, wherein the concentration of alpha fetoprotein antibody in the step 3) is 0.5mg/L and the addition amount is 500. Mu.L.
5. The method for detecting AFP according to claim 1, wherein the H in the step 6) is a sandwich electrochemical immunoassay format based on hyperbranched polyethyleneimine 2 O 2 The concentration of the solution is 3mol/L, and the pH value of the solution is 5.5.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101196523A (en) * | 2007-12-29 | 2008-06-11 | 华中农业大学 | Chicken infectious Fabricius bursa immune body immune colloidal gold fast detecting and its application |
| CN101526523A (en) * | 2009-03-27 | 2009-09-09 | 东南大学 | Preparation for cadmium antimonide quantum dot immune marker and detection method for electrochemical sandwich immune |
| WO2011041586A1 (en) * | 2009-09-30 | 2011-04-07 | Virogenomics, Inc. | Electromagnetic detection of analytes |
| JP2017019992A (en) * | 2015-07-10 | 2017-01-26 | 国立大学法人神戸大学 | Manufacturing method of molecular imprint polymer, molecular imprint polymer and detection method of target protein |
| CN111171808A (en) * | 2020-01-13 | 2020-05-19 | 重庆工程职业技术学院 | Preparation method of butyric acid-4-methylumbelliferone/citrulline carbon dot fluorescent probe, product and application thereof |
-
2021
- 2021-12-15 CN CN202111533030.5A patent/CN115248243B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101196523A (en) * | 2007-12-29 | 2008-06-11 | 华中农业大学 | Chicken infectious Fabricius bursa immune body immune colloidal gold fast detecting and its application |
| CN101526523A (en) * | 2009-03-27 | 2009-09-09 | 东南大学 | Preparation for cadmium antimonide quantum dot immune marker and detection method for electrochemical sandwich immune |
| WO2011041586A1 (en) * | 2009-09-30 | 2011-04-07 | Virogenomics, Inc. | Electromagnetic detection of analytes |
| JP2017019992A (en) * | 2015-07-10 | 2017-01-26 | 国立大学法人神戸大学 | Manufacturing method of molecular imprint polymer, molecular imprint polymer and detection method of target protein |
| CN111171808A (en) * | 2020-01-13 | 2020-05-19 | 重庆工程职业技术学院 | Preparation method of butyric acid-4-methylumbelliferone/citrulline carbon dot fluorescent probe, product and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| Molecular imprinting of proteins emerging as a tool for protein recogenition;Toshifumi takeuchi;Royal society of chemistry;20080630;2459-2467 * |
| 基于压力传感器及支持向量机的人体运动识别系统;王晋;;电子设计工程;20180920(第18期);145-150 * |
| 复合材料自动铺放表面缺陷检测技术研究进展;马少博;符合材料科学与工程;20201215;78-96 * |
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