CN102507953A - Preparation method of electrochemistry immunosensor for determining alpha fetoprotein - Google Patents
Preparation method of electrochemistry immunosensor for determining alpha fetoprotein Download PDFInfo
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- CN102507953A CN102507953A CN2011103198947A CN201110319894A CN102507953A CN 102507953 A CN102507953 A CN 102507953A CN 2011103198947 A CN2011103198947 A CN 2011103198947A CN 201110319894 A CN201110319894 A CN 201110319894A CN 102507953 A CN102507953 A CN 102507953A
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Abstract
The invention relates to a preparation method of an electrochemistry immunosensor for detecting alpha fetoprotein, in particular to a gold-composite nanometer mesoporous SiO2 composite-based preparation method of the electrochemistry immunosensor for determining the alpha fetoprotein. The immunosensor is employed to provide a method for determining the alpha fetoprotein. The preparation method is characterized by comprising the steps of: firstly synthetizing a nanometer mesoporous SiO2-nano-gold composite, fixing horseradish peroxidase and a secondary antibody on the composite to form a complex; fixing a primary antibody by using a thionine-graphene complex, thereby preparing the electrochemistry immunosensor for determining the alpha fetoprotein, wherein the immunosensor is successfully used for the determination of the alpha fetoprotein. The electrochemistry immunosensor disclosed by the invention has the advantages of high accuracy, high sensitivity, stability and repeatability, rapidness and convenience for immunoassay determination, can be used for clinical analysis and provides important basis for early diagnosis of cancers.
Description
Technical field
The present invention relates to a kind of preparation method who measures the electrochemical immunosensor of alpha-fetoprotein, especially based on the mesoporous nano SiO of golden hydridization
2The preparation method of the electrochemical immunosensor of the mensuration alpha-fetoprotein of compound substance.
Background technology
In recent years, nano material will obtain large-scale progress in the research of aspects such as the various probe techniques of single molecules level, nanometer integrated array device, electrode face finish.Wherein, nano material is introduced electrode surface, can effectively increase the specific surface area of electrode; Accelerate the transmission of electronics, the electric conductivity of intensifier electrode improves the for example fixed amount of enzyme of biomacromolecule; Strengthen the sensitivity and the stability of sensor, improve sensor's response speed etc.Mesoporous nano SiO
2Material (MSN) has advantages such as specific surface area is big, the aperture is big, biocompatibility is better, can be effectively fixing horseradish peroxidase (HRP), strengthen the catalytic activity of system, be the good material of assembling immunosensor.Golden nanometer particle has performances such as specific surface area is big, surface reaction activity is high, the surfactivity center is many, catalytic efficiency is high, high adsorption capacity, and excellent biological compatibility and stability can be used for immobilized and the mark biomolecule.MSN and golden nanometer particle (Au) are obtained the MSN-Au compound substance through the double salt method is synthetic, promptly kept both original advantages, can further improve electron transport efficient again, strengthen the activity of enzyme, can be used for the preparation of immunosensor.
(α-fetoprotein is the early stage a kind of main haemocyanin of embryonic development AFP) to alpha-fetoprotein, and diagnosis has great importance to primary carcinoma of liver in the rising of alpha-fetoprotein in the serum.The detection method of alpha-fetoprotein mainly contains enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), indirect hemase method and agar double immunodiffusion method etc. at present.These methods are sensitive, reliable, but enzyme is unstable, and the radiomaterial equipment requirements is high, and harm is bigger.Have report to adopt electrochemical method to combine with immune response to detect alpha-fetoprotein in recent years, this method equipment needed thereby is simple, with low cost, highly sensitive, and detection limit is low, and nano composite material is incorporated into electrochemical immunosensor, can strengthen the activity of enzyme.
CN101441218 has set up a kind of CdTe of utilization quantum dot and Fe
3O
4-Dextran nano particle detects the method for the alpha-fetoprotein content in the serum, and its step comprises: Fe
3O
4The preparation of the preparation of-Dextran-first antibody compound and CdTe-SA compound, according to the fluorescence intensity size measurement alpha-fetoprotein value of hepatocarcinoma patient.Magnetic immuno-chromatographic test paper strip of alpha-fetoprotein in a kind of detection by quantitative blood and preparation method thereof has been set up in the CN101566636A invention; This invention is incorporated into magnetic immuno-chromatographic technology and biotin Avidin system in the detection by quantitative of alpha-fetoprotein in the blood, has improved detection sensitivity greatly.
The present invention is based on MSN-Au is carrier material, has set up a kind of preparation method who measures the electrochemical immunosensor of alpha-fetoprotein, has improved the sensitivity that detects, and the range of linearity is wide.
Summary of the invention
The purpose of this invention is to provide a kind of preparation method who measures the electrochemical immunosensor of alpha-fetoprotein, its characteristic may further comprise the steps:
(1) (MSN-Au)-HRP-alpha-fetoprotein SA (Ab
2) preparation of compound;
(2) preparation of thionine (TH)-Graphene (GS) mixed solution;
(3) set up electrochemical immunosensor, measure alpha-fetoprotein, the drawing curve.
(MSN-Au)-HRP-Ab described in the present invention
2The preparation of compound is characterized in that process specifically may further comprise the steps:
(1) preparation of MSN-Au nano composite material:
With amido modified MSN and H
4AuCl
4According to 1.2: 1 mixed of quality, at room temperature, stir gently with magnetic stirring apparatus, add hydrochloric acid then and regulate acidity, and then stir, add 40mmo1L
-1Sodium borohydride, become black (purple, black) up to its color, centrifugal, the room temperature vacuum drying can obtain the MSN-Au nano composite material;
(2) (MSN-Au)-HRP+Ab
2The preparation of compound:
With 1.0mgmL
-1MSN-Au nano composite material solution and 1.0mgmL
-1HRP solution and 1.0mgmL
-1Ab
2Solution is according to 1～1.2: 1: 0.5～1.2 volume ratio is mixed, and slight wobble absorption 24h is centrifugal, and the material of gained is scattered in 0.05molL again
-1Subsequent use in the PBS buffer solution of pH=7.50.
The preparation of the TH-GS mixed solution described in the present invention may further comprise the steps: with 1.0mgmL
-1GS solution and 1.0mgmL
-1TH solution mixes and ultrasonic 20～30h.
Set up electrochemical immunosensor described in the present invention, measure alpha-fetoprotein, the drawing curve is characterized in that may further comprise the steps:
(1) glass-carbon electrode of 5mm diameter is used successively the alundum (Al burnishing powder polishing of 1.0,0.3 and 0.05 μ m, rinsed well with ultrapure water again, then electrode is placed 0.05mo1L
-1In the potassium ferricyanide solution, in-0.6～0.6V scanning, the peak current difference is less than 85mA;
(2) 3～8 μ L GS+TH solution are dripped to the glass-carbon electrode surface, wait to dry;
(3) drip and be coated with 3～8 μ L volume fractions to be that 3% glutaraldehyde solution keeps wetting, water cleans, and dries film forming;
(4) drip and be coated with 3～8 μ L 1.0mgmL
-1Alpha-fetoprotein first antibody (Ab1) to glass-carbon electrode surface, placement a period of time keeps wetting, and water cleans, and dries;
(5) with 3～8 μ L 1gL
-1Bovine serum albumin solution drips and is applied to the glass-carbon electrode surface, places a period of time, dries;
(6) the alpha-fetoprotein antigenic solution of variable concentrations is dripped be coated onto the glass-carbon electrode surface, reaction a period of time, measure its ORP and change;
(7) with 3～8 μ L (MSN-Au)-HRP-Ab
2Complex solution drips and is coated onto on the glass-carbon electrode that contains alpha-fetoprotein antigen, reaction 0.5～1.5h, then respectively 5mL pH=7.50PBS buffer solution with contain 1mmol H
2O
2Its ORP of measured in solution of 5mL pH=7.50PBS buffer solution change;
(8) linear according to gained electric current difference and alpha-fetoprotein antigen concentration, the drawing curve.
Graphene has good electric conductivity among the present invention.The MSN-Au compound substance has advantages such as specific surface area is big, the aperture is big, biocompatibility is better, can fix more HRP, strengthens the catalytic activity of system.Electrochemical immunosensor of the present invention has shown good accuracy, high sensitivity property, stability and reappearance, and it is rapid, convenient that immunoassay detects, and can be used for clinical analysis.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in the restriction scope of the present invention.
Embodiment 1
Step 1. is with amido modified mesoporous nano SiO
2And H
4AuCl
4According to 1.2: 1 mixed of quality, at room temperature, stir gently with magnetic stirring apparatus, add hydrochloric acid then and regulate acidity, and then stir, add 40mmolL
-1Sodium borohydride, become black (purple, black) up to its color, centrifugal, the room temperature vacuum drying can obtain the MSN-Au nano composite material;
Step 2. is with 1.0mL 1.0mgmL
-1MSN-Au nano composite material solution and 1.0mL 1.0mgmL
-1HRP solution and 1.0mL 1.0mgmL
-1Ab
2Solution mixes, and slight wobble absorption 24h is centrifugal, and the material of gained is scattered in 1.0mL 0.05molL again
-1Subsequent use in the PBS buffer solution of pH=7.50;
Step 3. is used the alundum (Al burnishing powder polishing of 1.0,0.3 and 0.05 μ m successively with the glass-carbon electrode of 5mm diameter, rinses well with secondary water again, then electrode is placed 0.05molL
-1In the potassium ferricyanide solution, in-0.6～0.6V scanning, the peak current difference is 80mA;
Step 4. is with 0.5mL 1mgmL
-1GS solution and 0.5mL 1mgmL
-1TH solution mixes and ultrasonic 24h, then 5 μ L gained GS+TH solution is dripped to glass-carbon electrode surface, air dry;
It is that 3% the about 1.0h of glutaraldehyde solution keeps wetting that step 5. droplet is coated with volume fraction, and ultrapure water cleans, and dries film forming;
Step 6. droplet is coated with 5 μ L 1.0mgmL
-1Alpha-fetoprotein first antibody to glass-carbon electrode surface, placement a period of time keeps wetting, and water cleans, and dries;
Step 7. is with 3 μ L 1gL
-1Bovine serum albumin solution drips and is applied to the glass-carbon electrode surface, places a period of time, dries;
Step 8. is with 0.01～16ngmL
-1The alpha-fetoprotein antigenic solution of variable concentrations drips to different electrode surfaces respectively, and reaction 1.0h measures its ORP and changes;
Step 9. is with 5 μ L (MSN-Au)-HRP-Ab
2Complex solution drips and is coated onto on the glass-carbon electrode that contains alpha-fetoprotein antigen, reaction 1.0h, then respectively 5mL pH=7.50PBS buffer solution with contain 1mmol H
2O
2Its ORP of measured in solution of 5mL pH=7.50PBS buffer solution change;
Step 10. is linear according to gained electric current difference and alpha-fetoprotein antigen concentration, the drawing curve; Mensuration result shows that alpha-fetoprotein is at 0.02～15ngmL
-1Linear in the scope, linearly dependent coefficient is 0.9991.Detect and be limited to 6pgmL
-1
Claims (4)
1. preparation method who measures the electrochemical immunosensor of alpha-fetoprotein is characterized in that may further comprise the steps:
(1) mesoporous nano SiO
2-metal/composite material (MSN-Au)-horseradish peroxidase (HRP)-alpha-fetoprotein SA (Ab
2) preparation of compound;
(2) preparation of thionine (TH)-Graphene (GS) mixed solution;
(3) set up electrochemical immunosensor, measure alpha-fetoprotein, the drawing curve.
2. according to said (the MSN-Au)-HRP-Ab of claim 1
2The preparation process of compound specifically may further comprise the steps, and it is characterized in that may further comprise the steps:
(1) preparation of MSN-Au nano composite material:
The preparation of MSN-Au nano composite material is with amido modified MSN and H
4AuCl
4According to 1.2: 1 mixed of quality, at room temperature, stir gently with magnetic stirring apparatus, add hydrochloric acid then and regulate acidity, and then stir, add 40mmolL
-1Sodium borohydride, become black (purple, black) up to its color, centrifugal, the room temperature vacuum drying can obtain the MSN-Au nano composite material;
(2) (MSN-Au)-HRP-Ab
2The preparation of compound:
With 1.0mgmL
-1MSN-Au nano composite material solution and 1.0mgmL
-1HRP solution and 1.0mgmL
-1Ab
2Solution is according to 1～1.2: 1: 0.5～1.2 volume ratio is mixed, and slight wobble absorption 24h is centrifugal, and the material of gained is scattered in 0.05molL again
-1Subsequent use in the PBS buffer solution of pH=7.50.
3. the preparation of TH-GS mixed solution according to claim 1 is characterized in that: with 1.0mgmL
-1GS solution and 1.0mgmL
-1TH solution mixes and ultrasonic 20～30h.
4. the alpha-fetoprotein electrochemical immunosensor of setting up according to claim 1 is measured alpha-fetoprotein, and the drawing curve is characterized in that may further comprise the steps:
(1) glass-carbon electrode of 5mm diameter is used successively the alundum (Al burnishing powder polishing of 1.0,0.3 and 0.05 μ m, rinsed well with ultrapure water again, then electrode is placed 0.05molL
-1In the potassium ferricyanide solution, in-0.6～0.6V scanning, the peak current difference is less than 85mA;
(2) 3～8 μ L GS+TH solution are dripped to the glass-carbon electrode surface, wait to dry;
(3) drip and be coated with 3～8 μ L volume fractions to be that 3% glutaraldehyde solution keeps wetting, water cleans, and dries film forming;
(4) drip and be coated with 3～8 μ L 1.0mgmL
-1Alpha-fetoprotein first antibody to glass-carbon electrode surface, placement a period of time keeps wetting, and water cleans, and dries;
(5) with 3～8 μ L 1gL
-1Bovine serum albumin solution drips and is applied to the glass-carbon electrode surface, places a period of time, dries;
(6) the alpha-fetoprotein antigenic solution of variable concentrations is dripped respectively be coated onto different glass-carbon electrodes surface, reaction a period of time, measure its ORP and change;
(7) with 3～8 μ L (MSN-Au)-HRP-Ab
2Complex solution drips and is coated onto on the glass-carbon electrode that contains alpha-fetoprotein antigen, reaction 0.5～1.5h, then respectively 5mL pH=7.50PBS buffer solution with contain 1mmol H
2O
2Its ORP of measured in solution of 5mLpH=7.50PBS buffer solution change;
(8) linear according to gained electric current difference and alpha-fetoprotein antigen concentration, the drawing curve.
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| CN102879582A (en) * | 2012-09-11 | 2013-01-16 | 宁波大学 | In-vitro sandwich immunoassay process with magnetic separation for detecting alpha fetoprotein |
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| CN113325060A (en) * | 2021-06-07 | 2021-08-31 | 内江师范学院 | Graphene magnetic nano-electrode, electrochemical immunosensor, preparation method and application |
| CN113325060B (en) * | 2021-06-07 | 2023-02-17 | 内江师范学院 | Graphene magnetic nano-electrode, electrochemical immunosensor, preparation method and application |
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