CN102507953B - 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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- CN102507953B CN102507953B CN 201110319894 CN201110319894A CN102507953B CN 102507953 B CN102507953 B CN 102507953B CN 201110319894 CN201110319894 CN 201110319894 CN 201110319894 A CN201110319894 A CN 201110319894A CN 102507953 B CN102507953 B CN 102507953B
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- 230000005518 electrochemistry Effects 0.000 title abstract 5
- 229910021389 graphene Inorganic materials 0.000 claims abstract description 4
- 239000002131 composite material Substances 0.000 claims abstract 5
- 239000000243 solution Substances 0.000 claims description 36
- 102100023635 Alpha-fetoprotein Human genes 0.000 claims description 33
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- 239000002114 nanocomposite Substances 0.000 claims description 9
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 6
- PNEYBMLMFCGWSK-UHFFFAOYSA-N aluminium oxide Inorganic materials [O-2].[O-2].[O-2].[Al+3].[Al+3] PNEYBMLMFCGWSK-UHFFFAOYSA-N 0.000 claims description 6
- 239000000427 antigen Substances 0.000 claims description 6
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- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 claims description 3
- 229910003771 Gold(I) chloride Inorganic materials 0.000 claims description 3
- 102000007562 Serum Albumin Human genes 0.000 claims description 3
- 108010071390 Serum Albumin Proteins 0.000 claims description 3
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- 108040007629 peroxidase activity proteins Proteins 0.000 claims 1
- ANRHNWWPFJCPAZ-UHFFFAOYSA-M thionine Chemical compound [Cl-].C1=CC(N)=CC2=[S+]C3=CC(N)=CC=C3N=C21 ANRHNWWPFJCPAZ-UHFFFAOYSA-M 0.000 claims 1
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- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 abstract 2
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- PVPBBTJXIKFICP-UHFFFAOYSA-N (7-aminophenothiazin-3-ylidene)azanium;chloride Chemical compound [Cl-].C1=CC(=[NH2+])C=C2SC3=CC(N)=CC=C3N=C21 PVPBBTJXIKFICP-UHFFFAOYSA-N 0.000 description 4
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
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 the 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 for example fixed amount of enzyme of biomacromolecule, strengthen sensitivity and the stability of sensor, improve the response speed of sensor etc.Mesoporous nano SiO
2Material (MSN) has the advantages such as specific surface area is large, the aperture is large, 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 the performances such as specific surface area is large, surface reaction activity is high, Active sites is many, catalytic efficiency is high, high adsorption capacity, and good biocompatibility and stability can be used for immobilized and the mark biomolecule.MSN and golden nanometer particle (Au) are obtained the MSN-Au compound substance by the double salt method is synthetic, namely kept both original advantages, can further improve the electronics transmission efficiency again, strengthen the activity of enzyme, can be used for the preparation of immunosensor.
Alpha-fetoprotein (α-fetoprotein, AFP) is the early stage a kind of main haemocyanin of embryonic development, and the rising of Serum Alpha Fetoprotein has great importance to primary hepatic carcinoma diagnosis.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 requirement is high, and harm is larger.Have in recent years report to adopt electrochemical method to be combined with immune response and detect alpha-fetoprotein, 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 α-Fetoprotein in the serum, and its step comprises: Fe
3O
4The preparation of the preparation of-Dextran-first antibody compound and CdTe-second antibody compound, according to the fluorescence intensity size measurement alpha-fetoprotein value of hepatocarcinoma patient.Magnetic immuno-chromatographic test paper strip of a kind of quantitative detection alpha-fetoprotein in 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 quantitative detection of alpha-fetoprotein in blood, has greatly improved detection sensitivity.
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 feature may further comprise the steps:
(1) (MSN-Au)-HRP-alpha-fetoprotein second antibody (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
4Mix according to 1.2: 1 ratio of quality, at room temperature, stir gently with magnetic stirring apparatus, then add hydrochloric acid and regulate acidity, and then stir, add 40mmo1L
-1Sodium borohydride, until its color becomes black (purple, black), 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
-1For subsequent 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 (Al2O3) 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, peak current is poor 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) is to the glass-carbon electrode surface, and 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 oxidation-reduction potential 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, and reaction 0.5～1.5h is then respectively at 5mL pH=7.50PBS buffer solution with contain 1mmol H
2O
2Its oxidation-reduction potential 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 the advantages such as specific surface area is large, the aperture is large, 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, 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 for explanation the present invention and be not used in and limit the scope of the invention.
Embodiment 1
Step 1. is with amido modified mesoporous nano SiO
2And H
4AuCl
4Mix according to 1.2: 1 ratio of quality, at room temperature, stir gently with magnetic stirring apparatus, then add hydrochloric acid and regulate acidity, and then stir, add 40mmolL
-1Sodium borohydride, until its color becomes black (purple, black), 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
-1For subsequent use in the PBS buffer solution of pH=7.50;
Step 3. is used the alundum (Al2O3) 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 intermediate water again, then electrode is placed 0.05molL
-1In the potassium ferricyanide solution, in-0.6～0.6V scanning, peak current is poor to be 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 the glass-carbon electrode surface, and is natural drying;
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
-1The alpha-fetoprotein first antibody is to the glass-carbon electrode surface, and 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 respectively the Different electrodes surface, and reaction 1.0h measures its oxidation-reduction potential 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, and reaction 1.0h is then respectively at 5mL pH=7.50PBS buffer solution with contain 1mmol H
2O
2Its oxidation-reduction potential 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; Measurement 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 (2)
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 second antibody Ab
2The preparation of compound;
(2) preparation of thionine TH-Graphene GS mixed solution: with 1.0 mgmL
-1GS solution and 1.0 mgmL
-1TH solution mixes and ultrasonic 20 ~ 30 h;
(3) set up electrochemical immunosensor, measure alpha-fetoprotein, the drawing curve;
Wherein, described (the MSN-Au)-HRP-Ab of step (1)
2The preparation process of compound is as follows:
1) preparation of MSN-Au nano composite material:
With amido modified MSN and H
4AuCl
4Mix according to 1.2: 1 ratio of quality, at room temperature, stir gently with magnetic stirring apparatus, then add hydrochloric acid and regulate acidity, and then stir, add 40 mmolL
-1Sodium borohydride, centrifugal until its color becomes purple or black, the room temperature vacuum drying can obtain the MSN-Au nano composite material;
2) (MSN-Au)-HRP-Ab
2The preparation of compound:
With 1.0 mgmL
-1MSN-Au nano composite material solution and 1.0 mgmL
-1HRP solution and 1.0 mgmL
-1Ab
2Solution is according to 1 ~ 1.2: 1: 0.5 ~ 1.2 volume ratio is mixed, and slight wobble is adsorbed 24 h, and is centrifugal, and the material of gained is scattered in 0.05 molL again
-1For subsequent use in the PBS buffer solution of pH=7.50.
2. 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 5 mm diameters is used successively the alundum (Al2O3) burnishing powder polishing of 1.0,0.3 and 0.05 mm, rinsed well with ultrapure water again, then electrode is placed 0.05 molL
-1In the potassium ferricyanide solution, in-0.6 ~ 0.6 V scanning, peak current is poor less than 85 mA;
(2) 3 ~ 8 μ L GS+TH solution are dripped to the glass-carbon electrode surface, wait to dry;
(3) drip that to be coated with 3 ~ 8 μ L volume fractions be that the glutaraldehyde solution of 3 % keeps wetting, water cleans, and dries film forming;
(4) drip and be coated with 3 ~ 8 μ L, 1.0 mgmL
-1The alpha-fetoprotein first antibody is to the glass-carbon electrode surface, and placement a period of time keeps wetting, and water cleans, and dries;
(5) with 3 ~ 8 μ L 1g L
-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 oxidation-reduction potential 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, and reaction 0.5 ~ 1.5 h is then respectively at 5 mL pH=7.50 PBS buffer solution with contain 1mmol H
2O
2Its oxidation-reduction potential of measured in solution of 5 mL pH=7.50 PBS buffer solution change;
(8) linear according to gained electric current difference and alpha-fetoprotein antigen concentration, the drawing curve.
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