CN104391117B - Preparation method and application of gastric cancer antigen electrogenerated chemiluminescence sensor based on PP<y>-NH2GO-Ag2Se@CdSe - Google Patents

Preparation method and application of gastric cancer antigen electrogenerated chemiluminescence sensor based on PP<y>-NH2GO-Ag2Se@CdSe Download PDF

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CN104391117B
CN104391117B CN201410623368.3A CN201410623368A CN104391117B CN 104391117 B CN104391117 B CN 104391117B CN 201410623368 A CN201410623368 A CN 201410623368A CN 104391117 B CN104391117 B CN 104391117B
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吕晓辉
魏琴
李月云
庞雪辉
曹伟
张勇
马洪敏
吴丹
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University of Jinan
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Abstract

The invention relates to a preparation method and the application of a gastric cancer antigen electrogenerated chemiluminescence sensor based on PP<y>-NH2GO-Ag2Se@CdSe, belonging to the field of an electrochemical luminescence sensor. An Ag2Se@CdSe nano pin is taken as an electrogenerated chemiluminescence signal source; the Ag2Se@CdSe nano pin is fixedly loaded on the surface of PP<y>-NH2GO by utilizing the large specific surface area of the PP<y>-NH2GO, so that an antibody capture substrate can be prepared; the luminescent signals of the sensor can be effectively enhanced by the excellent electrical conductivity of the PP<y>-NH2GO. The CA72-4 can be detected according to different electrogenerated chemiluminescence signal intensities for the gastric cancer antigen CA72-4 with different concentrations.

Description

A kind of based on PPy-NH 2gO-Ag 2the preparation method of the gastric cancer antigen Electrochemiluminescsensor sensor of Se@CdSe and application
Technical field
The present invention relates to a kind of based on PPy-NH 2gO-Ag 2the preparation method of Se@CdSe electrochemiluminescimmunosensor immunosensor and application.Be specifically related to a kind of Ag 2se@CdSe nanometer pin as luminescent material, PPy-NH 2gO-Ag 2se@CdSe, as the preparation and application of the electrochemiluminescimmunosensor immunosensor of capture antibody substrate, belongs to electrochemiluminescence detection technique field.
Background technology
Gastric cancer antigen CA724 is the chemical examination mark detecting cancer of the stomach and various alimentary tract cancer, and full name is correlativity glycoprotein, belongs to mucin carcinoid embryonal antigen, be present in 5% breast tissue and 85% ~ 95% colon, pancreas, stomach, lung and ovary tumour in.Therefore, the tumour for positions such as pancreas, ovary, stomaches has certain diagnosis and differential diagnosis meaning.
The immune analysis method of current mensuration CA72-4 mainly contains radio immunoassay, enzyme-linked immunosorbent assay and RNA isolation kit.Application number is the preparation method and the application that disclose carbohydrate PROTEIN C A72-4 quantitative determination reagent kit in the patent of 201110235595.5; Application number is disclose the preparation method and application that detect CA72-4 kit in the patent of 201320494726.6.But the reagent term of validity is short, and the operating cycle is long, complex steps.In order to overcome the shortcoming of above chromatogram analysis method, the present invention devises a kind of high specificity, highly sensitive, no radioactivity pollute, operates fast and convenient electrogenerated chemiluminescence immune analysis method.And electrochemiluminescimmunosensor immunosensor method detection carbohydrate PROTEIN C A72-4 has no disclosed patent and bibliographical information.
The present invention utilizes Ag 2se@CdSe nanometer pin is electrochemiluminescence signal source, with Ag 2se@CdSe nanometer pin is electrochemiluminescence signal source, utilizes PPy-NH 2the large specific surface area of GO is by Ag 2se@CdSe nanometer pin is immobilized at PPy-NH 2the obtained antibody capture substrate in GO surface, PPy-NH 2the excellent electric conductivity of GO effectively can strengthen the luminous signal of sensor.The chemiluminescence immunoassay sensor of sending a telegraph built in the present invention has simple to operate, and cost is low, no radioactivity pollute, highly sensitive, the advantages such as high specificity, overcomes the some shortcomings of radio immunoassay, enzyme-linked immunosorbent assay and RNA isolation kit.
Summary of the invention
The object of the invention is the problem for existing carbohydrate PROTEIN C A72-4 detection method, a kind of simple, quick, free of contamination PPy-NH is provided 2gO-Ag 2the preparation method of Se@CdSe electrochemiluminescimmunosensor immunosensor and application, realize detecting the rapid sensitive of CA72-4.
technical scheme of the present invention is as follows:
1. one kind based on PPy-NH 2gO-Ag 2the preparation method of the gastric cancer antigen Electrochemiluminescsensor sensor of Se@CdSe
(1) glass-carbon electrode of diameter 4 mm is taken turns doing polishing with 1.0 μm, 0.3 μm, 0.05 μm aluminum oxide polishing powder successively, with EtOH Sonicate cleaning, cleaner with ultrapure water;
(2) painting 4 μ L, 0.5 ~ 2.0 mg mL is dripped -1pPy-NH 2gO-Ag 2se@CdSe-Ab antibody hatching solution is in electrode surface, and 4 DEG C are saved to drying;
(3) drip 4 μ L, massfraction is the bovine serum albumin solution of 1 ~ 3%, with the nonspecific activity site on enclosed-electrode surface, rinse electrode surface with the PBS buffer solution of pH 6.4 ~ 8.4,4 DEG C are dried;
(4) drip the gastric cancer antigen CA72-4 solution of 4 μ L, rinse electrode surface with the PBS buffer solution of pH 6.4 ~ 8.4, be placed on hatching 3 ~ 12 h in the refrigerator of 4 DEG C and dry, obtained a kind of based on PPy-NH 2gO-Ag 2the gastric cancer antigen Electrochemiluminescsensor sensor of Se@CdSe.
2. PPy-NH described above 2gO-Ag 2the preparation of Se@CdSe-Ab antibody hatching solution
0.05 ~ 0.15 mL, 1 μ g/mL CA72-4 antibody A b join PPy-NH 2gO-Ag 2in Se@CdSe nano-complex solution, vibrate hatching 12 ~ 24 h at 4 DEG C, the CA72-4 antibody A b that centrifugal removing is excessive, is distributed to by product in the PBS of 1 mL, pH 7.4, obtained PPy-NH 2gO-Ag 2se@CdSe-Ab antibody hatching solution, for subsequent use at being stored in 4 DEG C.
3. PPy-NH described above 2gO-Ag 2the preparation of Se@CdSe nano-complex solution
(1) amination Graphene NH 2the preparation of GO
90 ~ 110 mg graphene oxides are distributed in 30 ~ 50 mL ethylene glycol, ultrasonic limit, limit drips 0.8 ~ 1.2 mL ammoniacal liquor, the deep brown solution obtained is proceeded in polytetrafluoroethyllining lining autoclave, 8 ~ 12 h are reacted at 170 ~ 190 DEG C, filter and use milli-Q water, 50 ~ 70 DEG C of drying 12 ~ 36 h;
(2) polypyrrole intercalation amination Graphene PPy-NH 2the preparation of GO
By amination Graphene NH 2gO is distributed in 20 ~ 30 mL ultrapure waters, ultrasonic 25 ~ 35 min, ultrasonic limit, limit adds the neopelex of 90 ~ 110 mL, 0.13 ~ 0.15 g/mL, after ultrasonic 0.5 ~ 1.5 h, cools the temperature to 0 DEG C, drip 200 μ L pyrroles, magnetic agitation 20 ~ 40 min, adds the liquor ferri trichloridi of 20 ~ 40 mL, 2 ~ 3 mol/L while stirring, stirring reaction 2 ~ 3 h, filtration washing is extremely neutral, 40 ~ 60 DEG C of drying 36 ~ 64 h;
(3) Ag 2the preparation of Se@CdSe nanometer pin solution
By 20 ~ 26 mg dichloride cadmiums, 10 ~ 12 mg selenium dioxide and 0.1 ~ 0.3 g polyvinylpyrrolidone k-30 dissolve in the dimethyl formamide of 8 ~ 12 mL, add 4 ~ 6 mL oleic acid while stirring, the silver nitrate aqueous solution of 0.1 ~ 0.3 mL, 4 mmol/L, the sodium hexametaphosphate of 0.06 ~ 0.09 g and the mercaptoacetic acid of 50 μ L, the mixed solution obtained is proceeded in polytetrafluoroethyllining lining autoclave, 15 h are reacted at 90 ~ 110 DEG C, centrifuging also uses washed with isopropyl alcohol, product is distributed in isopropyl alcohol, obtained Ag 2se@CdSe nanometer pin solution;
(4) PPy-NH 2gO-Ag 2the preparation of Se@CdSe nano-complex solution
At Ag 2the polypyrrole intercalation amination Graphene PPy-NH of 1 mL, 2 mg/mL is added in Se@CdSe nanometer pin solution 2gO solution, vibration hatching 12 ~ 24 h, obtained PPy-NH 2gO-Ag 2se@CdSe nano-complex solution.
4. the detection of CA72-4
(1) three-electrode system of electrochemical workstation is used to test, Ag/AgCl electrode is as contrast electrode, platinum electrode is to electrode, prepared gastric cancer antigen Electrochemiluminescsensor sensor is working electrode, electrochemical workstation and chemiluminescence detector are linked together the high pressure of photomultiplier is set to 800 V, scanning voltage is set to-1.7 ~ 0 V, sweep speed 0.1 V/s;
(2) 10 mL, pH 6.4 ~ 8.4 containing 50 ~ 60 mmol/L potassium persulfates PBS buffer solution in, by the gastric cancer antigen CA72-4 standard solution of electrogenerated chemiluminescence systems axiol-ogy to a series of variable concentrations of 0.01 pg/mL ~ 50 ng/mL, the electrochemiluminescence signal intensity produced, drawing curve;
(3) gastric cancer antigen CA72-4 standard solution is replaced by testing sample solution to detect.
useful achievement of the present invention
(1) electrochemiluminescimmunosensor immunosensor preparation method, with Ag 2se CdSe nanometer pin is luminescent material, utilizes the optical property that it is good, and the sensor of structure has higher sensitivity;
(2) PPy-NH is utilized 2the large specific surface area of GO is by Ag 2se@CdSe is immobilized at PPy-NH 2the obtained antibody capture substrate in GO surface, PPy-NH 2the excellent electric conductivity of GO effectively can strengthen the luminous signal of sensor;
(3)) electrochemiluminescimmunosensor immunosensor prepared of the present invention is for the detection of CA72-4, simple to operate, no radioactivity pollute, signal response wide ranges, the range of linearity is 0.1 pg/mL ~ 20 ng/mL, detects and is limited to 21 fg/mL, can realize simple, quick, highly sensitive detection.
embodiment 1a kind of based on PPy-NH 2gO-Ag 2the preparation method of the gastric cancer antigen Electrochemiluminescsensor sensor of Se@CdSe
(1) glass-carbon electrode of diameter 4 mm is taken turns doing polishing with 1.0 μm, 0.3 μm, 0.05 μm aluminum oxide polishing powder successively, with EtOH Sonicate cleaning, cleaner with ultrapure water;
(2) painting 4 μ L, 0.5 mg mL is dripped -1pPy-NH 2gO-Ag 2se@CdSe-Ab antibody hatching solution is in electrode surface, and 4 DEG C are saved to drying;
(3) drip 4 μ L, bovine serum albumin solution that massfraction is 1 %, with the nonspecific activity site on enclosed-electrode surface, rinse electrode surface with the PBS buffer solution of pH 6.4,4 DEG C are dried;
(4) drip the gastric cancer antigen CA72-4 solution of 4 μ L, rinse electrode surface with the PBS buffer solution of pH 6.4, be placed in the refrigerator of 4 DEG C and hatch 3 h and dry, obtained a kind of based on PPy-NH 2gO-Ag 2se@CdSe electrochemiluminescimmunosensor immunosensor.
embodiment 2a kind of based on PPy-NH 2gO-Ag 2the preparation method of the gastric cancer antigen Electrochemiluminescsensor sensor of Se@CdSe
(1) glass-carbon electrode of diameter 4 mm is taken turns doing polishing with 1.0 μm, 0.3 μm, 0.05 μm aluminum oxide polishing powder successively, with EtOH Sonicate cleaning, cleaner with ultrapure water;
(2) painting 4 μ L, 1 mg mL is dripped -1pPy-NH 2gO-Ag 2se@CdSe-Ab antibody hatching solution is in electrode surface, and 4 DEG C are saved to drying;
(3) drip 4 μ L, bovine serum albumin solution that massfraction is 2 %, with the nonspecific activity site on enclosed-electrode surface, rinse electrode surface with the PBS buffer solution of pH 7.4,4 DEG C are dried;
(4) drip the gastric cancer antigen CA72-4 solution of 4 μ L, rinse electrode surface with the PBS buffer solution of pH 7.4, be placed in the refrigerator of 4 DEG C and hatch 7 h and dry, obtained a kind of based on PPy-NH 2gO-Ag 2se@CdSe electrochemiluminescimmunosensor immunosensor.
embodiment 3a kind of based on PPy-NH 2gO-Ag 2the preparation method of the gastric cancer antigen Electrochemiluminescsensor sensor of Se@CdSe
(1) glass-carbon electrode of diameter 4 mm is taken turns doing polishing with 1.0 μm, 0.3 μm, 0.05 μm aluminum oxide polishing powder successively, with EtOH Sonicate cleaning, cleaner with ultrapure water;
(2) painting 4 μ L, 2.0 mg mL are dripped -1pPy-NH 2gO-Ag 2se@CdSe-Ab antibody hatching solution is in electrode surface, and 4 DEG C are saved to drying;
(3) drip 4 μ L, massfraction is the bovine serum albumin solution of 3%, with the nonspecific activity site on enclosed-electrode surface, rinse electrode surface with the PBS buffer solution of pH 8.4,4 DEG C are dried;
(4) drip the gastric cancer antigen CA72-4 solution of 4 μ L, rinse electrode surface with the PBS buffer solution of pH 8.4, be placed in the refrigerator of 4 DEG C and hatch 12 h and dry, obtained a kind of based on PPy-NH 2gO-Ag 2se@CdSe electrochemiluminescimmunosensor immunosensor.
embodiment 4pPy-NH 2gO-Ag 2the preparation of Se@CdSe-Ab antibody hatching solution
0.05 mL, 1 μ g/mL CA72-4 antibody A b join PPy-NH 2gO-Ag 2in Se@CdSe nano-complex solution, vibrate hatching 12 h at 4 DEG C, the CA72-4 antibody A b that centrifugal removing is excessive, is distributed to by product in the PBS of 1 mL, pH 7.4, obtained PPy-NH 2gO-Ag 2se@CdSe-Ab antibody hatching solution, for subsequent use at being stored in 4 DEG C.
embodiment 5pPy-NH 2gO-Ag 2the preparation of Se@CdSe-Ab antibody hatching solution
0.1 mL, 1 μ g/mL CA72-4 antibody A b join PPy-NH 2gO-Ag 2in Se@CdSe nano-complex solution, vibrate hatching 18 h at 4 DEG C, the CA72-4 antibody A b that centrifugal removing is excessive, is distributed to by product in the PBS of 1 mL, pH 7.4, obtained PPy-NH 2gO-Ag 2se@CdSe-Ab antibody hatching solution, for subsequent use at being stored in 4 DEG C.
embodiment 6pPy-NH 2gO-Ag 2the preparation of Se@CdSe-Ab antibody hatching solution
0.15 mL, 1 μ g/mL CA72-4 antibody A b join PPy-NH 2gO-Ag 2in Se@CdSe nano-complex solution, vibrate hatching 24 h at 4 DEG C, the CA72-4 antibody A b that centrifugal removing is excessive, is distributed to by product in the PBS of 1 mL, pH 7.4, obtained PPy-NH 2gO-Ag 2se@CdSe-Ab antibody hatching solution, for subsequent use at being stored in 4 DEG C.
embodiment 7pPy-NH 2gO-Ag 2the preparation of Se@CdSe nano-complex solution
(1) amination Graphene NH 2the preparation of GO
90 mg graphene oxides are distributed in 30 mL ethylene glycol, ultrasonic limit, limit drips 0.8 mL ammoniacal liquor, the deep brown solution obtained is proceeded in polytetrafluoroethyllining lining autoclave, reacts 8h at 170 DEG C, filter and use milli-Q water, 50 DEG C of drying 12 h;
(2) polypyrrole intercalation amination Graphene PPy-NH 2the preparation of GO
By amination Graphene NH 2gO is distributed in 20 mL ultrapure waters, ultrasonic 25 min, ultrasonic limit, limit adds the neopelex of 90 mL, 0.13 g/mL, after ultrasonic 0.5 h, cools the temperature to 0 DEG C, drip 200 μ L pyrroles, magnetic agitation 20 min, adds the liquor ferri trichloridi of 20 mL, 2 mol/L while stirring, stirring reaction 2 h, filtration washing is extremely neutral, 40 DEG C of drying 36 h;
(3) Ag 2the preparation of Se@CdSe nanometer pin solution
By 20 mg dichloride cadmiums, 10 mg selenium dioxide and 0.1 g polyvinylpyrrolidone k-30 dissolve in the dimethyl formamide of 8 mL, add 4 mL oleic acid while stirring, the silver nitrate aqueous solution of 0.1 mL, 4 mmol/L, the sodium hexametaphosphate of 0.06 g and the mercaptoacetic acid of 50 μ L, proceed to the mixed solution obtained in polytetrafluoroethyllining lining autoclave, 15 h are reacted at 90 DEG C, product also by washed with isopropyl alcohol, is distributed in isopropyl alcohol by centrifuging, obtained Ag 2se@CdSe nanometer pin solution;
(4) PPy-NH 2gO-Ag 2the preparation of Se@CdSe nano-complex solution
At Ag 2the polypyrrole intercalation amination Graphene PPy-NH of 1 mL, 2 mg/mL is added in Se@CdSe nanometer pin solution 2gO solution, vibration hatching 12 h, obtained PPy-NH 2gO-Ag 2se@CdSe nano-complex solution.
embodiment 8pPy-NH described above 2gO-Ag 2the preparation of Se@CdSe nano-complex solution
(1) amination Graphene NH 2the preparation of GO
100 mg graphene oxides are distributed in 40 mL ethylene glycol, ultrasonic limit, limit drips 1.0 mL ammoniacal liquor, the deep brown solution obtained is proceeded in polytetrafluoroethyllining lining autoclave, reacts 10 h at 180 DEG C, filter and use milli-Q water, 60 DEG C of drying 24 h;
(2) polypyrrole intercalation amination Graphene PPy-NH 2the preparation of GO
By amination Graphene NH 2gO is distributed in 25 mL ultrapure waters, ultrasonic 30 min, ultrasonic limit, limit adds the neopelex of 100 mL, 0.14 g/mL, after ultrasonic 1.0 h, cools the temperature to 0 DEG C, drip 200 μ L pyrroles, magnetic agitation 30 min, adds the liquor ferri trichloridi of 30 mL, 2.5 mol/L while stirring, stirring reaction 2.5 h, filtration washing is extremely neutral, 50 DEG C of drying 42 h;
(3) Ag 2the preparation of Se@CdSe nanometer pin solution
By 23 mg dichloride cadmiums, 11 mg selenium dioxide and 0.2 g polyvinylpyrrolidone k-30 dissolve in the dimethyl formamide of 10 mL, add 5 mL oleic acid while stirring, the silver nitrate aqueous solution of 0.2 mL, 4 mmol/L, the sodium hexametaphosphate of 0.07 g and the mercaptoacetic acid of 50 μ L, proceed to the mixed solution obtained in polytetrafluoroethyllining lining autoclave, 15 h are reacted at 100 DEG C, product also by washed with isopropyl alcohol, is distributed in isopropyl alcohol by centrifuging, obtained Ag 2se@CdSe nanometer pin solution;
(4) PPy-NH 2gO-Ag 2the preparation of Se@CdSe nano-complex solution
At Ag 2the polypyrrole intercalation amination Graphene PPy-NH of 1 mL, 2 mg/mL is added in Se@CdSe nanometer pin solution 2gO solution, vibration hatching 18 h, obtained PPy-NH 2gO-Ag 2se@CdSe nano-complex solution.
embodiment 9pPy-NH described above 2gO-Ag 2the preparation of Se@CdSe nano-complex solution
(1) amination Graphene NH 2the preparation of GO
110 mg graphene oxides are distributed in 50 mL ethylene glycol, ultrasonic limit, limit drips 1.2 mL ammoniacal liquor, the deep brown solution obtained is proceeded in polytetrafluoroethyllining lining autoclave, reacts 12 h at 190 DEG C, filter and use milli-Q water, 70 DEG C of drying 36 h;
(2) polypyrrole intercalation amination Graphene PPy-NH 2the preparation of GO
By amination Graphene NH 2gO is distributed in 30 mL ultrapure waters, ultrasonic 35 min, ultrasonic limit, limit adds the neopelex of 110 mL, 0.15 g/mL, after ultrasonic 1.5 h, cools the temperature to 0 DEG C, drip 200 μ L pyrroles, magnetic agitation 40 min, adds the liquor ferri trichloridi of 40 mL, 3 mol/L while stirring, stirring reaction 3 h, filtration washing is extremely neutral, 60 DEG C of drying 64 h;
(3) Ag 2the preparation of Se@CdSe nanometer pin solution
By 26 mg dichloride cadmiums, 12 mg selenium dioxide and 0.3 g polyvinylpyrrolidone k-30 dissolve in the dimethyl formamide of 12 mL, add 6 mL oleic acid while stirring, the silver nitrate aqueous solution of 0.3 mL, 4 mmol/L, the sodium hexametaphosphate of 0.09 g and the mercaptoacetic acid of 50 μ L, proceed to the mixed solution obtained in polytetrafluoroethyllining lining autoclave, 15 h are reacted at 110 DEG C, product also by washed with isopropyl alcohol, is distributed in isopropyl alcohol by centrifuging, obtained Ag 2se@CdSe nanometer pin solution;
(4) PPy-NH 2gO-Ag 2the preparation of Se@CdSe nano-complex solution
At Ag 2the polypyrrole intercalation amination Graphene PPy-NH of 1 mL, 2 mg/mL is added in Se@CdSe nanometer pin solution 2gO solution, vibration hatching 24 h, obtained PPy-NH 2gO-Ag 2se@CdSe nano-complex solution.
embodiment 10the detection of CA72-4
(1) three-electrode system of electrochemical workstation is used to test, Ag/AgCl electrode is as contrast electrode, platinum electrode is to electrode, prepared gastric cancer antigen Electrochemiluminescsensor sensor is working electrode, electrochemical workstation and chemiluminescence detector are linked together the high pressure of photomultiplier is set to 800 V, scanning voltage is set to-1.7 ~ 0 V, sweep speed 0.1 V/s;
(2) 10 mL, pH 6.4 ~ 8.4 containing 50 ~ 60 mmol/L potassium persulfates PBS buffer solution in, by the CA72-4 standard solution of electrogenerated chemiluminescence systems axiol-ogy to a series of variable concentrations of 0.01 pg/mL ~ 50 ng/mL, the electrochemiluminescence signal intensity produced, drawing curve;
(3) replaced by testing sample solution gastric cancer antigen CA72-4 standard solution to detect, recording the range of linearity is 0.1 pg/mL ~ 20 ng/mL, detects and is limited to 21 fg/mL.

Claims (2)

1. one kind based on PPy-NH 2gO-Ag 2the gastric cancer antigen CA72-4 Electrochemiluminescsensor sensor preparation method of Se@CdSe, is characterized in that, comprise the following steps:
(1) amination Graphene NH 2the preparation of GO
90 ~ 110 mg graphene oxides are distributed in 30 ~ 50 mL ethylene glycol, ultrasonic limit, limit drips 0.8 ~ 1.2 mL ammoniacal liquor, the deep brown solution obtained is proceeded in polytetrafluoroethyllining lining autoclave, 8 ~ 12 h are reacted at 170 ~ 190 DEG C, filter and use milli-Q water, 50 ~ 70 DEG C of drying 12 ~ 36 h, obtained amination Graphene NH 2gO;
(2) polypyrrole intercalation amination Graphene PPy-NH 2the preparation of GO
By amination Graphene NH 2gO is distributed in 20 ~ 30 mL ultrapure waters, ultrasonic 25 ~ 35 min, ultrasonic limit, limit adds the neopelex of 90 ~ 110 mL, 0.13 ~ 0.15 g/mL, ultrasonic 0.5 ~ 1.5 h, temperature is down to 0 DEG C, drip 200 μ L pyrroles, magnetic agitation 20 ~ 40 min, add the liquor ferri trichloridi of 20 ~ 40 mL, 2 ~ 3 mol/L while stirring, stirring reaction 2 ~ 3 h, filtration washing is to neutral, and 40 ~ 60 DEG C of drying 36 ~ 64 h, obtain polypyrrole intercalation amination Graphene PPy-NH 2gO;
(3) Ag 2the preparation of Se@CdSe nanometer pin solution
By 20 ~ 26 mg dichloride cadmiums, 10 ~ 12 mg selenium dioxide and 0.1 ~ 0.3 g polyvinylpyrrolidone k-30 dissolve in the dimethyl formamide of 8 ~ 12 mL, add 4 ~ 6 mL oleic acid while stirring, the silver nitrate aqueous solution of 0.1 ~ 0.3 mL, 4 mmol/L, the sodium hexametaphosphate of 0.06 ~ 0.09 g and the mercaptoacetic acid of 50 μ L, the mixed solution obtained is proceeded in polytetrafluoroethyllining lining autoclave, 15 h are reacted at 90 ~ 110 DEG C, centrifuging also uses washed with isopropyl alcohol, product is distributed in isopropyl alcohol, obtained Ag 2se@CdSe nanometer pin solution;
(4) PPy-NH 2gO-Ag 2the preparation of Se@CdSe nano-complex solution
At Ag 2the polypyrrole intercalation amination Graphene PPy-NH of 1 mL, 2 mg/mL is added in Se@CdSe nanometer pin solution 2gO solution, vibration hatching 12 ~ 24 h, obtained PPy-NH 2gO-Ag 2se@CdSe nano-complex solution;
(5) PPy-NH 2gO-Ag 2the preparation of Se@CdSe-Ab antibody hatching solution
0.05 ~ 0.15 mL, 1 μ g/mL CA72-4 antibody A b join PPy-NH 2gO-Ag 2in Se@CdSe nano-complex solution, vibrate hatching 12 ~ 24 h at 4 DEG C, and product is distributed in the PBS of 1 mL, pH7.4 by the CA72-4 antibody A b that centrifugal removing is excessive, obtained PPy-NH 2gO-Ag 2se@CdSe-Ab antibody hatching solution, for subsequent use at being stored in 4 DEG C;
(6) preparation of gastric cancer antigen CA72-4 Electrochemiluminescsensor sensor
The glass-carbon electrode of diameter 4 mm is taken turns doing polishing with 1.0 μm, 0.3 μm, 0.05 μm aluminum oxide polishing powder by a successively, with EtOH Sonicate cleaning, cleaner with ultrapure water;
B drips painting 4 μ L, 0.5 ~ 2.0 mg mL -1pPy-NH 2gO-Ag 2se@CdSe-Ab antibody hatching solution is in electrode surface, and 4 DEG C are saved to drying;
C drips 4 μ L, massfraction is the bovine serum albumin solution of 1 ~ 3%, and with the nonspecific activity site on enclosed-electrode surface, rinse electrode surface with the PBS buffer solution of pH6.4 ~ 8.4,4 DEG C are dried;
D drips the gastric cancer antigen CA72-4 solution of 4 μ L, rinses electrode surface with the PBS buffer solution of pH6.4 ~ 8.4, is placed on hatching 3 ~ 12 h in the refrigerator of 4 DEG C and dries, obtained a kind of based on PPy-NH 2gO-Ag 2the gastric cancer antigen CA72-4 Electrochemiluminescsensor sensor of Se@CdSe.
2. the detection method of Electrochemiluminescsensor sensor prepared of preparation method as claimed in claim 1, is characterized in that, comprise the steps:
(1) three-electrode system of electrochemical workstation is used to test, Ag/AgCl electrode is as contrast electrode, platinum electrode is to electrode, prepared Electrochemiluminescsensor sensor is working electrode, electrochemical workstation and chemiluminescence detector are linked together the high pressure of photomultiplier is set to 800 V, scanning voltage is set to-1.7 ~ 0 V, sweep speed 0.1 V/s;
(2) 10 mL, pH 6.4 ~ 8.4 containing 50 ~ 60 mmol/L potassium persulfates PBS buffer solution in, by the gastric cancer antigen CA72-4 standard solution of electrogenerated chemiluminescence systems axiol-ogy to a series of variable concentrations of 0.01 pg/mL ~ 50 ng/mL, the electrochemiluminescence signal intensity produced, drawing curve;
(3) gastric cancer antigen CA72-4 standard solution is replaced by testing sample solution to detect.
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