CN106770529A - A kind of preparation method and application of the interlayer type immunosensor based on silver load vulcanization copper nano-wire - Google Patents
A kind of preparation method and application of the interlayer type immunosensor based on silver load vulcanization copper nano-wire Download PDFInfo
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Abstract
The invention belongs to new function material and bio-sensing detection technique field, there is provided a kind of preparation method and application of the interlayer type immunosensor based on silver load vulcanization copper nano-wire；Specifically using the copper sulfide nano rice noodles of silver load as detection antibody label, the detection of lung cancer carcinoma marker CA125, CA153, SCCA is realized, with high specificity, sensitivity is high, test limit is low, has important scientific meaning and application value to the early detection of lung cancer.
Description
Technical field
The invention belongs to immunoassay and biosensor technique field, there is provided one kind is based on silver load vulcanization copper nano-wire
Interlayer type immunosensor preparation method and application.
Background technology
In many countries and regions, the incidence of disease of lung cancer accounts for the first place of malignant tumour, and the health to the mankind produces greatly danger
Evil.The Sensitive Detection of lung cancer tumor mark, it is the screening of tumor high-risk, good clinically for the early detection of tumour
Property and malignant tumour antidiastole, the judgement of tumor development degree, the O＆A and tumour of the therapeutic effect of tumour are answered
The prediction of hair and prognosis produces strong influence.
Current electrochemical immunosensor is widely used for the detection of lung cancer and various tumor markerses, interlayer type electrification
Immuno analytical method and highly sensitive electrochemical analysis techniques that immunosensor combines high specific are learned, with sensitivity
High, preparation is simple, detection is quick, low cost and other advantages, in clinical examination, environmental monitoring, food safety control, biological monitoring etc.
There is important application value in field.
The pattern of the copper sulfide used in the present invention is nano wire, with good stability and catalytic performance, with more
Many binding sites, can effectively adsorb immobilized tumor markers antibody.Copper sulfide nano rice noodles can be with silver nanoparticle by S-Ag keys
Particle-stabilised combination, because the concerted catalysis effect of the two is favorably improved the electrochemical properties of material.
The content of the invention
The invention provides it is a kind of based on silver load vulcanization copper nano-wire interlayer type immunosensor preparation method and
Using realizing the super sensitivity detection to lung cancer marker.
An object of the present invention is to provide a kind of interlayer type immunosensor based on silver load vulcanization copper nano-wire
Preparation method.
The second object of the present invention is by a kind of prepared immune biography of interlayer type based on silver load vulcanization copper nano-wire
Sensor is applied to highly sensitive, the specific detection of lung cancer marker.
Technical scheme, comprises the following steps.
1. a kind of preparation method of the interlayer type immunosensor based on silver load vulcanization copper nano-wire, step is as follows：
（1）By the glass-carbon electrode Al of a diameter of 4mm2O3Polishing powder is polished, and ultra-pure water is cleaned up；
（2）The L-cys@Ag drop coatings of 6 μ L, 1.0 ~ 3.0 mg/mL are taken to electrode surface, is dried at room temperature, use ultrapure water
Electrode surface, dries；
（3）Continue for the tumor markers of 6 μ L, 10 ~ 15 μ g/mL capture antibody to be added drop-wise to electrode surface, ultrapure water,
Dried in 4 °C of refrigerators；
（4）Continue for the bovine serum albumin(BSA) BSA solution of 3 μ L, 2.0 ~ 3.0 mg/mL to be added drop-wise to electrode surface, ultra-pure water punching
Electrode surface is washed, is dried in 4 °C of refrigerators；
（5）A series of tumor markers antigenic solution of various concentrations of 6 μ L, 0.1pg ~ 30 ng/mL, ultra-pure water punching is added dropwise
Electrode surface is washed, is dried in 4 °C of refrigerators；
（6）By 6 μ L, the silver load vulcanization copper nano-wire detection antibody incubation content solution of 1 ~ 3 mg/mL, drop coating is in electrode table
On face, it is placed in 4 °C of refrigerators and dries, a kind of interlayer type immunosensor based on silver load vulcanization copper nano-wire is obtained.
2. it is a kind of based on silver load vulcanization copper nano-wire interlayer type immunosensor preparation method, the L-cys Ag
Preparation, step is as follows：
Take 25 mL, 2 mmoL.L-1Cys L-cys 1 ~ 3 mL mass is sequentially added in beaker, while stirring
Fraction is 1% polyvinylpyrrolidone PVP, 1 ~ 3 mL, 0.1 moL.L-1Ascorbic acid and 2 ~ 4 mL, 0.1
moL.L-1AgNO3Solution, the at room temperature min of magnetic agitation 10；Centrifugation, milli-Q water three times, 65 °C of vacuum drying 6
~ 10h, is obtained L-cys@Ag；
3. it is a kind of based on silver load vulcanization copper nano-wire interlayer type immunosensor preparation method, it is described silver load copper sulfide
The preparation of nano wire detection antibody incubation content solution, step is as follows：
（1）The synthesis of copper sulfide nano rice noodles
Weigh 1 ~ 3 mmol CuCl2It is dissolved in 50 ml water, the cetyl of 1 ~ 3 mmol is sequentially added while stirring
Amine and 0.2 g glucose, are transferred to 60 °C of 4 ~ 5 h of reaction in 100 mL autoclaves after dissolving, be warmed up to 120 °C of reactions
3 ~ 4 h, centrifugation；Isolated solid is distributed to 50 mL, 0.2 moL.L-1Na2In S solution, addition 6 ~
10 mg PVP, continue to be transferred in autoclave after stirring 30 min, and 2 ~ 3 h are reacted under 120 °C, are cooled to room temperature,
10min is centrifuged in 12000 rpm, milli-Q water three times dries 12 ~ 14 h, copper sulfide is obtained in 50 °C of vacuum drying chambers
Nano wire；
（2）The synthesis of silver load vulcanization copper nano-wire
0.1 ~ 0.3 g copper sulfide nano rice noodles are scattered in 100 mL ultra-pure waters, 10 mL, 0.1 are added under stirring condition
moL.L-1AgNO3Solution and the PVP solution that 1.5 mL, mass fraction are 1%, after 10 min of stirring, add 10 mL, 0.1
moL.L-1Ascorbic acid, stir 30 min, centrifugation, milli-Q water three times, in 60 °C of vacuum drying chambers dry 12 ~
14 h, are obtained silver load vulcanization copper nano-wire；
（3）The preparation of silver load vulcanization copper nano-wire detection antibody incubation content solution
The silver load vulcanization copper nano-wire of 2 ~ 4 mg is dissolved in the PBS of pH 7.4 of 1 mL, concussion dissolving,
Add 100 μ L, the tumor markers detection antibody solution of 80 ~ 120 μ g/mL and 900 μ L, the pH 7.4 of 50 mmol/L
PBS, in 4 °C of constant-temperature shaking incubators vibrate, hatch 12 h, be obtained silver load vulcanization copper nano-wire detection
Antibody incubation content solution, saves backup under 4 °C.
4. it is a kind of that the interlayer type immunosensor for vulcanizing copper nano-wire is loaded based on silver, for the detection of tumor markers,
Step is as follows：
（1）Tested with three-electrode system using electrochemical workstation, saturated calomel electrode is reference electrode, platinum electrode is
Auxiliary electrode, prepared sensor is working electrode, in 10 mL, containing 4 mmoL.L-1The potassium ferricyanide and 0.1 moL.L-1
Potassium nitrate solution in tested；
（2）Analyte is detected with square wave voltammetry, initial potential is 0 V, termination current potential is 0.4 V, and current potential increment is
0.004 V, amplitude is 0.025 V, and time of repose is 2 s, record current change.
Tumor markers described above is selected from one of following：CA125、CA153、SCCA.
Raw materials of the present invention can be bought in chemical reagents corporation or biopharmaceutical company.
Useful achievement of the invention
（1）Nano silver grain L-cys@Ag are loaded as substrate present invention uses Cys, and nano-particle has big
Specific surface area, and Cys are hydroaropic substances, there is superior dispersiveness in water, and carboxyl can be anti-with tumor markers
Amino on body is effectively combined so that with reference to more firmly, can increase with antibody combination number and stability,；
（2）Using the copper sulfide nano rice noodles of silver load as capture antibody labeling thing, with big specific surface area, Ag nano-particles
With good catalytic performance, the copper sulfide nano rice noodles of silver load have good stability and catalytic performance, can effectively adsorb
Immobilized tumor markers antibody.Copper sulfide nano rice noodles by S-Ag keys can and Nano silver grain stabilization combination, due to the two
Concerted catalysis effect help to realize the effect for amplifying electrochemical signals, so as to improve the sensitivity of sensor, reduce
Test limit；
（3）A kind of interlayer type immunosensor based on silver load vulcanization copper nano-wire is used for the detection to lung cancer marker, its
To range of linearity 0.1pg ~ 30 ng/mL, minimum 0.03 pg/mL of test limit of SCCA detections, to the linear model of CA125 detections
0.1pg ~ 20 ng/mL, minimum 0.03 pg/mL of test limit are enclosed, to range of linearity 0.5pg ~ 30 ng/ of CA153 detections
ML, minimum 0.17 pg/mL of test limit, show that a kind of interlayer type immunosensor based on silver load vulcanization copper nano-wire can be with
Reach the purpose of Accurate Determining.
Specific embodiment
Now the present invention is further illustrated by specific embodiment, but not limited to this.
A kind of preparation method of the interlayer type immunosensor based on silver load vulcanization copper nano-wire of embodiment 1, step is such as
Under：
（1）By the glass-carbon electrode Al of a diameter of 4mm2O3Polishing powder is polished, and ultra-pure water is cleaned up；
（2）The L-cys@Ag drop coatings of 6 μ L, 1.0 mg/mL are taken to electrode surface, is dried at room temperature, with ultrapure water electrode table
Face, dries；
（3）Continue for the tumor markers capture antibody of 6 μ L, 10 μ g/mL to be added drop-wise to electrode surface, ultrapure water, 4 °C of ice
Dried in case；
（4）The bovine serum albumin(BSA) BSA solution of 3 μ L, 2.0 mg/mL is added drop-wise to electrode surface, ultrapure water electrode by continuation
Surface, dries in 4 °C of refrigerators；
（5）A series of tumor markers antigenic solution of various concentrations of 6 μ L, 0.1pg ~ 30 ng/mL, ultra-pure water punching is added dropwise
Electrode surface is washed, is dried in 4 °C of refrigerators；
（6）By the silver load vulcanization copper nano-wire detection antibody incubation content solution of 6 μ L, 1mg/mL, drop coating on electrode surface,
It is placed in 4 °C of refrigerators and dries, a kind of interlayer type immunosensor based on silver load vulcanization copper nano-wire is obtained.
A kind of preparation method of the interlayer type immunosensor based on silver load vulcanization copper nano-wire of embodiment 2, step
It is as follows：
（1）By the glass-carbon electrode Al of a diameter of 4mm2O3Polishing powder is polished, and ultra-pure water is cleaned up；
（2）The L-cys@Ag drop coatings of 6 μ L, 2.0 mg/mL are taken to electrode surface, is dried at room temperature, with ultrapure water electrode table
Face, dries；
（3）Continue for the tumor markers capture antibody of 6 μ L, 12 μ g/mL to be added drop-wise to electrode surface, ultrapure water, 4 °C of ice
Dried in case；
（4）The bovine serum albumin(BSA) BSA solution of 3 μ L, 2.5 mg/mL is added drop-wise to electrode surface, ultrapure water electrode by continuation
Surface, dries in 4 °C of refrigerators；
（5）A series of tumor markers antigenic solution of various concentrations of 6 μ L, 0.1pg ~ 30 ng/mL, ultra-pure water punching is added dropwise
Electrode surface is washed, is dried in 4 °C of refrigerators；
（6）By 6 μ L, the silver load vulcanization copper nano-wire detection antibody incubation content solution of 2 mg/mL, drop coating on electrode surface,
It is placed in 4 °C of refrigerators and dries, a kind of interlayer type immunosensor based on silver load vulcanization copper nano-wire is obtained.
A kind of preparation method of the interlayer type immunosensor based on silver load vulcanization copper nano-wire of embodiment 3, step is such as
Under：
（1）By the glass-carbon electrode Al of a diameter of 4mm2O3Polishing powder is polished, and ultra-pure water is cleaned up；
（2）The L-cys@Ag drop coatings of 6 μ L, 3.0 mg/mL are taken to electrode surface, is dried at room temperature, with ultrapure water electrode table
Face, dries；
（3）Continue for the tumor markers capture antibody of 6 μ L, 15 μ g/mL to be added drop-wise to electrode surface, ultrapure water, 4 °C of ice
Dried in case；
（4）The bovine serum albumin(BSA) BSA solution of 3 μ L, 3.0 mg/mL is added drop-wise to electrode surface, ultrapure water electrode by continuation
Surface, dries in 4 °C of refrigerators；
（5）A series of tumor markers antigenic solution of various concentrations of 6 μ L, 0.1pg ~ 30 ng/mL, ultra-pure water punching is added dropwise
Electrode surface is washed, is dried in 4 °C of refrigerators；
（6）By 6 μ L, the silver load vulcanization copper nano-wire detection antibody incubation content solution of 3 mg/mL, drop coating on electrode surface,
It is placed in 4 °C of refrigerators and dries, a kind of interlayer type immunosensor based on silver load vulcanization copper nano-wire is obtained.
The preparation of the L-cys@Ag of embodiment 4, step is as follows：
Take 25 mL, 2 mmoL.L-1Cys L-cys 1 mL mass fractions are sequentially added in beaker, while stirring
It is 1% polyvinylpyrrolidone PVP, 1mL, 0.1 moL.L-1Ascorbic acid and 2 mL, 0.1 moL.L-1AgNO3Solution,
The min of magnetic agitation 10 at room temperature；Centrifugation, milli-Q water three times, 65 °C of 6 h of vacuum drying are obtained L-cys@Ag.
The preparation of the L-cys@Ag of embodiment 5, step is as follows：
Take 25 mL, 2 mmoL.L-1Cys L-cys 2 mL mass fractions are sequentially added in beaker, while stirring
It is 1% polyvinylpyrrolidone PVP, 2 mL, 0.1 moL.L-1Ascorbic acid and 3 mL, 0.1 moL.L-1AgNO3It is molten
Liquid, the at room temperature min of magnetic agitation 10；Centrifugation, milli-Q water three times, 65 °C of 8 h of vacuum drying are obtained L-cys@Ag.
The preparation of the L-cys@Ag of embodiment 6, step is as follows：
Take 25 mL, 2 mmoL.L-1Cys L-cys 3 mL mass fractions are sequentially added in beaker, while stirring
It is 1% polyvinylpyrrolidone PVP, 3 mL, 0.1 moL.L-1Ascorbic acid and 4 mL, 0.1 moL.L-1AgNO3It is molten
Liquid, the at room temperature min of magnetic agitation 10；Centrifugation, milli-Q water three times, 65 °C of 10 h of vacuum drying are obtained L-cys@
Ag。
The preparation of the silver medal of embodiment 7 load vulcanization copper nano-wire detection antibody incubation content solution, step is as follows：
（1）The synthesis of copper sulfide nano rice noodles
Weigh 1 mmol CuCl2It is dissolved in 50 ml water, the hexadecylamine and 0.2 g of 1 mmol is sequentially added while stirring
Glucose, is transferred to 60 °C of 4 h of reaction in 100 mL autoclaves after dissolving, be warmed up to 120 °C of 3 h of reaction, centrifugation；
Isolated solid is distributed to 50 mL, 0.2 moL.L-1Na2In S solution, 6 mg PVP are added, continue to stir 30
It is transferred in autoclave after min, 2 h is reacted under 120 °C, be cooled to room temperature, 10min is centrifuged in 12000 rpm, it is ultrapure
Water washing three times, dries 12 h in 50 °C of vacuum drying chambers, copper sulfide nano rice noodles are obtained；
（2）The synthesis of silver load vulcanization copper nano-wire
0.1g copper sulfide nano rice noodles are scattered in 100 mL ultra-pure waters, 10 mL, 0.1 moL.L are added under stirring condition-1's
AgNO3Solution and the PVP solution that 1.5 mL, mass fraction are 1%, after 10 min of stirring, add 10 mL, 0.1 moL.L-1It is anti-
Bad hematic acid, stirs 30 min, and centrifugation, milli-Q water three times dries 12 h in 60 °C of vacuum drying chambers, silver load is obtained
Copper sulfide nano rice noodles；
（3）The preparation of silver load vulcanization copper nano-wire detection antibody incubation content solution
The silver load vulcanization copper nano-wire of 2 mg is dissolved in the PBS of pH 7.4 of 1 mL, concussion dissolving, then
Add 100 μ L, the tumor markers detection antibody solution of 80 μ g/mL and 900 μ L, the phosphate of the pH 7.4 of 50 mmol/L
Cushioning liquid, vibrates in 4 °C of constant-temperature shaking incubators, hatches 12 h, and silver load vulcanization copper nano-wire detection antibody hatching is obtained
Thing solution, saves backup under 4 °C.
The preparation of the silver medal of embodiment 8 load vulcanization copper nano-wire detection antibody incubation content solution, step is as follows：
（1）The synthesis of copper sulfide nano rice noodles
Weigh 2 mmol CuCl2It is dissolved in 50 ml water, the hexadecylamine and 0.2 g of 2 mmol is sequentially added while stirring
Glucose, is transferred to 60 °C of 4.5 h of reaction in 100 mL autoclaves after dissolving, be warmed up to 120 °C of 3.5 h of reaction, centrifugation
Separate；Isolated solid is distributed to 50 mL, 0.2 moL.L-1Na2In S solution, 8 mg PVP are added, continue to stir
It is transferred in autoclave after mixing 30 min, 2.5h is reacted under 120 °C, be cooled to room temperature, in 12000 rpm centrifugation
10min, milli-Q water three times dries 13 h in 50 °C of vacuum drying chambers, copper sulfide nano rice noodles are obtained；
（2）The synthesis of silver load vulcanization copper nano-wire
0.2 g copper sulfide nano rice noodles are scattered in 100 mL ultra-pure waters, 10 mL, 0.1 moL.L are added under stirring condition-1's
AgNO3Solution and the PVP solution that 1.5 mL, mass fraction are 1%, after 10 min of stirring, add 10 mL, 0.1 moL.L-1It is anti-
Bad hematic acid, stirs 30 min, and centrifugation, milli-Q water three times dries 13 h in 60 °C of vacuum drying chambers, silver load is obtained
Copper sulfide nano rice noodles；
（3）The preparation of silver load vulcanization copper nano-wire detection antibody incubation content solution
The silver load vulcanization copper nano-wire of 3 mg is dissolved in the PBS of pH 7.4 of 1 mL, concussion dissolving, then
Add 100 μ L, the tumor markers detection antibody solution of 100 μ g/mL and 900 μ L, the phosphoric acid of the pH 7.4 of 50 mmol/L
Salt buffer solution, vibrates in 4 °C of constant-temperature shaking incubators, hatches 12 h, silver load vulcanization copper nano-wire detection antibody is obtained and incubates
Compound solution, saves backup under 4 °C.
The preparation of the silver medal of embodiment 9 load vulcanization copper nano-wire detection antibody incubation content solution, step is as follows：
（1）The synthesis of copper sulfide nano rice noodles
Weigh 3 mmol CuCl2It is dissolved in 50 ml water, the hexadecylamine and 0.2 g of 3 mmol is sequentially added while stirring
Glucose, is transferred to 60 °C of 5 h of reaction in 100 mL autoclaves after dissolving, be warmed up to 120 °C of 4 h of reaction, centrifugation；
Isolated solid is distributed to 50 mL, 0.2 moL.L-1Na2In S solution, 10 mg PVP are added, continue to stir 30
It is transferred in autoclave after min, 3 h is reacted under 120 °C, be cooled to room temperature, 10min is centrifuged in 12000 rpm, it is ultrapure
Water washing three times, dries 14 h in 50 °C of vacuum drying chambers, copper sulfide nano rice noodles are obtained；
（2）The synthesis of silver load vulcanization copper nano-wire
0.3 g copper sulfide nano rice noodles are scattered in 100 mL ultra-pure waters, 10 mL, 0.1 moL.L are added under stirring condition-1's
AgNO3Solution and the PVP solution that 1.5 mL, mass fraction are 1%, after 10 min of stirring, add 10 mL, 0.1 moL.L-1It is anti-
Bad hematic acid, stirs 30 min, and centrifugation, milli-Q water three times dries 14 h in 60 °C of vacuum drying chambers, silver load is obtained
Copper sulfide nano rice noodles；
（3）The preparation of silver load vulcanization copper nano-wire detection antibody incubation content solution
The silver load vulcanization copper nano-wire of 4 mg is dissolved in the PBS of pH 7.4 of 1 mL, concussion dissolving, then
Add 100 μ L, the tumor markers detection antibody solution of 120 μ g/mL and 900 μ L, the phosphoric acid of the pH 7.4 of 50 mmol/L
Salt buffer solution, vibrates in 4 °C of constant-temperature shaking incubators, hatches 12 h, silver load vulcanization copper nano-wire detection antibody is obtained and incubates
Compound solution, saves backup under 4 °C.
The detection of the tumor markers CA125 of embodiment 10, step is as follows：
（1）Tested with three-electrode system using electrochemical workstation, saturated calomel electrode is reference electrode, platinum electrode is
Auxiliary electrode, prepared sensor is working electrode, in 10 mL, containing 4 mmoL.L-1The potassium ferricyanide and 0.1 moL.L-1
Potassium nitrate solution in tested；
（2）Analyte is detected with square wave voltammetry, initial potential is 0 V, termination current potential is 0.4 V, and current potential increment is
0.004 V, amplitude is 0.025 V, and time of repose is 2 s, record current change；
（3）According to the linear relationship between gained electric current and CA125 concentration, drawing curve measures the range of linearity for 0.1pg
~ 20 ng/mL, minimum 0.03 pg/mL of test limit.
The detection of the tumor markers CA153 of embodiment 11
Method according to embodiment 10 detects that its range of linearity is 0.5pg ~ 30 ng/mL to CA153, and detection is limited to
0.17 pg/mL。
The detection of the tumor markers SCCA of embodiment 12
Method according to embodiment 10 detects that its range of linearity 0.1pg ~ 30 ng/mL, test limit is minimum to SCCA
0.03 pg/mL。
Claims (5)
1. it is a kind of based on silver load vulcanization copper nano-wire interlayer type immunosensor preparation method, it is characterised in that step
It is as follows：
（1）By the glass-carbon electrode Al of a diameter of 4mm2O3Polishing powder is polished, and ultra-pure water is cleaned up；
（2）The L-cys@Ag drop coatings of 6 μ L, 1.0 ~ 3.0 mg/mL are taken to electrode surface, is dried at room temperature, use ultrapure water
Electrode surface, dries；
（3）Continue for the tumor markers of 6 μ L, 10 ~ 15 μ g/mL capture antibody to be added drop-wise to electrode surface, ultrapure water,
Dried in 4 °C of refrigerators；
（4）Continue for the bovine serum albumin(BSA) BSA solution of 3 μ L, 2.0 ~ 3.0 mg/mL to be added drop-wise to electrode surface, ultra-pure water punching
Electrode surface is washed, is dried in 4 °C of refrigerators；
（5）A series of tumor markers antigenic solution of various concentrations of 6 μ L, 0.1pg ~ 30 ng/mL, ultra-pure water punching is added dropwise
Electrode surface is washed, is dried in 4 °C of refrigerators；
（6）By 6 μ L, the silver load vulcanization copper nano-wire detection antibody incubation content solution of 1 ~ 3 mg/mL, drop coating is in electrode table
On face, it is placed in 4 °C of refrigerators and dries, a kind of interlayer type immunosensor based on silver load vulcanization copper nano-wire is obtained.
2. the preparation side of a kind of interlayer type immunosensor based on silver load vulcanization copper nano-wire as claimed in claim 1
Method, the preparation of the L-cys@Ag, step is as follows：
Take 25 mL, 2 mmoL.L-1Cys L-cys 1 ~ 3 mL mass point is sequentially added in beaker, while stirring
Number is 1% polyvinylpyrrolidone PVP, 1 ~ 3 mL, 0.1 moL.L-1Ascorbic acid and 2 ~ 4 mL, 0.1 moL.L-1
AgNO3Solution, the at room temperature min of magnetic agitation 10；Centrifugation, milli-Q water three times, 65 °C of 6 ~ 10h of vacuum drying,
L-cys@Ag are obtained.
3. the preparation side of a kind of interlayer type immunosensor based on silver load vulcanization copper nano-wire as claimed in claim 1
Method, the preparation of the silver load vulcanization copper nano-wire detection antibody incubation content solution, step is as follows：
（1）The synthesis of copper sulfide nano rice noodles
Weigh 1 ~ 3 mmol CuCl2It is dissolved in 50 ml water, the hexadecylamine of 1 ~ 3 mmol is sequentially added while stirring
With 0.2 g glucose, 60 °C of 4 ~ 5 h of reaction in 100 mL autoclaves are transferred to after dissolving, are warmed up to 120 °C of reactions 3
~ 4 h, centrifugation；Isolated solid is distributed to 50 mL, 0.2 moL.L-1Na2In S solution, addition 6 ~
10 mg PVP, continue to be transferred in autoclave after stirring 30 min, and 2 ~ 3 h are reacted under 120 °C, are cooled to room temperature,
10min is centrifuged in 12000 rpm, milli-Q water three times dries 12 ~ 14 h, copper sulfide is obtained in 50 °C of vacuum drying chambers
Nano wire；
（2）The synthesis of silver load vulcanization copper nano-wire
0.1 ~ 0.3 g copper sulfide nano rice noodles are scattered in 100 mL ultra-pure waters, 10 mL, 0.1 are added under stirring condition
moL.L-1AgNO3Solution and the PVP solution that 1.5 mL, mass fraction are 1%, after 10 min of stirring, add 10 mL, 0.1
moL.L-1Ascorbic acid, stir 30 min, centrifugation, milli-Q water three times, in 60 °C of vacuum drying chambers dry 12 ~
14 h, are obtained silver load vulcanization copper nano-wire；
（3）The preparation of silver load vulcanization copper nano-wire detection antibody incubation content solution
The silver load vulcanization copper nano-wire of 2 ~ 4 mg is dissolved in the PBS of pH 7.4 of 1 mL, concussion dissolving,
Add 100 μ L, the tumor markers detection antibody solution of 80 ~ 120 μ g/mL and 900 μ L, the pH 7.4 of 50 mmol/L
PBS, in 4 °C of constant-temperature shaking incubators vibrate, hatch 12 h, be obtained silver load vulcanization copper nano-wire detection
Antibody incubation content solution, saves backup under 4 °C.
4. a kind of interlayer type based on silver load vulcanization copper nano-wire that prepared by preparation method as claimed in claim 1 is immunized and passes
Sensor, for the detection of tumor markers, detecting step is as follows：
（1）Tested with three-electrode system using electrochemical workstation, saturated calomel electrode is reference electrode, platinum electrode is
Auxiliary electrode, prepared sensor is working electrode, in 10 mL, containing 4 mmoL.L-1The potassium ferricyanide and 0.1 moL.L-1
Potassium nitrate solution in tested；
（2）Analyte is detected with square wave voltammetry, initial potential is 0 V, termination current potential is 0.4 V, and current potential increment is
0.004 V, amplitude is 0.025 V, and time of repose is 2 s, record current change.
5. as described in claim 1,3 or 4 it is a kind of based on silver load vulcanization copper nano-wire interlayer type immunosensor system
Preparation Method and application, it is characterised in that the tumor markers is selected from one of following： CA125、CA153、SCCA.
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