CN108469461A - A kind of preparation method and application of interlayer type lung cancer marker electrochemical sensor - Google Patents
A kind of preparation method and application of interlayer type lung cancer marker electrochemical sensor Download PDFInfo
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Abstract
The invention belongs to immunoassays and biosensor technique field, provide a kind of preparation method and application of interlayer type lung cancer marker electrochemical sensor.The present invention utilizes the graphene-supported Cu of the bimetal nano materials A u@Pt functionalization N dopings of nucleocapsid2+As catalysis material, it is used as marker with after detection antibody hatching, simultaneously using the gold nanoparticle of poly-dopamine functionalization as electrode modified material, to realize the detection of lung cancer marker CEA, CA125, SCCA, has high sensitivity, high specificity, detection limit is low, has important scientific meaning and application value to the early detection of lung cancer.
Description
Technical field
The invention belongs to immunoassays and biosensor technique field, are related to a kind of interlayer type lung cancer marker electrochemistry biography
The preparation method and application of sensor.Nano-function thin films are specifically used to mark detection antibody, realization pair as catalyst
The Sensitive Detection of lung cancer marker CEA, CA125, SCCA.
Background technology
Lung cancer has higher incidence, not noticeable, and the speed of growth and the transfer of tumour is fast, becomes strong to crowd
One of health and the maximum malignant tumour of life threat.Therefore, the Sensitive Detection for realizing lung cancer marker sends out the early stage of lung cancer
It is now extremely important with early treatment.Currently, having much for the detection method of lung cancer marker, such as enzyme-linked immunization, radiation is exempted from
Epidemic disease method etc., but the problems such as these methods may need large-scale instrument and equipment in detection process, and there are radioactive pollution, therefore,
A kind of high specificity is invented, high sensitivity, detection speed is fast, and lung cancer tumor marker sensor easy to operate is particularly significant.
Electrochemical immunosensor rely on antigen and its corresponding antibodies specific binding, have been widely used for lung cancer and
The detection of various tumor markers generally can be divided into two kinds of interlayer type immunosensor and unmarked type immunosensor.It is sandwich
Type electrochemical immunosensor combines the immuno analytical method of high specific and highly sensitive electrochemical analysis techniques, has spirit
The advantages that sensitivity is high, detection range is wide, Monitoring lower-cut is low, detection is quick, is widely used to environmental monitoring, food security control
The fields such as system, biological monitoring, clinical examination.
The bimetal nano composite material of nucleocapsid exists at present because of its unique optics, electricity and chemical property
It is widely applied in production, life and scientific research.Nitrogen-doped graphene has big surface area, good electric conductivity and biology
Compatibility is widely used in the fields such as battery, sensor.The present invention is using the gold nanoparticle of poly-dopamine functionalization as base
Bottom material, Au@Pt DNs/NG/Cu2+It as catalysis material, and carries out hatching with detection antibody and is used as marker, construct and be used for
The interlayer type electrochemical immunosensor of lung cancer marker analyte detection.
Invention content
The present invention provides a kind of preparation method and application of interlayer type lung cancer marker electrochemical sensor, realize pair
The super sensitivity detection of lung cancer marker.
An object of the present invention is to provide a kind of preparation method of interlayer type lung cancer marker electrochemical sensor.
The second object of the present invention is to by prepared Au@Pt DNs/NG/Cu2+The sensor application of label is in lung cancer mark
Highly sensitive, the specific detection of will object.
Technical scheme of the present invention includes the following steps.
1. a kind of preparation method of interlayer type lung cancer marker electrochemical sensor, steps are as follows：
（1）By the glass-carbon electrode Al of a diameter of 4 mm2O3Polishing powder is polished, and ultra-pure water cleans up；
（2）Take the golden nanometer particle dispersion liquid drop coating of the poly-dopamine functionalization of 6 μ L, 1.0 ~ 3.0 mg/mL to electrode table
Face is dried at room temperature, and ultrapure water electrode surface dries；
（3）By the lung cancer marker capture antibody A b of 6 μ L, 8 ~ 12 μ g/mL1It is added drop-wise to electrode surface, ultrapure water, 4
It is dry in DEG C refrigerator；
（4）Continue the bovine serum albumin(BSA) BSA solution of 3 μ L, 0.5 ~ 1.5 mg/mL being added drop-wise to electrode surface, ultra-pure water punching
Electrode surface is washed, is dried in 4 DEG C of refrigerators；
（5）A series of lung cancer marker antigen A g solution of various concentrations of 6 μ L, 0.0001 ~ 100 ng/mL is added dropwise, it is ultrapure
Water rinses electrode surface, is dried in 4 DEG C of refrigerators；
（6）By 6 μ L, 1.5 ~ 3.5 mg/mL detection antibody incubation content Au@Pt DNs/NG/Cu2+-Ab2Solution drops to electrode table
Face is placed in 4 DEG C of refrigerators and hatches 40 min, ultrapure water electrode surface, is dried in 4 DEG C of refrigerators, and a kind of interlayer type lung is made
Carcinoma marker electrochemical sensor.
2. the preparation of the gold nanoparticle of the poly-dopamine functionalization, steps are as follows：
（1）The preparation of solution of gold nanoparticles
Will 1 ~ 2 mL, mass fraction be 1% gold chloride be added 99 mL ultra-pure waters in, be heated to boiling after be added 1.5 ~
3.5 mL, the sodium citrate solution that mass fraction is 1%, flow back 10 ~ 30 min, is cooled to room temperature, obtains the Jenner of claret
Rice corpuscles solution；
（2）The preparation of the gold nanoparticle of poly-dopamine functionalization
It takes 10 ~ 20 mg dopamines to be added in the Tris-HCl buffer solutions of pH=8.5 of 10 mL, 10 mmol/L, is added
The solution of gold nanoparticles of the above-mentioned preparations of 10 mL, vibrates the gold nanoparticle for obtaining poly-dopamine functionalization for 24 hours at room temperature.
3. the Au@Pt DNs/NG/Cu2+Preparation, steps are as follows：
（1）The preparation of Au@Pt DNs
It is water-soluble containing 0.25 mmol/L gold chlorides and 75 mmol/L cetyl trimethylammonium bromides that 10 mL are prepared in flask
Liquid is vigorously stirred down and rapidly joins 0.6 mL, 10 mmol/L sodium borohydrides, and Jinsui River colloidal sol is made after being slowly stirred 3h at room temperature；
It measures Jinsui River colloidal sol ultra-pure water made from 1 mL and is diluted to 100 mL, gold seed-solution is made.
By 0.1 ~ 0.5 mL, 10 mmol/L gold chlorides, 2 ~ 3 mL, 0.2 mol/L cetyl trimethylammonium bromides and
The aqueous solution mixing of 1.5 ~ 2.0mL, 0.1 mol/L ascorbic acid, it is molten to be added 0.3 ~ 0.5 mL Jin Jings kind in mixed liquor
Liquid, ultra-pure water are diluted to 25mL, vibrate 8h at room temperature, and the solution of cube gold nanoparticle is made；In a cube gold nanoparticle
Solution in sequentially add 1 ~ 3 mL, 0.1 mol/L ascorbic acid and 1 mL, 10 mmol/L potassium chloroplatinites, in 60 DEG C
12h is reacted in water-bath,；Centrifugation, milli-Q water 3 times are dried in 40 DEG C of vacuum drying chambers, and Au@Pt DNs are made；Weigh one
Determine that the use of Au@Pt DNs dispersion liquids is made in the Au@Pt DNs ultrasonic disperses to the ultra-pure water of certain volume of quality；
（2）The preparation of nitrogen-doped graphene NG
It weighs 20 ~ 40 mg propionaldoximes to be placed in 500 mL three-necked flasks, sequentially adds 200 mL concentrated ammonia liquors and 15 mL oxidations
After ultrasonic disperse is uniform, three-necked flask is transferred in oil bath pan for graphene suspension, under agitation 90 DEG C of reaction 4h,
Cooled to room temperature；It centrifuges, with milli-Q water to filtrate at neutrality, NG is made in freeze-drying；
The suspended liquid and preparation method thereof of graphene oxide is as follows：2.0 g graphite powders are weighed, the 96 mL concentrated sulfuric acids are added, in stirring bar
20 min are reacted under part, and 12 g potassium permanganate are then added, 1.5h is stirred under the conditions of ice-water bath, is then heated to 55 DEG C, instead
Answer 5h；After being slowly added to 100 mL ultra-pure waters, the hydrogen peroxide of 10 mL, mass fraction 30% is added, stirs 30 min, centrifugation point
From supernatant is graphene oxide suspension；
（3）Au@Pt DNs/NG/Cu2+Preparation
The NG for weighing the above-mentioned preparations of 10 mg is dispersed in the Au@Pt DNs dispersion liquids of 10 ~ 20 mL, 1 mg/mL, ultrasonic 2h,
It centrifuges, is dried at 40 DEG C and obtain Au@Pt DNs/NG；The Au@Pt DNs/NG of 10 mg are taken to be dispersed in 10 mL ultra-pure waters,
1 ~ 3 mmol/L copper chlorides are added, is stirred at room temperature for 24 hours, centrifuges, dried at 40 DEG C, Au@Pt DNs/NG/ are made
Cu2+；
（4）Au@Pt DNs/NG/Cu2+-Ab2Preparation
Weigh the Au@Pt DNs/NG/Cu of 3 ~ 7 mg2+It is distributed in 1 mL ultra-pure waters, 100 μ L, 80 ~ 120 μ g/ is added
The detection antibody incubation content Au@Pt DNs/NG/Cu of mL2+-Ab2The phosphoric acid of pH=6.8 of solution and 900 μ L, 50 mmol/L
Salt buffer solution, oscillation hatching 12h, centrifuges in 4 DEG C of constant-temperature shaking incubators, is scattered in 1 mL, 50 mmol/L again
Au@Pt DNs/NG/Cu are made in the phosphate buffer solution of pH=6.82+Detect antibody A b2Incubation content solution preserves at 4 DEG C
It is spare.
4. the detection of lung cancer marker, steps are as follows：
（1）It is tested with three-electrode system using electrochemical workstation, saturated calomel electrode is reference electrode, and platinum electrode is
Auxiliary electrode, prepared sensor are working electrode, are carried out in the pH of 10 mL is 5.8 ~ 8.0 phosphate buffer solutions
Test；
（2）Analyte is detected with chronoamperometry, input current potential is -0.4 V, and sampling interval is 0.1 s, when operation
Between be 400 s；
（3）After background current tends towards stability, 10 μ are injected in 6.8 phosphate buffer solutions every 50 s to the pH of 10 mL
L, the hydrogen peroxide of 5 mol/L records the curent change corresponding to the lung cancer marker antigen under various concentration；
（4）Using working curve method, the concentration of lung cancer marker antigen in sample to be tested is obtained.
Above-mentioned tumor markers are selected from one of following：CEA、CA125、SCCA.
Raw materials of the present invention can be bought in chemical reagents corporation or biopharmaceutical company.
The useful achievement of the present invention
（1）Using the gold nanoparticle of poly-dopamine functionalization as base material, there is unique electric conductivity and adhesiveness, it can
To combine more multispecific antibody.Au@Pt DNs are dendritic crystalline nanocomposite, have good water-soluble and catalytic performance, and deposit
In more binding sites, can effectively adsorb and immobilized antibody.NG has excellent electric conductivity, big specific surface area, Ke Yijie
More Au@Pt DNs are closed, Adsorption of Cu is passed through2+, since synergistic effect can further increase the catalytic performance of material, realize pair
Signal amplification is measured, the sensitivity of tumor-marker analyte detection is improved, reduces detection limit；
（2）A kind of interlayer type lung cancer marker electrochemical sensor, realizes the Sensitive Detection to lung cancer marker, wherein right
The measurement of CEA, the range of linearity are 0.0005 ~ 50 ng/mL, and detection is limited to 0.167pg/mL；It is linear to the measurement of CA125
Ranging from 0.0001 ~ 50 ng/mL, detection are limited to 0.033pg/mL；To the measurement of SCCA, the range of linearity is 0.0005 ~
100 ng/mL, detection are limited to 0.167pg/mL, show that a kind of interlayer type lung cancer marker electrochemical sensor can reach accurate
The purpose of measurement.
Specific implementation mode
Now the present invention is further illustrated by specific implementation mode, but not limited to this.
A kind of preparation method of 1 interlayer type lung cancer marker electrochemical sensor of embodiment, steps are as follows：
（1）By the glass-carbon electrode Al of a diameter of 4 mm2O3Polishing powder is polished, and ultra-pure water cleans up；
（2）Take the golden nanometer particle dispersion liquid drop coating of the poly-dopamine functionalization of 6 μ L, 1.0 mg/mL to electrode surface, room temperature
Under dry, ultrapure water electrode surface dries；
（3）By the lung cancer marker capture antibody A b of 6 μ L, 8 μ g/mL1It is added drop-wise to electrode surface, ultrapure water, 4 DEG C of refrigerators
Middle drying；
（4）Continue the bovine serum albumin(BSA) BSA solution of 3 μ L, 0.5 mg/mL being added drop-wise to electrode surface, ultrapure water electrode
Surface is dried in 4 DEG C of refrigerators；
（5）A series of lung cancer marker antigen A g solution of various concentrations of 6 μ L, 0.0001 ~ 100 ng/mL is added dropwise, it is ultrapure
Water rinses electrode surface, is dried in 4 DEG C of refrigerators；
（6）By 6 μ L, 1.5 mg/mL detection antibody incubation content Au@Pt DNs/NG/Cu2+-Ab2Solution drops to electrode surface, sets
Hatch 40 min in 4 DEG C of refrigerators, ultrapure water electrode surface dries in 4 DEG C of refrigerators, a kind of interlayer type lung cancer mark is made
Object electrochemical sensor.
A kind of preparation method of 2 interlayer type lung cancer marker electrochemical sensor of embodiment, steps are as follows：
（1）By the glass-carbon electrode Al of a diameter of 4 mm2O3Polishing powder is polished, and ultra-pure water cleans up；
（2）Take the golden nanometer particle dispersion liquid drop coating of the poly-dopamine functionalization of 6 μ L, 2.0 mg/mL to electrode surface, room temperature
Under dry, ultrapure water electrode surface dries；
（3）By the lung cancer marker capture antibody A b of 6 μ L, 10 μ g/mL1It is added drop-wise to electrode surface, ultrapure water, 4 DEG C of ice
It is dry in case；
（4）Continue the bovine serum albumin(BSA) BSA solution of 3 μ L, 1.0 mg/mL being added drop-wise to electrode surface, ultrapure water electrode
Surface is dried in 4 DEG C of refrigerators；
（5）A series of lung cancer marker antigen A g solution of various concentrations of 6 μ L, 0.0001 ~ 100 ng/mL is added dropwise, it is ultrapure
Water rinses electrode surface, is dried in 4 DEG C of refrigerators；
（6）By 6 μ L, 2.5 mg/mL detection antibody incubation content Au@Pt DNs/NG/Cu2+-Ab2Solution drops to electrode surface, sets
Hatch 40 min in 4 DEG C of refrigerators, ultrapure water electrode surface dries in 4 DEG C of refrigerators, a kind of interlayer type lung cancer mark is made
Object electrochemical sensor.
A kind of preparation method of 3 interlayer type lung cancer marker electrochemical sensor of embodiment, steps are as follows：
（1）By the glass-carbon electrode Al of a diameter of 4 mm2O3Polishing powder is polished, and ultra-pure water cleans up；
（2）Take the golden nanometer particle dispersion liquid drop coating of the poly-dopamine functionalization of 6 μ L, 3.0 mg/mL to electrode surface, room temperature
Under dry, ultrapure water electrode surface dries；
（3）By the lung cancer marker capture antibody A b of 6 μ L, 12 μ g/mL1It is added drop-wise to electrode surface, ultrapure water, 4 DEG C of ice
It is dry in case；
（4）Continue the bovine serum albumin(BSA) BSA solution of 3 μ L, 1.5 mg/mL being added drop-wise to electrode surface, ultrapure water electrode
Surface is dried in 4 DEG C of refrigerators；
（5）A series of lung cancer marker antigen A g solution of various concentrations of 6 μ L, 0.0001 ~ 100 ng/mL is added dropwise, it is ultrapure
Water rinses electrode surface, is dried in 4 DEG C of refrigerators；
（6）By 6 μ L, 3.5 mg/mL detection antibody incubation content Au@Pt DNs/NG/Cu2+-Ab2Solution drops to electrode surface, sets
Hatch 40 min in 4 DEG C of refrigerators, ultrapure water electrode surface dries in 4 DEG C of refrigerators, a kind of interlayer type lung cancer mark is made
Object electrochemical sensor.
The preparation of the gold nanoparticle of poly-dopamine functionalization described in embodiment 4, steps are as follows：
（1）The preparation of solution of gold nanoparticles
1 mL, the gold chloride that mass fraction is 1% are added in 99 mL ultra-pure waters, 1.5 mL, quality is added after being heated to boiling
The sodium citrate solution that score is 1%, flow back 10 min, is cooled to room temperature, obtains the solution of gold nanoparticles of claret；
（2）The preparation of the gold nanoparticle of poly-dopamine functionalization
It takes 10 mg dopamines to be added in the Tris-HCl buffer solutions of pH=8.5 of 10 mL, 10 mmol/L, 10 mL is added
The solution of gold nanoparticles of above-mentioned preparation vibrates the gold nanoparticle for obtaining poly-dopamine functionalization for 24 hours at room temperature.
The preparation of the gold nanoparticle of poly-dopamine functionalization described in embodiment 5, steps are as follows：
（1）The preparation of solution of gold nanoparticles
1.5 mL, the gold chloride that mass fraction is 1% are added in 99 mL ultra-pure waters, 2.5 mL, matter is added after being heated to boiling
The sodium citrate solution that score is 1% is measured, flow back 20 min, is cooled to room temperature, obtains the solution of gold nanoparticles of claret；
（2）The preparation of the gold nanoparticle of poly-dopamine functionalization
It takes 15 mg dopamines to be added in the Tris-HCl buffer solutions of pH=8.5 of 10 mL, 10 mmol/L, 10 mL is added
The solution of gold nanoparticles of above-mentioned preparation vibrates the gold nanoparticle for obtaining poly-dopamine functionalization for 24 hours at room temperature.
The preparation of the gold nanoparticle of poly-dopamine functionalization described in embodiment 6, steps are as follows：
（1）The preparation of solution of gold nanoparticles
2 mL, the gold chloride that mass fraction is 1% are added in 99 mL ultra-pure waters, 3.5 mL, quality is added after being heated to boiling
The sodium citrate solution that score is 1%, flow back 30 min, is cooled to room temperature, obtains the solution of gold nanoparticles of claret；
（2）The preparation of the gold nanoparticle of poly-dopamine functionalization
It takes 20 mg dopamines to be added in the Tris-HCl buffer solutions of pH=8.5 of 10 mL, 10 mmol/L, 10 mL is added
The solution of gold nanoparticles of above-mentioned preparation vibrates the gold nanoparticle for obtaining poly-dopamine functionalization for 24 hours at room temperature.
Au@Pt DNs/NG/Cu described in embodiment 72+Preparation, steps are as follows：
（1）The preparation of Au@Pt DNs
It is water-soluble containing 0.25 mmol/L gold chlorides and 75 mmol/L cetyl trimethylammonium bromides that 10 mL are prepared in flask
Liquid is vigorously stirred down and rapidly joins 0.6 mL, 10 mmol/L sodium borohydrides, and Jinsui River colloidal sol is made after being slowly stirred 3h at room temperature；
It measures Jinsui River colloidal sol ultra-pure water made from 1 mL and is diluted to 100 mL, gold seed-solution is made.
By 0.1 mL, 10 mmol/L gold chlorides, 2 mL, 0.2 mol/L cetyl trimethylammonium bromides and 1.5mL,
The aqueous solution of 0.1 mol/L ascorbic acid mixes, and 0.3 mL gold seed-solutions is added in mixed liquor, ultra-pure water is diluted to
25mL vibrates 8h at room temperature, and the solution of cube gold nanoparticle is made；
1mL, 0.1 mol/L ascorbic acid and 1 mL, 10 mmol/L chlorine Asias are sequentially added in the solution of cube gold nanoparticle
Potassium platinate reacts 12h in 60 DEG C of water-bath,；Centrifugation, milli-Q water 3 times are dried in 40 DEG C of vacuum drying chambers, are made
Au@Pt DNs；It weighs and DNs points of Au@Pt is made in the Au@Pt DNs ultrasonic disperses to the ultra-pure water of certain volume of certain mass
Dispersion liquid uses；
（2）The preparation of nitrogen-doped graphene NG
It weighs 20mg propionaldoximes to be placed in 500 mL three-necked flasks, sequentially adds 200 mL concentrated ammonia liquors and 15 mL graphene oxides
After ultrasonic disperse is uniform, three-necked flask is transferred in oil bath pan for suspension, reacts 4h for 90 DEG C under agitation, naturally cold
But it to room temperature, centrifuges, with milli-Q water to filtrate at neutrality, NG is made in freeze-drying；
The suspended liquid and preparation method thereof of graphene oxide is as follows：2.0 g graphite powders are weighed, the 96 mL concentrated sulfuric acids are added, in stirring bar
20 min are reacted under part, and 12 g potassium permanganate are then added, 1.5h is stirred under the conditions of ice-water bath, is then heated to 55 DEG C, instead
Answer 5h；After being slowly added to 100 mL ultra-pure waters, the hydrogen peroxide of 10 mL, mass fraction 30% is added, stirs 30 min, centrifugation point
From supernatant is graphene oxide suspension；
（3）Au@Pt DNs/NG/Cu2+Preparation
The NG for weighing the above-mentioned preparations of 10 mg is dispersed in the Au@Pt DNs dispersion liquids of 10 mL, 1 mg/mL, ultrasonic 2h, centrifugation
It detaches, is dried at 40 DEG C and obtain Au@Pt DNs/NG.It takes the Au@Pt DNs/NG of 10 mg to be dispersed in 10 mL ultra-pure waters, is added
1 mmol/L copper chlorides are stirred at room temperature for 24 hours, centrifuge, dried at 40 DEG C, Au@Pt DNs/NG/Cu are made2+；
（4）Au@Pt DNs/NG/Cu2+-Ab2Preparation
Weigh the Au@Pt DNs/NG/Cu of 3 mg2+It is distributed in 1 mL ultra-pure waters, 100 μ L is added, the detection of 80 μ g/mL resists
Body incubation content Au@Pt DNs/NG/Cu2+-Ab2The phosphate buffer solution of pH=6.8 of solution and 900 μ L, 50 mmol/L,
Oscillation hatching 12h, centrifuges, is scattered in pH=6.8 of 1 mL, 50 mmol/L again in 4 DEG C of constant-temperature shaking incubators
Au@Pt DNs/NG/Cu are made in phosphate buffer solution2+Detect antibody A b2Incubation content solution saves backup at 4 DEG C.
Au@Pt DNs/NG/Cu described in embodiment 82+Preparation, steps are as follows：
（1）The preparation of Au@Pt DNs
It is water-soluble containing 0.25 mmol/L gold chlorides and 75 mmol/L cetyl trimethylammonium bromides that 10 mL are prepared in flask
Liquid is vigorously stirred down and rapidly joins 0.6 mL, 10 mmol/L sodium borohydrides, and Jinsui River colloidal sol is made after being slowly stirred 3h at room temperature；
It measures Jinsui River colloidal sol ultra-pure water made from 1 mL and is diluted to 100 mL, gold seed-solution is made.
By 0.3 mL, 10 mmol/L gold chlorides, 2.5 mL, 0.2 mol/L cetyl trimethylammonium bromides and 1.8mL,
The aqueous solution of 0.1 mol/L ascorbic acid mixes, and 0.4 mL gold seed-solutions is added in mixed liquor, ultra-pure water is diluted to
25mL vibrates 8h at room temperature, and the solution of cube gold nanoparticle is made；It is sequentially added in the solution of cube gold nanoparticle
2 mL, 0.1 mol/L ascorbic acid and 1 mL, 10 mmol/L potassium chloroplatinites, react 12h in 60 DEG C of water-bath,；Centrifugation,
Milli-Q water 3 times, is dried in 40 DEG C of vacuum drying chambers, and Au@Pt DNs are made；The Au@Pt DNs for weighing certain mass are super
Sound, which is distributed in the ultra-pure water of certain volume, is made the use of Au@Pt DNs dispersion liquids；
（2）The preparation of nitrogen-doped graphene NG
It weighs 30 mg propionaldoximes to be placed in 500 mL three-necked flasks, sequentially adds 200 mL concentrated ammonia liquors and 15 mL graphene oxides
After ultrasonic disperse is uniform, three-necked flask is transferred in oil bath pan for suspension, reacts 4h for 90 DEG C under agitation, naturally cold
But it to room temperature, centrifuges, with milli-Q water to filtrate at neutrality, NG is made in freeze-drying；
The suspended liquid and preparation method thereof of graphene oxide is the same as embodiment 7.
（3）Au@Pt DNs/NG/Cu2+Preparation
The NG for weighing the above-mentioned preparations of 10 mg is dispersed in the Au@Pt DNs dispersion liquids of 15 mL, 1 mg/mL, ultrasonic 2h, centrifugation
It detaches, is dried at 40 DEG C and obtain Au@Pt DNs/NG；It takes the Au@Pt DNs/NG of 10 mg to be dispersed in 10 mL ultra-pure waters, is added
1.5 mmol/L copper chlorides are stirred at room temperature for 24 hours, centrifuge, dried at 40 DEG C, Au@Pt DNs/NG/Cu are made2+；
（4）Au@Pt DNs/NG/Cu2+-Ab2Preparation
Weigh the Au@Pt DNs/NG/Cu of 5 mg2+It is distributed in 1 mL ultra-pure waters, the detection of 100 μ L, 100 μ g/mL is added
Antibody incubation content Au@Pt DNs/NG/Cu2+-Ab2The phosphate-buffered of pH=6.8 of solution and 900 μ L, 50 mmol/L is molten
Liquid, oscillation hatching 12h, centrifuges, is scattered in pH=6.8 of 1 mL, 50 mmol/L again in 4 DEG C of constant-temperature shaking incubators
Phosphate buffer solution, Au@Pt DNs/NG/Cu are made2+Detect antibody A b2Incubation content solution saves backup at 4 DEG C.
Au@Pt DNs/NG/Cu described in embodiment 92+Preparation, steps are as follows：
（1）The preparation of Au@Pt DNs
It is water-soluble containing 0.25 mmol/L gold chlorides and 75 mmol/L cetyl trimethylammonium bromides that 10 mL are prepared in flask
Liquid is vigorously stirred down and rapidly joins 0.6 mL, 10 mmol/L sodium borohydrides, and Jinsui River colloidal sol is made after being slowly stirred 3h at room temperature；
It measures Jinsui River colloidal sol ultra-pure water made from 1 mL and is diluted to 100 mL, gold seed-solution is made.
By 0.5 mL, 10 mmol/L gold chlorides, 3 mL, 0.2 mol/L cetyl trimethylammonium bromides and 2.0mL,
The aqueous solution of 0.1 mol/L ascorbic acid mixes, and 0.5 mL gold seed-solutions is added in mixed liquor, ultra-pure water is diluted to
25mL vibrates 8h at room temperature, and the solution of cube gold nanoparticle is made；It is sequentially added in the solution of cube gold nanoparticle
3 mL, 0.1 mol/L ascorbic acid and 1 mL, 10 mmol/L potassium chloroplatinites, react 12h in 60 DEG C of water-bath,；Centrifugation,
Milli-Q water 3 times, is dried in 40 DEG C of vacuum drying chambers, and Au@Pt DNs are made；The Au@Pt DNs for weighing certain mass are super
Sound, which is distributed in the ultra-pure water of certain volume, is made the use of Au@Pt DNs dispersion liquids；
（2）The preparation of nitrogen-doped graphene NG
It weighs 40 mg propionaldoximes to be placed in 500 mL three-necked flasks, sequentially adds 200 mL concentrated ammonia liquors and 15 mL graphene oxides
After ultrasonic disperse is uniform, three-necked flask is transferred in oil bath pan for suspension, reacts 4h for 90 DEG C under agitation, naturally cold
But it to room temperature, centrifuges, with milli-Q water to filtrate at neutrality, NG is made in freeze-drying；
The suspended liquid and preparation method thereof of graphene oxide is the same as embodiment 7；
（3）Au@Pt DNs/NG/Cu2+Preparation
The NG for weighing the above-mentioned preparations of 10 mg is dispersed in the Au@Pt DNs dispersion liquids of 20 mL, 1 mg/mL, ultrasonic 2h, centrifugation
It detaches, is dried at 40 DEG C and obtain Au@Pt DNs/NG；It takes the Au@Pt DNs/NG of 10 mg to be dispersed in 10 mL ultra-pure waters, is added
3 mmol/L copper chlorides are stirred at room temperature for 24 hours, centrifuge, dried at 40 DEG C, Au@Pt DNs/NG/Cu are made2+；
（4）Au@Pt DNs/NG/Cu2+-Ab2Preparation
Weigh the Au@Pt DNs/NG/Cu of 7 mg2+It is distributed in 1 mL ultra-pure waters, the detection of 100 μ L, 120 μ g/mL is added
Antibody incubation content Au@Pt DNs/NG/Cu2+-Ab2The phosphate-buffered of pH=6.8 of solution and 900 μ L, 50 mmol/L is molten
Liquid, oscillation hatching 12h, centrifuges, is scattered in pH=6.8 of 1 mL, 50 mmol/L again in 4 DEG C of constant-temperature shaking incubators
Phosphate buffer solution, Au@Pt DNs/NG/Cu are made2+Detect antibody A b2Incubation content solution saves backup at 4 DEG C.
The detection of 10 lung cancer marker CEA of embodiment, steps are as follows：
（1）It is tested with three-electrode system using electrochemical workstation, saturated calomel electrode is reference electrode, and platinum electrode is
Auxiliary electrode, prepared sensor are working electrode, are surveyed in the pH of 10mL is 5.8 ~ 8.0 phosphate buffer solutions
Examination；
（2）Analyte is detected with chronoamperometry, input current potential is -0.4 V, and sampling interval is 0.1 s, run time
For 400 s；
（3）After background current tends towards stability, 10 μ are injected in 6.8 phosphate buffer solutions every 50 s to the pH of 10 mL
L, the hydrogen peroxide of 5 M records the curent change corresponding to the tumor markers antigen under various concentration；
（4）Using working curve method, the concentration of tumor markers antigen in sample to be tested is obtained.
（5）According to the linear relationship between gained current strength and CEA concentration, drawing curve measures the range of linearity
For 0.0005 ~ 50 ng/mL, detection is limited to 0.167pg/mL.
The detection of 11 lung cancer marker CA125 of embodiment
CA125 in sample is detected according to the method for embodiment 10, the range of linearity is 0.0001 ~ 50 ng/mL, inspection
Survey is limited to 0.033pg/mL.
The detection of 12 lung cancer marker SCCA of embodiment
SCCA in sample is detected according to the method for embodiment 10,0.0005 ~ 100 ng/mL of the range of linearity, detection limit
For 0.167pg/mL.
Claims (5)
1. a kind of preparation method of interlayer type lung cancer marker electrochemical sensor, which is characterized in that steps are as follows：
（1）By the glass-carbon electrode Al of a diameter of 4 mm2O3Polishing powder is polished, and ultra-pure water cleans up；
（2）Take the golden nanometer particle dispersion liquid drop coating of the poly-dopamine functionalization of 6 μ L, 1.0 ~ 3.0 mg/mL to electrode table
Face is dried at room temperature, and ultrapure water electrode surface dries；
（3）By the lung cancer marker capture antibody A b of 6 μ L, 8 ~ 12 μ g/mL1It is added drop-wise to electrode surface, ultrapure water, 4
It is dry in DEG C refrigerator；
（4）Continue the bovine serum albumin(BSA) BSA solution of 3 μ L, 0.5 ~ 1.5 mg/mL being added drop-wise to electrode surface, ultra-pure water punching
Electrode surface is washed, is dried in 4 DEG C of refrigerators；
（5）A series of lung cancer marker antigen A g solution of various concentrations of 6 μ L, 0.0001 ~ 100 ng/mL is added dropwise, it is ultrapure
Water rinses electrode surface, is dried in 4 DEG C of refrigerators；
（6）By 6 μ L, 1.5 ~ 3.5 mg/mL detection antibody incubation content Au@Pt DNs/NG/Cu2+-Ab2Solution drops to electrode table
Face is placed in 4 DEG C of refrigerators and hatches 40 min, ultrapure water electrode surface, is dried in 4 DEG C of refrigerators, and a kind of interlayer type lung is made
Carcinoma marker electrochemical sensor.
2. a kind of preparation method of interlayer type lung cancer marker electrochemical sensor as described in claim 1, the poly- DOPA
The preparation of the gold nanoparticle of amino-functionalization, steps are as follows：
（1）The preparation of solution of gold nanoparticles
Will 1 ~ 2 mL, mass fraction be 1% gold chloride be added 99 mL ultra-pure waters in, be heated to boiling after be added 1.5 ~
3.5 mL, the sodium citrate solution that mass fraction is 1%, flow back 10 ~ 30 min, is cooled to room temperature, obtains the Jenner of claret
Rice corpuscles solution；
（2）The preparation of the gold nanoparticle of poly-dopamine functionalization
It takes 10 ~ 20 mg dopamines to be added in the Tris-HCl buffer solutions of pH=8.5 of 10 mL, 10 mmol/L, is added
The solution of gold nanoparticles of the above-mentioned preparations of 10 mL, vibrates the gold nanoparticle for obtaining poly-dopamine functionalization for 24 hours at room temperature.
3. a kind of preparation method of interlayer type lung cancer marker electrochemical sensor as described in claim 1, the Au@Pt
DNs/NG/Cu2+Preparation, steps are as follows：
（1）The preparation of Au@Pt DNs
It is water-soluble containing 0.25 mmol/L gold chlorides and 75 mmol/L cetyl trimethylammonium bromides that 10 mL are prepared in flask
Liquid is vigorously stirred down and rapidly joins 0.6 mL, 10 mmol/L sodium borohydrides, and Jinsui River colloidal sol is made after being slowly stirred 3h at room temperature；
It measures Jinsui River colloidal sol ultra-pure water made from 1 mL and is diluted to 100 mL, gold seed-solution is made；
By 0.1 ~ 0.5 mL, 10 mmol/L gold chlorides, 2 ~ 3 mL, 0.2 mol/L cetyl trimethylammonium bromides and 1.5 ~
2.0mL, the mixing of the aqueous solution of 0.1 mol/L ascorbic acid, 0.3 ~ 0.5 mL gold seed-solutions are added in mixed liquor, it is ultrapure
Water is diluted to 25mL, vibrates 8h at room temperature, and the solution of cube gold nanoparticle is made；In the solution of cube gold nanoparticle
1 ~ 3 mL, 0.1 mol/L ascorbic acid and 1 mL, 10 mmol/L potassium chloroplatinites are sequentially added, in 60 DEG C of water-bath
12h is reacted,；Centrifugation, milli-Q water 3 times are dried in 40 DEG C of vacuum drying chambers, and Au@Pt DNs are made；Weigh certain matter
The use of Au@Pt DNs dispersion liquids is made in the Au@Pt DNs ultrasonic disperses to the ultra-pure water of certain volume of amount；
（2）The preparation of nitrogen-doped graphene NG
It weighs 20 ~ 40 mg propionaldoximes to be placed in 500 mL three-necked flasks, sequentially adds 200 mL concentrated ammonia liquors and 15 mL oxidations
After ultrasonic disperse is uniform, three-necked flask is transferred in oil bath pan for graphene suspension, under agitation 90 DEG C of reaction 4h,
Cooled to room temperature centrifuges, and with milli-Q water to filtrate at neutrality, NG is made in freeze-drying；
The suspended liquid and preparation method thereof of graphene oxide is as follows：2.0 g graphite powders are weighed, the 96 mL concentrated sulfuric acids are added, in stirring bar
20 min are reacted under part, and 12 g potassium permanganate are then added, 1.5h is stirred under the conditions of ice-water bath, is then heated to 55 DEG C, instead
Answer 5h；After being slowly added to 100 mL ultra-pure waters, the hydrogen peroxide of 10 mL, mass fraction 30% is added, stirs 30 min, centrifugation point
From supernatant is graphene oxide suspension；
（3）Au@Pt DNs/NG/Cu2+Preparation
The NG for weighing the above-mentioned preparations of 10 mg is dispersed in the Au@Pt DNs dispersion liquids of 10 ~ 20 mL, 1 mg/mL, ultrasonic 2h,
It centrifuges, is dried at 40 DEG C and obtain Au@Pt DNs/NG；The Au@Pt DNs/NG of 10 mg are taken to be dispersed in 10 mL ultra-pure waters
In, 1 ~ 3 mmol/L copper chlorides are added, is stirred at room temperature for 24 hours, centrifuges, dried at 40 DEG C, Au@Pt DNs/ are made
NG/Cu2+；
（4）Au@Pt DNs/NG/Cu2+-Ab2Preparation
Weigh the Au@Pt DNs/NG/Cu of 3 ~ 7 mg2+It is distributed in 1 mL ultra-pure waters, 100 μ L, 80 ~ 120 μ g/ is added
The detection antibody incubation content Au@Pt DNs/NG/Cu of mL2+-Ab2The phosphoric acid of pH=6.8 of solution and 900 μ L, 50 mmol/L
Salt buffer solution, oscillation hatching 12h, centrifuges in 4 DEG C of constant-temperature shaking incubators, is scattered in 1 mL, 50 mmol/L again
Au@Pt DNs/NG/Cu are made in the phosphate buffer solution of pH=6.82+Detect antibody A b2Incubation content solution preserves at 4 DEG C
It is spare.
4. a kind of interlayer type lung cancer marker electrochemical sensor prepared by preparation method as described in claim 1 is used for lung
The detection of carcinoma marker, steps are as follows：
（1）It is tested with three-electrode system using electrochemical workstation, saturated calomel electrode is reference electrode, and platinum electrode is
Auxiliary electrode, prepared sensor are working electrode, are carried out in the pH of 10 mL is 5.8 ~ 8.0 phosphate buffer solutions
Test；
（2）Analyte is detected with chronoamperometry, input current potential is -0.4 V, and sampling interval is 0.1 s, when operation
Between be 400 s；
（3）After background current tends towards stability, 10 μ are injected in 6.8 phosphate buffer solutions every 50 s to the pH of 10 mL
L, the hydrogen peroxide of 5 mol/L records the curent change corresponding to the lung cancer marker antigen under various concentration；
（4）Using working curve method, the concentration of lung cancer marker antigen in sample to be tested is obtained.
5. the lung cancer marker as described in claim 1,2,3,4, which is characterized in that the lung cancer marker be selected from it is following it
One：CEA、CA125、SCCA.
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| CN109709188A (en) * | 2019-02-27 | 2019-05-03 | 山东理工大学 | A kind of preparation method and application of the interlayer type immunosensor of nitrogen sulphur codope graphene oxide label |
| CN112858420A (en) * | 2021-03-30 | 2021-05-28 | 济南大学 | Preparation method and application of sandwich type electrochemical sensor constructed based on vanadium selenide/gold nanoparticles |
| CN113325179A (en) * | 2021-04-14 | 2021-08-31 | 中国农业科学院烟草研究所(中国烟草总公司青州烟草研究所) | Immunochromatographic test strip based on Au @ Pt enzyme and preparation method thereof |
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