CN105928997A - Preparation method and application of immunosensor based on Au-GQD@PtPd - Google Patents
Preparation method and application of immunosensor based on Au-GQD@PtPd Download PDFInfo
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- CN105928997A CN105928997A CN201610539821.1A CN201610539821A CN105928997A CN 105928997 A CN105928997 A CN 105928997A CN 201610539821 A CN201610539821 A CN 201610539821A CN 105928997 A CN105928997 A CN 105928997A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/308—Electrodes, e.g. test electrodes; Half-cells at least partially made of carbon
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
- G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
- G01N27/3278—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction involving nanosized elements, e.g. nanogaps or nanoparticles
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57473—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving carcinoembryonic antigen, i.e. CEA
Abstract
The invention belongs to the technical field of nano functional materials, immunoassay and biosensing, and provides a preparation method and application of an immunosensor based on Au-GQD@PtPd. A sandwich type electrochemical immunosensor for carcino-embryonic antigens is prepared by taking Au-GQD@PtPd as a detection antibody marker; due to excellent biocompatibility and high catalysis property of Au-GQD@PtPd, the prepared sensor has the advantages of high specificity, high sensitivity, low detection limit and the like.
Description
Technical field
The present invention relates to the preparation method and application of a kind of immunosensor based on Au-GQD@PtPd.Specifically use Au-GQD@PtPd as detection antibody labeling thing, preparation one sandwich type electrochemical immunosensor, belong to new function material, immunoassay and bio-sensing detection technique field.
Background technology
The mankind are perplexed by numerous diseases, and such as pulmonary carcinoma, ovarian cancer, breast carcinoma and cystadenocarcinoma, sickness rate is high, grow and transfer velocity is fast, and the health of the mankind is had harm greatly.Carcinoembryonic antigen is originally found in colon cancer and fetal gut tissue, the rising of the content of carcinoembryonic antigen in human serum, shows that human body is in the state of a kind of non-health.The meaning of carcinoembryonic antigen is that it can reflect the existence of kinds of tumors, can be used for the Outcome measure to colorectal cancer, breast carcinoma and pulmonary carcinoma, PD, monitoring and prognosis and estimates.Carcinoembryonic antigen raises and is common in colorectal cancer, cancer of pancreas, gastric cancer, breast carcinoma, medullary thyroid carcinoma etc..Therefore, in clinical research, the method developing quick, easy, sensitive detection carcinoembryonic antigen is particularly significant.
The most existing detection method for carcinoembryonic antigen is a lot, radioimmunology, enzyme-linked immune analytic method, fluorescence immune analysis method and Electrogenerated chemiluminescent immunoassay method etc. are mainly included as being usually used in detecting the immune analysis method of carcinoembryonic antigen at present, but most detection methods are loaded down with trivial details, operation complexity, somewhat expensive, detection limit for height, therefore, sets up a kind of quick, easy, sensitive detection method significant.
Immunosensor is a kind of biosensor combined with analytical chemistry method by immunological method, specific binding by between antigen and antibody so that it have highly sensitive, selectivity good, simple in construction, easy and simple to handle, be prone to miniaturization, can the advantage such as detection analysis continuous, rapid automatized.And the key building electrochemical immunosensor has 2 points: the first uses simply, fast and effectively method the biomolecule such as antigen-antibody are fixed on electrode surface；Its two be exploitation sensor signal amplification technique.
Deposited Au has good electric conductivity, its excellent biocompatibility enables good immobilized antibody, ordered mesopore carbon can be good at immobilized golden nanometer particle and chromium ion with its big specific surface area, it is allowed to that there is good catalytic performance, can speed up electron transmission, for improving transducer sensitivity, there is important function.
Therefore the present invention is prepared for a kind of applying deposited Au as base material and the immunosensor with Au-GQD@PtPd as label, it is achieved that the super sensitivity detection to carcinoembryonic antigen.
Summary of the invention
An object of the present invention is as detection antibody labeling thing based on Au-GQD@PtPd, constructs the sandwich type electrochemical immunosensor of a kind of fast super sensitivity.
The two of the purpose of the present invention are the detections that this sandwich type electrochemical immunosensor is applied to carcinoembryonic antigen.
Technical scheme is as follows:
1. the preparation method of an immunosensor based on Au-GQD PtPd, it is characterised in that step is as follows:
(1) by a diameter of 3 ~ 5
The glass-carbon electrode Al of mm2O3Polishing powder is polished, and ultra-pure water cleans up；
(2) by HAuCl4
(1 wt%) is electrodeposited into electrode surface with chronoamperometry from-0.2V ~ 0.2V, dries, and with ultrapure water, dries；
(3) drop coating 6 L, 8 ~
The anti-Ab of tumor markers one of 12 g/mL1Solution in electrode surface, 4oC refrigerator is dried；
(4) ultrapure water falls unconjugated capture antibody A b1After, drip 3 L, 0.5 ~ 1.5 mg/mL bovine serum albumin BSA solution in electrode surface, 4oC refrigerator dries；
(5) after ultrapure water falls unconjugated BSA, drip 6 L, 1 × 10-6
The carcinoembryonic antigen solution of a series of variable concentrations of ~ 10 ng/mL, incubation at room temperature 1h, ultra-pure water cleans, is placed in 4oC refrigerator is dried；
(6) the detection antibody of 6 L, 1.5 ~ 5.0 mg/mL is hatched thing Au-GQD@PtPd-Ab2Solution drop coating is on electrode surface, and incubation at room temperature 1h, ultra-pure water cleans up, and is placed in 4oC refrigerator dries, prepares a kind of carcinoembryonic antigen sensor based on Au-GQD@PtPd.
A kind of preparation method of immunosensor based on Au-GQD@PtPd, described described Au-GQD@PtPd
-Ab2The two anti-preparations incubating compound solution, it is characterised in that preparation process is as follows:
(1) preparation of graphene quantum dot
First by 2.0 g citric acids and 1.0 g
Dicyandiamide joins in 5mL ultra-pure water, is heated to 180 DEG C and lasting 12 h in mixture is transferred to 25 mL autoclaves afterwards；Product is dispersed in 100 mL ultra-pure waters, in centrifuge, is centrifuged 10 min with the rotating speed of 10000 r/min, take supernatant ambient temperature in vacuum and be dried 12 h, prepare graphene quantum dot；
(2) preparation of GQD@PtPd
By 0.158 mL chloroplatinic acid (20mmol/L), 0.233 mL chlorine palladium acid (20mmol/L) and 20mg graphene quantum dot join in 15.0 mL ultra-pure waters under magnetic stirring, pH to 10 is regulated with NaOH, subsequently mixed solution is transferred in autoclave, at 160 DEG C, reacts 6h, centrifugation, and with milli-Q water, until supernatant is colourless, ambient temperature in vacuum is dried 12 h, prepares black powder GQD@PtPd；
(3) preparation of golden nanometer particle
1.0 mL gold chlorides (1 wt%) are joined in 99 mL ultra-pure waters, adds the sodium citrate (1 wt%) of 2.5 mL while stirring, 100oBackflow 15 under C
Min, centrifugation, discard precipitation, the supernatant of finely dispersed claret is golden nanometer particle seed solution；Take 1 mL golden nanometer particle seed solution and bis-water of 25 mL and mix homogeneously, add 100 μ L, the hydroxylamine hydrochloride solution of 0.2 mol/L, at room temperature high-speed stirred mixing, then 3.0 mL gold chlorides (1 wt%) solution it is added dropwise over, addition along with gold chloride, the color of solution is gradually converted into peony, and prepared particle diameter is the solution of gold nanoparticles of 50 ± 5 nm；
(4) preparation of Au-GQD@PtPd
First 1.0 mL solution of gold nanoparticles and 2.0 mL ethylenediamines are dispersed in 5.0 mL ultra-pure waters, ultrasonic 1h, then magnetic agitation 5h, centrifugation, and with milli-Q water, abandon supernatant, obtain the golden nanometer particle of amino functional, the most accurately add 4 ~ 6 mg GQD@PtPd, and add 1.0 mL ultra-pure waters, room temperature reaction 12h, centrifugation, and with milli-Q water, until supernatant is colourless, 35oC is vacuum dried, and prepares Au-GQD@PtPd；
(5) detection antibody hatching thing Au-GQD@PtPd-Ab2The preparation of solution
The anti-label of Au-GQD@PtPd bis-of 1.5 ~ 5 mg is distributed in 1 mL ultra-pure water, adds the anti-Ab of tumor markers two of 100 μ L, 80 ~ 120 μ g/mL2Solution and 900 μ L, pH 7.4 phosphate buffered solution of 50 mmol/L, vibration hatching 12 h in 4 DEG C of constant-temperature shaking incubators；After centrifugation, add the pH=7.4 phosphate buffered solution of 1 mL 50 mmol/L, obtain detecting antibody hatching thing Au-GQD@PtPd-Ab2Solution, saves backup at 4 DEG C.
3. a kind of based on Au-GQD@PtPd immunosensor that prepared by preparation method as claimed in claim 1 is for the detection of carcinoembryonic antigen, and step is as follows:
(1) electrochemical workstation is used to test with three-electrode system, saturated calomel electrode is reference electrode, platinum electrode is auxiliary electrode, and prepared sensor is working electrode, 10 mL, 50 mmol/L pH 5.91 ~ 8.04 phosphate buffered solution in test；
(2) detecting carcinoembryonic antigen with chronoamperometry, input voltage is-0.4
V, sampling interval 0.1 s, run time 400 s；
(3) after background current tends towards stability, every 50 s to 10 mL, the pH=7.4 of 50 mmol/L phosphate buffered solution in add 10 L, the hydrogen peroxide solution of 5 mol/L, record current changes.
The preparation method of a kind of immunosensor based on Au-GQD@PtPd, described carcinoembryonic antigen is selected from CEA.
The useful achievement of the present invention
(1) in the middle of the preparation of the electrochemical immunosensor that Au-GQD@PtPd nanoparticle is incorporated into carcinoembryonic antigen as detection antibody labeling thing by the present inventor first, utilize biocompatibility excellent for Au-GQD@PtPd and high catalytic performance, made sensor is made to have high sensitivity and wide detection range, the detection CEA range of linearity is 0.001 pg/mL～10 ng/mL, and detection is limited to 0.0003 pg/mL.
(2) employ and there is good catalytic effect deposited Au as base material, can either good immobilized capture antibody, again can be with increasing specific surface area and electron transfer rate, it is achieved that the amplification of electrochemical signals, further increase the sensitivity of sensor.
(3) identical nano material and method of modifying are used, utilize the specific binding of antigen and antibody, only need to change immune globulin classes and can realize highly sensitive, the specific detection of different immunoglobulin, the method is simple to operate, detection speed is fast, the mensuration of a large amount of sample, the beneficially commercialization of immunosensor can be realized at short notice.
(4) Au-GQD@PtPd nanoparticle is directly hatched with hCEA detection antibody, it is required for enzyme in the label of detection antibody, avoid the detection error caused because of inactivation and the leakage of enzyme, simplify the making step of detection antibody labeling thing, significantly improve repeatability and the stability of electrochemical immunosensor.
Detailed description of the invention
Now the present invention is further illustrated by detailed description of the invention, but be not limited to this
Embodiment 1.The preparation method of a kind of immunosensor based on Au-GQD@PtPd, it is characterised in that step is as follows:
(1) by the glass-carbon electrode Al of a diameter of 3 mm2O3Polishing powder is polished, and ultra-pure water cleans up；
(2) by HAuCl4
(1 wt%) is electrodeposited into electrode surface with chronoamperometry from-0.2V, dries, and with ultrapure water, dries；
(3) carcinoembryonic antigen capture antibody A b of drop coating 6 L, 8 g/mL1Solution in electrode surface, 4oC refrigerator is dried；
(4) ultrapure water falls unconjugated capture antibody A b1After, drip 3 L, 0.5 mg/mL bovine serum albumin BSA solution in electrode surface, 4oC refrigerator dries；
(5) after ultrapure water falls unconjugated BSA, drip 6 L, 1 × 10-6
Human normal immunoglobulin's solution of a series of variable concentrations of ~ 10 ng/mL, incubation at room temperature 1h, ultra-pure water cleans, is placed in 4oC refrigerator is dried；
(6) the detection antibody of 6 L, 1.5 mg/mL is hatched thing Au-GQD@PtPd-Ab2Solution drop coating is on electrode surface, and incubation at room temperature 1h, ultra-pure water cleans up, and is placed in 4oC refrigerator dries, prepares a kind of carcinoembryonic antigen sensor based on Au-GQD@PtPd.
Embodiment 2.The preparation method of a kind of immunosensor based on Au-GQD@PtPd, it is characterised in that step is 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) by HAuCl4
(1 wt%) is electrodeposited into electrode surface with chronoamperometry from 0 V, dries, and with ultrapure water, dries；
(3) drop coating 6 L, 10
Carcinoembryonic antigen capture antibody A b of g/mL1Solution in electrode surface, 4oC refrigerator is dried；
(4) ultrapure water falls unconjugated capture antibody A b1After, drip 3 L, 1.0 mg/mL bovine serum albumin BSA solution in electrode surface, 4oC refrigerator dries；
(5) after ultrapure water falls unconjugated BSA, drip 6 L, 1 × 10-6
Human normal immunoglobulin's solution of a series of variable concentrations of ~ 10 ng/mL, incubation at room temperature 1h, ultra-pure water cleans, is placed in 4oC refrigerator is dried；
(6) the detection antibody of 6 L, 3.0 mg/mL is hatched thing Au-GQD@PtPd-Ab2Solution drop coating is on electrode surface, and incubation at room temperature 1h, ultra-pure water cleans up, and is placed in 4oC refrigerator dries, prepares a kind of carcinoembryonic antigen sensor based on Au-GQD@PtPd.
Embodiment 3.The preparation method of a kind of immunosensor based on Au-GQD@PtPd, it is characterised in that step is as follows:
(1) by the glass-carbon electrode Al of a diameter of 5 mm2O3Polishing powder is polished, and ultra-pure water cleans up；
(2) by HAuCl4
(1 wt%) is electrodeposited into electrode surface with chronoamperometry from 0.2V, dries, and with ultrapure water, dries；
(3) drop coating 6 L, 12
Carcinoembryonic antigen capture antibody A b of g/mL1Solution in electrode surface, 4oC refrigerator is dried；
(4) ultrapure water falls unconjugated capture antibody A b1After, drip 3 L, 1.5 mg/mL bovine serum albumin BSA solution in electrode surface, 4oC refrigerator dries；
(5) after ultrapure water falls unconjugated BSA, drip 6 L, 1 × 10-6
Human normal immunoglobulin's solution of a series of variable concentrations of ~ 10 ng/mL, incubation at room temperature 1h, ultra-pure water cleans, is placed in 4oC refrigerator is dried；
(6) the detection antibody of 6 L, 5.0 mg/mL is hatched thing Au-GQD@PtPd-Ab2Solution drop coating is on electrode surface, and incubation at room temperature 1h, ultra-pure water cleans up, and is placed in 4oC refrigerator dries, prepares a kind of carcinoembryonic antigen sensor based on Au-GQD@PtPd.
Embodiment 4. Build the preparation method of the various sensing materials of carcinoembryonic antigen immunosensor, comprise the following steps:
(1) preparation of graphene quantum dot
First by 2.0 g citric acids and 1.0 g
Dicyandiamide joins in 5mL ultra-pure water, is heated to 180 DEG C and lasting 12 h in mixture is transferred to 25 mL autoclaves afterwards；Product is dispersed in 100 mL ultra-pure waters, in centrifuge, is centrifuged 10 min with the rotating speed of 10000 r/min, take supernatant ambient temperature in vacuum and be dried 12 h, prepare graphene quantum dot；
(2) preparation of GQD@PtPd
By 0.158 mL chloroplatinic acid (20mmol/L), 0.233 mL chlorine palladium acid (20mmol/L) and 20mg graphene quantum dot join in 15.0 mL ultra-pure waters under magnetic stirring, pH to 10 is regulated with NaOH, subsequently mixed solution is transferred in autoclave, at 160 DEG C, reacts 6h, centrifugation, and with milli-Q water, until supernatant is colourless, ambient temperature in vacuum is dried 12 h, prepares black powder GQD@PtPd；
(3) preparation of golden nanometer particle
1.0 mL gold chlorides (1 wt%) are joined in 99 mL ultra-pure waters, adds the sodium citrate (1 wt%) of 2.5 mL while stirring, 100oBackflow 15 under C
Min, centrifugation, discard precipitation, the supernatant of finely dispersed claret is golden nanometer particle seed solution；Take 1 mL golden nanometer particle seed solution and bis-water of 25 mL and mix homogeneously, add 100 μ L, the hydroxylamine hydrochloride solution of 0.2 mol/L, at room temperature high-speed stirred mixing, then 3.0 mL gold chlorides (1 wt%) solution it is added dropwise over, addition along with gold chloride, the color of solution is gradually converted into peony, and prepared particle diameter is the solution of gold nanoparticles of 50 ± 5 nm；
(4) preparation of Au-GQD@PtPd
First 1.0 mL solution of gold nanoparticles and 2.0 mL ethylenediamines are dispersed in 5.0 mL ultra-pure waters, ultrasonic 1h, then magnetic agitation 5h, centrifugation, and with milli-Q water, abandon supernatant, obtain the golden nanometer particle of amino functional, the most accurately add 4 mg GQD@PtPd, and add 1.0 mL ultra-pure waters, room temperature reaction 12h, centrifugation, and with milli-Q water, until supernatant is colourless, 35oC is vacuum dried, and prepares Au-GQD@PtPd；
(5) detection antibody hatching thing Au-GQD@PtPd-Ab2The preparation of solution
The anti-label of Au-GQD@PtPd bis-of 1.5 mg is distributed in 1 mL ultra-pure water, adds the anti-Ab of tumor markers two of 100 μ L, 80 μ g/mL2Solution and 900 μ L, pH 7.4 phosphate buffered solution of 50 mmol/L, vibration hatching 12 h in 4 DEG C of constant-temperature shaking incubators；After centrifugation, add the pH=7.4 phosphate buffered solution of 1 mL 50 mmol/L, obtain detecting antibody hatching thing Au-GQD@PtPd-Ab2Solution, saves backup at 4 DEG C.
Embodiment 5. Build the preparation method of the various sensing materials of carcinoembryonic antigen immunosensor, comprise the following steps:
(1) preparation of graphene quantum dot
First by 2.0 g citric acids and 1.0 g
Dicyandiamide joins in 5mL ultra-pure water, is heated to 180 DEG C and lasting 12 h in mixture is transferred to 25 mL autoclaves afterwards；Product is dispersed in 100 mL ultra-pure waters, in centrifuge, is centrifuged 10 min with the rotating speed of 10000 r/min, take supernatant ambient temperature in vacuum and be dried 12 h, prepare graphene quantum dot；
(2) preparation of GQD@PtPd
By 0.158 mL chloroplatinic acid (20mmol/L), 0.233 mL chlorine palladium acid (20mmol/L) and 20mg graphene quantum dot join in 15.0 mL ultra-pure waters under magnetic stirring, pH to 10 is regulated with NaOH, subsequently mixed solution is transferred in autoclave, at 160 DEG C, reacts 6h, centrifugation, and with milli-Q water, until supernatant is colourless, ambient temperature in vacuum is dried 12 h, prepares black powder GQD@PtPd；
(3) preparation of golden nanometer particle
1.0 mL gold chlorides (1 wt%) are joined in 99 mL ultra-pure waters, adds the sodium citrate (1 wt%) of 2.5 mL while stirring, 100oBackflow 15 under C
Min, centrifugation, discard precipitation, the supernatant of finely dispersed claret is golden nanometer particle seed solution；Take 1 mL golden nanometer particle seed solution and bis-water of 25 mL and mix homogeneously, add 100 μ L, the hydroxylamine hydrochloride solution of 0.2 mol/L, at room temperature high-speed stirred mixing, then 3.0 mL gold chlorides (1 wt%) solution it is added dropwise over, addition along with gold chloride, the color of solution is gradually converted into peony, and prepared particle diameter is the solution of gold nanoparticles of 50 ± 5 nm；
(4) preparation of Au-GQD@PtPd
First 1.0 mL solution of gold nanoparticles and 2.0 mL ethylenediamines are dispersed in 5.0 mL ultra-pure waters, ultrasonic 1h, then magnetic agitation 5h, centrifugation, and with milli-Q water, abandon supernatant, obtain the golden nanometer particle of amino functional, the most accurately add 5 mg GQD@PtPd, and add 1.0 mL ultra-pure waters, room temperature reaction 12h, centrifugation, and with milli-Q water, until supernatant is colourless, 35oC is vacuum dried, and prepares Au-GQD@PtPd；
(5) detection antibody hatching thing Au-GQD@PtPd-Ab2The preparation of solution
The anti-label of Au-GQD@PtPd bis-of 3 mg is distributed in 1 mL ultra-pure water, adds the anti-Ab of tumor markers two of 100 μ L, 100 μ g/mL2Solution and 900 μ L, pH 7.4 phosphate buffered solution of 50 mmol/L, vibration hatching 12 h in 4 DEG C of constant-temperature shaking incubators；After centrifugation, add the pH=7.4 phosphate buffered solution of 1 mL 50 mmol/L, obtain detecting antibody hatching thing Au-GQD@PtPd-Ab2Solution, saves backup at 4 DEG C.
Embodiment 6.Build the preparation method of the various sensing materials of carcinoembryonic antigen immunosensor, comprise the following steps:
(1) preparation of graphene quantum dot
First by 2.0 g citric acids and 1.0 g
Dicyandiamide joins in 5mL ultra-pure water, is heated to 180 DEG C and lasting 12 h in mixture is transferred to 25 mL autoclaves afterwards；Product is dispersed in 100 mL ultra-pure waters, in centrifuge, is centrifuged 10 min with the rotating speed of 10000 r/min, take supernatant ambient temperature in vacuum and be dried 12 h, prepare graphene quantum dot；
(2) preparation of GQD@PtPd
By 0.158 mL chloroplatinic acid (20 mmol/L), 0.233 mL chlorine palladium acid (20 mmol/L) and 20mg graphene quantum dot join in 15.0 mL ultra-pure waters under magnetic stirring, pH to 10 is regulated with NaOH, subsequently mixed solution is transferred in autoclave, at 160 DEG C, reacts 6h, centrifugation, and with milli-Q water, until supernatant is colourless, ambient temperature in vacuum is dried 12 h, prepares black powder GQD@PtPd；
(3) preparation of golden nanometer particle
1.0 mL gold chlorides (1 wt%) are joined in 99 mL ultra-pure waters, adds the sodium citrate (1 wt%) of 2.5 mL while stirring, 100oBackflow 15 under C
Min, centrifugation, discard precipitation, the supernatant of finely dispersed claret is golden nanometer particle seed solution；Take 1 mL golden nanometer particle seed solution and bis-water of 25 mL and mix homogeneously, add 100 μ L, the hydroxylamine hydrochloride solution of 0.2 mol/L, at room temperature high-speed stirred mixing, then 3.0 mL gold chlorides (1 wt%) solution it is added dropwise over, addition along with gold chloride, the color of solution is gradually converted into peony, and prepared particle diameter is the solution of gold nanoparticles of 50 ± 5 nm；
(4) preparation of Au-GQD@PtPd
First 1.0 mL solution of gold nanoparticles and 2.0 mL ethylenediamines are dispersed in 5.0 mL ultra-pure waters, ultrasonic 1h, then magnetic agitation 5h, centrifugation, and with milli-Q water, abandon supernatant, obtain the golden nanometer particle of amino functional, the most accurately add 6 mg GQD@PtPd, and add 1.0 mL ultra-pure waters, room temperature reaction 12h, centrifugation, and with milli-Q water, until supernatant is colourless, 35oC is vacuum dried, and prepares Au-GQD@PtPd；
(5) detection antibody hatching thing Au-GQD@PtPd-Ab2The preparation of solution
The anti-label of Au-GQD@PtPd bis-of 5 mg is distributed in 1 mL ultra-pure water, adds the anti-Ab of tumor markers two of 100 μ L, 120 μ g/mL2Solution and 900 μ L, pH 7.4 phosphate buffered solution of 50 mmol/L, vibration hatching 12 h in 4 DEG C of constant-temperature shaking incubators；After centrifugation, add the pH=7.4 phosphate buffered solution of 1 mL 50 mmol/L, obtain detecting antibody hatching thing Au-GQD@PtPd-Ab2Solution, saves backup at 4 DEG C.
Embodiment 7. Constructed immunosensor, for the detection of CEA, detecting step is as follows:
(1) electrochemical workstation is used to test with three-electrode system, saturated calomel electrode is reference electrode, platinum electrode is auxiliary electrode, and prepared sensor is working electrode, 10 mL, 50 mmol/L pH 5.59 ~ 8.02 phosphate buffered solution in test；
(2) detecting CEA with chronoamperometry, input voltage is-0.4 V, sampling interval 0.1 s, runs time 400 s；
(3) after background current tends towards stability, every 50 s to 10 mL, the pH=7.4 of 50 mmol/L phosphate buffered solution in add 10 L, the hydrogen peroxide solution of 5 mol/L, record current changes；
(4) according to the linear relationship between gained current intensity and CEA concentration, drawing curve, recording its range of linearity is 0.001 pg/mL～100 ng/mL, and detection is limited to 0.0003 pg/mL.
Claims (4)
1. the preparation method of an immunosensor based on Au-GQD PtPd, it is characterised in that step is as follows:
(1) by the glass-carbon electrode Al of a diameter of 3 ~ 5 mm2O3Polishing powder is polished, and ultra-pure water cleans up；
(2) by HAuCl4 (1 wt%) is electrodeposited into electrode surface with chronoamperometry from-0.2V ~ 0.2V, dries, and with ultrapure water, dries；
(3) the anti-Ab of tumor markers one of drop coating 6 L, 8 ~ 12 g/mL1Solution in electrode surface, 4oC refrigerator is dried；
(4) ultrapure water falls unconjugated capture antibody A b1After, drip 3 L, 0.5 ~ 1.5 mg/mL bovine serum albumin BSA solution in electrode surface, 4oC refrigerator dries；
(5) after ultrapure water falls unconjugated BSA, drip 6 L, 1 × 10-6The carcinoembryonic antigen solution of a series of variable concentrations of ~ 10 ng/mL, incubation at room temperature 1h, ultra-pure water cleans, is placed in 4oC refrigerator is dried；
(6) the detection antibody of 6 L, 1.5 ~ 5.0 mg/mL is hatched thing Au-GQD@PtPd-Ab2Solution drop coating is on electrode surface, and incubation at room temperature 1h, ultra-pure water cleans up, and is placed in 4oC refrigerator dries, prepares a kind of carcinoembryonic antigen sensor based on Au-GQD@PtPd.
A kind of preparation method of immunosensor based on Au-GQD@PtPd, described described Au-GQD@PtPd-Ab2The two anti-preparations incubating compound solution, it is characterised in that preparation process is as follows:
(1) preparation of graphene quantum dot
First 2.0 g citric acids and 1.0 g dicyandiamides are joined in 5mL ultra-pure water, in afterwards mixture is transferred to 25 mL autoclaves, be heated to 180 DEG C and lasting 12 h；Product is dispersed in 100 mL ultra-pure waters, in centrifuge, is centrifuged 10 min with the rotating speed of 10000 r/min, take supernatant ambient temperature in vacuum and be dried 12 h, prepare graphene quantum dot；
(2) preparation of GQD@PtPd
By 0.158 mL chloroplatinic acid (20mmol/L), 0.233 mL chlorine palladium acid (20mmol/L) and 20mg graphene quantum dot join in 15.0 mL ultra-pure waters under magnetic stirring, pH to 10 is regulated with NaOH, subsequently mixed solution is transferred in autoclave, at 160 DEG C, reacts 6h, centrifugation, and with milli-Q water, until supernatant is colourless, ambient temperature in vacuum is dried 12 h, prepares black powder GQD@PtPd；
(3) preparation of golden nanometer particle
1.0 mL gold chlorides (1 wt%) are joined in 99 mL ultra-pure waters, adds the sodium citrate (1 wt%) of 2.5 mL while stirring, 100oReflux under C 15 min, centrifugation, discards precipitation, and the supernatant of finely dispersed claret is golden nanometer particle seed solution；Take 1 mL golden nanometer particle seed solution and bis-water of 25 mL and mix homogeneously, add 100 μ L, the hydroxylamine hydrochloride solution of 0.2 mol/L, at room temperature high-speed stirred mixing, then 3.0 mL gold chlorides (1 wt%) solution it is added dropwise over, addition along with gold chloride, the color of solution is gradually converted into peony, and prepared particle diameter is the solution of gold nanoparticles of 50 ± 5 nm；
(4) preparation of Au-GQD@PtPd
First 1.0 mL solution of gold nanoparticles and 2.0 mL ethylenediamines are dispersed in 5.0 mL ultra-pure waters, ultrasonic 1h, then magnetic agitation 5h, centrifugation, and with milli-Q water, abandon supernatant, obtain the golden nanometer particle of amino functional, the most accurately add 4 ~ 6 mg GQD@PtPd, and add 1.0 mL ultra-pure waters, room temperature reaction 12h, centrifugation, and with milli-Q water, until supernatant is colourless, 35oC is vacuum dried, and prepares Au-GQD@PtPd；
(5) detection antibody hatching thing Au-GQD@PtPd
-Ab2The preparation of solution
The anti-label of Au-GQD@PtPd bis-of 1.5 ~ 5 mg is distributed in 1 mL ultra-pure water, adds the anti-Ab of tumor markers two of 100 μ L, 80 ~ 120 μ g/mL2Solution and 900 μ L, pH 7.4 phosphate buffered solution of 50 mmol/L, vibration hatching 12 h in 4 DEG C of constant-temperature shaking incubators；After centrifugation, add 1 mL
The pH=7.4 phosphate buffered solution of 50 mmol/L, obtains detecting antibody hatching thing Au-GQD@PtPd-Ab2Solution, saves backup at 4 DEG C.
3. a kind of based on Au-GQD@PtPd immunosensor that prepared by preparation method as claimed in claim 1 is for the detection of carcinoembryonic antigen, and step is as follows:
(1) electrochemical workstation is used to test with three-electrode system, saturated calomel electrode is reference electrode, platinum electrode is auxiliary electrode, and prepared sensor is working electrode, 10 mL, 50 mmol/L pH 5.91 ~ 8.04 phosphate buffered solution in test；
(2) detecting carcinoembryonic antigen with chronoamperometry, input voltage is-0.4 V, sampling interval 0.1 s, runs time 400 s；
(3) after background current tends towards stability, every 50 s to 10 mL, the pH=7.4 of 50 mmol/L phosphate buffered solution in add 10 L, the hydrogen peroxide solution of 5 mol/L, record current changes.
The preparation method of a kind of immunosensor based on Au-GQD@PtPd, described carcinoembryonic antigen is selected from CEA.
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