CN106093396A - A kind of preparation method and application of immunosensor based on Au GQD@PtPd - Google Patents

Abstract

The invention belongs to nano-functional material, immunoassay and biosensor technique field, it is provided that 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, prepare the sandwich type electrochemical immunosensor of a kind of carcinoembryonic antigen, utilize biocompatibility excellent for Au GQD@PtPd and high catalytic performance, made sensor is made to have high specificity, the advantages such as highly sensitive and detection limit is low.

Description

A kind of preparation method and application of immunosensor based on Au-GQD@PtPd

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, belongs to new function material 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 shift Speed is fast, and the health of the mankind is had harm greatly.Carcinoembryonic antigen is originally found in colon cancer and fetal gut tissue, human body blood In clear, the rising of the content of carcinoembryonic antigen, shows that human body is in the state of a kind of non-health.The meaning of carcinoembryonic antigen is that it The existence of kinds of tumors can be reflected, can be used for the Outcome measure to colorectal cancer, breast carcinoma and pulmonary carcinoma, PD, monitoring and Prognosis is estimated.Carcinoembryonic antigen raises and is common in colorectal cancer, cancer of pancreas, gastric cancer, breast carcinoma, medullary thyroid carcinoma etc..Therefore facing In bed research, the method developing quick, easy, sensitive detection carcinoembryonic antigen is particularly significant.

The most existing detection method for carcinoembryonic antigen is a lot, divides as being usually used in detecting the immunity of carcinoembryonic antigen at present Analysis method mainly includes radioimmunology, enzyme-linked immune analytic method, fluorescence immune analysis method and electrochemiluminescence immunity Analysis method etc., but most detection method is loaded down with trivial details, operation complexity, somewhat expensive, detects limit for height, therefore, sets up a kind of quick, simple Just, sensitive detection method is significant.

Immunosensor is a kind of biosensor combined with analytical chemistry method by immunological method, passes through antigen And specific binding between antibody so that it have highly sensitive, selectivity good, simple in construction, easy and simple to handle, be prone to small-sized Change, can the advantage such as detection analysis continuous, rapid automatized.And the key building electrochemical immunosensor has 2 points: the first Use 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, and 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, is allowed to have good catalysis Performance, it is possible to accelerate electron transmission, has important function for improving transducer sensitivity.

Therefore the present invention is prepared for a kind of applying deposited Au as base material with Au-GQD@PtPd as label Immunosensor, 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 a kind of super Sensitive sandwich type electrochemical immunosensor.

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 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, uses ultra-pure water Rinse, dry；

(3) the anti-Ab of tumor markers one of drop coating 6 L, 8 ~ 12 g/mL₁Solution in electrode surface, 4^oC refrigerator is dried；

(4) ultrapure water falls unconjugated capture antibody A b₁After, drip 3 L, the bovine serum albumin of 0.5 ~ 1.5 mg/mL BSA solution is in electrode surface in vain, and 4^oC refrigerator dries；

(5) after ultrapure water falls unconjugated BSA, drip 6 L, 1 × 10^-6A series of variable concentrations of ~ 10 ng/mL Carcinoembryonic antigen solution, incubation at room temperature 1h, ultra-pure water clean, be placed in 4^oC 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 in electrode surface On, incubation at room temperature 1h, ultra-pure water cleans up, and is placed in 4^oC refrigerator dries, prepares a kind of cancer embryo based on Au-GQD@PtPd Antigen sensor.

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, afterwards mixture is transferred to 25 180 DEG C and lasting 12 h it are heated in mL autoclave；Product is dispersed in 100 mL ultra-pure waters, with 10000 in centrifuge The rotating speed of r/min is centrifuged 10 min, takes supernatant ambient temperature in vacuum and is dried 12 h, prepares 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 exist Join in 15.0 mL ultra-pure waters under magnetic agitation, regulate pH to 10 with NaOH, subsequently mixed solution is transferred to reaction under high pressure In still, at 160 DEG C, react 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 of 2.5 mL while stirring Wt%), 100^oReflux under C 15 min, centrifugation, discards precipitation, and the supernatant of finely dispersed claret is Jenner Rice corpuscles 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, is then added dropwise over 3.0 mL gold chlorides (1 wt%) Solution, along with the addition of gold chloride, the color of solution is gradually converted into peony, and prepared particle diameter is Jenner's grain of rice of 50 ± 5 nm Sub-solution；

(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, and obtain the golden nanometer particle of amino functional, then accurate Really 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, 35^oC 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 100 μ L, 80 ~ 120 μ g/ The anti-Ab of tumor markers two of mL₂Solution and 900 μ L, pH 7.4 phosphate buffered solution of 50 mmol/L, 4 DEG C of constant temperature oscillations Vibration hatching 12 h in incubator；After centrifugation, add the pH=7.4 phosphate buffered solution of 1 mL 50 mmol/L, obtain Detection 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 for The detection of carcinoembryonic antigen, step is as follows:

(1) using electrochemical workstation to test with three-electrode system, saturated calomel electrode is reference electrode, and platinum electrode is Auxiliary electrode, prepared sensor is working electrode, in 10 mL, pH 5.91 ~ 8.04 phosphate-buffered of 50 mmol/L Solution is tested；

(2) detecting carcinoembryonic antigen with chronoamperometry, input voltage is-0.4 V, sampling interval 0.1 s, runs the time 400 s；

(3) after background current tends towards stability, every 50 s to 10 mL, the phosphate buffered solution of the pH=7.4 of 50 mmol/L Middle addition 10 L, the hydrogen peroxide solution of 5 mol/L, record current changes.

A kind of preparation method of immunosensor based on Au-GQD@PtPd, described cancer embryo Antigen is selected from CEA.

The useful achievement of the present invention

(1) Au-GQD@PtPd nanoparticle is incorporated into cancer embryo resists as detection antibody labeling thing by the present inventor first In the middle of the preparation of former electrochemical immunosensor, utilize biocompatibility excellent for Au-GQD@PtPd and high catalytic performance, Making made sensor have high sensitivity and wide detection range, the detection CEA range of linearity is 0.001 pg/mL～10 Ng/mL, 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 resist Body, again can be with increasing specific surface area and electron transfer rate, it is achieved that the amplification of electrochemical signals, further increases sensor Sensitivity.

(3) use identical nano material and method of modifying, utilize the specific binding of antigen and antibody, only need to change Becoming immune globulin classes and can realize highly sensitive, the specific detection of different immunoglobulin, the method is simple to operate, detection Speed is fast, can realize the mensuration of a large amount of sample, the beneficially commercialization of immunosensor at short notice.

(4) Au-GQD@PtPd nanoparticle is directly hatched, at the labelling of detection antibody with hCEA detection antibody Thing is required for enzyme, it is to avoid the detection error that causes because of inactivation and the leakage of enzyme, simplifies the system of detection antibody labeling thing Make step, 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

The preparation method of embodiment 1. 1 kinds 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, with ultrapure water, Dry；

(3) carcinoembryonic antigen capture antibody A b of drop coating 6 L, 8 g/mL₁Solution in electrode surface, 4^oC refrigerator is dried；

(4) ultrapure water falls unconjugated capture antibody A b₁After, drip the bovine serum albumin BSA of 3 L, 0.5 mg/mL Solution in electrode surface, 4^oC refrigerator dries；

(5) after ultrapure water falls unconjugated BSA, drip 6 L, 1 × 10^-6A series of variable concentrations of ~ 10 ng/mL Human normal immunoglobulin's solution, incubation at room temperature 1h, ultra-pure water clean, be placed in 4^oC refrigerator is dried；

(6) the detection antibody of 6 L, 1.5 mg/mL is hatched thing Au-GQD@PtPd-Ab₂Solution drop coating on electrode surface, room Temperature hatching 1h, ultra-pure water cleans up, and is placed in 4^oC refrigerator dries, prepares a kind of carcinoembryonic antigen based on Au-GQD@PtPd and pass Sensor.

The preparation method of embodiment 2. 1 kinds immunosensor based on Au-GQD PtPd, it is characterised in that step is such as Under:

(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 in the air Dry；

(3) carcinoembryonic antigen capture antibody A b of drop coating 6 L, 10 g/mL₁Solution in electrode surface, 4^oC refrigerator is dried；

(4) ultrapure water falls unconjugated capture antibody A b₁After, drip the bovine serum albumin BSA of 3 L, 1.0 mg/mL Solution in electrode surface, 4^oC refrigerator dries；

(5) after ultrapure water falls unconjugated BSA, drip 6 L, 1 × 10^-6A series of variable concentrations of ~ 10 ng/mL Human normal immunoglobulin's solution, incubation at room temperature 1h, ultra-pure water clean, be placed in 4^oC refrigerator is dried；

(6) the detection antibody of 6 L, 3.0 mg/mL is hatched thing Au-GQD@PtPd-Ab₂Solution drop coating on electrode surface, room Temperature hatching 1h, ultra-pure water cleans up, and is placed in 4^oC refrigerator dries, prepares a kind of carcinoembryonic antigen based on Au-GQD@PtPd and pass Sensor.

The preparation method of embodiment 3. 1 kinds immunosensor based on Au-GQD PtPd, it is characterised in that step is such as Under:

(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, with ultrapure water, Dry；

(3) carcinoembryonic antigen capture antibody A b of drop coating 6 L, 12 g/mL₁Solution in electrode surface, 4^oC refrigerator is dried；

(4) ultrapure water falls unconjugated capture antibody A b₁After, drip the bovine serum albumin BSA of 3 L, 1.5 mg/mL Solution in electrode surface, 4^oC refrigerator dries；

(5) after ultrapure water falls unconjugated BSA, drip 6 L, 1 × 10^-6A series of variable concentrations of ~ 10 ng/mL Human normal immunoglobulin's solution, incubation at room temperature 1h, ultra-pure water clean, be placed in 4^oC refrigerator is dried；

(6) the detection antibody of 6 L, 5.0 mg/mL is hatched thing Au-GQD@PtPd-Ab₂Solution drop coating on electrode surface, room Temperature hatching 1h, ultra-pure water cleans up, and is placed in 4^oC refrigerator dries, prepares a kind of carcinoembryonic antigen based on Au-GQD@PtPd and pass Sensor.

Embodiment 4. builds the preparation method of the various sensing materials of carcinoembryonic antigen immunosensor, comprises the following steps:

(1) preparation of graphene quantum dot

First 2.0 g citric acids and 1.0 g dicyandiamides are joined in 5mL ultra-pure water, afterwards mixture is transferred to 25 180 DEG C and lasting 12 h it are heated in mL autoclave；Product is dispersed in 100 mL ultra-pure waters, with 10000 in centrifuge The rotating speed of r/min is centrifuged 10 min, takes supernatant ambient temperature in vacuum and is dried 12 h, prepares 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 exist Join in 15.0 mL ultra-pure waters under magnetic agitation, regulate pH to 10 with NaOH, subsequently mixed solution is transferred to reaction under high pressure In still, at 160 DEG C, react 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 of 2.5 mL while stirring Wt%), 100^oReflux under C 15 min, centrifugation, discards precipitation, and the supernatant of finely dispersed claret is Jenner Rice corpuscles 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, is then added dropwise over 3.0 mL gold chlorides (1 wt%) Solution, along with the addition of gold chloride, the color of solution is gradually converted into peony, and prepared particle diameter is Jenner's grain of rice of 50 ± 5 nm Sub-solution；

(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, and obtain the golden nanometer particle of amino functional, then accurate Really add 4 mg GQD@PtPd, and add 1.0 mL ultra-pure waters, room temperature reaction 12h, centrifugation, and with milli-Q water, directly Colourless to supernatant, 35^oC 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, add 100 μ L, 80 μ g/mL swollen The anti-Ab of tumor markers two₂Solution and 900 μ L, pH 7.4 phosphate buffered solution of 50 mmol/L, 4 DEG C of constant-temperature shaking incubators Middle vibration hatching 12 h；After centrifugation, add the pH=7.4 phosphate buffered solution of 1 mL 50 mmol/L, obtain detection anti- Body hatching thing Au-GQD@PtPd-Ab₂Solution, saves backup at 4 DEG C.

Embodiment 5. builds the preparation method of the various sensing materials of carcinoembryonic antigen immunosensor, comprises the following steps:

(1) preparation of graphene quantum dot

First 2.0 g citric acids and 1.0 g dicyandiamides are joined in 5mL ultra-pure water, afterwards mixture is transferred to 25 mL 180 DEG C and lasting 12 h it are heated in autoclave；Product is dispersed in 100 mL ultra-pure waters, centrifuge is used 10000 r/ The rotating speed of min is centrifuged 10 min, takes supernatant ambient temperature in vacuum and is dried 12 h, prepares 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 exist Join in 15.0 mL ultra-pure waters under magnetic agitation, regulate pH to 10 with NaOH, subsequently mixed solution is transferred to reaction under high pressure In still, at 160 DEG C, react 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 of 2.5 mL while stirring Wt%), 100^oReflux under C 15 min, centrifugation, discards precipitation, and the supernatant of finely dispersed claret is Jenner Rice corpuscles 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, is then added dropwise over 3.0 mL gold chlorides (1 wt%) Solution, along with the addition of gold chloride, the color of solution is gradually converted into peony, and prepared particle diameter is Jenner's grain of rice of 50 ± 5 nm Sub-solution；

(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, and obtain the golden nanometer particle of amino functional, then accurate Really add 5 mg GQD@PtPd, and add 1.0 mL ultra-pure waters, room temperature reaction 12h, centrifugation, and with milli-Q water, directly Colourless to supernatant, 35^oC 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, add 100 μ L, 100 μ g/mL swollen The anti-Ab of tumor markers two₂Solution and 900 μ L, pH 7.4 phosphate buffered solution of 50 mmol/L, 4 DEG C of constant-temperature shaking incubators Middle vibration hatching 12 h；After centrifugation, add the pH=7.4 phosphate buffered solution of 1 mL 50 mmol/L, obtain detection anti- Body hatching thing Au-GQD@PtPd-Ab₂Solution, saves backup at 4 DEG C.

Embodiment 6. builds the preparation method of the various sensing materials of carcinoembryonic antigen immunosensor, comprises the following steps:

(1) preparation of graphene quantum dot

First 2.0 g citric acids and 1.0 g dicyandiamides are joined in 5mL ultra-pure water, afterwards mixture is transferred to 25 mL 180 DEG C and lasting 12 h it are heated in autoclave；Product is dispersed in 100 mL ultra-pure waters, centrifuge is used 10000 r/ The rotating speed of min is centrifuged 10 min, takes supernatant ambient temperature in vacuum and is dried 12 h, prepares 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, regulate pH to 10 with NaOH, subsequently mixed solution is transferred to high pressure anti- Answer in still, at 160 DEG C, react 6h, centrifugation, and with milli-Q water, until supernatant is colourless, ambient temperature in vacuum is dried 12 h, prepare 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 of 2.5 mL while stirring Wt%), 100^oReflux under C 15 min, centrifugation, discards precipitation, and the supernatant of finely dispersed claret is Jenner Rice corpuscles 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, is then added dropwise over 3.0 mL gold chlorides (1 wt%) Solution, along with the addition of gold chloride, the color of solution is gradually converted into peony, and prepared particle diameter is Jenner's grain of rice of 50 ± 5 nm Sub-solution；

(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, and obtain the golden nanometer particle of amino functional, then accurate Really add 6 mg GQD@PtPd, and add 1.0 mL ultra-pure waters, room temperature reaction 12h, centrifugation, and with milli-Q water, directly Colourless to supernatant, 35^oC 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, add 100 μ L, 120 μ g/mL swollen The anti-Ab of tumor markers two₂Solution and 900 μ L, pH 7.4 phosphate buffered solution of 50 mmol/L, 4 DEG C of constant-temperature shaking incubators Middle vibration hatching 12 h；After centrifugation, add the pH=7.4 phosphate buffered solution of 1 mL 50 mmol/L, obtain detection anti- Body hatching thing Au-GQD@PtPd-Ab₂Solution, saves backup at 4 DEG C.

Immunosensor constructed by embodiment 7., for the detection of CEA, detecting step is as follows:

(1) using electrochemical workstation to test with three-electrode system, saturated calomel electrode is reference electrode, and platinum electrode is Auxiliary electrode, prepared sensor is working electrode, in 10 mL, pH 5.59 ~ 8.02 phosphate-buffered of 50 mmol/L Solution is tested；

(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 phosphate buffered solution of the pH=7.4 of 50 mmol/L Middle addition 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, 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, uses ultra-pure water Rinse, dry；

(3) the anti-Ab of tumor markers one of drop coating 6 L, 8 ~ 12 g/mL₁Solution in electrode surface, 4^oC refrigerator is dried；

(4) ultrapure water falls unconjugated capture antibody A b₁After, drip 3 L, the bovine serum albumin of 0.5 ~ 1.5 mg/mL BSA solution is in electrode surface in vain, and 4^oC refrigerator dries；

(5) after ultrapure water falls unconjugated BSA, drip 6 L, 1 × 10^-6A series of variable concentrations of ~ 10 ng/mL Carcinoembryonic antigen solution, incubation at room temperature 1h, ultra-pure water cleans, is placed in 4^oC 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 in electrode surface On, incubation at room temperature 1h, ultra-pure water cleans up, and is placed in 4^oC refrigerator dries, prepares a kind of cancer embryo based on Au-GQD@PtPd Antigen sensor.

The preparation method of a kind of immunosensor based on Au-GQD@PtPd, described is 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, afterwards mixture is transferred to 25 180 DEG C and lasting 12 h it are heated in mL autoclave；Product is dispersed in 100 mL ultra-pure waters, with 10000 in centrifuge The rotating speed of r/min is centrifuged 10 min, takes supernatant ambient temperature in vacuum and is dried 12 h, prepares 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 exist Join in 15.0 mL ultra-pure waters under magnetic agitation, regulate pH to 10 with NaOH, subsequently mixed solution is transferred to reaction under high pressure In still, at 160 DEG C, react 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 of 2.5 mL while stirring Wt%), 100^oReflux under C 15 min, centrifugation, discards precipitation, and the supernatant of finely dispersed claret is Jenner Rice corpuscles 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, is then added dropwise over 3.0 mL gold chlorides (1 wt%) Solution, along with the addition of gold chloride, the color of solution is gradually converted into peony, and prepared particle diameter is Jenner's grain of rice of 50 ± 5 nm Sub-solution；

(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, and obtain the golden nanometer particle of amino functional, then accurate Really 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, 35^oC 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 100 μ L, 80 ~ 120 μ g/ The anti-Ab of tumor markers two of mL₂Solution and 900 μ L, pH 7.4 phosphate buffered solution of 50 mmol/L, 4 DEG C of constant temperature oscillations Vibration hatching 12 h in incubator；After centrifugation, add the pH=7.4 phosphate buffered solution of 1 mL 50 mmol/L, obtain Detection 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 cancer embryo The detection of antigen, step is as follows:

(1) using electrochemical workstation to test with three-electrode system, saturated calomel electrode is reference electrode, and platinum electrode is Auxiliary electrode, prepared sensor is working electrode, in 10 mL, pH 5.91 ~ 8.04 phosphate-buffered of 50 mmol/L Solution is tested；

(2) detecting carcinoembryonic antigen with chronoamperometry, input voltage is-0.4 V, sampling interval 0.1 s, runs the time 400 s；

(3) after background current tends towards stability, every 50 s to 10 mL, the phosphate buffered solution of the pH=7.4 of 50 mmol/L Middle addition 10 L, the hydrogen peroxide solution of 5 mol/L, record current changes.

A kind of preparation method of immunosensor based on Au-GQD@PtPd, described carcinoembryonic antigen Selected from CEA.