CN107727858B - A kind of preparation method and application based on Rh@Pt nanodendrites composite material immunosensor - Google Patents

A kind of preparation method and application based on Rh@Pt nanodendrites composite material immunosensor Download PDF

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CN107727858B
CN107727858B CN201710924116.8A CN201710924116A CN107727858B CN 107727858 B CN107727858 B CN 107727858B CN 201710924116 A CN201710924116 A CN 201710924116A CN 107727858 B CN107727858 B CN 107727858B
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王平
裴福彬
李明党
马恩慧
余昊轩
董云会
李月云
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Shandong University of Technology
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Abstract

The invention belongs to new function materials and bio-sensing detection technique field, provide a kind of preparation method and application based on Rh@Pt nanodendrites composite material immunosensor.Specifically using Rh@Pt nanodendrites composite material as marker, polypyrrole-gold nanoparticle is prepared for a kind of electrochemical immunosensor and the detection applied to tumor markers antigen as substrate.

Description

A kind of preparation method based on Rh@Pt nanodendrites composite material immunosensor and Using
Technical field
The invention belongs to new function materials and bio-sensing detection technique field, provide a kind of based on Pt nanometers of Rh@ The preparation method and application of dendrite composite material immunosensor.
Background technique
The disease incidence of tumour is high, and not noticeable, and China's case load is quite huge, accounts for the 55% of whole world case load, and swollen The speed of growth and the transfer of tumor is fast, generates high risks to the health of the mankind, if however tumour can make a definite diagnosis healing in early days Rate or very high.Tumor markers and tumour have close relationship, are contained by tumor markers in detection human serum It measures to speculate that whether suffering from tumour has become a kind of method that people are widely recognized as.
Electrochemical immunosensor is widely used for the detection of tumor markers, interlayer type electrochemistry immuno-sensing at present Device combine high specific immuno analytical method and highly sensitive electrochemical analysis techniques, have high sensitivity, preparation it is simple, The advantages that quick, low in cost is detected, has weight in fields such as clinical examination, environmental monitoring, food safety control, biological detections The application value wanted.
Rh@Pt nanodendrites used in the present invention not only have the bimetallic synergistic effect of nucleocapsid shape, also have abundant Edge and corner atom, this make Rh@Pt nanodendrites have excellent catalytic.Amination graphene nanometer sheet tool There is biggish specific surface area, a large amount of Rh@Pt nanodendrites can be loaded, while amination graphene also has excellent conduction Property, electronics caused by Rh Pt nanodendrites catalysis reduction hydrogen peroxide can be dispersed, to prevent from causing since electronics is assembled Catalysis reaction slows down.In addition, the introducing of Rh@Pt nanodendrites not only makes the electric conductivity of composite material obtain significantly mentioning It is high, moreover it is possible to effectively prevent the stacking of graphene.Therefore, Rh@Pt nanodendrites composite material can be mentioned as signal amplifier The sensitivity of high sensor.Polypyrrole can accelerate electronics to conduct as conducting polymer, form polypyrrole-gold nanoparticle Not only it is also provided with good biocompatibility, can be good at immobilized capture antibody, together with the high conductivity of polypyrrole afterwards When gold nanoparticle addition further improve the electric conductivity of polypyrrole.
Summary of the invention
The present invention provides a kind of preparation method and application based on Rh@Pt nanodendrites composite material immunosensor, Realize the quantitative detection to tumor markers.
An object of the present invention is to provide a kind of preparation side based on Rh@Pt nanodendrites composite material immunosensor Method.
The second object of the present invention is to prepared Rh@Pt nanodendrites composite material immunosensor is applied to tumour Specificity, the quantitative detection of marker.
Technical solution of the present invention includes the following steps.
1. a kind of preparation method based on Rh@Pt nanodendrites composite material immunosensor, steps are as follows:
(1) the glass-carbon electrode Al for being 3 ~ 5 mm by diameter2O3Polishing powder polishing, ultrapure water, ethyl alcohol clean up；
(2) it takes polypyrrole-gold nanoparticle of 6 μ L, 1 ~ 3 mg/mL to modify to electrode surface, dries at room temperature, use Ultrapure water electrode surface, dries；
(3) continue the tumor markers of 6 μ L, 8 ~ 12 μ g/mL capturing antibody A b1It is added drop-wise to electrode surface, 4 DEG C of ice It is dry in case；
(4) continue the bovine serum albumin(BSA) BSA solution of 3 μ L, 0.5 ~ 1 wt% being added drop-wise to electrode surface, be with pH 7.4 phosphate buffer solution rinses electrode surface, dries in 4 DEG C of refrigerators；
(5) a series of tumor markers antigen A g solution of various concentrations of 6 μ L, 0.0001 ~ 5 ng/mL is added dropwise, uses The phosphate buffer solution that pH is 7.4 rinses electrode surface, drying in 4 DEG C of refrigerators；
(6) by the Rh Pt nanodendrites composite material tumor-marker analyte detection antibody Rh Pt/ of 6 μ L, 1 ~ 3 mg/mL NH2-GS/Ab2Solution, drop coating on electrode surface, be placed in 4 DEG C of refrigerators hatch 40 min after with pH be 7.4 phosphate-buffered Solution cleaning electrode, it is dry in 4 DEG C of refrigerators, a kind of immunosensor based on Rh@Pt nanodendrites composite material is made.
Polypyrrole-gold nanoparticle used in 2., Rh Pt nanodendrites composite material tumor-marker analyte detection antibody Rh Pt/NH2-GS/Ab2The preparation of solution
(1) polypyrrole-gold nanoparticle preparation
1. the synthesis of polypyrrole
It takes 0.5 ~ 1.5 mL pyrroles to be dispersed in the hydrochloric acid of 25 mL, 0.5 ~ 1.5 mol/L, obtains pyrroles and hydrochloric acid Mixed liquor；The mixed liquor of 10 mL pyrroles and hydrochloric acid is taken, the ammonium persulfate of 2 mL, 1 mol/L are added under stirring condition, ice bath is anti- 4 ~ 6 h are answered, are centrifugated, drying at room temperature obtains polypyrrole；
2. the synthesis of polypyrrole-gold nanoparticle
Take 1 mL, the chlorauric acid solution of 0.8 ~ 1.2 wt% is added in 99 mL ultrapure waters, be heated to it is slightly boiled, be added The sodium citrate solution of 2.5 mL, 0.5 ~ 1.5 wt% are cooled to room temperature after keeping 15 min of slight boiling condition, obtain gold nano Particle dispersion is transferred at 4 DEG C and saves backup；The golden nanometer particle dispersion liquid of the above-mentioned preparation of 20 ~ 30 mL is centrifuged Separation, is dispersed in again in the ultrapure water of 10 mL, and 10 mg polypyrroles are added, and 12 h, centrifugation point are vibrated in shaken cultivation case From drying at room temperature obtains polypyrrole-gold nanoparticle；
(2) prepared by Rh@Pt nanodendrites composite material
1. the synthesis of Rh@Pt nanodendrites
0.21 g sodium citrate and 0.2 ~ 0.3 g hexadecyltrimethylammonium chloride is taken to be added in 9 mL ultrapure waters, 5 min are stirred, the rhodium chloride of 0.1 mL, 0.1 mol/L, the chlorine platinum of 0.9 mL, 0.08 ~ 0.12 mol/L are sequentially added Acid after stirring 15 min, is added 1 mL formaldehyde, mixed liquor is transferred in autoclave, reacts 40 under 180 DEG C of condition ~ 60 min, are cooled to room temperature, and are centrifugated, and freeze-drying obtains Rh@Pt nanodendrites；
2. Rh@Pt nanodendrites composite material synthesizes
Take the concentrated sulfuric acid and 10 mL concentrated phosphoric acids that 0.8 g graphite powder adds 90 ~ 98 mL, mass fraction is 98 wt%, stirring After 25 min, 4 ~ 5 g potassium permanganate are added, after stirring 15 min, is heated to 45 ~ 60 DEG C, after reacting 12 h, adds 40 mL The ice that ultrapure water is frozen into, then plus 0.5 mL, the hydrogen peroxide of 20 ~ 30 wt% stir centrifuge washing after 20 min, add 30 mL super Supernatant is freeze-dried to obtain graphene oxide by pure water, 40 min of ultrasound, centrifuge separation；0.1 g graphene oxide is taken to be put into In small beaker, adding 40 mL ethylene glycol, 30 min of ultrasound add 0.8 ~ 1 mL concentrated ammonia liquor, mixture are transferred to autoclave, 5 h are reacted at 180 DEG C, are cooled to room temperature, are centrifugated, at room temperature dry amination graphene；Take 15 mg amino graphites The Rh@Pt nanodendrites of alkene and 5 ~ 10 mg are dispersed in 10 mL ultrapure waters, and 1 h of ultrasound is centrifugated to obtain Pt nanometers of branches of Rh@ Crystal composite material；
(3) Rh Pt nanodendrites composite material tumor-marker analyte detection antibody Rh Pt/NH2 -GS/Ab2The preparation of solution
The amination graphene of the load Rh@Pt nanodendrites of 1 ~ 4 mg is distributed in 1 mL ultrapure water, is added 100 The tumor-marker analyte detection antibody A b of μ L, 80 ~ 120 μ g/mL2The phosphoric acid that the pH of solution and 900 μ L, 50 mmol/L are 7.4 Salt buffer solution, 12 h of oscillation hatching in 4 DEG C of constant-temperature shaking incubators；At 4 DEG C, 10 min are centrifuged under 6000 rpm revolving speeds, Lower sediment is obtained, 1 mL is added, the phosphate buffer solution centrifuge washing that the pH of 50 mmol/L is 7.4, obtains lower sediment, most 1 mL is added afterwards, the phosphate buffer solution that the pH of 50 mmol/L is 7.4, Rh@Pt nanodendrites composite material tumour mark is made Will analyte detection antibody Rh@Pt/NH2-GS/Ab2Solution saves backup at 4 DEG C.
3. the detection of tumor markers
(1) it is tested using electrochemical workstation with three-electrode system, saturated calomel electrode is reference electrode, platinum filament electricity Extremely auxiliary electrode, prepared sensor are working electrode, the phosphoric acid for being 5.3 ~ 8.0 in the pH of 10 mL, 50 mmol/L It is tested in salt buffer solution；
(2) used time m- current method detects analyte, and input voltage is -0.4 V, 0.1 s of sampling interval, operation 400 s of time；
(3) after background current tends towards stability, delay every 50 s to the phosphate that the pH of 10 mL, 50 mmol/L are 7.4 It rushes in solution and injects the hydrogen peroxide solution of 10 μ L, 5 mol/L, record current variation.
Tumor markers described above are selected from one of following: AFP, CEA, PSA.
Raw materials of the present invention can be bought in chemical reagents corporation or biopharmaceutical company.
Beneficial achievement of the invention
(1) present invention uses polypyrrole-gold nanoparticle, polypyrrole is cheap, has excellent electric conductivity, can Accelerate electronics transfer, gold nanoparticle can further increase the electric conductivity of polypyrrole, while flake is presented in polypyrrole, has Biggish specific surface area can load more gold nanoparticle, this makes polypyrrole-gold nanoparticle not only and have excellent Electric conductivity is also provided with good biocompatibility, while the addition of gold nanoparticle also increases point of polypyrrole in water Dissipate property；
(2) using Rh@Pt nanodendrites composite material as detection antibody marker, Rh@Pt nanodendrites can be prevented The stacking of graphene improves the dispersibility of graphene in water, and furthermore Rh@Pt nanodendrites have more edge and turning, Specific surface area is increased, there is more active site, is capable of the reduction of preferably catalyzing hydrogen peroxide, while being based on platinum-ammonia Between effect Rh@Pt nanodendrites can be good at immobilized antibody, realize amplification electrochemical signals effect, to improve The sensitivity of sensor reduces detection limit；
(3) a kind of detection based on Rh@Pt nanodendrites composite material immunosensor to tumor markers, linear model 0.0001 ~ 5 ng/mL is enclosed, detection limits minimum 0.033 pg/mL, shows a kind of immune based on Rh@Pt nanodendrites composite material Sensor can achieve the purpose of quantitative detection.
Specific embodiment
Now the present invention is further illustrated by specific embodiment, but not limited to this.
A kind of preparation based on Rh@Pt nanodendrites composite material immunosensor of embodiment 1
(1) the glass-carbon electrode Al for being 3 mm by diameter2O3Polishing powder polishing, ultrapure water, ethyl alcohol clean up；
(2) it takes polypyrrole-gold nanoparticle of 6 μ L, 1 mg/mL to modify to electrode surface, dries at room temperature, use is ultrapure Water rinses electrode surface, dries；
(3) continue the tumor markers of 6 μ L, 8 μ g/mL capturing antibody A b1Electrode surface is added drop-wise to, in 4 DEG C of refrigerators It is dry；
(4) continue the bovine serum albumin(BSA) BSA solution of 3 μ L, 0.5 wt% being added drop-wise to electrode surface, be 7.4 with pH Phosphate buffer solution rinses electrode surface, dries in 4 DEG C of refrigerators；
(5) a series of tumor markers antigen A g solution of various concentrations of 6 μ L, 0.0001 ~ 5 ng/mL is added dropwise, uses The phosphate buffer solution that pH is 7.4 rinses electrode surface, drying in 4 DEG C of refrigerators；
(6) by the Rh Pt nanodendrites composite material tumor-marker analyte detection antibody Rh Pt/NH of 6 μ L, 1 mg/mL2- GS/Ab2Solution, drop coating on electrode surface, be placed in 4 DEG C of refrigerators hatch 40 min after with pH be 7.4 phosphate-buffered it is molten Liquid cleaning electrode, it is dry in 4 DEG C of refrigerators, it is made a kind of and is based on Rh@Pt nanodendrites composite material immunosensor.
A kind of preparation based on Rh@Pt nanodendrites composite material immunosensor of embodiment 2
(1) the glass-carbon electrode Al for being 4 mm by diameter2O3Polishing powder polishing, ultrapure water, ethyl alcohol clean up；
(2) it takes polypyrrole-gold nanoparticle of 6 μ L, 2 mg/mL to modify to electrode surface, dries at room temperature, use is ultrapure Water rinses electrode surface, dries；
(3) continue the tumor markers of 6 μ L, 10 μ g/mL capturing antibody A b1Electrode surface is added drop-wise to, in 4 DEG C of refrigerators It is dry；
(4) continue the bovine serum albumin(BSA) BSA solution of 3 μ L, 0.8 wt% being added drop-wise to electrode surface, be 7.4 with pH Phosphate buffer solution rinses electrode surface, dries in 4 DEG C of refrigerators；
(5) a series of tumor markers antigen A g solution of various concentrations of 6 μ L, 0.0001 ~ 5 ng/mL is added dropwise, uses The phosphate buffer solution that pH is 7.4 rinses electrode surface, drying in 4 DEG C of refrigerators；
(6) by the Rh Pt nanodendrites composite material tumor-marker analyte detection antibody Rh Pt/NH of 6 μ L, 2 mg/mL2- GS/Ab2Solution, drop coating on electrode surface, be placed in 4 DEG C of refrigerators hatch 40 min after with pH be 7.4 phosphate-buffered it is molten Liquid cleaning electrode, it is dry in 4 DEG C of refrigerators, it is made a kind of and is based on Rh@Pt nanodendrites composite material immunosensor.
A kind of preparation based on Rh@Pt nanodendrites composite material immunosensor of embodiment 3
(1) the glass-carbon electrode Al for being 5 mm by diameter2O3Polishing powder polishing, ultrapure water, ethyl alcohol clean up；
(2) it takes polypyrrole-gold nanoparticle of 6 μ L, 3 mg/mL to modify to electrode surface, dries at room temperature, use is ultrapure Water rinses electrode surface, dries；
(3) continue the tumor markers of 6 μ L, 12 μ g/mL capturing antibody A b1Electrode surface is added drop-wise to, in 4 DEG C of refrigerators It is dry；
(4) continue the bovine serum albumin(BSA) BSA solution of 3 μ L, 1 wt% being added drop-wise to electrode surface, the phosphorus for being 7.4 with pH Hydrochlorate buffer solution rinses electrode surface, dries in 4 DEG C of refrigerators；
(5) a series of tumor markers antigen A g solution of various concentrations of 6 μ L, 0.0001 ~ 5 ng/mL is added dropwise, uses The phosphate buffer solution that pH is 7.4 rinses electrode surface, drying in 4 DEG C of refrigerators；
(6) by the Rh Pt nanodendrites composite material tumor-marker analyte detection antibody Rh Pt/NH of 6 μ L, 3 mg/mL2- GS/Ab2Solution, drop coating on electrode surface, be placed in 4 DEG C of refrigerators hatch 40 min after with pH be 7.4 phosphate-buffered it is molten Liquid cleaning electrode, it is dry in 4 DEG C of refrigerators, it is made a kind of and is based on Rh@Pt nanodendrites composite material immunosensor.
The preparation of 4 polypyrroles of embodiment-gold nanoparticle
(1) synthesis of polypyrrole
It takes 0.5 mL pyrroles to be dispersed in the hydrochloric acid of 25 mL, 0.5 mol/L, obtains the mixed liquor of pyrroles and hydrochloric acid；Take 10 The ammonium persulfate solution of 2 mL, 1 mol/L are added under stirring condition for the mixed liquor of mL pyrroles and hydrochloric acid, and ice bath reacts 4 h, from Heart separation, drying at room temperature obtain polypyrrole；
(2) preparation of golden nanometer particle dispersion liquid
Take 1 mL, the chlorauric acid solution of 0.8 wt% is added in 99 mL ultrapure waters, be heated to it is slightly boiled, be added 2.5 mL, The sodium citrate solution of 0.5 wt% is cooled to room temperature after keeping 15 min of slight boiling condition, obtains golden nanometer particle dispersion liquid, will It is transferred at 4 DEG C and saves backup；
(3) polypyrrole-gold nanoparticle synthesis
The golden nanometer particle dispersion liquid of the above-mentioned preparation of 20 mL is centrifugated, is dispersed in the ultrapure water of 10 mL again, 10 mg polypyrroles are added, 12 h are vibrated in shaken cultivation case, are centrifugated, drying at room temperature obtains polypyrrole-gold nanoparticle.
The preparation of 5 polypyrroles of embodiment-gold nanoparticle
(1) synthesis of polypyrrole
It takes 1 mL pyrroles to be dispersed in the hydrochloric acid of 25 mL, 1 mol/L, obtains the mixed liquor of pyrroles and hydrochloric acid；Take 10 mL pyrroles The mixed liquor with hydrochloric acid is coughed up, the ammonium persulfate solution of 2 mL, 1 mol/L are added under stirring condition, ice bath reacts 5 h, centrifugation point From drying at room temperature obtains polypyrrole；
(2) preparation of golden nanometer particle dispersion liquid
Take 1 mL, the chlorauric acid solution of 1 wt% is added in 99 mL ultrapure waters, be heated to it is slightly boiled, be added 2.5 mL, 1 The sodium citrate solution of wt% is cooled to room temperature after keeping 15 min of slight boiling condition, obtains golden nanometer particle dispersion liquid, by its turn It moves on at 4 DEG C and saves backup；
(3) polypyrrole-gold nanoparticle synthesis
The golden nanometer particle dispersion liquid of the above-mentioned preparation of 25 mL is centrifugated, is dispersed in the ultrapure water of 10 mL again, 10 mg polypyrroles are added, 12 h are vibrated in shaken cultivation case, are centrifugated, drying at room temperature obtains polypyrrole-gold nanoparticle.
The preparation of 6 polypyrroles of embodiment-gold nanoparticle
(1) synthesis of polypyrrole
It takes 1.5 mL pyrroles to be dispersed in the hydrochloric acid of 25 mL, 1.5 mol/L, obtains the mixed liquor of pyrroles and hydrochloric acid；Take 10 The ammonium persulfate solution of 2 mL, 1 mol/L are added under stirring condition for the mixed liquor of mL pyrroles and hydrochloric acid, and ice bath reacts 6 h, from Heart separation, drying at room temperature obtain polypyrrole；
(2) preparation of golden nanometer particle dispersion liquid
Take 1 mL, the chlorauric acid solution of 1.2 wt% is added in 99 mL ultrapure waters, be heated to it is slightly boiled, be added 2.5 mL, The sodium citrate solution of 1.5 wt% is cooled to room temperature after keeping 15 min of slight boiling condition, obtains golden nanometer particle dispersion liquid, will It is transferred at 4 DEG C and saves backup；
(3) polypyrrole-gold nanoparticle synthesis
The golden nanometer particle dispersion liquid of the above-mentioned preparation of 30 mL is centrifugated, is dispersed in the ultrapure water of 10 mL again, 10 mg polypyrroles are added, 12 h are vibrated in shaken cultivation case, are centrifugated, drying at room temperature obtains polypyrrole-gold nanoparticle.
Embodiment 7Rh Pt nanodendrites composite material tumor-marker analyte detection antibody Rh Pt/NH2-GS/Ab2The system of solution It is standby
(1) synthesis of Rh@Pt nanodendrites
0.21 g sodium citrate and 0.2 g hexadecyltrimethylammonium chloride is taken to be added in 9 mL ultrapure waters, stirring 5 Min, sequentially adds the rhodium chloride of 0.1 mL, 0.1 mol/L, and the chloroplatinic acid of 0.9 mL, 0.08 mol/L stir 15 min Afterwards, 1 mL formaldehyde is added, mixed liquor is transferred in autoclave, 40 min are reacted under 180 DEG C of condition, are cooled to room Temperature is centrifugated, and freeze-drying obtains Rh@Pt nanodendrites；
(2) synthesis of amination graphene
It takes 0.8 g graphite powder to add 90 mL, the concentrated sulfuric acid of 98 wt% and 10 mL concentrated phosphoric acids, after stirring 25 min, 4 g is added Potassium permanganate is heated to 45 DEG C after stirring 15 min, after reacting 12 h, the ice for adding 40 mL ultrapure waters to be frozen into, then plus 0.5 mL, The hydrogen peroxide of 20 wt% stirs centrifuge washing after 20 min, adds 30 mL ultrapure waters, 40 min of ultrasound, centrifuge separation, by supernatant Liquid is freeze-dried to obtain graphene oxide；It takes 0.1 g graphene oxide to be put into small beaker, adds 40 mL ethylene glycol, ultrasound 30 Min adds 0.8 mL concentrated ammonia liquor, and mixture is transferred to autoclave, 5 h are reacted at 180 DEG C, are cooled to room temperature, centrifuge separation, Dry amination graphene at room temperature；
(3) Rh@Pt nanodendrites composite material synthesizes
The Rh@Pt nanodendrites of 15 mg amination graphenes and 5 mg are taken to be dispersed in 10 mL ultrapure waters, 1 h of ultrasound, It is centrifugated to obtain Rh@Pt nanodendrites composite material；
(4) Rh Pt nanodendrites composite material tumor-marker analyte detection antibody Rh Pt/NH2 -GS/Ab2The preparation of solution
The Rh@Pt nanodendrites composite material of 1 mg is distributed in 1 mL ultrapure water, 100 μ L, 80 μ g/mL are added Tumor-marker analyte detection antibody A b2The phosphate buffer solution that the pH of solution and 900 μ L, 50 mmol/L are 7.4,4 DEG C of constant temperature 12 h of oscillation hatching in shaken cultivation case；At 4 DEG C, 10 min are centrifuged under 6000 rpm revolving speeds, obtain lower sediment, are added 1 ML, 50 mmol/L pH be 7.4 phosphate buffer solution centrifuge washing, obtain lower sediment, be eventually adding 1 mL, 50 Rh Pt nanodendrites composite material tumor-marker analyte detection antibody is made in the phosphate buffer solution that the pH of mmol/L is 7.4 Rh@Pt/NH2-GS/Ab2Solution saves backup at 4 DEG C.
Embodiment 8Rh Pt nanodendrites composite material tumor-marker analyte detection antibody Rh Pt/NH2-GS/Ab2The system of solution It is standby
(1) synthesis of Rh@Pt nanodendrites
0.21 g sodium citrate and 0.25 g hexadecyltrimethylammonium chloride is taken to be added in 9 mL ultrapure waters, stirring 5 Min, sequentially adds the rhodium chloride of 0.1 mL, 0.1 mol/L, the chloroplatinic acid of 0.9 mL, 0.1 mol/L, after stirring 15 min, 1 mL formaldehyde is added, mixed liquor is transferred in autoclave, 50 min are reacted under 180 DEG C of condition, are cooled to room temperature, Centrifuge separation, freeze-drying, obtains Rh@Pt nanodendrites；
(2) synthesis of amination graphene
It takes 0.8 g graphite powder to add 90 ~ 98 mL, the concentrated sulfuric acid of 98 wt% and 10 mL concentrated phosphoric acids, after stirring 25 min, adds Enter 4.5 g potassium permanganate, after stirring 15 min, is heated to 50 DEG C, after reacting 12 h, the ice for adding 40 mL ultrapure waters to be frozen into, then plus 0.5 mL, the hydrogen peroxide of 25 wt% stir centrifuge washing after 20 min, add 30 mL ultrapure waters, and 40 min of ultrasound are centrifugated, Supernatant is freeze-dried to obtain graphene oxide；It takes 0.1 g graphene oxide to be put into small beaker, adds 40 mL ethylene glycol, surpass 30 min of sound adds 0.9 mL concentrated ammonia liquor, and mixture is transferred to autoclave, 5 h are reacted at 180 DEG C, are cooled to room temperature, centrifugation Separation, at room temperature dry amination graphene；
(3) Rh@Pt nanodendrites composite material synthesizes
The Rh@Pt nanodendrites of 15 mg amination graphenes and 8 mg are taken to be dispersed in 10 mL ultrapure waters, 1 h of ultrasound, It is centrifugated to obtain Rh@Pt nanodendrites composite material；
(4) Rh Pt nanodendrites composite material tumor-marker analyte detection antibody Rh Pt/NH2 -GS/Ab2The preparation of solution
The Rh@Pt nanodendrites composite material of 2 mg is distributed in 1 mL ultrapure water, 100 μ L, 100 μ g/mL are added Tumor-marker analyte detection antibody A b2The phosphate buffer solution that the pH of solution and 900 μ L, 50 mmol/L are 7.4,4 DEG C of perseverances 12 h of oscillation hatching in warm shaken cultivation case；At 4 DEG C, 10 min are centrifuged under 6000 rpm revolving speeds, obtain lower sediment, are added 1 ML, 50 mmol/L pH be 7.4 phosphate buffer solution centrifuge washing, obtain lower sediment, be eventually adding 1 mL, 50 Rh Pt nanodendrites composite material tumor-marker analyte detection antibody is made in the phosphate buffer solution that the pH of mmol/L is 7.4 Rh@Pt/NH2-GS/Ab2Solution saves backup at 4 DEG C.
Embodiment 9Rh Pt nanodendrites composite material tumor-marker analyte detection antibody Rh Pt/NH2-GS/Ab2The system of solution It is standby
(1) synthesis of Rh@Pt nanodendrites
0.21 g sodium citrate and 0.3 g hexadecyltrimethylammonium chloride is taken to be added in 9 mL ultrapure waters, stirring 5 Min, sequentially adds the rhodium chloride of 0.1 mL, 0.1 mol/L, and the chloroplatinic acid of 0.9 mL, 0.12 mol/L stir 15 min Afterwards, 1 mL formaldehyde is added, mixed liquor is transferred in autoclave, 60 min are reacted under 180 DEG C of condition, are cooled to room Temperature is centrifugated, and freeze-drying obtains Rh@Pt nanodendrites；
(2) synthesis of amination graphene
It takes 0.8 g graphite powder to add 98 mL, the concentrated sulfuric acid of 98 wt% and 10 mL concentrated phosphoric acids, after stirring 25 min, 5 g is added Potassium permanganate is heated to 60 DEG C after stirring 15 min, after reacting 12 h, the ice for adding 40 mL ultrapure waters to be frozen into, then plus 0.5 mL, The hydrogen peroxide of 30 wt% stirs centrifuge washing after 20 min, adds 30 mL ultrapure waters, 40 min of ultrasound, centrifuge separation, by supernatant Liquid is freeze-dried to obtain graphene oxide；It takes 0.1 g graphene oxide to be put into small beaker, adds 40 mL ethylene glycol, ultrasound 30 Min adds 1 mL concentrated ammonia liquor, and mixture is transferred to autoclave, 5 h are reacted at 180 DEG C, are cooled to room temperature, centrifuge separation, room Dry amination graphene under temperature；
(3) Rh@Pt nanodendrites composite material synthesizes
The Rh@Pt nanodendrites of 15 mg amination graphenes and 10 mg are taken to be dispersed in 10 mL ultrapure waters, 1 h of ultrasound, It is centrifugated to obtain Rh@Pt nanodendrites composite material；
(4) Rh Pt nanodendrites composite material tumor-marker analyte detection antibody Rh Pt/NH2 -GS/Ab2The preparation of solution
The Rh@Pt nanodendrites composite material of 4 mg is distributed in 1 mL ultrapure water, 100 μ L, 120 μ g/mL are added Tumor-marker analyte detection antibody A b2The phosphate buffer solution that the pH of solution and 900 μ L, 50 mmol/L are 7.4,4 DEG C of perseverances 12 h of oscillation hatching in warm shaken cultivation case；At 4 DEG C, 10 min are centrifuged under 6000 rpm revolving speeds, obtain lower sediment, are added 1 ML, 50 mmol/L pH be 7.4 phosphate buffer solution centrifuge washing, obtain lower sediment, be eventually adding 1 mL, 50 Rh Pt nanodendrites composite material tumor-marker analyte detection antibody is made in the phosphate buffer solution that the pH of mmol/L is 7.4 Rh@Pt/NH2-GS/Ab2Solution saves backup at 4 DEG C.
The detection of 10 tumor markers AFP of embodiment
(1) it is tested using electrochemical workstation with three-electrode system, saturated calomel electrode is reference electrode, platinum filament electricity Extremely auxiliary electrode, prepared sensor are working electrode, the phosphate-buffered for being 5.3 in the pH of 10 mL, 50 mmol/L It is tested in solution；
(2) used time m- current method detects analyte, and input voltage is -0.4 V, 0.1 s of sampling interval, operation 400 s of time；
(3) after background current tends towards stability, delay every 50 s to the phosphate that the pH of 10 mL, 50 mmol/L are 7.4 It rushes in solution and injects the hydrogen peroxide solution of 10 μ L, 5 mol/L, record current variation；
(4) according to the linear relationship between gained current strength and AFP concentration, working curve is drawn, the range of linearity is measured For 0.0001 ~ 5 ng/mL, detection is limited to 0.033 pg/mL.
The detection of 11 tumor markers CEA of embodiment
CEA in sample is detected according to the method for embodiment 10, the range of linearity is 0.001 ~ 5 ng/mL, inspection Survey is limited to 0.3 pg/mL.
The detection of 12 tumor markers PSA of embodiment
PSA in sample is detected according to the method for embodiment 10, the range of linearity is 0.0005 ~ 5 ng/mL, inspection Survey is limited to 0.1 pg/mL.

Claims (4)

1. a kind of preparation method of the immunosensor of the amination graphene based on load Rh@Pt nanodendrites, feature exist In steps are as follows:
(1) the glass-carbon electrode Al for being 3 ~ 5 mm by diameter2O3Polishing powder polishing, ultrapure water, ethyl alcohol clean up；
(2) it takes polypyrrole-gold nanoparticle of 6 μ L, 1 ~ 3 mg/mL to modify to electrode surface, dries at room temperature, use is ultrapure Water rinses electrode surface, dries；
(3) continue the tumor markers of 6 μ L, 8 ~ 12 μ g/mL capturing antibody A b1Electrode surface is added drop-wise to, in 4 °C of refrigerators It is dry；
(4) continue the bovine serum albumin(BSA) BSA solution of 3 μ L, 0.5 ~ 1 wt% being added drop-wise to electrode surface, be 7.4 with pH Phosphate buffer solution rinses electrode surface, dries in 4 °C of refrigerators；
(5) a series of tumor markers antigen A g solution of various concentrations of 6 μ L, 0.0001 ~ 5 ng/mL is added dropwise, is with pH 7.4 phosphate buffer solution rinses electrode surface, drying in 4 °C of refrigerators；
(6) the tumor-marker analyte detection of the amination graphene of the load Rh Pt nanodendrites of 6 μ L, 1 ~ 3 mg/mL is anti- Body Rh@Pt/NH2-GS/Ab2Solution, drop coating on electrode surface, be placed in 4 °C of refrigerators hatch 40 min after with pH be 7.4 Phosphate buffer solution cleaning electrode, it is dry in 4 °C of refrigerators, a kind of amino fossil based on load Rh Pt nanodendrites is made The immunosensor of black alkene；
The preparation of the polypyrrole-gold nanoparticle, steps are as follows:
(1) synthesis of polypyrrole
It takes 0.5 ~ 1.5 mL pyrroles to be dispersed in the hydrochloric acid of 25 mL, 0.5 ~ 1.5 mol/L, obtains the mixing of pyrroles and hydrochloric acid Liquid；The mixed liquor of 10 mL pyrroles and hydrochloric acid is taken, the ammonium persulfate solution of 2 mL, 1 mol/L are added under stirring condition, ice bath is anti- 4 ~ 6 h are answered, are centrifugated, drying at room temperature obtains polypyrrole；
(2) preparation of golden nanometer particle dispersion liquid
Take 1 mL, the chlorauric acid solution of 0.8 ~ 1.2 wt% is added in 99 mL ultrapure waters, be heated to it is slightly boiled, be added 2.5 The sodium citrate solution of mL, 0.5 ~ 1.5 wt% are cooled to room temperature after keeping 15 min of slight boiling condition, obtain gold nanoparticle Dispersion liquid is transferred under 4 °C and saves backup；
(3) polypyrrole-gold nanoparticle synthesis
The golden nanometer particle dispersion liquid of the above-mentioned preparation of 20 ~ 30 mL is centrifugated, is dispersed in the ultrapure water of 10 mL again, 10 mg polypyrroles are added, 12 h are vibrated in shaken cultivation case, are centrifugated, drying at room temperature obtains polypyrrole-gold nanoparticle；
The tumor-marker analyte detection antibody of the preparation method and application of the amination graphene of the load Rh Pt nanodendrites Rh@Pt/NH2-GS/Ab2The preparation of solution, steps are as follows:
(1) synthesis of Rh@Pt nanodendrites
0.21 g sodium citrate and 0.2 ~ 0.3 g hexadecyltrimethylammonium chloride is taken to be added in 9 mL ultrapure waters, stirring 5 Min sequentially adds the rhodium chloride of 0.1 mL, 0.1 mol/L, the chloroplatinic acid of 0.9 mL, 0.08 ~ 0.12 mol/L, stirring After 15 min, 1 mL formaldehyde is added, mixed liquor is transferred in autoclave, 40 ~ 60 are reacted under 180 °C of condition Min is cooled to room temperature, and is centrifugated, and freeze-drying obtains Rh@Pt nanodendrites；
(2) synthesis of amination graphene
It takes 0.8 g graphite powder to add 90 ~ 98 mL, the concentrated sulfuric acid of 98 wt% and 10 mL concentrated phosphoric acids, after stirring 25 min, is added 4 ~ 5 g potassium permanganate after stirring 15 min, are heated to 45 ~ 60 °C, after reacting 12 h, the ice for adding 40 mL ultrapure waters to be frozen into, Again plus 0.5 mL, the hydrogen peroxide of 20 ~ 30 wt% stirs centrifuge washing after 20 min, adds 30 mL ultrapure waters, 40 min of ultrasound, Centrifuge separation, supernatant is freeze-dried to obtain graphene oxide；It takes 0.1 g graphene oxide to be put into small beaker, adds 40 mL Ethylene glycol, 30 min of ultrasound, adds 0.8 ~ 1 mL concentrated ammonia liquor, and mixture is transferred to autoclave, 5 h are reacted under 180 °C, It is cooled to room temperature, is centrifugated, at room temperature dry amination graphene；
(3) synthesis of the amination graphene of Rh@Pt nanodendrites is loaded
The Rh@Pt nanodendrites of 15 mg amination graphenes and 5 ~ 10 mg are taken to be dispersed in 10 mL ultrapure waters, 1 h of ultrasound, It is centrifugated to load the amination graphene of Rh@Pt nanodendrites；
(4) the tumor-marker analyte detection antibody Rh Pt/NH of the amination graphene of Rh Pt nanodendrites is loaded2 -GS/Ab2It is molten The preparation of liquid
The amination graphene of the load Rh@Pt nanodendrites of 1 ~ 4 mg is distributed in 1 mL ultrapure water, 100 μ L of addition, The tumor-marker analyte detection antibody A b of 80 ~ 120 μ g/mL2The pH of solution and 900 μ L, 50 mmol/L are slow for 7.4 phosphate Solution is rushed, 12 h of oscillation hatching in 4 °C of constant-temperature shaking incubators；Under 4 °C, 10 min are centrifuged under 6000 rpm revolving speeds, are obtained Lower sediment, be added 1 mL, 50 mmol/L pH be 7.4 phosphate buffer solution centrifuge washings, obtain lower sediment, finally plus Enter 1 mL, the pH of 50 mmol/L is 7.4 phosphate buffer solutions, the amination graphene of load Rh@Pt nanodendrites is made Tumor-marker analyte detection antibody Rh Pt/NH2-GS/Ab2Solution saves backup under 4 °C.
2. a kind of immunosensor of amination graphene based on load Rh@Pt nanodendrites as described in claim 1 Preparation method, which is characterized in that the tumor markers are selected from one of following: AFP, CEA, PSA.
3. a kind of amination graphene based on load Rh@Pt nanodendrites of preparation method preparation as described in claim 1 Immunosensor, for the detection of tumor markers, detecting step is as follows:
(1) it is tested using electrochemical workstation with three-electrode system, saturated calomel electrode is reference electrode, and platinum electrode is Auxiliary electrode, prepared sensor are working electrode, are 5.3 ~ 8.0 phosphate-buffereds in the pH of 10 mL, 50 mmol/L It is tested in solution；
(2) used time m- current method detects analyte, and input voltage is -0.4 V, 0.1 s of sampling interval, runing time 400 s；
(3) molten for 7.4 phosphate-buffered to the pH of 10 mL, 50 mmol/L every 50 s after background current tends towards stability The hydrogen peroxide solution of 10 μ L, 5 mol/L, record current variation are injected in liquid.
4. a kind of immunosensor of amination graphene based on load Rh@Pt nanodendrites as claimed in claim 3, It is characterized in that, the tumor markers are selected from one of following: AFP, CEA, PSA.
CN201710924116.8A 2017-09-30 2017-09-30 A kind of preparation method and application based on Rh@Pt nanodendrites composite material immunosensor Expired - Fee Related CN107727858B (en)

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