CN109613244B - Preparation method and application of Ag @ Pt-CuS labeled immunosensor - Google Patents

Preparation method and application of Ag @ Pt-CuS labeled immunosensor Download PDF

Info

Publication number
CN109613244B
CN109613244B CN201811058947.2A CN201811058947A CN109613244B CN 109613244 B CN109613244 B CN 109613244B CN 201811058947 A CN201811058947 A CN 201811058947A CN 109613244 B CN109613244 B CN 109613244B
Authority
CN
China
Prior art keywords
cus
solution
electrode
ultrapure water
preparation
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN201811058947.2A
Other languages
Chinese (zh)
Other versions
CN109613244A (en
Inventor
李月云
贾翌雷
张春燕
张栓
禹晓东
徐振
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shandong University of Technology
Original Assignee
Shandong University of Technology
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Shandong University of Technology filed Critical Shandong University of Technology
Priority to CN201811058947.2A priority Critical patent/CN109613244B/en
Publication of CN109613244A publication Critical patent/CN109613244A/en
Application granted granted Critical
Publication of CN109613244B publication Critical patent/CN109613244B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • 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
    • 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

Abstract

The invention belongs to the technical field of immunoassay and biosensing, and provides a preparation method and application of an Ag @ Pt-CuS labeled immunosensor. The invention uses Au NPs @ MWCNTs-SO3H is used as a substrate material, and Ag @ Pt nano particle loaded aminated CuS microspheres and detection antibody incubation are used as signal markers to enhance the catalytic performance of the immunosensor, so that the Ag @ Pt-CuS labeled immunosensor is successfully constructed, the tumor markers CEA and AFP are detected, and the immunosensor has the advantages of low detection limit, high sensitivity, strong specificity and the like, and has important scientific significance and application value for early detection of tumors.

Description

Preparation method and application of Ag @ Pt-CuS labeled immunosensor
Technical Field
The invention belongs to the technical field of nano functional materials, immunoassay and biosensing, and relates to a preparation method and application of an Ag @ Pt-CuS labeled immunosensor. In particular, Ag @ Pt-CuS is used as a detection antibody marker, so that the tumor markers CEA and AFP can be sensitively detected.
Background
Tumors are heteroproliferative cells transformed from normal cells, which can appear in many organs and tissues of the human body and are not easily detected by people at an early stage, and since the growth and metastasis rates of tumors are very fast, they are often detected late, which seriously damages people's health and endangers life. The development of a sensitive detection of relevant biomarkers is crucial for many biomedical and diagnostic studies. At present, there are many methods for detecting tumor markers, such as enzyme-linked immunosorbent assay, fluorescence immunoassay, radioimmunoassay, etc., and besides these techniques, electrochemical immunosensors have attracted extensive attention because of their high sensitivity, low detection limit, fast detection speed, and simple operation.
The electrochemical immunosensor combines an immunoassay technology and a biosensing technology, and is used for measuring changes of current and potential to carry out analysis. The electrochemical immunosensor reflects the change of an electrochemical signal caused by specific binding of an antigen and an antibody, and is generally divided into a labeled immunosensor and an unlabeled immunosensor. The labeled immunosensor has the advantages of high sensitivity, low detection limit, high specificity and the like, and is widely applied to various fields.
Au NPs@MWCNTs-SO3The H is used as a substrate material, so that the specific surface area of the immunosensor can be increased, the characteristic of strong conductivity of the multi-walled carbon nanotube can be played, the conductivity of the electrode is improved, Au NPs are a material with biological affinity, more antibodies can be combined, meanwhile, the conductivity of the Au NPs is very good, and the transfer and transmission of electrons can be accelerated. Ag @ Pt-CuS is used as a detection antibody marker, so that the sensitivity of the immunosensor can be increased, and the catalytic performance of the immunosensor can be improved. The CuS is used as a transition metal sulfide semiconductor, and has good biocompatibility, high electron transport performance and good conductivity. The Ag @ Pt nano particle has strong catalytic performance and can further improve the sensitivity of the immunosensor. The invention adopts gold nanoparticle-loaded sulfonated multi-walled carbon nanotubes as a substrate material, and Ag @ Pt-CuS as a labeled immunosensor constructed by detecting antibody markers, realizes the detection of the tumor marker CEA, has the advantages of low detection limit, high sensitivity, good repeatability, selectivity, good stability and the like, and realizes the accurate detection of the tumor marker in clinical application.
Disclosure of Invention
The invention provides a preparation method and application of an Ag @ Pt-CuS labeled immunosensor, and realizes sensitive detection of tumor markers.
The invention aims to provide a preparation method of an Ag @ Pt-CuS labeled immunosensor.
The second purpose of the invention is to apply the prepared Ag @ Pt-CuS labeled immunosensor to the high-sensitivity and specific detection of tumor markers.
The technical scheme of the invention comprises the following steps.
1. A preparation method of an Ag @ Pt-CuS labeled immunosensor comprises the following steps:
(1) al for glassy carbon electrode with diameter of 4 mm2O3Polishing the polishing powder into a mirror surface, and cleaning with ultrapure water;
(2) au NPs @ MWCNTs-SO of 6 mu L and 1.0-3.0 mg/mL3H solution is dripped on the surface of the electrode, and is dried at room temperature, and the surface of the electrode is washed by ultrapure water and is dried;
(3) continuing to capture the antibody Ab of the tumor marker of 6 mu L and 8-12 mu g/mL1Dropwise adding the solution to the surface of an electrode, washing with ultrapure water, and drying in a refrigerator at 4 ℃;
(4) continuously dropwise adding bovine serum albumin BSA solution with the mass fraction of 1% to the surface of the electrode by 3 muL, 0.5-1.5 mg/mL to seal non-specific active sites on the surface of the electrode, washing the surface of the electrode with ultrapure water, removing unbound BSA, and drying in a refrigerator at 4 ℃;
(5) continuously dropwise adding a series of tumor marker antigen Ag solutions with different concentrations, namely 6 muL and 10 pg/mL-100 ng/mL, washing the surface of the electrode with ultrapure water, and airing in a refrigerator at 4 ℃;
(6) continuously dropwise adding the detection antibody hatching Ag @ Pt-CuS-Ab of 6 mu L and 1.5-3.5 mg/mL2And putting the solution on the surface of an electrode, incubating for 40min in a refrigerator at 4 ℃, washing with ultrapure water, and drying to prepare the Ag @ Pt-CuS labeled immunosensor.
2. The Au NPs @ MWCNTs-SO3H solution preparation, the steps are as follows:
(1) preparation of Au NPs solution
Mixing 1-3 mL of chloroauric acid with the mass fraction of 1% and 99 mL of ultrapure water, refluxing for 15min at 100 ℃, then adding 1-3 mL of 10mg/mL sodium citrate solution, and keeping stirring for 10-30 min to prepare uniformly dispersed wine red Au NPs solution;
（2）MWCNTs-SO3preparation of H
Firstly, MWCNTs are dried in a vacuum drying oven at 60 ℃ for 24H, and then 1g of MWCNTs and 30mL of concentrated H are sequentially added into a three-neck flask2SO4And 10mL concentrated HNO3Uniformly dispersing by ultrasonic treatment for 10min, refluxing for 20-40 min at 120 ℃ while stirring, cooling to room temperature, and filtering; washing and filtering black precipitate ultrapure water to be neutral, drying in a vacuum drying oven at 60 ℃ for 8-12 h to obtain MWCNTs-SO3H；
（3）Au NPs@MWCNTs-SO3Preparation of H
Adding 5-15 mgMWCNTs-SO3Dispersing H in 5-15 mL of Au NPs solution, ultrasonically dispersing for 1H, carrying out suction filtration on a polytetrafluoroethylene filter membrane, and drying a product in a vacuum drying oven at the temperature of 30 ℃ for 8-12H to obtain Au NPs @ MWCNTs-SO3H。
3. The detection antibody hatching Ag @ Pt-CuS-Ab2The solution was prepared by the following steps:
(1) preparation of Ag @ Pt nanoparticles
Stirring the mixture, adding 1 mol/L of 0.05 to 0.15mL-12-4 mL of KOH, 0.01 mol/L of ascorbic acid and 0.05g of PVP are added into 2mL of ultrapure water, and then 4mL of 0.002 mol/L of AgNO is added3Dropwise adding the mixture into the solution, and then continuously dropwise adding 4mL of 0.002 mol/L H2PtCl6Stirring for 1h at 60 ℃, centrifugally washing the product for three times by using an absolute ethanol ultrapure water mixed solution with the volume ratio of 1:1, and ultrasonically dispersing the product in 5mL of ultrapure water to prepare an Ag @ Pt nanoparticle dispersion solution for later use;
(2) preparation of CuS microspheres
At room temperature, 1g of PVP was dissolved in 40mL of ultrapure water, and 0.5g of Cu (NO) was added3)▪3H2Mixing O, continuously adding 20-40 mL of ethylene glycol and 0.2-0.4 g of thiourea in sequence, continuously stirring for 1h, transferring the product into a three-neck flask, refluxing for 2h at 160 ℃ under the stirring state to obtain a black suspension, naturally cooling to room temperature, centrifuging, washing with absolute ethyl alcohol for three times, and drying in a vacuum drying oven at 60 DEG CPreparing CuS microspheres for 8-12 h;
(3) preparation of Ag @ Pt-CuS
Sequentially adding 0.1g of CuS microspheres and 0.1-0.3 g of LAPTES into 5-15 mL of absolute ethyl alcohol, heating to 90 ℃ in an oil bath pot, and keeping refluxing for 1-3 h; cooling to room temperature, centrifuging, washing with absolute ethyl alcohol and ultrapure water for three times, and drying in a vacuum drying oven at 50 ℃ to obtain aminated CuS microspheres;
adding 3mg of aminated CuS microspheres into 3mLAg @ Pt nanoparticle dispersion liquid, carrying out ultrasonic treatment for 1h, centrifuging, washing with absolute ethyl alcohol for three times, and drying in a vacuum drying oven at 50 ℃ for 12 h;
(4) detection of antibody hatching Ag @ Pt-CuS-Ab2Preparation of the solution
Adding 1-3 mL and 2mg/mL of Ag @ Pt-CuS dispersion into 0.5-1.5 mL and 10 mu g/mL of tumor marker detection antibody Ab2In the solution, oscillating and incubating for 12h in a constant-temperature oscillation box; centrifugally separating, and re-dispersing into 1-3 mL of phosphoric acid buffer solution with pH =7.38 to prepare the detection antibody hatching Ag @ Pt-CuS-Ab2The solution was stored at 4 ℃ until use.
4. Detecting a tumor marker by the following steps:
(1) testing by using an electrochemical workstation in a three-electrode system, taking a saturated calomel electrode as a reference electrode, taking a platinum wire electrode as an auxiliary electrode, taking the prepared sensor as a working electrode, and testing in 10mL of potassium ferricyanide solution containing 10 mmol/L;
(2) detecting the tumor marker by a time-lapse current method, selecting-0.4V as the detection of current measurement, wherein the input voltage is-0.4V, the sampling interval is 0.1 s, and the running time is 300 s;
(3) when the background current tended to be stable, 10 μ L of 5 mol/L hydrogen peroxide solution was injected into 10mL of 50 mmol/L phosphate buffer solution with pH =7.38 every 50 s, and the change in current was recorded.
The tumor marker is selected from one of the following: CEA, AFP.
The raw materials used in the present invention are all available from chemical agents companies or biopharmaceutical companies.
Advantageous results of the invention
(1) Gold nanoparticle-loaded sulfonated multi-walled carbon nanotube Au NPs @ MWCNTs-SO3The H is used as a substrate material, so that the specific surface area of the immunosensor can be increased, the characteristic of strong conductivity of the multi-walled carbon nanotube can be played, the conductivity of the electrode is improved, Au NPs are a material with biological affinity, more antibodies can be combined, meanwhile, the conductivity of the Au NPs is very good, and the transfer and transmission of electrons can be accelerated. Ag @ Pt-CuS is used as a detection antibody marker, so that the sensitivity of the immunosensor can be increased, and the catalytic performance of the immunosensor can be improved. The CuS is used as a transition metal sulfide semiconductor, and has good biocompatibility, high electron transport performance and good conductivity. The Ag @ Pt nano particle has strong catalytic performance and can further improve the sensitivity of the immunosensor. The invention adopts Au NPs @ MWCNTs-SO3The sandwich type immunosensor is constructed by taking H as a substrate material and Ag @ Pt-CuS as a detection antibody marker, so that the amplification of a measurement signal is realized, the detection sensitivity of a tumor marker is improved, and the detection limit is reduced;
(2) the Ag @ Pt-CuS mark-based immunosensor realizes the detection of a tumor marker CEA, the linear range of the immunosensor is 10 fg-80 ng/mL, the detection limit is minimum 3.33fg/mL, the detection of a tumor marker AFP is realized, the linear range of the immunosensor is 50 fg-100 ng/mL, the detection limit is minimum 16.7fg/mL, and the purpose of accurate determination can be achieved.
Detailed Description
The present invention will now be further illustrated by, but not limited to, specific embodiments thereof.
Example 1 a method for preparing an Ag @ Pt-CuS labeled immunosensor, comprising the steps of:
(1) al for glassy carbon electrode with diameter of 4 mm2O3Polishing the polishing powder into a mirror surface, and cleaning with ultrapure water;
(2) au NPs @ MWCNTs-SO of 6 mu L and 1.0 mg/mL3H solution is dripped on the surface of the electrode, and is dried at room temperature, and the surface of the electrode is washed by ultrapure water and is dried;
(3) continuously capturing and resisting tumor markers of 6 mu L and 8 mu g/mLBody Ab1Dropwise adding the solution to the surface of an electrode, washing with ultrapure water, and drying in a refrigerator at 4 ℃;
(4) continuously dropwise adding bovine serum albumin BSA solution with the mass fraction of 1% to the surface of the electrode by 3 muL and 0.5 mg/mL to seal the non-specific active sites on the surface of the electrode, washing the surface of the electrode with ultrapure water, removing unbound BSA, and drying in a refrigerator at 4 ℃;
(5) continuously dropwise adding a series of tumor marker antigen Ag solutions with different concentrations, namely 6 muL and 10 pg/mL-100 ng/mL, washing the surface of the electrode with ultrapure water, and airing in a refrigerator at 4 ℃;
(6) continuously dropwise adding detection antibody hatching Ag @ Pt-CuS-Ab of 6 mu L and 1.5 mg/mL2And putting the solution on the surface of an electrode, incubating for 40min in a refrigerator at 4 ℃, washing with ultrapure water, and drying to prepare the Ag @ Pt-CuS labeled immunosensor.
Embodiment 2 a method for preparing an Ag @ Pt-CuS labeled immunosensor, comprising the steps of:
(1) al for glassy carbon electrode with diameter of 4 mm2O3Polishing the polishing powder into a mirror surface, and cleaning with ultrapure water;
(2) au NPs @ MWCNTs-SO of 6 mu L and 2.0 mg/mL3H solution is dripped on the surface of the electrode, and is dried at room temperature, and the surface of the electrode is washed by ultrapure water and is dried;
(3) continuing to capture the antibody Ab of the tumor marker of 6 mu L and 10 mu g/mL1Dropwise adding the solution to the surface of an electrode, washing with ultrapure water, and drying in a refrigerator at 4 ℃;
(4) continuously dropwise adding bovine serum albumin BSA solution with the mass fraction of 1% to the surface of the electrode by 3 muL and 1.0 mg/mL to seal the non-specific active sites on the surface of the electrode, washing the surface of the electrode with ultrapure water, removing unbound BSA, and drying in a refrigerator at 4 ℃;
(5) continuously dropwise adding a series of tumor marker antigen Ag solutions with different concentrations, namely 6 muL and 10 pg/mL-100 ng/mL, washing the surface of the electrode with ultrapure water, and airing in a refrigerator at 4 ℃;
(6) continuously dropwise adding detection antibody hatching Ag @ Pt-CuS-Ab of 6 mu L and 2.5 mg/mL2Solution in electrode meterAnd (3) putting the mixture into a refrigerator at 4 ℃ for incubation for 40min, washing with ultrapure water, and drying in the air to prepare the Ag @ Pt-CuS labeled immunosensor.
Embodiment 3 a method for preparing an Ag @ Pt-CuS labeled immunosensor, comprising the steps of:
(1) al for glassy carbon electrode with diameter of 4 mm2O3Polishing the polishing powder into a mirror surface, and cleaning with ultrapure water;
(2) au NPs @ MWCNTs-SO of 6 mu L and 3.0 mg/mL3H solution is dripped on the surface of the electrode, and is dried at room temperature, and the surface of the electrode is washed by ultrapure water and is dried;
(3) continuing to capture the antibody Ab of the tumor marker of 6 mu L and 12 mu g/mL1Dropwise adding the solution to the surface of an electrode, washing with ultrapure water, and drying in a refrigerator at 4 ℃;
(4) continuously dropwise adding bovine serum albumin BSA solution with the mass fraction of 1% to the surface of the electrode by 3 muL and 1.5 mg/mL to seal the non-specific active sites on the surface of the electrode, washing the surface of the electrode with ultrapure water, removing unbound BSA, and drying in a refrigerator at 4 ℃;
(5) continuously dropwise adding a series of tumor marker antigen Ag solutions with different concentrations, namely 6 muL and 10 pg/mL-100 ng/mL, washing the surface of the electrode with ultrapure water, and airing in a refrigerator at 4 ℃;
(6) continuously dropwise adding detection antibody hatching Ag @ Pt-CuS-Ab of 6 mu L and 3.5 mg/mL2And putting the solution on the surface of an electrode, incubating for 40min in a refrigerator at 4 ℃, washing with ultrapure water, and drying to prepare the Ag @ Pt-CuS labeled immunosensor.
Au NPs @ MWCNTs-SO as described in example 43H solution preparation, the steps are as follows:
(1) preparation of Au NPs solution
Mixing 1mL of chloroauric acid with the mass fraction of 1% and 99 mL of ultrapure water, refluxing for 15min at 100 ℃, then adding 1mL of sodium citrate solution with the mass fraction of 10mg/mL, and keeping stirring for 10min to prepare uniformly dispersed wine red Au NPs solution;
（2）MWCNTs-SO3preparation of H
Firstly, MWCNTs are put in a vacuum drying box at 60 DEG CDrying for 24H, then adding 1g MWCNTs and 30mL concentrated H in sequence into a three-neck flask2SO4And 10mL concentrated HNO3Performing ultrasonic treatment for 10min to disperse uniformly, refluxing at 120 ℃ for 20min under stirring, cooling to room temperature, and filtering; washing black precipitate ultrapure water, filtering to neutrality, drying in a vacuum drying oven at 60 ℃ for 8h to obtain MWCNTs-SO3H；
（3）Au NPs@MWCNTs-SO3Preparation of H
5mgMWCNTs-SO3Dispersing H in 5mL of Au NPs solution, ultrasonically dispersing for 1H, carrying out suction filtration on a polytetrafluoroethylene filter membrane, and drying a product in a vacuum drying oven at the temperature of 30 ℃ for 8H to obtain Au NPs @ MWCNTs-SO3H。
Au NPs @ MWCNTs-SO described in example 53H solution preparation, the steps are as follows:
(1) preparation of Au NPs solution
Mixing 2mL of chloroauric acid with the mass fraction of 1% and 99 mL of ultrapure water, refluxing for 15min at 100 ℃, then adding 2mL of sodium citrate solution with the mass fraction of 10mg/mL, and keeping stirring for 20min to prepare uniformly dispersed wine red Au NPs solution;
（2）MWCNTs-SO3preparation of H
Firstly, MWCNTs are dried in a vacuum drying oven at 60 ℃ for 24H, and then 1g of MWCNTs and 30mL of concentrated H are sequentially added into a three-neck flask2SO4And 10mL concentrated HNO3Performing ultrasonic treatment for 10min to disperse uniformly, refluxing at 120 ℃ for 30min under stirring, cooling to room temperature, and filtering; washing black precipitate ultrapure water, filtering to neutrality, drying in a vacuum drying oven at 60 ℃ for 10h to obtain MWCNTs-SO3H；
（3）Au NPs@MWCNTs-SO3Preparation of H
Mixing 10mgMWCNTs-SO3Dispersing H in 10mL of Au NPs solution, ultrasonically dispersing for 1H, carrying out suction filtration on a polytetrafluoroethylene filter membrane, and drying a product in a vacuum drying oven at the temperature of 30 ℃ for 10H to obtain Au NPs @ MWCNTs-SO3H。
Au NPs @ MWCNTs-SO described in example 63H solution preparation, the steps are as follows:
(1) preparation of Au NPs solution
Mixing 3mL of chloroauric acid with the mass fraction of 1% and 99 mL of ultrapure water, refluxing for 15min at 100 ℃, then adding 3mL of sodium citrate solution with the mass fraction of 10mg/mL, and keeping stirring for 30min to prepare uniformly dispersed wine red Au NPs solution;
（2）MWCNTs-SO3preparation of H
Firstly, MWCNTs are dried in a vacuum drying oven at 60 ℃ for 24H, and then 1g of MWCNTs and 30mL of concentrated H are sequentially added into a three-neck flask2SO4And 10mL concentrated HNO3Performing ultrasonic treatment for 10min to disperse uniformly, refluxing at 120 ℃ for 40min under stirring, cooling to room temperature, and filtering; washing black precipitate ultrapure water, filtering to neutrality, drying in a vacuum drying oven at 60 ℃ for 12h to obtain MWCNTs-SO3H；
（3）Au NPs@MWCNTs-SO3Preparation of H
15mgMWCNTs-SO3Dispersing H in 15mL of Au NPs solution, ultrasonically dispersing for 1H, carrying out suction filtration on a polytetrafluoroethylene filter membrane, and drying a product in a vacuum drying oven at the temperature of 30 ℃ for 12H to obtain Au NPs @ MWCNTs-SO3H。
Detection of antibody hatching Ag @ Pt-CuS-Ab as described in example 72The solution was prepared by the following steps:
(1) preparation of Ag @ Pt nanoparticles
While stirring, add 0.05mL of 1 mol. L-12mL of KOH, 0.01 mol/L of ascorbic acid and 0.05g of PVP were added to 2mL of ultrapure water, and then 4mL of 0.002 mol/L of AgNO was added3Dropwise adding the mixture into the solution, and then continuously dropwise adding 4mL of 0.002 mol/L H2PtCl6Stirring for 1h at 60 ℃, centrifugally washing the product for three times by using an absolute ethanol ultrapure water mixed solution with the volume ratio of 1:1, and ultrasonically dispersing the product in 5mL of ultrapure water to prepare an Ag @ Pt nanoparticle dispersion solution for later use;
(2) preparation of CuS microspheres
At room temperature, 1g of PVP was dissolved in 40mL of ultrapure water, and 0.5g of Cu (NO) was added3)▪3H2Mixing O, continuously adding 20 mL of ethylene glycol and 0.2 g of thiourea in sequence, continuously stirring for 1h, transferring the product into a three-neck flask, refluxing at 160 ℃ for 2h under the stirring state to obtain a black suspension, and naturally cooling to room temperatureWarming, centrifuging, washing with absolute ethyl alcohol for three times, and drying in a vacuum drying oven at 60 ℃ for 8h to obtain CuS microspheres;
(3) preparation of Ag @ Pt-CuS
Sequentially adding 0.1g of CuS microspheres and 0.1g of LAPTES into 5mL of absolute ethyl alcohol, heating to 90 ℃ in an oil bath pot, and keeping refluxing for 1 h; cooling to room temperature, centrifuging, washing with absolute ethyl alcohol and ultrapure water for three times, and drying in a vacuum drying oven at 50 ℃ to obtain aminated CuS microspheres;
adding 3mg of aminated CuS microspheres into 3mLAg @ Pt nanoparticle dispersion liquid, carrying out ultrasonic treatment for 1h, centrifuging, washing with absolute ethyl alcohol for three times, and drying in a vacuum drying oven at 50 ℃ for 12 h;
(4) detection of antibody hatching Ag @ Pt-CuS-Ab2Preparation of the solution
1mL of 2mg/mL Ag @ Pt-CuS dispersion was added to 0.5mL of 10. mu.g/mL of the tumor marker-detecting antibody Ab2In the solution, oscillating and incubating for 12h in a constant-temperature oscillation box; centrifugally separating, and re-dispersing into 1mL of phosphate buffer solution with pH =7.38 to prepare the detection antibody hatching material Ag @ Pt-CuS-Ab2The solution was stored at 4 ℃ until use.
Detection of antibody hatching Ag @ Pt-CuS-Ab as described in example 82The solution was prepared by the following steps:
(1) preparation of Ag @ Pt nanoparticles
While stirring, add 0.10mL of 1 mol. L-13mL of 0.01 mol/L ascorbic acid and 0.05g of PVP was added to 2mL of ultrapure water, and then 4mL of 0.002 mol/L AgNO was added3Dropwise adding the mixture into the solution, and then continuously dropwise adding 4mL of 0.002 mol/L H2PtCl6Stirring for 1h at 60 ℃, centrifugally washing the product for three times by using an absolute ethanol ultrapure water mixed solution with the volume ratio of 1:1, and ultrasonically dispersing the product in 5mL of ultrapure water to prepare an Ag @ Pt nanoparticle dispersion solution for later use;
(2) preparation of CuS microspheres
At room temperature, 1g of PVP was dissolved in 40mL of ultrapure water, and 0.5g of Cu (NO) was added3)▪3H2Mixing O, continuously adding 30mL of ethylene glycol and 0.3 g of thiourea in sequence, continuously stirring for 1h, transferring the product into a three-neck flask,refluxing for 2h at 160 ℃ under a stirring state to obtain a black suspension, naturally cooling to room temperature, centrifuging, washing with absolute ethyl alcohol for three times, and drying in a vacuum drying oven at 60 ℃ for 10h to obtain CuS microspheres;
(3) preparation of Ag @ Pt-CuS
Sequentially adding 0.1g of CuS microspheres and 0.2 g of LAPTES into 10mL of absolute ethyl alcohol, heating to 90 ℃ in an oil bath pot, and keeping refluxing for 2 h; cooling to room temperature, centrifuging, washing with absolute ethyl alcohol and ultrapure water for three times, and drying in a vacuum drying oven at 50 ℃ to obtain aminated CuS microspheres;
adding 3mg of aminated CuS microspheres into 3mLAg @ Pt nanoparticle dispersion liquid, carrying out ultrasonic treatment for 1h, centrifuging, washing with absolute ethyl alcohol for three times, and drying in a vacuum drying oven at 50 ℃ for 12 h;
(4) detection of antibody hatching Ag @ Pt-CuS-Ab2Preparation of the solution
2mL of 2mg/mL Ag @ Pt-CuS dispersion was added to 1.0mL of 10. mu.g/mL of the tumor marker-detecting antibody Ab2In the solution, oscillating and incubating for 12h in a constant-temperature oscillation box; centrifugally separating, and re-dispersing into 2mL of phosphate buffer solution with pH =7.38 to prepare the detection antibody hatching material Ag @ Pt-CuS-Ab2The solution was stored at 4 ℃ until use.
Detection of antibody hatching Ag @ Pt-CuS-Ab as described in example 92The solution was prepared by the following steps:
(1) preparation of Ag @ Pt nanoparticles
While stirring, add 0.15mL of 1 mol. L-1KOH, 4mL, 0.01 mol/L ascorbic acid and 0.05g PVP was added to 2mL of ultrapure water, followed by 4mL, 0.002 mol/L AgNO3Dropwise adding the mixture into the solution, and then continuously dropwise adding 4mL of 0.002 mol/L H2PtCl6Stirring for 1h at 60 ℃, centrifugally washing the product for three times by using an absolute ethanol ultrapure water mixed solution with the volume ratio of 1:1, and ultrasonically dispersing the product in 5mL of ultrapure water to prepare an Ag @ Pt nanoparticle dispersion solution for later use;
(2) preparation of CuS microspheres
At room temperature, 1g of PVP was dissolved in 40mL of ultrapure water, and 0.5g of Cu (NO) was added3)▪3H2O mixing, and adding 40mL of ethanediolContinuously stirring alcohol and 0.4 g of thiourea for 1h, transferring the product into a three-neck flask, refluxing for 2h at 160 ℃ under the stirring state to obtain a black suspension, naturally cooling to room temperature, centrifuging, washing with absolute ethyl alcohol for three times, and drying for 12h in a vacuum drying oven at 60 ℃ to obtain the CuS microspheres;
(3) preparation of Ag @ Pt-CuS
Sequentially adding 0.1g of CuS microspheres and 0.3 g of LAPTES into 15mL of absolute ethyl alcohol, heating to 90 ℃ in an oil bath pot, and keeping refluxing for 3 h; cooling to room temperature, centrifuging, washing with absolute ethyl alcohol and ultrapure water for three times, and drying in a vacuum drying oven at 50 ℃ to obtain aminated CuS microspheres;
adding 3mg of aminated CuS microspheres into 3mLAg @ Pt nanoparticle dispersion liquid, carrying out ultrasonic treatment for 1h, centrifuging, washing with absolute ethyl alcohol for three times, and drying in a vacuum drying oven at 50 ℃ for 12 h;
(4) detection of antibody hatching Ag @ Pt-CuS-Ab2Preparation of the solution
3mL of 2mg/mL Ag @ Pt-CuS dispersion was added to 1.5mL of 10. mu.g/mL of the tumor marker-detecting antibody Ab2In the solution, oscillating and incubating for 12h in a constant-temperature oscillation box; centrifugally separating, and re-dispersing into 3mL of phosphate buffer solution with pH =7.38 to prepare the detection antibody hatching material Ag @ Pt-CuS-Ab2The solution was stored at 4 ℃ until use.
Example 10 detection of the tumor marker CEA, the procedure is as follows:
(1) an electrochemical workstation is used for testing by a three-electrode system, a saturated calomel electrode is used as a reference electrode, a platinum wire electrode is used as an auxiliary electrode, the prepared sensor is used as a working electrode, and the test is carried out in 10mL of phosphate buffer solution containing 10 mmol/L of potassium ferricyanide and having the pH value of 7.38;
(2) detecting the tumor marker by a time-lapse current method, selecting-0.4V as the detection of current measurement, wherein the input voltage is-0.4V, the sampling interval is 0.1 s, and the running time is 300 s;
(3) recording current values corresponding to the tumor marker antigens under different concentrations;
(4) obtaining the concentration of the tumor marker antigen in the sample to be detected by using a working curve method;
(5) and drawing a working curve according to the linear relation between the obtained current intensity and the CEA concentration, wherein the measured linear range is 10 fg-80 ng/mL, and the detection limit is 3.3 fg/mL.
Example 11 detection of tumor marker AFP
AFP in the sample was detected according to the method of example 10, with a linear range of 50fg to 100 ng/mL and a detection limit of 16.7 fg/mL.

Claims (5)

1. A preparation method of an Ag @ Pt-CuS labeled immunosensor comprises the following steps:
(1) al for glassy carbon electrode with diameter of 4 mm2O3Polishing the polishing powder into a mirror surface, and cleaning with ultrapure water;
(2) au NPs @ MWCNTs-SO of 6 mu L and 1.0-3.0 mg/mL3H solution is dripped on the surface of the electrode, and is dried at room temperature, and the surface of the electrode is washed by ultrapure water and is dried;
(3) continuing to capture the antibody Ab of the tumor marker of 6 mu L and 8-12 mu g/mL1Dropwise adding the solution to the surface of an electrode, washing with ultrapure water, and drying in a refrigerator at 4 ℃;
(4) continuously dropwise adding bovine serum albumin BSA solution with the mass fraction of 1% to the surface of the electrode by 3 muL, 0.5-1.5 mg/mL to seal non-specific active sites on the surface of the electrode, washing the surface of the electrode with ultrapure water, removing unbound BSA, and drying in a refrigerator at 4 ℃;
(5) continuously dropwise adding a series of tumor marker antigen solutions with different concentrations, namely 6 muL and 10 pg/mL-100 ng/mL, washing the surface of the electrode with ultrapure water, and airing in a refrigerator at 4 ℃;
(6) continuously dropwise adding the detection antibody hatching Ag @ Pt-CuS-Ab of 6 mu L and 1.5-3.5 mg/mL2Placing the solution on the surface of an electrode, placing the electrode in a refrigerator at 4 ℃ for incubation for 40min, washing with ultrapure water, and drying in the air to prepare the Ag @ Pt-CuS labeled immunosensor;
the detection antibody hatching Ag @ Pt-CuS-Ab2The solution was prepared by the following steps:
(a) preparation of Ag @ Pt nanoparticles
Under the condition of stirring, the mixture is stirred,mixing 0.05-0.15 mL of 1 mol. L-12-4 mL of KOH, 0.01 mol/L of ascorbic acid and 0.05g of PVP are added into 2mL of ultrapure water, and then 4mL of 0.002 mol/L of AgNO is added3Dropwise adding the mixture into the solution, and then continuously dropwise adding 4mL of 0.002 mol/L H2PtCl6Stirring for 1h at 60 ℃, centrifugally washing the product for three times by using an absolute ethanol ultrapure water mixed solution with the volume ratio of 1:1, and ultrasonically dispersing the product in 5mL of ultrapure water to prepare an Ag @ Pt nanoparticle dispersion solution for later use;
(b) preparation of CuS microspheres
1g PVP was dissolved in 40mL of ultrapure water at room temperature, and 0.5g Cu (NO) was added3)▪3H2Mixing O, continuously adding 20-40 mL of ethylene glycol and 0.2-0.4 g of thiourea in sequence, continuously stirring for 1h, transferring the product into a three-neck flask, refluxing for 2h at 160 ℃ under a stirring state to obtain a black suspension, naturally cooling to room temperature, centrifuging, washing with absolute ethyl alcohol for three times, and drying in a vacuum drying oven at 60 ℃ for 8-12 h to obtain the CuS microsphere;
(c) preparation of Ag @ Pt-CuS
Sequentially adding 0.1g of CuS microspheres and 0.1-0.3 mL of APTES into 5-15 mL of absolute ethyl alcohol, heating to 90 ℃ in an oil bath pot, and keeping refluxing for 1-3 h; cooling to room temperature, centrifuging, washing with absolute ethyl alcohol and ultrapure water for three times, and drying in a vacuum drying oven at 50 ℃ to obtain aminated CuS microspheres;
adding 3mg of aminated CuS microspheres into 3mL of Ag @ Pt nanoparticle dispersion liquid, carrying out ultrasonic treatment for 1h, centrifuging, washing with absolute ethyl alcohol for three times, and drying in a vacuum drying oven at 50 ℃ for 12 h;
(d) detection of antibody hatching Ag @ Pt-CuS-Ab2Preparation of the solution
Adding 1-3 mL and 2mg/mL of Ag @ Pt-CuS dispersion into 0.5-1.5 mL and 10 mu g/mL of tumor marker detection antibody Ab2In the solution, oscillating and incubating for 12h in a constant-temperature oscillation box; centrifugally separating, and re-dispersing into 1-3 mL of phosphoric acid buffer solution with pH =7.38 to prepare the detection antibody hatching Ag @ Pt-CuS-Ab2The solution was stored at 4 ℃ until use.
2. As in claimThe method for preparing an Ag @ Pt-CuS labeled immunosensor, described in claim 1, said Au NPs @ MWCNTs-SO3H solution preparation, the steps are as follows:
(1) preparation of Au NPs solution
Mixing 1-3 mL of chloroauric acid with the mass fraction of 1% and 99 mL of ultrapure water, refluxing for 15min at 100 ℃, then adding 1-3 mL of 10mg/mL sodium citrate solution, and keeping stirring for 10-30 min to prepare uniformly dispersed wine red Au NPs solution;
（2）MWCNTs-SO3preparation of H
Firstly, MWCNTs are dried in a vacuum drying oven at 60 ℃ for 24H, and then 1g of MWCNTs and 30mL of concentrated H are sequentially added into a three-neck flask2SO4And 10mL concentrated HNO3Uniformly dispersing by ultrasonic treatment for 10min, refluxing for 20-40 min at 120 ℃ while stirring, cooling to room temperature, and filtering; washing and filtering black precipitate ultrapure water to be neutral, drying in a vacuum drying oven at 60 ℃ for 8-12 h to obtain MWCNTs-SO3H；
（3）Au NPs@MWCNTs-SO3Preparation of H
5-15 mg of MWCNTs-SO3Dispersing H in 5-15 mL of Au NPs solution, ultrasonically dispersing for 1H, carrying out suction filtration on a polytetrafluoroethylene filter membrane, and drying a product in a vacuum drying oven at the temperature of 30 ℃ for 8-12H to obtain Au NPs @ MWCNTs-SO3H。
3. The method of claim 1, wherein the tumor marker is selected from the group consisting of: CEA, AFP.
4. The application of the Ag @ Pt-CuS labeled immunosensor prepared by the preparation method of claim 1 in tumor marker detection comprises the following steps:
(1) testing by using an electrochemical workstation in a three-electrode system, taking a saturated calomel electrode as a reference electrode, taking a platinum wire electrode as an auxiliary electrode, taking the prepared sensor as a working electrode, and testing in 10mL of potassium ferricyanide solution containing 10 mmol/L;
(2) detecting the tumor marker by a time-lapse current method, selecting-0.4V as the detection of current measurement, wherein the input voltage is-0.4V, the sampling interval is 0.1 s, and the running time is 300 s;
(3) when the background current tended to be stable, 10 μ L of 5 mol/L hydrogen peroxide solution was injected into 10mL of 50 mmol/L phosphate buffer solution with pH =7.38 every 50 s, and the change in current was recorded.
5. The use of an Ag @ Pt-CuS labeled immunosensor prepared by the preparation method of claim 4 in the detection of a tumor marker, wherein the tumor marker is selected from one of the following: CEA, AFP.
CN201811058947.2A 2018-09-12 2018-09-12 Preparation method and application of Ag @ Pt-CuS labeled immunosensor Active CN109613244B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201811058947.2A CN109613244B (en) 2018-09-12 2018-09-12 Preparation method and application of Ag @ Pt-CuS labeled immunosensor

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201811058947.2A CN109613244B (en) 2018-09-12 2018-09-12 Preparation method and application of Ag @ Pt-CuS labeled immunosensor

Publications (2)

Publication Number Publication Date
CN109613244A CN109613244A (en) 2019-04-12
CN109613244B true CN109613244B (en) 2021-09-24

Family

ID=66001551

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201811058947.2A Active CN109613244B (en) 2018-09-12 2018-09-12 Preparation method and application of Ag @ Pt-CuS labeled immunosensor

Country Status (1)

Country Link
CN (1) CN109613244B (en)

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110687168B (en) * 2019-10-31 2022-02-11 上海应用技术大学 Ag @ Pt nanocrystal and preparation method thereof, formaldehyde sensor and preparation and application thereof
CN111735861A (en) * 2020-07-08 2020-10-02 山东理工大学 Preparation method and application of photoelectrochemical biosensor for detecting beta-lactoglobulin
CN113295867B (en) * 2021-05-28 2023-09-12 山东理工大学 Preparation method and application of electrochemical immunosensor for amplifying palladium-silver copper mesoporous nanosphere signals
CN113866414B (en) * 2021-10-12 2023-11-07 济南大学 Bi-based 2 MoO 6 Preparation method and application of dual-mode electrochemical immunosensor of-Ag

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106442994A (en) * 2016-09-14 2017-02-22 山东理工大学 Preparation method and application of electrochemical immunosensor based on Ag@Au nanocomposite
CN106770529A (en) * 2017-02-13 2017-05-31 山东理工大学 A kind of preparation method and application of the interlayer type immunosensor based on silver load vulcanization copper nano-wire
CN107525835A (en) * 2017-08-09 2017-12-29 山东理工大学 A kind of preparation method and application of the immunosensor of the phenolic resin micropore carbon ball of functionalization based on Au AgNPs

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP5473382B2 (en) * 2008-04-17 2014-04-16 キヤノン株式会社 Immunoassay method
CA2978159A1 (en) * 2016-08-31 2018-02-28 Mcmaster University Carbon nanotube-based magnetic bio-ink and biosensors and methods of making and using

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106442994A (en) * 2016-09-14 2017-02-22 山东理工大学 Preparation method and application of electrochemical immunosensor based on Ag@Au nanocomposite
CN106770529A (en) * 2017-02-13 2017-05-31 山东理工大学 A kind of preparation method and application of the interlayer type immunosensor based on silver load vulcanization copper nano-wire
CN107525835A (en) * 2017-08-09 2017-12-29 山东理工大学 A kind of preparation method and application of the immunosensor of the phenolic resin micropore carbon ball of functionalization based on Au AgNPs

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
Fabrication of an electrochemical sensor based on gold nanoparticles/carbon nanotubes as nanocomposite materials: determination of myricetin in some drinks;Reza Hajian 等;《PLoS One》;20140508;第9卷(第5期);第1-7页 *
Role of Au(NPs) in the enhanced response of Au(NPs)-decorated MWCNT electrochemical biosensor;Shahid Mehmood 等;《Int J Nanomedicine》;20180417;第13卷;第2093-2106页 *
三种基于碳纳米复合材料的亚硝酸盐电化学传感器的构置及其应用;李博强;《中国优秀硕士学位论文全文数据库 工程科技Ⅰ辑》;20151015(第10期);第B014-175页 *
蛋白质介导Pt/Au/CuS纳米粒子的合成及其在生物成像与肿瘤治疗中的应用;王智明;《中国优秀硕士学位论文全文数据库 医药卫生科技辑》;20180215(第02期);第E072-152页 *

Also Published As

Publication number Publication date
CN109613244A (en) 2019-04-12

Similar Documents

Publication Publication Date Title
CN109613244B (en) Preparation method and application of Ag @ Pt-CuS labeled immunosensor
CN109507174B (en) Preparation of curcumin composite ZnO nanoparticle based quenching luminol electrochemical luminescence sensor
Li et al. Simultaneous electrochemical immunoassay of three liver cancer biomarkers using distinguishable redox probes as signal tags and gold nanoparticles coated carbon nanotubes as signal enhancers
CN106442994B (en) A kind of preparation method and application of the electrochemical immunosensor based on Ag@Au nano composite materials
CN108802133A (en) A kind of preparation method and application of detection stomach neoplasms tumor markers interlayer type immunosensor
CN105572356B (en) A kind of preparation method and application of breast cancer tumour marker immunosensor
CN109839501B (en) Electrochemiluminescence immunosensor for measuring circulating tumor cells and preparation method and application thereof
Xiang et al. A redox cycling-amplified electrochemical immunosensor for α-fetoprotein sensitive detection via polydopamine nanolabels
CN110794017B (en) Preparation method of electrochemical immunosensor for detecting procalcitonin
CN111693571B (en) Method for detecting GPC3 based on light addressing potential sensor
CN108663424B (en) Preparation method and application of immunosensor based on sea urchin-shaped core-shell gold @ palladium nanospheres
CN110687182A (en) Preparation method of electrochemical immunosensor for detecting prostate specific antigen
CN108896638B (en) Preparation method and application of immunosensor based on titanium dioxide doped graphene loaded sea cucumber-like gold-palladium core-shell nanoparticles
CN108918853B (en) Pd @ Ag @ CeO2Preparation method and application of labeled immunosensor
CN108469461B (en) Preparation method and application of sandwich type lung cancer marker electrochemical sensor
CN110133252A (en) For detecting kit and detection method and its application of carcinomebryonic antigen
CN109115855A (en) A kind of preparation method and application for the electrochemical immunosensor detecting Alzheimer's disease marker
CN110441294B (en) Co-coated based on ferritin3O4Preparation method of biosensor with core-shell structure
CN106093396A (en) A kind of preparation method and application of immunosensor based on Au GQD@PtPd
CN110687175A (en) Construction method of electrochemical luminescence sensor based on cerium dioxide and nano-silver dual-enhanced perylene tetracarboxylic acid luminescence
CN109991298B (en) Preparation method and application of Pt @ MOF-GO labeled electrochemical sensor
CN106770530B (en) A kind of preparation method and application of squamous cell carcinoma marker interlayer type immunosensor
CN111198222B (en) Preparation and use methods of sandwich type electrochemical immunosensor for detecting prostate specific antigen
CN109100400A (en) Sensor and its preparation method and application for detecting concanavalin A
CN109709188B (en) Preparation method and application of sandwich type immunosensor marked by nitrogen-sulfur double-doped graphene oxide

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant