CN110441528A - One kind being based on core-shell structure Mo2The building of the cTnI immunosensor of C@C nano ball - Google Patents
One kind being based on core-shell structure Mo2The building of the cTnI immunosensor of C@C nano ball Download PDFInfo
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
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- G01N27/28—Electrolytic cell components
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- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
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- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
- G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
- G01N27/3278—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction involving nanosized elements, e.g. nanogaps or nanoparticles
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- 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
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- 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6887—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids from muscle, cartilage or connective tissue
Abstract
The present invention relates to one kind to be based on core-shell structure Mo2The building of the cTnI immunosensor of C@C nano ball, belongs to novel sensor constructing technology field.Based on specificity good between antigen-antibody, which utilizes amido modified multi-walled carbon nanotube@perfluorinated sulfonic acid-teflon-copolymers MWCNTs-NH2@Nafion solution has the core-shell structure Mo of polyethyleneimine PEI as base material, load2C@C nano ball is secondary antibody marker, and boron nitride quantum dot BNQDs is coreagent, constructs cTnI immunosensor by LBL self-assembly.The electrochemiluminescimmunosensor immunosensor that the present invention constructs has wider detection range, higher sensitivity and lower detection limit, has great importance to the detection of cTnI.
Description
Technical field
The present invention relates to a kind of preparation method and application of transition metal nano material electrochemiluminescimmunosensor immunosensor,
More specifically, the present invention is using MWCNTs-NH2@Nafion solution has polyethyleneimine PEI as base material, load
Core-shell structure Mo2C@C nano ball is secondary antibody marker, and BNQDs is coreagent, the myocardium calcium constructed by LBL self-assembly
The specific detection to cTnI may be implemented in protein I immunosensor, belongs to novel sensor constructing technology field.
Background technique
In early days, accurately treatment myocardial damage be it is very useful, the irreversible lesion to heart can be prevented, especially
Patients of acute myocardial infarction, and survival rate can be significantly improved.And cardiac troponin is a kind of regulatory protein of myocardial contraction, position
In the bifilar certain position of myocardium actin.When myocardial damage occurs, cTnI can be promptly released into blood,
Wherein it is directly proportional to cTnI to be discharged into the amount in blood for the degree of myocardial damage.Currently, cTnI is public
Think the marker of diagnosis acute myocardial infarction AMI and acute coronary syndrome.Up to the present, a variety of inspections are had been set up
The analysis method of thought-read Troponin I, such as liquid chromatogram, capillary electrophoresis and surface plasma resonance detection method etc., but
The instrument and equipment that most methods need is expensive, and time-consuming for preparation of samples, is unsuitable for the screening of a large amount of samples.Therefore, it is necessary to establish
A kind of high sensitivity, fast response time and cTnI measurement method easy to operate.Electrogenerated chemiluminescence immune sensing
Device, especially interlayer type electrochemiluminescimmunosensor immunosensor with its high sensitivity, the at low cost and response time is fast the advantages that, In
Extensive interest is caused in bioanalysis.
The present invention is based on nano-functional materials to construct a kind of novel electrochemiluminescimmunosensor immunosensor, for cardiac muscle
The detection of calcium protein I.Utilize MWCNTs-NH2@Nafion solution has the nucleocapsid of polyethyleneimine PEI as base material, load
Structure Mo2C@C nano ball is secondary antibody marker, and boron nitride quantum dot BNQDs is coreagent.Test result shows electroluminescentization
It is high to learn electrochemiluminescent immunoassay transducer sensitivity, stability is good, and detection limit is low, is based on above-mentioned discovery, and inventor completes the present invention.
Summary of the invention
An object of the present invention is with MWCNTs-NH2@Nafion solution utilizes antigen-antibody as base material
Specific binding, constructs that a kind of selectivity is good, quick and overdelicate electrochemiluminescimmunosensor immunosensor, realizes myocardium calcium egg
Simple, the Sensitive Detection of white I.
The second object of the present invention is to load the core-shell structure Mo for having polyethyleneimine PEI2C@C nano ball is secondary antibody mark
Remember object, provides a kind of based on transition metal material core-shell structure Mo2The electrochemiluminescimmunosensor immunosensor of C@C nano ball
Preparation method, the sensor stability of this method preparation is good, selectivity good, high sensitivity and favorable reproducibility.
The third object of the present invention is constructed a kind of novel based on core-shell structure Mo using BNQDs as coreagent2C@C
The cTnI immunosensor of nanosphere.
Technical solution of the present invention
1. one kind is based on core-shell structure Mo2The building of the cTnI immunosensor of C@C nano ball
(1) Al is used2O3Polishing powder polishes diameter as the glass-carbon electrode of 4 mm, and successively with dehydrated alcohol and ultrapure water, ultrasound is clear respectively
30 min are washed, by 6 μ L, 0.1 ~ 1.0 mg mL-1Amido modified multi-walled carbon nanotube@perfluorinated sulfonic acid-polytetrafluoroethylene (PTFE)
Copolymer MWCNTs-NH2@Nafion solution is added drop-wise to electrode surface, dries at room temperature；
(2) by 6 μ L, 10 mmol L-1Terpyridyl ruthenium solution drop coating to electrode surface, dry at room temperature；
(3) 1- ethyl -3- (3- dimethyl aminopropyl)-carbodiimides/n-hydroxysuccinimide EDC/NHS of 3 μ L is molten
Drop is added to electrode surface, to activate MWCNTs-NH2Amino in@Nafion, the phosphate buffer solution PBS for being 7.4 with pH
Electrode is rinsed, is dried at room temperature；
(4) by 6 μ L, 5 ~ 15 μ g mL-1CTnI capture antibody cTnI-Ab1, 3 μ L, mass fraction 0.5%
~ 2.0% bovine serum albumin(BSA) BSA solution is added drop-wise to electrode surface, rinses electrode with the PBS that pH is 7.4, dries at room temperature；
(5) 6 μ L, 10 are added dropwise-4 ~ 50 ng mL-1A series of various concentrations cTnI antigen to electrode surface,
Hatch 2 h, rinses electrode with the PBS that pH is 7.4, dry at room temperature；
(6) the core-shell structure Mo that 6 μ L load has polyethyleneimine PEI is added dropwise2The secondary antibody marker Mo of C@C nano ball2C@C/
PEI-Ab2Solution dries at room temperature, is made a kind of and is based on core-shell structure Mo2The cTnI immune sensing of C@C nano ball
Device.
2. MWCNTs-NH2The preparation of@Nafion solution
The carbon nanotube MWCNTs-COOH of 0.4 g, bis- ring ethylcarbodiimine DCC and 20 mg carboxylated are added to 2.5
In mL ethylenediamine EDA solution, heating reaction 4 days, by being centrifugally separating to obtain sediment, and use ultrapure water respectively at 120 DEG C
Washing precipitate obtains aminated carbon nano tube MWCNTs-NH repeatedly with dehydrated alcohol2, it places it at 35 DEG C and is dried in vacuo,
Then weigh the MWCNTs-NH of 0.04 ~ 0.4 mg2Sample dispersion is gathered in 0.4 mL, the perfluorinated sulfonic acid-that mass fraction is 0.5%
In TFE copolymer Nafion solution, by obtaining uniform MWCNTs-NH after 20 min of ultrasonic treatment2@Nafion is molten
Liquid.
3. the core-shell structure Mo that load has polyethyleneimine PEI2The secondary antibody marker Mo of C@C nano ball2C@C/PEI-Ab2
Solution preparation
(1) core-shell structure Mo2The preparation of C@C nano ball
12 ~ 30 mg ammonium molybdates and 500 ~ 1200 mg glucose are successively dissolved in 45 mL ultrapure waters, are then transferred into high pressure
In reaction kettle, make it in 180 DEG C of 10 h of reaction, after reaction, place the product in being centrifuged in centrifuge tube, with anhydrous second
Pure and mild ultrapure water washs three times respectively, is subsequently placed at 25 DEG C and is dried in vacuo 12 h, and obtained sample is ground, and
Using tube furnace 800 DEG C of 2 h of calcining under argon gas, core-shell structure Mo is obtained2C@C nano ball；
(2) load has the core-shell structure Mo of polyethyleneimine PEI2The secondary antibody marker Mo of C@C nano ball2C@C/PEI-Ab2Solution
Preparation
By 3.0 mL, 1.0 mg mL-1 Core-shell structure Mo2C@C nano ball is dispersed in 12 mL and contains 0.625 mol L-1Chlorination
Potassium and 1.25 mg mL-1In the mixed solution of PEI, 12 h are shaken after 2 h of ultrasound, uniform dark solution is obtained, is collected by centrifugation
Solid product three times and is scattered in the ultrapure water of 6 mL with ultrapure washing and obtains Mo2C@C/PEI solution, then pipettes 1
The above-mentioned solution of mL, and 200 μ L cTnIs are added and detect antibody cTnI-Ab2, 12 ~ 24 h of concussion hatching at 4 DEG C
After be centrifuged, disperse the solid in the PBS that 1 mL, pH is 7.4 again and obtain loading have the nucleocapsid knot of polyethyleneimine PEI
Structure Mo2The secondary antibody marker Mo of C@C nano ball2C@C/PEI-Ab2。
4. the detection of cTnI
(1) by reference electrode-Ag/AgCl electrode, be connected to chemical hair to electrode-platinum electrode and the working electrode modified
In the magazine of optical detector, the mixed solution of Tri-n-Propylamine solution and BNQDs that 10 mL contain NaCl is added in electrolytic cell,
The volume ratio of the two is 5:1, and wherein the concentration of Tri-n-Propylamine solution is 1 ~ 5 mmol L-1；
(2) it is detected with cTnI standard solution of the cyclic voltammetry to various concentration, the high pressure of photomultiplier tube is set
800 V are set to, scanning voltage is set as 0 ~ 1.0 V；
(3) according to the linear relationship of resulting electrochemical luminescence intensity value and cTnI log concentration, it is bent to draw work
Line.
Beneficial achievement of the invention
(1) the present inventor is by MWCNTs-NH2@Nafion solution is as base material, due to MWCNTs-NH2@
Nafion has good electric conductivity and big specific surface area, improves the sensitivity and stability of sensor；
(2) load is had the core-shell structure Mo of PEI by the present invention2The secondary antibody marker Mo of C@C nano ball2C@C/PEI-Ab2It is applied to
In the preparation of electrochemiluminescimmunosensor immunosensor, due to core-shell structure Mo2C@C nano ball has good electric conductivity and steady
Fixed chemical property improves the stability and reproducibility of sensor；
(3) present invention uses BNQDs for coreagent, constructs one based on core-shell structure Mo2The myocardium calcium egg of C@C nano ball
White I immunosensor, and effective detection has been carried out to cTnI, the method operation is relatively simple；
(4) electrochemiluminescimmunosensor immunosensor prepared by the present invention is used for the detection of cTnI, the electrogenerated chemiluminescence
Immunosensor stability is high, and favorable reproducibility, high sensitivity, the range of linearity is wide, may be implemented simple, quick, highly sensitive and special
Opposite sex detection.
Specific embodiment
Embodiment 1 is a kind of to be based on core-shell structure Mo2The building of the cTnI immunosensor of C@C nano ball
(1) Al is used2O3Polishing powder polishes diameter as the glass-carbon electrode of 4 mm, and successively with dehydrated alcohol and ultrapure water, ultrasound is clear respectively
30 min are washed, by 6 μ L, 0.1 mg mL-1Amido modified multi-walled carbon nanotube@perfluorinated sulfonic acid-teflon-copolymers
MWCNTs-NH2@Nafion solution is added drop-wise to electrode surface, dries at room temperature；
(2) by 6 μ L, 10 mmol L-1Terpyridyl ruthenium solution drop coating to electrode surface, dry at room temperature；
(3) 1- ethyl -3- (3- dimethyl aminopropyl)-carbodiimides/n-hydroxysuccinimide EDC/NHS of 3 μ L is molten
Drop is coated onto electrode surface, to activate MWCNTs-NH2Amino in@Nafion rinses electrode, room temperature with the PBS that pH is 7.4
Under dry；
(4) by 6 μ L, 5 μ g mL-1CTnI capture antibody cTnI-Ab1, 3 μ L, the ox blood that mass fraction is 0.5%
Pure protein B SA solution drop coating rinses electrode to electrode surface, with the PBS that pH is 7.4, dries at room temperature；
(5) 6 μ L, 10 are added dropwise-4 ~ 50 ng mL-1A series of various concentrations cTnI antigen to electrode surface,
Hatch 2 h, rinses electrode with the PBS that pH is 7.4, dry at room temperature；
(6) the core-shell structure Mo that 6 μ L load has polyethyleneimine PEI is added dropwise2The secondary antibody marker Mo of C@C nano ball2C@C/
PEI-Ab2Solution dries at room temperature, is made a kind of and is based on core-shell structure Mo2The cTnI immune sensing of C@C nano ball
Device.
Embodiment 2 is a kind of to be based on core-shell structure Mo2The building of the cTnI immunosensor of C@C nano ball
(1) Al is used2O3Polishing powder polishes diameter as the glass-carbon electrode of 4 mm, and successively with dehydrated alcohol and ultrapure water, ultrasound is clear respectively
30 min are washed, by 6 μ L, 0.5 mg mL-1Amido modified multi-walled carbon nanotube@perfluorinated sulfonic acid-teflon-copolymers
MWCNTs-NH2@Nafion solution is added drop-wise to electrode surface, dries at room temperature；
(2) by 6 μ L, 10 mmol L-1Terpyridyl ruthenium solution drop coating to electrode surface, dry at room temperature；
(3) 1- ethyl -3- (3- dimethyl aminopropyl)-carbodiimides/n-hydroxysuccinimide EDC/NHS of 3 μ L is molten
Drop is coated onto electrode surface, to activate MWCNTs-NH2Amino in@Nafion rinses electrode, room temperature with the PBS that pH is 7.4
Under dry；
(4) by 6 μ L, 10 μ g mL-1CTnI capture antibody cTnI-Ab1, 3 μ L, the ox blood that mass fraction is 1%
Pure protein B SA solution is added drop-wise to electrode surface, rinses electrode with the PBS that pH is 7.4, dries at room temperature；
(5) 6 μ L, 10 are added dropwise-4 ~ 50 ng mL-1A series of various concentrations cTnI antigen to electrode surface,
Hatch 2 h, rinses electrode with the PBS that pH is 7.4, dry at room temperature；
(6) the core-shell structure Mo that 6 μ L load has polyethyleneimine PEI is added dropwise2The secondary antibody marker Mo of C@C nano ball2C@C/
PEI-Ab2Solution dries at room temperature, is made a kind of and is based on core-shell structure Mo2The cTnI immune sensing of C@C nano ball
Device.
Embodiment 3 is a kind of to be based on core-shell structure Mo2The building of the cTnI immunosensor of C@C nano ball
(1) Al is used2O3Polishing powder polishes diameter as the glass-carbon electrode of 4 mm, and successively with dehydrated alcohol and ultrapure water, ultrasound is clear respectively
30 min are washed, by 6 μ L, 1.0 mg mL-1Amido modified multi-walled carbon nanotube@perfluorinated sulfonic acid-teflon-copolymers
MWCNTs-NH2@Nafion solution is added drop-wise to electrode surface, dries at room temperature；
(2) by 6 μ L, 10 mmol L-1Terpyridyl ruthenium solution drop coating to electrode surface, dry at room temperature；
(3) 1- ethyl -3- (3- dimethyl aminopropyl)-carbodiimides/n-hydroxysuccinimide EDC/NHS of 3 μ L is molten
Drop is coated onto electrode surface, to activate MWCNTs-NH2Amino in@Nafion rinses electrode, room temperature with the PBS that pH is 7.4
Under dry；
(4) by 6 μ L, 15 μ g mL-1CTnI capture antibody cTnI-Ab1, 3 μ L, the ox blood that mass fraction is 2%
Pure protein B SA solution is added drop-wise to electrode surface, rinses electrode with the PBS that pH is 7.4, dries at room temperature；
(5) 6 μ L, 10 are added dropwise-4 ~ 50 ng mL-1A series of various concentrations cTnI antigen to electrode surface,
Hatch 2 h, rinses electrode with the PBS that pH is 7.4, dry at room temperature；
(6) the core-shell structure Mo that 6 μ L load has polyethyleneimine PEI is added dropwise2The secondary antibody marker Mo of C@C nano ball2C@C/
PEI-Ab2Solution dries at room temperature, is made a kind of and is based on core-shell structure Mo2The cTnI immune sensing of C@C nano ball
Device.
4 MWCNTs-NH of embodiment2The preparation of@Nafion solution
The carbon nanotube MWCNTs-COOH of 0.4 g, bis- ring ethylcarbodiimine DCC and 20 mg carboxylated are added to 2.5
In mL ethylenediamine EDA solution, heating reaction 4 days, by being centrifugally separating to obtain sediment, and use ultrapure water respectively at 120 DEG C
Washing precipitate obtains aminated carbon nano tube MWCNTs-NH repeatedly with dehydrated alcohol2, it places it at 35 DEG C and is dried in vacuo,
Then weigh the MWCNTs-NH of 0.04 mg2Sample dispersion is in 0.4 mL, perfluorinated sulfonic acid-polytetrafluoroethyl-ne that mass fraction is 0.5%
In alkene copolymer Nafion solution, by obtaining uniform MWCNTs-NH after 20 min of ultrasonic treatment2@Nafion solution.
5 MWCNTs-NH of embodiment2The preparation of@Nafion solution
The carbon nanotube MWCNTs-COOH of 0.4 g, bis- ring ethylcarbodiimine DCC and 20 mg carboxylated are added to 2.5
In mL ethylenediamine EDA solution, heating reaction 4 days, by being centrifugally separating to obtain sediment, and use ultrapure water respectively at 120 DEG C
Washing precipitate obtains aminated carbon nano tube MWCNTs-NH repeatedly with dehydrated alcohol2, it places it at 35 DEG C and is dried in vacuo,
Then weigh the MWCNTs-NH of 0.1 mg2Sample dispersion is in 0.4 mL, perfluorinated sulfonic acid-polytetrafluoroethyl-ne that mass fraction is 0.5%
In alkene copolymer Nafion solution, by obtaining uniform MWCNTs-NH after 20 min of ultrasonic treatment2@Nafion solution.
6 MWCNTs-NH of embodiment2The preparation of@Nafion solution
The carbon nanotube MWCNTs-COOH of 0.4 g, bis- ring ethylcarbodiimine DCC and 20 mg carboxylated are added to 2.5
In mL ethylenediamine EDA solution, heating reaction 4 days, by being centrifugally separating to obtain sediment, and use ultrapure water respectively at 120 DEG C
Washing precipitate obtains aminated carbon nano tube MWCNTs-NH repeatedly with dehydrated alcohol2, it places it at 35 DEG C and is dried in vacuo,
Then weigh the MWCNTs-NH of 0.4 mg2Sample dispersion is in 0.4 mL, perfluorinated sulfonic acid-polytetrafluoroethyl-ne that mass fraction is 0.5%
In alkene copolymer Nafion solution, by obtaining uniform MWCNTs-NH after 20 min of ultrasonic treatment2@Nafion solution.
Embodiment 7 loads the core-shell structure Mo for having polyethyleneimine PEI2The secondary antibody marker Mo of C@C nano ball2C@C/
PEI-Ab2Solution preparation
(1) core-shell structure Mo2The preparation of C@C nano ball
12 mg ammonium molybdates and 500 mg glucose are successively dissolved in 45 mL ultrapure waters, is then transferred into autoclave, makes
Product is placed in centrifuge tube and is centrifuged after reaction in 180 DEG C of 10 h of reaction by it, with dehydrated alcohol and ultrapure moisture
It does not wash three times, is subsequently placed at 25 DEG C and is dried in vacuo 12 h, obtained sample is ground, and existed using tube furnace
Argon gas 2 h of lower 800 DEG C of calcinings, obtain core-shell structure Mo2C@C nano ball；
(2) load has the core-shell structure Mo of polyethyleneimine PEI2The secondary antibody marker Mo of C@C nano ball2C@C/PEI-Ab2Solution
Preparation
By 3.0 mL, 1.0 mg mL-1Core-shell structure Mo2C@C nano ball is dispersed in 12 mL and contains 0.625 mol L-1Chlorination
Potassium and 1.25 mg mL-1In the mixed solution of PEI, 12 h are shaken after 2 h of ultrasound, uniform dark solution is obtained, is collected by centrifugation
Solid product three times and is scattered in the ultrapure water of 6 mL with ultrapure washing and obtains Mo2C@C/PEI solution, then pipettes 1
The above-mentioned solution of mL, and 200 μ L cTnIs are added and detect antibody cTnI-Ab2, at 4 DEG C concussion hatching 12 h after from
The heart disperses the solid in the core-shell structure for obtaining load in the PBS that 1 mL, pH is 7.4 and having polyethyleneimine PEI again
Mo2The secondary antibody marker Mo of C@C nano ball2C@C/PEI-Ab2。
Embodiment 8 loads the core-shell structure Mo for having polyethyleneimine PEI2The secondary antibody marker Mo of C@C nano ball2C@C/
PEI-Ab2Solution preparation
(1) core-shell structure Mo2The preparation of C@C nano ball
23 mg ammonium molybdates and 1000 mg glucose are successively dissolved in 45 mL ultrapure waters, is then transferred into autoclave, makes
It reacts 10 h at 180 DEG C, after reaction, product is placed in centrifuge tube and is centrifuged, with dehydrated alcohol and ultrapure water
It washs respectively three times, is subsequently placed at 25 DEG C and is dried in vacuo 12 h, obtained sample is ground, and use tube furnace
800 DEG C of 2 h of calcining under argon gas, obtain core-shell structure Mo2C@C nano ball；
(2) load has the core-shell structure Mo of polyethyleneimine PEI2The secondary antibody marker Mo of C@C nano ball2C@C/PEI-Ab2Solution
Preparation
By 3.0 mL, 1.0 mg mL-1Core-shell structure Mo2C@C nano ball is dispersed in 12 mL and contains 0.625 mol L-1Chlorination
Potassium and 1.25 mg mL-1In the mixed solution of PEI, 12 h are shaken after 2 h of ultrasound, uniform dark solution is obtained, is collected by centrifugation
Solid product three times and is scattered in the ultrapure water of 6 mL with ultrapure washing and obtains Mo2C@C/PEI solution, then pipettes 1
The above-mentioned solution of mL, and 200 μ L cTnIs are added and detect antibody cTnI-Ab2, at 4 DEG C concussion hatching 18 h after from
The heart disperses the solid in the core-shell structure for obtaining load in the PBS that 1 mL, pH is 7.4 and having polyethyleneimine PEI again
Mo2The secondary antibody marker Mo of C@C nano ball2C@C/PEI-Ab2。
Embodiment 9 loads the core-shell structure Mo for having polyethyleneimine PEI2The secondary antibody marker Mo of C@C nano ball2C@C/
PEI-Ab2Solution preparation
(1) core-shell structure Mo2The preparation of C@C nano ball
30 mg ammonium molybdates and 1200 mg glucose are successively dissolved in 45 mL ultrapure waters, is then transferred into autoclave, makes
It is in 180 DEG C of 10 h of reaction, after reaction, place the product in being centrifuged in centrifuge tube, with dehydrated alcohol and ultrapure moisture
It does not wash three times, is subsequently placed at 25 DEG C and is dried in vacuo 12 h, obtained sample is ground, and existed using tube furnace
Argon gas 2 h of lower 800 DEG C of calcinings, obtain core-shell structure Mo2C@C nano ball；
(2) load has the core-shell structure Mo of polyethyleneimine PEI2The secondary antibody marker Mo of C@C nano ball2C@C/PEI-Ab2Solution
Preparation
By 3.0 mL, 1.0 mg mL-1Core-shell structure Mo2C@C nano ball is dispersed in 12 mL and contains 0.625 mol L-1Chlorination
Potassium and 1.25 mg mL-1In the mixed solution of PEI, 12 h are shaken after 2 h of ultrasound, uniform dark solution is obtained, is collected by centrifugation
Solid product three times and is scattered in the ultrapure water of 6 mL with ultrapure washing and obtains Mo2C@C/PEI solution, then pipettes 1
The above-mentioned solution of mL, and 200 μ L cTnIs are added and detect antibody cTnI-Ab2, at 4 DEG C concussion hatching 24 h after from
The heart disperses the solid in the core-shell structure for obtaining load in the PBS that 1 mL, pH is 7.4 and having polyethyleneimine PEI again
Mo2The secondary antibody marker Mo of C@C nano ball2C@C/PEI-Ab2。
The detection of 10 cTnI of embodiment
(1) by reference electrode-Ag/AgCl electrode, be connected to chemical hair to electrode-platinum electrode and the working electrode modified
In the magazine of optical detector, the mixed solution of Tri-n-Propylamine solution and BNQDs that 10 mL contain NaCl is added in electrolytic cell,
The volume ratio of the two is 5:1, and wherein the concentration of Tri-n-Propylamine solution is 1 mmol L-1；
(2) it is detected with cTnI standard solution of the cyclic voltammetry to various concentration, the high pressure of photomultiplier tube is set
800 V are set to, scanning voltage is set as 0 ~ 1.0 V；
(3) according to the linear relationship of resulting electrochemical luminescence intensity value and cTnI log concentration, it is bent to draw work
Line.
The detection of 11 cTnI of embodiment
(1) by reference electrode-Ag/AgCl electrode, to electrode-platinum electrode and the working electrode modified, it is connected to chemistry
In the magazine of luminometer, the mixing that Tri-n-Propylamine solution and BNQDs that 10 mL contain NaCl are added in electrolytic cell is molten
Liquid, the volume ratio of the two are 5:1, and wherein the concentration of Tri-n-Propylamine solution is 2 mmol L-1；
(2) it is detected with cTnI standard solution of the cyclic voltammetry to various concentration, the high pressure of photomultiplier tube is set
800 V are set to, scanning voltage is set as 0 ~ 1.0 V；
(3) according to the linear relationship of resulting electrochemical luminescence intensity value and cTnI log concentration, it is bent to draw work
Line.
The detection of 12 cTnI of embodiment
(1) by reference electrode-Ag/AgCl electrode, to electrode-platinum electrode and the working electrode modified, it is connected to chemistry
In the magazine of luminometer, the mixing that Tri-n-Propylamine solution and BNQDs that 10 mL contain NaCl are added in electrolytic cell is molten
Liquid, the volume ratio of the two are 5:1, and wherein the concentration of Tri-n-Propylamine solution is 5 mmol L-1；
(2) it is detected with cTnI standard solution of the cyclic voltammetry to various concentration, the high pressure of photomultiplier tube is set
800 V are set to, scanning voltage is set as 0 ~ 1.0 V；
(3) according to the linear relationship of resulting electrochemical luminescence intensity value and cTnI log concentration, it is bent to draw work
Line.
Claims (4)
1. one kind is based on core-shell structure Mo2The method of the cTnI immunosensor building of C@C nano ball, feature exist
In, comprising the following steps:
(1) Al is used2O3Polishing powder polishes diameter as the glass-carbon electrode of 4 mm, and successively with dehydrated alcohol and ultrapure water, ultrasound is clear respectively
30 min are washed, by 6 μ L, 0.1 ~ 1.0 mg mL-1Amido modified multi-walled carbon nanotube@perfluorinated sulfonic acid-polytetrafluoroethylene (PTFE)
Copolymer MWCNTs-NH2@Nafion solution is added drop-wise to electrode surface, dries at room temperature；
(2) by 6 μ L, 10 mmol L-1Terpyridyl ruthenium solution drop coating to electrode surface, dry at room temperature；
(3) 1- ethyl -3- (3- dimethyl aminopropyl)-carbodiimides/n-hydroxysuccinimide EDC/NHS of 3 μ L is molten
Drop is coated onto electrode surface, to activate MWCNTs-NH2Amino in@Nafion, the phosphate buffer solution PBS for being 7.4 with pH
Electrode is rinsed, is dried at room temperature；
(4) by 6 μ L, 5 ~ 15 μ g mL-1CTnI capture antibody cTnI-Ab1, 3 μ L, mass fraction 0.5%
~ 2% bovine serum albumin(BSA) BSA solution is added drop-wise to electrode surface, rinses electrode with the PBS that pH is 7.4, dries at room temperature；
(5) 6 μ L, 10 are added dropwise-4 ~ 50 ng mL-1A series of various concentrations cTnI antigen to electrode surface, incubate
Change 2 h, rinses electrode with the PBS that pH is 7.4, dry at room temperature；
(6) the core-shell structure Mo that 6 μ L load has polyethyleneimine PEI is added dropwise2The secondary antibody marker Mo of C@C nano ball2C@C/
PEI-Ab2Solution dries at room temperature, is made a kind of and is based on core-shell structure Mo2The cTnI immune sensing of C@C nano ball
Device.
2. as described in claim 1 a kind of based on core-shell structure Mo2The cTnI immunosensor of C@C nano ball constructs
Method, which is characterized in that the MWCNTs-NH2The preparation step of@Nafion solution is as follows:
The carbon nanotube MWCNTs-COOH of 0.4 g, bis- ring ethylcarbodiimine DCC and 20 mg carboxylated are added to 2.5
In mL ethylenediamine EDA solution, heating reaction 4 days, by being centrifugally separating to obtain sediment, and use ultrapure water respectively at 120 DEG C
Washing precipitate obtains aminated carbon nano tube MWCNTs-NH repeatedly with dehydrated alcohol2, it places it at 35 DEG C and is dried in vacuo,
Then weigh the MWCNTs-NH of 0.04 ~ 0.4 mg2Sample dispersion is gathered in 0.4 mL, the perfluorinated sulfonic acid-that mass fraction is 0.5%
In TFE copolymer Nafion solution, by obtaining uniform MWCNTs-NH after 20 min of ultrasonic treatment2@Nafion is molten
Liquid.
3. as described in claim 1 a kind of based on core-shell structure Mo2The cTnI immunosensor of C@C nano ball constructs
Method, which is characterized in that the load has the core-shell structure Mo of polyethyleneimine PEI2The secondary antibody marker of C@C nano ball
Mo2C@C/PEI-Ab2Solution preparation step is as follows:
(1) core-shell structure Mo2The preparation of C@C nano ball
12 ~ 30 mg ammonium molybdates and 500 ~ 1200 mg glucose are successively dissolved in 45 mL ultrapure waters, are then transferred into high pressure
In reaction kettle, it is made to react 10 h at 180 DEG C, after reaction, place the product in being centrifuged in centrifuge tube, use is anhydrous
Ethyl alcohol and ultrapure water wash three times respectively, are subsequently placed at 25 DEG C and are dried in vacuo 12 h, and obtained sample is ground,
And tube furnace 800 DEG C of 2 h of calcining under argon gas are used, obtain core-shell structure Mo2C@C nano ball；
(2) load has the core-shell structure Mo of polyethyleneimine PEI2The secondary antibody marker Mo of C@C nano ball2C@C/PEI-Ab2Solution
Preparation
By 3.0 mL, 1.0 mg mL-1Core-shell structure Mo2C@C nano ball is dispersed in 12 mL and contains 0.625 mol L-1 Chlorination
Potassium and 1.25 mg mL-1In the mixed solution of PEI, 12 h are shaken after 2 h of ultrasound, uniform dark solution is obtained, is collected by centrifugation
Solid product three times and is scattered in the ultrapure water of 6 mL with ultrapure washing and obtains Mo2C@C/PEI solution, then pipettes 1
The above-mentioned solution of mL, and 200 μ L cTnIs are added and detect antibody cTnI-Ab2, 12 ~ 24 h of concussion hatching at 4 DEG C
After be centrifuged, disperse the solid in the PBS that 1 mL, pH is 7.4 again and obtain loading have the nucleocapsid knot of polyethyleneimine PEI
Structure Mo2The secondary antibody marker Mo of C@C nano ball2C@C/PEI-Ab2。
4. as described in claim 1 a kind of based on core-shell structure Mo2The cTnI immunosensor of C@C nano ball constructs
Method, which is characterized in that for the detection of cTnI, preparation step is as follows:
(1) by reference electrode-Ag/AgCl electrode, be connected to chemical hair to electrode-platinum electrode and the working electrode modified
In the magazine of optical detector, the Tri-n-Propylamine solution and boron nitride quantum dot BNQDs that 10 mL contain NaCl are added in electrolytic cell
Mixed solution, the volume ratio of the two is 5:1, and wherein the concentration of Tri-n-Propylamine solution is 1 ~ 5 mmol L-1；
(2) it is detected with cTnI standard solution of the cyclic voltammetry to various concentration, the high pressure of photomultiplier tube
800 V are set as, scanning voltage is set as 0 ~ 1.0 V；
(3) according to the linear relationship of resulting electrochemical luminescence intensity value and cTnI log concentration, it is bent to draw work
Line.
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| CN111203186A (en) * | 2020-01-14 | 2020-05-29 | 中国计量科学研究院 | Efficient purifying agent with self-separation function and preparation method and application thereof |
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