CN104833718A - Preparation method and application of pH release-type immunosensor - Google Patents
Preparation method and application of pH release-type immunosensor Download PDFInfo
- Publication number
- CN104833718A CN104833718A CN201510248742.0A CN201510248742A CN104833718A CN 104833718 A CN104833718 A CN 104833718A CN 201510248742 A CN201510248742 A CN 201510248742A CN 104833718 A CN104833718 A CN 104833718A
- Authority
- CN
- China
- Prior art keywords
- solution
- silicon oxide
- mesoporous silicon
- preparation
- electrode
- 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.)
- Granted
Links
Abstract
The invention relates to a preparation method and application of a pH release-type immunosensor, belonging to a novel biosensing and detecting technology. The preparation method concretely comprises the steps of packaging oxidation reduction probe thionine into a mesopore of mesoporous silica-Au to obtain mesoporous silica-Au serving as a detecting antibody marker, and preparing to obtain a sandwich-type electrochemical immunosensor for detecting tumor markers. The invention opens up a novel detecting method for detecting tumor markers.
Description
Technical field
The present invention relates to a kind of preparation method and application of pH release type immunosensor.Specifically redox probe thionine is encapsulated in mesoporous silicon oxide-Au, the redox characteristic of thionine is utilized to detect tumor markers, prepare a kind of release type immunosensor detecting tumor markers, belong to new function material and bio-sensing detection technique field.
Background technology
Current electrochemical immunosensor has been widely used in the detection of tumor markers, and release type immunosensor is a kind of method of more novel detection tumor markers, is therefore subject to paying close attention to more widely.
In recent years, mesoporous silicon material has larger specific surface area due to it, and higher pore volume and adjustable aperture, receive the extensive concern in the fields such as absorption, sensing.Therefore, utilize it to have good absorption property to adsorb thionine, and with carboxyl-functional gold Au-COOH, its duct is closed, thionine is made to be stabilized in the duct of mesoporous silicon oxide, mesoporous silicon oxide-Au is as the detection antibody labeling thing in sensor production, the obtained immunosensor detecting tumor markers, utilizes pH to control the Kai Heguan in duct, reaches the object of detection.
Summary of the invention
An object of the present invention is a kind of pH release type immunosensor of preparation.
Two of object of the present invention is the detections this pH release type immunosensor being applied to tumor markers.
technical scheme of the present invention
1. the preparation method of a pH release type immunosensor
(1) with the alundum (Al2O3) burnishing powder of 1.0,0.3,0.05 μm, polishing is carried out to carbon electrode successively, clean up with ultrapure water, then electrode is placed in 5 mmol/L potassium ferricyanide solutions, scans under-0.2 ~ 0.6 V current potential, make spike potential difference be less than 110 mV;
(2) get 6 μ L, the amination tri-iron tetroxide solution of 0.5 ~ 3 mg/mL is added drop-wise to electrode surface, dry under room temperature;
(3) mixed liquor of 1-(3-the dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride EDC solution and N-hydroxy-succinamide NHS solution that drip 3 μ L is to electrode surface, continue the tumor markers capture antibody of dropping 6 μ L, 5 ~ 10 μ g/mL again, dry at 4 DEG C, ultrapure water;
The concentration of described 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride EDC solution and N-hydroxy-succinamide NHS solution is 0.1 mol/L, and its volume ratio is 1 ~ 4: 1;
(4) close nonspecific activity site with the bovine serum albumin solution that 3 μ L, massfraction are 0.5 ~ 1%, dry at 4 DEG C, ultrapure water;
(5) the tumor markers solution of a series of variable concentrations of 6 μ L is added drop-wise to electrode surface, for the specific recognition with antibody, incubated at room temperature 1 h, ultrapure water;
(6) continue dropping 4 ~ 6 μ L and detect antibody hatching thing-mesoporous silicon oxide-Au-Ab
2solution, to electrode surface, is placed in 4 DEG C of refrigerators and hatches 1 h, after cleaning, dry, obtained a kind of pH release type immunosensor;
2. detect antibody hatching thing-mesoporous silicon oxide-Au-Ab
2the preparation of solution
(1) preparation of mesoporous silicon oxide
The CTAB of 0.3 ~ 0.7 g is dissolved in the ultrapure water of 200 mL, add 1 ~ 2 mL, the NaOH of 2.0 mol/L, in 80 DEG C of back flow reaction 20 min, add the tetraethoxysilane of 1.5 ~ 3.0 mL subsequently, continue reaction 2 h and generate white precipitate, in order to remove CTAB residual in solution, the precipitation obtained is dispersed in 1.5 mL again, massfraction is in the mixed liquor of the HCl of 37% and the methyl alcohol of 75 ~ 100 mL, reflux at 80 DEG C 10 ~ 12 h, potpourri is centrifugal, through water and methanol wash several, vacuum drying at 35 ~ 45 DEG C, obtained mesoporous silicon oxide, particle size is about 100 nm, pore size is about 5 nm,
(2) preparation of the mesoporous silicon oxide MSN-Aetal of acetal linker modification
Be scattered in by the mesoporous silicon oxide of 0.1 ~ 0.3 g in 20 mL ethanol, add the 3-aminopropyl triethoxysilane of 0.2 ~ 1 mL, 80 DEG C of back flow reaction 12 h, potpourri, through centrifugal, washing, vacuum drying at 45 DEG C, obtains amidized mesoporous silicon oxide;
The amidized mesoporous silicon oxide of 0.1 ~ 0.3 g is added in the dimethyl sulphoxide solution of 10 mL, add 50 ~ 100 mg succinic anhydrides and 50 ~ 100 mg triethylamines subsequently, react 48 h at 40 DEG C, the solid obtained washs through ethanol, and vacuum drying obtains carboxylated mesoporous silicon oxide;
Respectively the N-hydroxy-succinamide NHS of 1-(3-the dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride EDC and 10 ~ 30 mg of 20 ~ 80 mg is dissolved in 3 mL ultrapure waters, add the mesoporous silicon oxide that 50 ~ 100 mg are carboxylated, 3 of 80 ~ 130 mg, two (the 3-aminopropyl)-2 of 9-, 4,8,10-tetra-oxaspiro [5.5] undecane, stirred at ambient temperature 8 h, precipitates the mesoporous silicon oxide MSN-Aetal obtaining the modification of acetal linker through washing drying;
(3) preparation of carboxyl-functional gold Au-COOH
By 20 mL, 1% HAuCl
4be scattered in the deoxidation methyl alcohol of 85 mL with 0.5 mmol mercapto succinic acid, under agitation, by the NaBH of 25 mL, 0.2 mol/L
4solution is added drop-wise in above-mentioned solution, and the dark brown deposit obtained is after ethanol washing, and vacuum drying, obtains carboxyl-functional gold Au-COOH;
(4) preparation of antibody labeling thing-mesoporous silicon oxide-Au is detected
The mesoporous silicon oxide MSN-Aetal that the acetal linker of 10 ~ 20 mg is modified is joined 0.5 ~ 3mmol/L, in the thionine aqueous solution of 1 mL, stirred at ambient temperature 12 h, add the carboxyl-functional gold Au-COOH of 4 ~ 10 mg, the N-hydroxy-succinamide NHS of 1-(3-the dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride EDC and 5 mg of 10 mg, continue stirring 8 h, potpourri through centrifuge washing until supernatant be colourless after, precipitation is dispersed in the ultrapure water of 1 mL again, successfully thionine is encapsulated in mesoporous silicon oxide-Au, obtained detection antibody labeling thing mesoporous silicon oxide-Au,
(5) antibody hatching thing-mesoporous silicon oxide-Au-Ab is detected
2the preparation of solution
After the detection antibody labeling thing mesoporous silicon oxide-Au of 1 mL is mixed with the detection antibody-solutions of 1 mL, 10 μ g/mL, 12 h are hatched under the condition of 4 DEG C, after centrifugal, precipitation be dispersed in the phosphate buffered solution of 1 mL, pH=7.4 again, be kept in the refrigerator of 4 DEG C for subsequent use.
3. the detection method of tumor markers
(1) immunosensor prepared by claim 1 is immersed in acetate buffer solution 12 h of 0.5 mL, pH=3.7;
(2) three-electrode system is adopted, Ag/AgCl electrode is as contrast electrode, platinum electrode is as auxiliary electrode, electro-conductive glass is as working electrode, Differential Pulse Voltammetry is adopted to carry out sweep test to above-mentioned soak solution, voltage is set to-0.4 ~ 0.6 V, the change of record current, drawing curve;
(3) tumor markers antigen standard solution is replaced by testing sample solution to test.
Above-mentioned tumor markers is selected from one of following: CD146, AFP, CA153, CA125.
useful achievement of the present invention
(1) what substrate adopted is the magnetic amination tri-iron tetroxide of tool, can very stable being adsorbed on carbon electrode, and it has good biocompatibility and stability.
(2) mesoporous silicon oxide has large specific surface area, good adsorptive power and biocompatibility, a large amount of thionines is conducive to be encapsulated in mesoporous silicon oxide-Au, prevent the leakage of thionine, utilize and regulate the switch of pH to duct to control, improve the sensitivity of sensor.
(3) electrochemical immunosensor prepared of the present invention is for the detection of tumor markers, and the response time is short, and detectability is low, and the range of linearity is wide, can realize simple, quick, highly sensitive and specific detection.
Embodiment
embodiment 1a kind of preparation method of pH release type immunosensor
(1) with the alundum (Al2O3) burnishing powder of 1.0,0.3,0.05 μm, polishing is carried out to carbon electrode successively, clean up with ultrapure water, then electrode is placed in 5 mmol/L potassium ferricyanide solutions, scans under-0.2 ~ 0.6 V current potential, make spike potential difference be less than 110 mV;
(2) get 6 μ L, the amination tri-iron tetroxide solution of 0.5 mg/mL is added drop-wise to electrode surface, dry under room temperature;
(3) mixed liquor of 1-(3-the dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride EDC solution and N-hydroxy-succinamide NHS solution that drip 3 μ L is to electrode surface, continue the tumor markers capture antibody of dropping 6 μ L, 5 μ g/mL again, dry at 4 DEG C, ultrapure water;
The concentration of described 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride EDC solution and N-hydroxy-succinamide NHS solution is 0.1 mol/L, and its volume ratio is 1: 1;
(4) close nonspecific activity site with the bovine serum albumin solution that 3 μ L, massfraction are 0.5%, dry at 4 DEG C, ultrapure water;
(5) the tumor markers solution of a series of variable concentrations of 6 μ L is added drop-wise to electrode surface, for the specific recognition with antibody, incubated at room temperature 1 h, ultrapure water;
(6) continue dropping 4 μ L and detect antibody hatching thing-mesoporous silicon oxide-Au-Ab
2solution, to electrode surface, is placed in 4 DEG C of refrigerators and hatches 1 h, after cleaning, dry, obtained a kind of pH release type immunosensor;
embodiment 2a kind of preparation method of pH release type immunosensor
(1) with the alundum (Al2O3) burnishing powder of 1.0,0.3,0.05 μm, polishing is carried out to carbon electrode successively, clean up with ultrapure water, then electrode is placed in 5 mmol/L potassium ferricyanide solutions, scans under-0.2 ~ 0.6 V current potential, make spike potential difference be less than 110 mV;
(2) get 6 μ L, the amination tri-iron tetroxide solution of 1 mg/mL is added drop-wise to electrode surface, dry under room temperature;
(3) mixed liquor of 1-(3-the dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride EDC solution and N-hydroxy-succinamide NHS solution that drip 3 μ L is to electrode surface, continue the tumor markers capture antibody of dropping 6 μ L, 8 μ g/mL again, dry at 4 DEG C, ultrapure water;
The concentration of described 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride EDC solution and N-hydroxy-succinamide NHS solution is 0.1 mol/L, and its volume ratio is 3: 1;
(4) close nonspecific activity site with the bovine serum albumin solution that 3 μ L, massfraction are 0.8%, dry at 4 DEG C, ultrapure water;
(5) the tumor markers solution of a series of variable concentrations of 6 μ L is added drop-wise to electrode surface, for the specific recognition with antibody, incubated at room temperature 1 h, ultrapure water;
(6) continue dropping 5 μ L and detect antibody hatching thing-mesoporous silicon oxide-Au-Ab
2solution, to electrode surface, is placed in 4 DEG C of refrigerators and hatches 1 h, after cleaning, dry, obtained a kind of pH release type immunosensor;
embodiment 3a kind of preparation method of pH release type immunosensor
(1) with the alundum (Al2O3) burnishing powder of 1.0,0.3,0.05 μm, polishing is carried out to carbon electrode successively, clean up with ultrapure water, then electrode is placed in 5 mmol/L potassium ferricyanide solutions, scans under-0.2 ~ 0.6 V current potential, make spike potential difference be less than 110 mV;
(2) get 6 μ L, the amination tri-iron tetroxide solution of 3 mg/mL is added drop-wise to electrode surface, dry under room temperature;
(3) mixed liquor of 1-(3-the dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride EDC solution and N-hydroxy-succinamide NHS solution that drip 3 μ L is to electrode surface, continue the tumor markers capture antibody of dropping 6 μ L, 10 μ g/mL again, dry at 4 DEG C, ultrapure water;
The concentration of described 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride EDC solution and N-hydroxy-succinamide NHS solution is 0.1 mol/L, and its volume ratio is 4: 1;
(4) close nonspecific activity site with the bovine serum albumin solution that 3 μ L, massfraction are 1%, dry at 4 DEG C, ultrapure water;
(5) the tumor markers solution of a series of variable concentrations of 6 μ L is added drop-wise to electrode surface, for the specific recognition with antibody, incubated at room temperature 1 h, ultrapure water;
(6) continue dropping 6 μ L and detect antibody hatching thing-mesoporous silicon oxide-Au-Ab
2solution, to electrode surface, is placed in 4 DEG C of refrigerators and hatches 1 h, after cleaning, dry, obtained a kind of pH release type immunosensor;
embodiment 4detect antibody hatching thing-mesoporous silicon oxide-Au-Ab
2the preparation of solution
(1) preparation of mesoporous silicon oxide
The CTAB of 0.3 g is dissolved in the ultrapure water of 200 mL, add 1 mL, the NaOH of 2.0 mol/L, in 80 DEG C of back flow reaction 20 min, add the tetraethoxysilane of 1.5 mL subsequently, continue reaction 2 h and generate white precipitate, , in order to remove CTAB residual in solution, the precipitation obtained is dispersed in 1.5 mL again, massfraction is in the mixed liquor of the HCl of 37% and the methyl alcohol of 75 mL, reflux at 80 DEG C 10 h, potpourri is centrifugal, through water and methanol wash several, vacuum drying at 35 DEG C, obtained mesoporous silicon oxide, particle size is about 100 nm, pore size is about 5 nm,
(2) preparation of the mesoporous silicon oxide MSN-Aetal of acetal linker modification
Be scattered in by the mesoporous silicon oxide of 0.1 g in 20 mL ethanol, add the 3-aminopropyl triethoxysilane of 0.2 mL, 80 DEG C of back flow reaction 12 h, potpourri, through centrifugal, washing, vacuum drying at 45 DEG C, obtains amidized mesoporous silicon oxide;
The amidized mesoporous silicon oxide of 0.1 g is added in the dimethyl sulphoxide solution of 10 mL, add 50 mg succinic anhydrides and 50 mg triethylamines subsequently, react 48 h at 40 DEG C, the solid obtained washs through ethanol, and vacuum drying obtains carboxylated mesoporous silicon oxide;
Respectively the N-hydroxy-succinamide NHS of 1-(3-the dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride EDC and 10 mg of 20 mg is dissolved in 3 mL ultrapure waters, add the mesoporous silicon oxide that 50 mg are carboxylated, 3 of 80 mg, two (the 3-aminopropyl)-2 of 9-, 4,8,10-tetra-oxaspiro [5.5] undecane, stirred at ambient temperature 8 h, precipitates the mesoporous silicon oxide MSN-Aetal obtaining the modification of acetal linker through washing drying;
(3) preparation of carboxyl-functional gold Au-COOH
By 20 mL, 1% HAuCl
4be scattered in the deoxidation methyl alcohol of 85 mL with 0.5 mmol mercapto succinic acid, under agitation, by the NaBH of 25 mL, 0.2 mol/L
4solution is added drop-wise in above-mentioned solution, and the dark brown deposit obtained is after ethanol washing, and vacuum drying, obtains carboxyl-functional gold Au-COOH;
(4) preparation of antibody labeling thing-mesoporous silicon oxide-Au is detected
The mesoporous silicon oxide MSN-Aetal that the acetal linker of 10 mg is modified is joined 0.5 mmol/L, in the thionine aqueous solution of 1 mL, stirred at ambient temperature 12 h, add the carboxyl-functional gold Au-COOH of 4 mg, the N-hydroxy-succinamide NHS of 1-(3-the dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride EDC and 5 mg of 10 mg, continue stirring 8 h, potpourri through centrifuge washing until supernatant be colourless after, precipitation is dispersed in the ultrapure water of 1 mL again, successfully thionine is encapsulated in mesoporous silicon oxide-Au, obtained detection antibody labeling thing mesoporous silicon oxide-Au,
(5) antibody hatching thing-mesoporous silicon oxide-Au-Ab is detected
2the preparation of solution
After the detection antibody labeling thing mesoporous silicon oxide-Au of 1 mL is mixed with the detection antibody-solutions of 1 mL, 10 μ g/mL, 12 h are hatched under the condition of 4 DEG C, after centrifugal, precipitation be dispersed in the phosphate buffered solution of 1 mL, pH=7.4 again, be kept in the refrigerator of 4 DEG C for subsequent use.
embodiment 5detect antibody hatching thing-mesoporous silicon oxide-Au-Ab
2the preparation of solution
(1) preparation of mesoporous silicon oxide
The CTAB of 0.5 g is dissolved in the ultrapure water of 200 mL, add 1.5 mL, the NaOH of 2.0 mol/L, in 80 DEG C of back flow reaction 20 min, add the tetraethoxysilane of 2.0 mL subsequently, continue reaction 2 h and generate white precipitate, , in order to remove CTAB residual in solution, the precipitation obtained is dispersed in 1.5 mL again, massfraction is in the mixed liquor of the HCl of 37% and the methyl alcohol of 85 mL, reflux at 80 DEG C 11 h, potpourri is centrifugal, through water and methanol wash several, vacuum drying at 40 DEG C, obtained mesoporous silicon oxide, particle size is about 100 nm, pore size is about 5 nm,
(2) preparation of the mesoporous silicon oxide MSN-Aetal of acetal linker modification
Be scattered in by the mesoporous silicon oxide of 0.2 g in 20 mL ethanol, add the 3-aminopropyl triethoxysilane of 0.7 mL, 80 DEG C of back flow reaction 12 h, potpourri, through centrifugal, washing, vacuum drying at 45 DEG C, obtains amidized mesoporous silicon oxide;
The amidized mesoporous silicon oxide of 0.2 g is added in the dimethyl sulphoxide solution of 10 mL, add 80 mg succinic anhydrides and 80 mg triethylamines subsequently, react 48 h at 40 DEG C, the solid obtained washs through ethanol, and vacuum drying obtains carboxylated mesoporous silicon oxide;
Respectively the N-hydroxy-succinamide NHS of 1-(3-the dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride EDC and 20 mg of 50 mg is dissolved in 3 mL ultrapure waters, add the mesoporous silicon oxide that 80 mg are carboxylated, 3 of 100 mg, two (the 3-aminopropyl)-2 of 9-, 4,8,10-tetra-oxaspiro [5.5] undecane, stirred at ambient temperature 8 h, precipitates the mesoporous silicon oxide MSN-Aetal obtaining the modification of acetal linker through washing drying;
(3) preparation of carboxyl-functional gold Au-COOH
By 20 mL, 1% HAuCl
4be scattered in the deoxidation methyl alcohol of 85 mL with 0.5 mmol mercapto succinic acid, under agitation, by the NaBH of 25 mL, 0.2 mol/L
4solution is added drop-wise in above-mentioned solution, and the dark brown deposit obtained is after ethanol washing, and vacuum drying, obtains carboxyl-functional gold Au-COOH;
(4) preparation of antibody labeling thing-mesoporous silicon oxide-Au is detected
The mesoporous silicon oxide MSN-Aetal that the acetal linker of 15 mg is modified is joined 1mmol/L, in the thionine aqueous solution of 1 mL, stirred at ambient temperature 12 h, add the carboxyl-functional gold Au-COOH of 8 mg, the N-hydroxy-succinamide NHS of 1-(3-the dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride EDC and 5 mg of 10 mg, continue stirring 8 h, potpourri through centrifuge washing until supernatant be colourless after, precipitation is dispersed in the ultrapure water of 1 mL again, successfully thionine is encapsulated in mesoporous silicon oxide-Au, obtained detection antibody labeling thing mesoporous silicon oxide-Au,
(5) antibody hatching thing-mesoporous silicon oxide-Au-Ab is detected
2the preparation of solution
After the detection antibody labeling thing mesoporous silicon oxide-Au of 1 mL is mixed with the detection antibody-solutions of 1 mL, 10 μ g/mL, 12 h are hatched under the condition of 4 DEG C, after centrifugal, precipitation be dispersed in the phosphate buffered solution of 1 mL, pH=7.4 again, be kept in the refrigerator of 4 DEG C for subsequent use.
embodiment 6detect antibody hatching thing-mesoporous silicon oxide-Au-Ab
2the preparation of solution
(1) preparation of mesoporous silicon oxide
The CTAB of 0.7 g is dissolved in the ultrapure water of 200 mL, add 2 mL, the NaOH of 2.0 mol/L, in 80 DEG C of back flow reaction 20 min, add the tetraethoxysilane of 3.0 mL subsequently, continue reaction 2 h and generate white precipitate, , in order to remove CTAB residual in solution, the precipitation obtained is dispersed in 1.5 mL again, massfraction is in the mixed liquor of the HCl of 37% and the methyl alcohol of 100 mL, reflux at 80 DEG C 12 h, potpourri is centrifugal, through water and methanol wash several, vacuum drying at 45 DEG C, obtained mesoporous silicon oxide, particle size is about 100 nm, pore size is about 5 nm,
(2) preparation of the mesoporous silicon oxide MSN-Aetal of acetal linker modification
Be scattered in by the mesoporous silicon oxide of 0.3 g in 20 mL ethanol, add the 3-aminopropyl triethoxysilane of 1 mL, 80 DEG C of back flow reaction 12 h, potpourri, through centrifugal, washing, vacuum drying at 45 DEG C, obtains amidized mesoporous silicon oxide;
The amidized mesoporous silicon oxide of 0.3 g is added in the dimethyl sulphoxide solution of 10 mL, add 100 mg succinic anhydrides and 100 mg triethylamines subsequently, react 48 h at 40 DEG C, the solid obtained washs through ethanol, and vacuum drying obtains carboxylated mesoporous silicon oxide;
Respectively the N-hydroxy-succinamide NHS of 1-(3-the dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride EDC and 30 mg of 80 mg is dissolved in 3 mL ultrapure waters, add the mesoporous silicon oxide that 100 mg are carboxylated, 3 of 130 mg, two (the 3-aminopropyl)-2 of 9-, 4,8,10-tetra-oxaspiro [5.5] undecane, stirred at ambient temperature 8 h, precipitates the mesoporous silicon oxide MSN-Aetal obtaining the modification of acetal linker through washing drying;
(3) preparation of carboxyl-functional gold Au-COOH
By 20 mL, 1% HAuCl
4be scattered in the deoxidation methyl alcohol of 85 mL with 0.5 mmol mercapto succinic acid, under agitation, by the NaBH of 25 mL, 0.2 mol/L
4solution is added drop-wise in above-mentioned solution, and the dark brown deposit obtained is after ethanol washing, and vacuum drying, obtains carboxyl-functional gold Au-COOH;
(4) preparation of antibody labeling thing-mesoporous silicon oxide-Au is detected
The mesoporous silicon oxide MSN-Aetal that the acetal linker of 20 mg is modified is joined 3 mmol/L, in the thionine aqueous solution of 1 mL, stirred at ambient temperature 12 h, add the carboxyl-functional gold Au-COOH of 10 mg, the N-hydroxy-succinamide NHS of 1-(3-the dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride EDC and 5 mg of 10 mg, continue stirring 8 h, potpourri through centrifuge washing until supernatant be colourless after, precipitation is dispersed in the ultrapure water of 1 mL again, successfully thionine is encapsulated in mesoporous silicon oxide-Au, obtained detection antibody labeling thing mesoporous silicon oxide-Au,
(5) antibody hatching thing-mesoporous silicon oxide-Au-Ab is detected
2the preparation of solution
After the detection antibody labeling thing mesoporous silicon oxide-Au of 1 mL is mixed with the detection antibody-solutions of 1 mL, 10 μ g/mL, 12 h are hatched under the condition of 4 DEG C, after centrifugal, precipitation be dispersed in the phosphate buffered solution of 1 mL, pH=7.4 again, be kept in the refrigerator of 4 DEG C for subsequent use.
embodiment 7the detection of tumor markers CD146
(1) immunosensor prepared by claim 1 is immersed in acetate buffer solution 12 h of 0.5 mL, pH=3.7;
(2) three-electrode system is adopted, Ag/AgCl electrode is as contrast electrode, platinum electrode is as auxiliary electrode, electro-conductive glass is as working electrode, Differential Pulse Voltammetry is adopted to carry out sweep test to above-mentioned soak solution, voltage is set to-0.4 ~ 0.6V, the change of record current, drawing curve;
(3) tumor markers antigen standard solution is replaced by testing sample solution to test;
(4) this electrochemical immunosensor detects the range of linearity to tumor markers CD146 is 0.001 ~ 10 ng/mL, detects and is limited to 0.21 pg/mL.
embodiment 8according to the method for embodiment 7, AFP, CA153, CA125 are detected, record the range of linearity and be respectively 0.001 ~ 10 ng/mL, 0.001 ~ 15 ng/mL and 0.002 ~ 10 ng/mL; Detectability is respectively 0.17 pg/mL, 0.31 pg/mL and 0.54 pg/mL.
Claims (4)
1. a preparation method for pH release type immunosensor, is characterized in that, comprises the following steps:
(1) with the alundum (Al2O3) burnishing powder of 1.0,0.3,0.05 μm, polishing is carried out to carbon electrode successively, clean up with ultrapure water, then electrode is placed in 5 mmol/L potassium ferricyanide solutions, scans under-0.2 ~ 0.6 V current potential, make spike potential difference be less than 110 mV;
(2) get 6 μ L, the amination tri-iron tetroxide solution of 0.5 ~ 3 mg/mL is added drop-wise to electrode surface, dry under room temperature;
(3) mixed liquor of 1-(3-the dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride EDC solution and N-hydroxy-succinamide NHS solution that drip 3 μ L is to electrode surface, continue the tumor markers capture antibody of dropping 6 μ L, 5 ~ 10 μ g/mL again, dry at 4 DEG C, ultrapure water;
The concentration of described 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride EDC solution and N-hydroxy-succinamide NHS solution is 0.1 mol/L, and its volume ratio is 1 ~ 4: 1;
(4) close nonspecific activity site with the bovine serum albumin solution that 3 μ L, massfraction are 0.5 ~ 1%, dry at 4 DEG C, ultrapure water;
(5) the tumor markers solution of a series of variable concentrations of 6 μ L is added drop-wise to electrode surface, for the specific recognition with antibody, incubated at room temperature 1 h, ultrapure water;
(6) continue dropping 4 ~ 6 μ L and detect antibody hatching thing-mesoporous silicon oxide-Au-Ab
2solution, to electrode surface, is placed in 4 DEG C of refrigerators and hatches 1 h, after cleaning, dry, obtained a kind of pH release type immunosensor.
2. the preparation method of a kind of pH release type immunosensor as claimed in claim 1, described detection antibody hatching thing-mesoporous silicon oxide-Au-Ab
2the preparation of solution, is characterized in that, comprises the following steps:
(1) preparation of mesoporous silicon oxide
The CTAB of 0.3 ~ 0.7 g is dissolved in the ultrapure water of 200 mL, add 1 ~ 2 mL, the NaOH of 2.0 mol/L, in 80 DEG C of back flow reaction 20 min, add the tetraethoxysilane of 1.5 ~ 3.0 mL subsequently, continue reaction 2 h and generate white precipitate, in order to remove CTAB residual in solution, the precipitation obtained is dispersed in 1.5 mL again, massfraction is in the mixed liquor of the HCl of 37% and the methyl alcohol of 75 ~ 100 mL, reflux at 80 DEG C 10 ~ 12 h, potpourri is centrifugal, through water and methanol wash several, vacuum drying at 35 ~ 45 DEG C, obtained mesoporous silicon oxide, particle size is about 100 nm, pore size is about 5 nm,
(2) preparation of the mesoporous silicon oxide MSN-Aetal of acetal linker modification
Be scattered in by the mesoporous silicon oxide of 0.1 ~ 0.3 g in 20 mL ethanol, add the 3-aminopropyl triethoxysilane of 0.2 ~ 1 mL, 80 DEG C of back flow reaction 12 h, potpourri, through centrifugal, washing, vacuum drying at 45 DEG C, obtains amidized mesoporous silicon oxide;
The amidized mesoporous silicon oxide of 0.1 ~ 0.3 g is added in the dimethyl sulphoxide solution of 10 mL, add 50 ~ 100 mg succinic anhydrides and 50 ~ 100 mg triethylamines subsequently, react 48 h at 40 DEG C, the solid obtained washs through ethanol, and vacuum drying obtains carboxylated mesoporous silicon oxide;
Respectively the N-hydroxy-succinamide NHS of 1-(3-the dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride EDC and 10 ~ 30 mg of 20 ~ 80 mg is dissolved in 3 mL ultrapure waters, add the mesoporous silicon oxide that 50 ~ 100 mg are carboxylated, 3 of 80 ~ 130 mg, two (the 3-aminopropyl)-2 of 9-, 4,8,10-tetra-oxaspiro [5.5] undecane, stirred at ambient temperature 8 h, precipitates the mesoporous silicon oxide MSN-Aetal obtaining the modification of acetal linker through washing drying;
(3) preparation of carboxyl-functional gold Au-COOH
By 20 mL, 1% HAuCl
4be scattered in the deoxidation methyl alcohol of 85 mL with 0.5 mmol mercapto succinic acid, under agitation, by the NaBH of 25 mL, 0.2 mol/L
4solution is added drop-wise in above-mentioned solution, and the dark brown deposit obtained is after ethanol washing, and vacuum drying, obtains carboxyl-functional gold Au-COOH;
(4) preparation of antibody labeling thing-mesoporous silicon oxide-Au is detected
The mesoporous silicon oxide MSN-Aetal that the acetal linker of 10 ~ 20 mg is modified is joined 0.5 ~ 3mmol/L, in the thionine aqueous solution of 1 mL, stirred at ambient temperature 12 h, add the carboxyl-functional gold Au-COOH of 4 ~ 10 mg, the N-hydroxy-succinamide NHS of 1-(3-the dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride EDC and 5 mg of 10 mg, continue stirring 8 h, potpourri through centrifuge washing until supernatant be colourless after, precipitation is dispersed in the ultrapure water of 1 mL again, successfully thionine is encapsulated in mesoporous silicon oxide-Au, obtained detection antibody labeling thing mesoporous silicon oxide-Au,
(5) antibody hatching thing-mesoporous silicon oxide-Au-Ab is detected
2the preparation of solution
After the detection antibody labeling thing mesoporous silicon oxide-Au of 1 mL is mixed with the detection antibody-solutions of 1 mL, 10 μ g/mL, 12 h are hatched under the condition of 4 DEG C, after centrifugal, precipitation be dispersed in the phosphate buffered solution of 1 mL, pH=7.4 again, be kept in the refrigerator of 4 DEG C for subsequent use.
3. the immunosensor prepared of preparation method as claimed in claim 1, for the detection of tumor markers, is characterized in that, comprises following analytical procedure:
(1) immunosensor prepared by claim 1 is immersed in acetate buffer solution 12 h of 0.5 mL, pH=3.7;
(2) three-electrode system is adopted, Ag/AgCl electrode is as contrast electrode, platinum electrode is as auxiliary electrode, electro-conductive glass is as working electrode, Differential Pulse Voltammetry is adopted to carry out sweep test to above-mentioned soak solution, voltage is set to-0.4 ~ 0.6 V, the change of record current, drawing curve;
(3) tumor markers antigen standard solution is replaced by testing sample solution to test.
4. the preparation method of a kind of pH release type immunosensor as claimed in claim 1, described tumor markers is selected from one of following: CD146, AFP, CA153, CA125.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510248742.0A CN104833718B (en) | 2015-05-16 | 2015-05-16 | A kind of preparation method of pH release type immunosensor and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510248742.0A CN104833718B (en) | 2015-05-16 | 2015-05-16 | A kind of preparation method of pH release type immunosensor and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104833718A true CN104833718A (en) | 2015-08-12 |
| CN104833718B CN104833718B (en) | 2016-04-20 |
Family
ID=53811732
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510248742.0A Expired - Fee Related CN104833718B (en) | 2015-05-16 | 2015-05-16 | A kind of preparation method of pH release type immunosensor and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104833718B (en) |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106596942A (en) * | 2016-12-21 | 2017-04-26 | 山东理工大学 | Construction method and application of sandwich-type hepatitis B virus marker immunosensor |
| CN109030450A (en) * | 2018-05-25 | 2018-12-18 | 苏州大学 | A method of in the two-way controllable self assembly difference charged metal nanoparticle of substrate surface |
| CN109709176A (en) * | 2019-02-26 | 2019-05-03 | 济南大学 | A kind of building of the photic electrochemical sensor of alpha-fetoprotein |
| CN109799229A (en) * | 2018-12-24 | 2019-05-24 | 济南大学 | One kind being based on [Ru (bpy)3]2+The preparation method of the immunosensor of/Au@NiFe MOFs |
| CN110208530A (en) * | 2019-07-16 | 2019-09-06 | 何金星 | A kind of diagnostic preparation and preparation method thereof measuring immune level |
| CN110428964A (en) * | 2019-06-26 | 2019-11-08 | 江苏诺鬲生物科技有限公司 | The preparation method and application of nanometer magnetic bead for enriching and purifying mycobacterium tuberculosis |
| CN113049651A (en) * | 2021-03-15 | 2021-06-29 | 重庆大学 | In-situ electrochemical immunosensor for simultaneously detecting four breast cancer markers |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3838033A (en) * | 1971-09-09 | 1974-09-24 | Hoffmann La Roche | Enzyme electrode |
| CN101915792A (en) * | 2010-08-13 | 2010-12-15 | 上海交通大学 | Preparation method of electrochemical immunosensor for detecting salbutamol quickly |
| CN103235145A (en) * | 2013-04-19 | 2013-08-07 | 济南大学 | Preparation method and application of two-channel environmental estrogen immunosensor |
-
2015
- 2015-05-16 CN CN201510248742.0A patent/CN104833718B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3838033A (en) * | 1971-09-09 | 1974-09-24 | Hoffmann La Roche | Enzyme electrode |
| CN101915792A (en) * | 2010-08-13 | 2010-12-15 | 上海交通大学 | Preparation method of electrochemical immunosensor for detecting salbutamol quickly |
| CN103235145A (en) * | 2013-04-19 | 2013-08-07 | 济南大学 | Preparation method and application of two-channel environmental estrogen immunosensor |
Non-Patent Citations (1)
| Title |
|---|
| 李灿鹏，杨龙，庞芳，杨周，王光灿: "钯-四氧化三铁纳米复合物生物传感器的制备及检测有机磷农药的研究", 《分析试验室》 * |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106596942A (en) * | 2016-12-21 | 2017-04-26 | 山东理工大学 | Construction method and application of sandwich-type hepatitis B virus marker immunosensor |
| CN109030450A (en) * | 2018-05-25 | 2018-12-18 | 苏州大学 | A method of in the two-way controllable self assembly difference charged metal nanoparticle of substrate surface |
| CN109799229A (en) * | 2018-12-24 | 2019-05-24 | 济南大学 | One kind being based on [Ru (bpy)3]2+The preparation method of the immunosensor of/Au@NiFe MOFs |
| CN109799229B (en) * | 2018-12-24 | 2021-04-30 | 济南大学 | Based on [ Ru (bpy)3]2+Preparation method of/Au @ NiFe MOFs (metal-organic frameworks) immunosensor |
| CN109709176A (en) * | 2019-02-26 | 2019-05-03 | 济南大学 | A kind of building of the photic electrochemical sensor of alpha-fetoprotein |
| CN110428964A (en) * | 2019-06-26 | 2019-11-08 | 江苏诺鬲生物科技有限公司 | The preparation method and application of nanometer magnetic bead for enriching and purifying mycobacterium tuberculosis |
| CN110208530A (en) * | 2019-07-16 | 2019-09-06 | 何金星 | A kind of diagnostic preparation and preparation method thereof measuring immune level |
| CN112083162A (en) * | 2019-07-16 | 2020-12-15 | 何金星 | Diagnostic preparation for determining immunity level |
| CN113049651A (en) * | 2021-03-15 | 2021-06-29 | 重庆大学 | In-situ electrochemical immunosensor for simultaneously detecting four breast cancer markers |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104833718B (en) | 2016-04-20 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104833718B (en) | A kind of preparation method of pH release type immunosensor and application | |
| CN103116023B (en) | ECL (electrochemiluminescence) immunosensor for detecting tumor markers and preparation method and applications thereof | |
| Zhang et al. | GoldMag nanocomposite-functionalized graphene sensing platform for one-step electrochemical immunoassay of alpha-fetoprotein | |
| CN102207502B (en) | Mercury ion test paper and preparation method thereof | |
| CN110794017B (en) | Preparation method of electrochemical immunosensor for detecting procalcitonin | |
| CN104459132B (en) | A kind of is preparation method and the application of the cancer of pancreas immunosensor of label based on golden electro-deposition and Au@Ag/CuO-GS | |
| CN104297305A (en) | Making method and application of CdS sensitized TiO2 environmental estrogen photoelectrochemical sensor | |
| CN110308286B (en) | Enhanced thyroglobulin electrochemiluminescence immunosensor based on photothermal release signals | |
| Zhang et al. | Label-free immunosensor based on Au@ Ag2S nanoparticles/magnetic chitosan matrix for sensitive determination of ractopamine | |
| CN109613244B (en) | Preparation method and application of Ag @ Pt-CuS labeled immunosensor | |
| CN104931698A (en) | Preparation method and application of NP-NiGd@Au-based gastric cancer marker gold nano-cluster electrogenerated chemiluminescence sensor | |
| CN105115961A (en) | Method for preparing electrochemical luminescence sensor made of nano-composites | |
| CN108709996B (en) | Preparation method and application of gold-palladium composite nanoenzyme immunosensor | |
| CN103472123A (en) | In-situ anode dissolving-out volt-ampere analytical method based on metal marking and biology affinity | |
| CN108918853B (en) | Pd @ Ag @ CeO2Preparation method and application of labeled immunosensor | |
| CN104297495A (en) | Making method and application of CdS sensitized TiO2 environmental estrogen photoelectrochemical sensor | |
| CN110618180B (en) | Preparation method and application of non-labeled electrochemical immunosensor for detecting prostate specific antigen | |
| CN112432981A (en) | Single-cell electrochemical sensor based on functionalized nanoprobe and application thereof | |
| CN110441535B (en) | Preparation method of electrochemical immunosensor for detecting procalcitonin based on Pd NCs functionalized CuInOS | |
| CN106483281A (en) | Renewable electrochemical immunosensor preparation method for sCD40L detection | |
| CN103698509B (en) | Method for detecting mercaptoacetic acid by utilizing electrochemical immunosensor sensor based on nano porous gold electrode | |
| Wang et al. | Highly efficient quenching of electrochemiluminescence from CdS nanocrystal film based on biocatalytic deposition | |
| CN108802390B (en) | Preparation of pancreatic cancer tumor marker immunosensor based on graphene-gold-palladium nanocomposite | |
| Xiao et al. | Electrochemiluminescence immunosensor using poly (l-histidine)-protected glucose dehydrogenase on Pt/Au bimetallic nanoparticles to generate an in situ co-reactant | |
| CN106404862B (en) | Electrochemical sensing of lead ion check with high sensitivity and preparation method thereof and application method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| EXSB | Decision made by sipo to initiate substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20160420 |