CN104833718B - A kind of preparation method of pH release type immunosensor and application - Google Patents

A kind of preparation method of pH release type immunosensor and application Download PDF

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CN104833718B
CN104833718B CN201510248742.0A CN201510248742A CN104833718B CN 104833718 B CN104833718 B CN 104833718B CN 201510248742 A CN201510248742 A CN 201510248742A CN 104833718 B CN104833718 B CN 104833718B
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silicon oxide
mesoporous silicon
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electrode
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CN104833718A (en
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李娜
魏琴
马洪敏
王超
刘振
闫涛
吴丹
庞雪辉
李贺
胡丽华
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University of Jinan
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Abstract

<b/> the present invention relates to a kind of preparation method and application of pH release type immunosensor, belongs to new bio sensing detection technology.Specifically redox probe thionine be encapsulated in mesoporous silicon oxide-Au mesoporous in, obtain detecting antibody labeling thing---mesoporous silicon oxide-Au, prepare a kind of sandwich type electrochemical immunosensor detecting tumor markers.New detection method is opened for detecting tumor markers.

Description

A kind of preparation method of pH release type immunosensor and application
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 5mmol/L potassium ferricyanide solution, scans under-0.2 ~ 0.6V current potential, make spike potential difference be less than 110mV;
(2) get 6 μ L, the amination tri-iron tetroxide solution of 0.5 ~ 3mg/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 0.1mol/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 1h, 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 1h, 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 ~ 0.7g is dissolved in the ultrapure water of 200mL, add 1 ~ 2mL, the NaOH of 2.0mol/L, in 80 DEG C of back flow reaction 20min, add the tetraethoxysilane of 1.5 ~ 3.0mL subsequently, continue reaction 2h and generate white precipitate, in order to remove CTAB residual in solution, the precipitation obtained is dispersed in 1.5mL again, massfraction is in the mixed liquor of the HCl of 37% and the methyl alcohol of 75 ~ 100mL, reflux at 80 DEG C 10 ~ 12h, potpourri is centrifugal, through water and methanol wash several, vacuum drying at 35 ~ 45 DEG C, obtained mesoporous silicon oxide, particle size is about 100nm, pore size is about 5nm,
(2) preparation of the mesoporous silicon oxide MSN-Aetal of acetal linker modification
Be scattered in 20mL ethanol by the mesoporous silicon oxide of 0.1 ~ 0.3g, add the 3-aminopropyl triethoxysilane of 0.2 ~ 1mL, 80 DEG C of back flow reaction 12h, potpourri, through centrifugal, washing, vacuum drying at 45 DEG C, obtains amidized mesoporous silicon oxide;
Amidized for 0.1 ~ 0.3g mesoporous silicon oxide is added in the dimethyl sulphoxide solution of 10mL, add 50 ~ 100mg succinic anhydride and 50 ~ 100mg triethylamine subsequently, react 48h 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 ~ 30mg of 20 ~ 80mg is dissolved in 3mL ultrapure water, add the mesoporous silicon oxide that 50 ~ 100mg is carboxylated, 3 of 80 ~ 130mg, two (the 3-aminopropyl)-2 of 9-, 4,8,10-tetra-oxaspiro [5.5] undecane, stirred at ambient temperature 8h, 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 20mL, 1% HAuCl 4be scattered in the deoxidation methyl alcohol of 85mL with 0.5mmol mercapto succinic acid, under agitation, by the NaBH of 25mL, 0.2mol/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 ~ 20mg is modified is joined 0.5 ~ 3mmol/L, in the thionine aqueous solution of 1mL, stirred at ambient temperature 12h, add the carboxyl-functional gold Au-COOH of 4 ~ 10mg, the N-hydroxy-succinamide NHS of 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride EDC and 5mg of 10mg, continue to stir 8h, potpourri through centrifuge washing until supernatant be colourless after, precipitation is dispersed in the ultrapure water of 1mL 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 1mL is mixed with the detection antibody-solutions of 1mL, 10 μ g/mL, 12h is hatched under the condition of 4 DEG C, after centrifugal, precipitation be dispersed in the phosphate buffered solution of 1mL, 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 the acetate buffer solution 12h of 0.5mL, 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.
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 5mmol/L potassium ferricyanide solution, scans under-0.2 ~ 0.6V current potential, make spike potential difference be less than 110mV;
(2) the amination tri-iron tetroxide solution getting 6 μ L, 0.5mg/mL is added drop-wise to electrode surface, dries 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 0.1mol/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 1h, 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 1h, 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 5mmol/L potassium ferricyanide solution, scans under-0.2 ~ 0.6V current potential, make spike potential difference be less than 110mV;
(2) the amination tri-iron tetroxide solution getting 6 μ L, 1mg/mL is added drop-wise to electrode surface, dries 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 0.1mol/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 1h, 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 1h, 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 5mmol/L potassium ferricyanide solution, scans under-0.2 ~ 0.6V current potential, make spike potential difference be less than 110mV;
(2) the amination tri-iron tetroxide solution getting 6 μ L, 3mg/mL is added drop-wise to electrode surface, dries 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 0.1mol/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 1h, 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 1h, 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 0.3g is dissolved in the ultrapure water of 200mL, add 1mL, the NaOH of 2.0mol/L, in 80 DEG C of back flow reaction 20min, add the tetraethoxysilane of 1.5mL subsequently, continue reaction 2h and generate white precipitate, , in order to remove CTAB residual in solution, the precipitation obtained is dispersed in 1.5mL again, massfraction is in the mixed liquor of the methyl alcohol of HCl and 75mL of 37%, reflux at 80 DEG C 10h, potpourri is centrifugal, through water and methanol wash several, vacuum drying at 35 DEG C, obtained mesoporous silicon oxide, particle size is about 100nm, pore size is about 5nm,
(2) preparation of the mesoporous silicon oxide MSN-Aetal of acetal linker modification
Be scattered in 20mL ethanol by the mesoporous silicon oxide of 0.1g, add the 3-aminopropyl triethoxysilane of 0.2mL, 80 DEG C of back flow reaction 12h, potpourri, through centrifugal, washing, vacuum drying at 45 DEG C, obtains amidized mesoporous silicon oxide;
Be added in the dimethyl sulphoxide solution of 10mL by amidized for 0.1g mesoporous silicon oxide, add 50mg succinic anhydride and 50mg triethylamine subsequently, react 48h 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-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride EDC and 10mg of 20mg is dissolved in 3mL ultrapure water, add the mesoporous silicon oxide that 50mg is carboxylated, 3 of 80mg, two (the 3-aminopropyl)-2 of 9-, 4,8,10-tetra-oxaspiro [5.5] undecane, stirred at ambient temperature 8h, 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 20mL, 1% HAuCl 4be scattered in the deoxidation methyl alcohol of 85mL with 0.5mmol mercapto succinic acid, under agitation, by the NaBH of 25mL, 0.2mol/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 10mg is modified is joined 0.5mmol/L, in the thionine aqueous solution of 1mL, stirred at ambient temperature 12h, add the carboxyl-functional gold Au-COOH of 4mg, the N-hydroxy-succinamide NHS of 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride EDC and 5mg of 10mg, continue to stir 8h, potpourri through centrifuge washing until supernatant be colourless after, precipitation is dispersed in the ultrapure water of 1mL 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 1mL is mixed with the detection antibody-solutions of 1mL, 10 μ g/mL, 12h is hatched under the condition of 4 DEG C, after centrifugal, precipitation be dispersed in the phosphate buffered solution of 1mL, 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 0.5g is dissolved in the ultrapure water of 200mL, add 1.5mL, the NaOH of 2.0mol/L, in 80 DEG C of back flow reaction 20min, add the tetraethoxysilane of 2.0mL subsequently, continue reaction 2h and generate white precipitate, , in order to remove CTAB residual in solution, the precipitation obtained is dispersed in 1.5mL again, massfraction is in the mixed liquor of the methyl alcohol of HCl and 85mL of 37%, reflux at 80 DEG C 11h, potpourri is centrifugal, through water and methanol wash several, vacuum drying at 40 DEG C, obtained mesoporous silicon oxide, particle size is about 100nm, pore size is about 5nm,
(2) preparation of the mesoporous silicon oxide MSN-Aetal of acetal linker modification
Be scattered in 20mL ethanol by the mesoporous silicon oxide of 0.2g, add the 3-aminopropyl triethoxysilane of 0.7mL, 80 DEG C of back flow reaction 12h, potpourri, through centrifugal, washing, vacuum drying at 45 DEG C, obtains amidized mesoporous silicon oxide;
Be added in the dimethyl sulphoxide solution of 10mL by amidized for 0.2g mesoporous silicon oxide, add 80mg succinic anhydride and 80mg triethylamine subsequently, react 48h 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-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride EDC and 20mg of 50mg is dissolved in 3mL ultrapure water, add the mesoporous silicon oxide that 80mg is carboxylated, 3 of 100mg, two (the 3-aminopropyl)-2 of 9-, 4,8,10-tetra-oxaspiro [5.5] undecane, stirred at ambient temperature 8h, 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 20mL, 1% HAuCl 4be scattered in the deoxidation methyl alcohol of 85mL with 0.5mmol mercapto succinic acid, under agitation, by the NaBH of 25mL, 0.2mol/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 15mg is modified is joined 1mmol/L, in the thionine aqueous solution of 1mL, stirred at ambient temperature 12h, add the carboxyl-functional gold Au-COOH of 8mg, the N-hydroxy-succinamide NHS of 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride EDC and 5mg of 10mg, continue to stir 8h, potpourri through centrifuge washing until supernatant be colourless after, precipitation is dispersed in the ultrapure water of 1mL 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 1mL is mixed with the detection antibody-solutions of 1mL, 10 μ g/mL, 12h is hatched under the condition of 4 DEG C, after centrifugal, precipitation be dispersed in the phosphate buffered solution of 1mL, 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 0.7g is dissolved in the ultrapure water of 200mL, add 2mL, the NaOH of 2.0mol/L, in 80 DEG C of back flow reaction 20min, add the tetraethoxysilane of 3.0mL subsequently, continue reaction 2h and generate white precipitate, , in order to remove CTAB residual in solution, the precipitation obtained is dispersed in 1.5mL again, massfraction is in the mixed liquor of the methyl alcohol of HCl and 100mL of 37%, reflux at 80 DEG C 12h, potpourri is centrifugal, through water and methanol wash several, vacuum drying at 45 DEG C, obtained mesoporous silicon oxide, particle size is about 100nm, pore size is about 5nm,
(2) preparation of the mesoporous silicon oxide MSN-Aetal of acetal linker modification
Be scattered in 20mL ethanol by the mesoporous silicon oxide of 0.3g, add the 3-aminopropyl triethoxysilane of 1mL, 80 DEG C of back flow reaction 12h, potpourri, through centrifugal, washing, vacuum drying at 45 DEG C, obtains amidized mesoporous silicon oxide;
Be added in the dimethyl sulphoxide solution of 10mL by amidized for 0.3g mesoporous silicon oxide, add 100mg succinic anhydride and 100mg triethylamine subsequently, react 48h 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-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride EDC and 30mg of 80mg is dissolved in 3mL ultrapure water, add the mesoporous silicon oxide that 100mg is carboxylated, 3 of 130mg, two (the 3-aminopropyl)-2 of 9-, 4,8,10-tetra-oxaspiro [5.5] undecane, stirred at ambient temperature 8h, 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 20mL, 1% HAuCl 4be scattered in the deoxidation methyl alcohol of 85mL with 0.5mmol mercapto succinic acid, under agitation, by the NaBH of 25mL, 0.2mol/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 20mg is modified is joined 3mmol/L, in the thionine aqueous solution of 1mL, stirred at ambient temperature 12h, add the carboxyl-functional gold Au-COOH of 10mg, the N-hydroxy-succinamide NHS of 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride EDC and 5mg of 10mg, continue to stir 8h, potpourri through centrifuge washing until supernatant be colourless after, precipitation is dispersed in the ultrapure water of 1mL 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 1mL is mixed with the detection antibody-solutions of 1mL, 10 μ g/mL, 12h is hatched under the condition of 4 DEG C, after centrifugal, precipitation be dispersed in the phosphate buffered solution of 1mL, 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 the acetate buffer solution 12h of 0.5mL, 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 ~ 10ng/mL, detects and is limited to 0.21pg/mL.
embodiment 8according to the method for embodiment 7, AFP, CA153, CA125 are detected, record the range of linearity and be respectively 0.001 ~ 10ng/mL, 0.001 ~ 15ng/mL and 0.002 ~ 10ng/mL; Detectability is respectively 0.17pg/mL, 0.31pg/mL and 0.54pg/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 5mmol/L potassium ferricyanide solution, scans under-0.2 ~ 0.6V current potential, make spike potential difference be less than 110mV;
(2) get 6 μ L, the amination tri-iron tetroxide solution of 0.5 ~ 3mg/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 0.1mol/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 multiple electrode surface respectively, for the specific recognition with antibody, incubated at room temperature 1h, 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 1h, 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 ~ 0.7g is dissolved in the ultrapure water of 200mL, add 1 ~ 2mL, the NaOH of 2.0mol/L, in 80 DEG C of back flow reaction 20min, add the tetraethoxysilane of 1.5 ~ 3.0mL subsequently, continue reaction 2h and generate white precipitate, in order to remove CTAB residual in solution, the precipitation obtained is dispersed in 1.5mL again, massfraction is in the mixed liquor of the HCl of 37% and the methyl alcohol of 75 ~ 100mL, reflux at 80 DEG C 10 ~ 12h, potpourri is centrifugal, through water and methanol wash several, vacuum drying at 35 ~ 45 DEG C, obtained mesoporous silicon oxide, particle size is about 100nm, pore size is about 5nm,
(2) preparation of the mesoporous silicon oxide MSN-Aetal of acetal linker modification
Be scattered in 20mL ethanol by the mesoporous silicon oxide of 0.1 ~ 0.3g, add the 3-aminopropyl triethoxysilane of 0.2 ~ 1mL, 80 DEG C of back flow reaction 12h, potpourri, through centrifugal, washing, vacuum drying at 45 DEG C, obtains amidized mesoporous silicon oxide;
Amidized for 0.1 ~ 0.3g mesoporous silicon oxide is added in the dimethyl sulphoxide solution of 10mL, add 50 ~ 100mg succinic anhydride and 50 ~ 100mg triethylamine subsequently, react 48h 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 ~ 30mg of 20 ~ 80mg is dissolved in 3mL ultrapure water, add the mesoporous silicon oxide that 50 ~ 100mg is carboxylated, 3 of 80 ~ 130mg, two (the 3-aminopropyl)-2 of 9-, 4,8,10-tetra-oxaspiro [5.5] undecane, stirred at ambient temperature 8h, 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 20mL, 1% HAuCl 4be scattered in the deoxidation methyl alcohol of 85mL with 0.5mmol mercapto succinic acid, under agitation, by the NaBH of 25mL, 0.2mol/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 ~ 20mg is modified is joined 0.5 ~ 3mmol/L, in the thionine aqueous solution of 1mL, stirred at ambient temperature 12h, add the carboxyl-functional gold Au-COOH of 4 ~ 10mg, the N-hydroxy-succinamide NHS of 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride EDC and 5mg of 10mg, continue to stir 8h, potpourri through centrifuge washing until supernatant be colourless after, precipitation is dispersed in the ultrapure water of 1mL 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 1mL is mixed with the detection antibody-solutions of 1mL, 10 μ g/mL, 12h is hatched under the condition of 4 DEG C, after centrifugal, precipitation be dispersed in the phosphate buffered solution of 1mL, pH=7.4 again, be kept in the refrigerator of 4 DEG C for subsequent use.
3. 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.
4. 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 the acetate buffer solution 12h of 0.5mL, 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.
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