CN104297480A - Preparation method and application of sandwich type immunosensor for prostate specific antigens - Google Patents
Preparation method and application of sandwich type immunosensor for prostate specific antigens Download PDFInfo
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Abstract
The invention relates to a preparation method and an application of a sandwich type immunosensor for prostate specific antigens, and belongs to the technical field of novel functional materials and biological sensing detection. On the basis of the advantages of larger specific surface area, higher catalytic efficiency and the like of a GS-CNT-Pt nano material with a three-dimensional structure, the preparation method has the advantages that the sensitivity for detection of prostate-cancer specific antigens is improved and the detection limit is reduced; and the preparation method has important significance in detecting the prostate-cancer specific antigens.
Description
Technical field
The present invention relates to a kind of prostate specific antigen sandwich type immunosensor preparation method and application.Specifically adopt the GS-CNT-Pt of three-dimensional structure to prepare a kind of electrochemical immunosensor detecting prostate cancer marker, belong to new function material and bio-sensing detection technique field.
Background technology
Multiple method is had, such as DNA test, chemiluminescence immune assay, enzyme-linked immuno assay etc. at present to the detection of prostate cancer marker; Immunology principle combines with Electroanalytical Chemistry by the present invention, has prepared that a kind of sensitivity is high, selectivity good, simple and rapid for detecting the sensor of prostate specific antigen.
Graphene nano layer, as a kind of two-dimensional material, has larger specific surface area, and catalytic performance is good, the features such as good biocompatibility; Golden nanometer particle has the features such as high, the long-term dispersiveness of catalytic performance is stable, Bc is strong; Amination Graphene compares Graphene can add the combination with gold, Au-NH to a greater degree
2-GS nano composite material can increase antibody in the charge capacity on its surface and the dispersiveness in water, and the application of this compound substance has the sensitivity and stability that improve immunosensor, reduces the detection limit of sensor, strengthens the advantages such as electrochemical signals.The present invention has prepared a kind of GS-CNT nano material with three-dimensional structure, inserts Pt nano particle in its three-D space structure, impel antibody more easily, firmly with its combination, thus significantly improve the catalytic performance of this material.Preparation process of the present invention is simple, detects quick, the series of advantages such as sensitivity is high, and detection limit is low.
Summary of the invention
An object of the present invention, based on the GS-CNT-Pt nano composite material with three-dimensional structure, prepares a kind of prostate specific antigen sandwich type immunosensor; Object of the present invention two by highly sensitive, the quick specific detection of this sensor application in PSA.
technical scheme of the present invention is as follows:
1. a prostate specific antigen sandwich type immunosensor preparation method, step is as follows:
(1) by diameter be the glass-carbon electrode of the 4 mm Al of 0.05 μm
2o
3burnishing powder is polished, and cleans up with ultrapure water; By the Au-NH of 4 ~ 6 μ L, 0.5 ~ 2 mg/mL
2-GS solution is added drop-wise on the bare electrode handled well, places in 4 DEG C of refrigerators and uses ultrapure water again after drying, dry in 4 DEG C of refrigerators;
(2) by the prostate cancer marker primary antibodie Ab of 4 ~ 6 μ L, 8 ~ 12 μ g/mL
1solution is added drop-wise to electrode surface, places in 4 DEG C of refrigerators and uses ultrapure water again after drying, dry in 4 DEG C of refrigerators;
(3) be that the bovine serum albumin(BSA) BSA solution of 0.5 ~ 3.0 % is added drop-wise to electrode surface by 3 μ L, massfraction, with enclosed-electrode nonspecific activity site on the surface, place in 4 DEG C of refrigerators and use ultrapure water again after drying, dry in 4 DEG C of refrigerators;
(4) the prostate cancer marker antigen standard solution of a series of variable concentrations of dropping 4 ~ 6 μ L, 0.5 pg/mL ~ 15 ng/mL is to electrode surface, places in 4 DEG C of refrigerators and uses ultrapure water again after drying, dry in 4 DEG C of refrigerators;
(5) by the GS-CNT-Pt/Ab of 4 ~ 6 μ L, 3 ~ 6 mg/mL
2two anti-compound solution of incubating drip and are applied on electrode surface, are placed in after 4 DEG C of refrigerators dry, ultrapure water, obtained prostate specific antigen sandwich type immunosensor.
2. Au-NH
2the preparation of-GS Nanocomposite solution, step is as follows:
(1) preparation of solution of gold nanoparticles
Be the HAuCl of 0.01 ~ 0.02% by 100 mL, massfraction
4solution is heated to boil, and dropping 2.0 ~ 3.0 mL, massfraction are the sodium citrate aqueous solution of 0.5 ~ 1.5%, and solution colour becomes rufous, is cooled to room temperature;
(2) amination Graphene NH
2the preparation of-GS
0.05 ~ 0.2 g graphene dispersion in the ethanol of 5 ~ 15 mL, ultrasonic agitation 1 h, add 0.1 ~ 0.3 mL, massfraction be 99% 3-aminopropyl triethoxysilane, under 70 DEG C of conditions, add thermal agitation 1.0 ~ 2.0 h; Add 0.1 mL, massfraction is the hydrazine hydrate of 80%, 95 DEG C of heating 1 h; Centrifugal 20 ~ 40 min under the rotating speed of 9000 rpm; Dry 8 ~ 12 h under vacuum, obtained amination Graphene NH
2-GS
;
(3) Au-NH
2the preparation of-GS Nanocomposite solution
Take the amination Graphene NH of 10 ~ 20 mg
2-GS is placed in small beaker, adds 10 ~ 20 mL gold nano solution, stirs 10 ~ 14 h, after centrifugal at vacuum condition 35 ~ 45 DEG C dry 12h, obtained Au-NH
2-GS nano composite material, is mixed with the Au-NH of 0.5 ~ 2 mg/mL with water
2-GS Nanocomposite solution.
3. GS-CNT-Pt/Ab
2two anti-preparations of incubating compound solution, step is as follows:
(1) preparation of graphene oxide
By the graphite of 0.3 g and the mixing of 1.5 ~ 2.0 g potassium permanganate, put into the there-necked flask of 500 mL with magneton, add sulfuric acid and phosphoric acid mixed liquor that volume ratio is 8 ~ 10: 1, there-necked flask is put into oil bath pan, is heated to 50 DEG C, reaction 10 ~ 12 h; Sample is poured into 40 mL on ice, under magnetic agitation, drip the hydrogen peroxide of 0.5 ~ 1.0 mL, solution becomes khaki, centrifugal 0.5 h under the rotating speed of 8000 rpm, use the absolute ethyl alcohol centrifuge washing 3 times of the hydrochloric acid of 30 mL, 0.2 mol/L and 30 mL successively, abandoning supernatant, with ether centrifuge washing 1 time, abandoning supernatant, the solid sample obtained is placed in dry 12 h of vacuum drying chamber of 30 ~ 40 DEG C, obtained graphene oxide yellow-brown solid powder;
(2) preparation of the GS-CNT nano composite material of three-dimensional structure
The carbon nano-tube CNT of 20 ~ 40 mg is joined 30 mL, 1 ~ 3 mg/mL graphene oxide uniform dispersion in, ultrasonic 30 min, the potpourri obtained is put into the autoclave of 50 mL, be heated to 160 ~ 190 DEG C, and keep 12 h, be cooled to room temperature, by the potpourri freeze drying obtained;
(3) preparation of the GS-CNT-Pt nano-complex of three-dimensional structure
Under the condition of vigorous stirring, by the K of 1 mL, 5 ~ 15 mmol/L
2ptCl
4the GS-CNT nano composite material of the three-dimensional structure of solution and 5 mg joins in the ultrapure water of 10 mL, ice bath 30 min, centrifuging 5 min under 7000 rpm, the GS-CNT-Pt nano-complex of obtained three-dimensional structure, wash with water, centrifuging 3 times, vacuum drying 12h at 35 ~ 45 DEG C;
(4) GS-CNT-Pt/Ab
2two anti-preparations of incubating compound solution
The GS-CNT-Pt nano-complex of the three-dimensional structure of 3 ~ 6 mg is joined in the phosphate buffered solution of 3 ~ 6 mL, pH=7.4, adds the anti-Ab of prostate cancer marker two of 150 ~ 300 μ L, 10 μ g/mL
2solution, vibrate in 4 DEG C of constant-temperature shaking incubators hatching 24 h, and centrifugal 10 min under the rotating speed of 3000 rpm, add 1 mL, pH=7.4 phosphate buffered solution, obtained GS-CNT-Pt/Ab
2two anti-incubate compound solution, save backup at 4 DEG C.
4. the detection method of prostate cancer marker, step is as follows:
(1) electrochemical workstation is used to test with three-electrode system, saturated calomel electrode is contrast electrode, platinum electrode is auxiliary electrode, and prepared sensor is working electrode, tests in pH=5.9 ~ 8.0 phosphate buffered solution of 10 mL, 30 ~ 50 mmol/L;
(2) detect prostate specific antigen standard solution with chronoamperometry, input voltage is-0.4 V, sample interval 0.1 s, working time 300 s;
(3) after background current tends towards stability, inject the hydrogen peroxide solution of 10 μ L, 5 ~ 10 mol/L every 50 s in pH=5.9 ~ 8.0 phosphate buffered solution of 10 mL, 30 ~ 50 mmol/L, record current changes, drawing curve;
(4) replace prostate specific antigen standard solution with blood serum sample solution, the method for drawing according to working curve detects.
useful achievement of the present invention
(1) Au-NH
2-GS nano composite material increases antibody in the charge capacity on its surface and the dispersiveness in water compared with Au-GS compound substance, thus improves sensitivity and the stability of sensor.
(2) will introduce in redox graphene in carbon nano-tube, form unique tridimensional network, increase specific surface area, the more Pt nano particle of load, make antibody easily and its combination.
(3) by three-dimensional GS-CNT-Pt nano-complex and tumor markers two is anti-directly hatches, utilize the biocompatibility of noble metal excellence and higher catalytic performance, enzyme need not be used in two marks resisted, avoid because of enzyme inactivation and leak the metrical error that causes, significantly improve reappearance and the stability of electrochemical immunosensor.
(4) the present invention utilizes the immune response of antigen, antibody, improves the specificity of detection method.
(5) electrochemical immunosensor prepared of the present invention is for the detection of PSA mark, there is the response time short, detectability is low, the range of linearity is wide, the advantages such as simple, quick, highly sensitive and specific detection can be realized, the range of linearity is 0.50 pg/mL ~ 15 ng/mL, detects and is limited to 0.12 pg/mL.
Embodiment
embodiment 1a kind of prostate specific antigen sandwich type immunosensor preparation method
(1) by diameter be the glass-carbon electrode of the 4 mm Al of 0.05 μm
2o
3burnishing powder is polished, and cleans up with ultrapure water; By the Au-NH of 4 μ L, 0.5 mg/mL
2-GS solution is added drop-wise on the bare electrode handled well, places in 4 DEG C of refrigerators and uses ultrapure water again after drying, dry in 4 DEG C of refrigerators;
(2) by the prostate cancer marker primary antibodie Ab of 4 μ L, 8 μ g/mL
1solution is added drop-wise to electrode surface, places in 4 DEG C of refrigerators and uses ultrapure water again after drying, dry in 4 DEG C of refrigerators;
(3) be that the bovine serum albumin(BSA) BSA solution of 0.5 % is added drop-wise to electrode surface by 3 μ L, massfraction, with enclosed-electrode nonspecific activity site on the surface, place in 4 DEG C of refrigerators and use ultrapure water again after drying, dry in 4 DEG C of refrigerators;
(4) drip 4 μ L, 0.5 pg/mL ~ 15 ng/mL the prostate cancer marker antigen standard solution of a series of variable concentrations to electrode surface, place in 4 DEG C of refrigerators and use ultrapure water again after drying, dry in 4 DEG C of refrigerators;
(5) by the GS-CNT-Pt/Ab of 4 μ L, 3 mg/mL
2two anti-compound solution of incubating drip and are applied on electrode surface, are placed in after 4 DEG C of refrigerators dry, ultrapure water, obtained prostate specific antigen sandwich type immunosensor.
embodiment 2a kind of prostate specific antigen sandwich type immunosensor preparation method
(1) by diameter be the glass-carbon electrode of the 4 mm Al of 0.05 μm
2o
3burnishing powder is polished, and cleans up with ultrapure water; By the Au-NH of 5 μ L, 1.0 mg/mL
2-GS solution is added drop-wise on the bare electrode handled well, places in 4 DEG C of refrigerators and uses ultrapure water again after drying, dry in 4 DEG C of refrigerators;
(2) by the prostate cancer marker primary antibodie Ab of 5 μ L, 10 μ g/mL
1solution is added drop-wise to electrode surface, places in 4 DEG C of refrigerators and uses ultrapure water again after drying, dry in 4 DEG C of refrigerators;
(3) by 3 μ L, massfraction be 2.0% bovine serum albumin(BSA) BSA solution be added drop-wise to electrode surface, with enclosed-electrode nonspecific activity site on the surface, place in 4 DEG C of refrigerators and use ultrapure water again after drying, dry in 4 DEG C of refrigerators;
(4) drip 5 μ L, 0.5 pg/mL ~ 15 ng/mL the prostate cancer marker antigen standard solution of a series of variable concentrations to electrode surface, place in 4 DEG C of refrigerators and use ultrapure water again after drying, dry in 4 DEG C of refrigerators;
(5) by the GS-CNT-Pt/Ab of 5 μ L, 5 mg/mL
2two anti-compound solution of incubating drip and are applied on electrode surface, are placed in after 4 DEG C of refrigerators dry, ultrapure water, obtained prostate specific antigen sandwich type immunosensor.
embodiment 3a kind of prostate specific antigen sandwich type immunosensor preparation method
(1) by diameter be the glass-carbon electrode of the 4 mm Al of 0.05 μm
2o
3burnishing powder is polished, and cleans up with ultrapure water; By the Au-NH of 6 μ L, 2 mg/mL
2-GS solution is added drop-wise on the bare electrode handled well, places in 4 DEG C of refrigerators and uses ultrapure water again after drying, dry in 4 DEG C of refrigerators;
(2) by the prostate cancer marker primary antibodie Ab of 6 μ L, 12 μ g/mL
1solution is added drop-wise to electrode surface, places in 4 DEG C of refrigerators and uses ultrapure water again after drying, dry in 4 DEG C of refrigerators;
(3) be that the bovine serum albumin(BSA) BSA solution of 3.0 % is added drop-wise to electrode surface by 3 μ L, massfraction, with enclosed-electrode nonspecific activity site on the surface, place in 4 DEG C of refrigerators and use ultrapure water again after drying, dry in 4 DEG C of refrigerators;
(4) drip 6 μ L, 0.5 pg/mL ~ 15 ng/mL the prostate cancer marker antigen standard solution of a series of variable concentrations to electrode surface, place in 4 DEG C of refrigerators and use ultrapure water again after drying, dry in 4 DEG C of refrigerators;
(5) by the GS-CNT-Pt/Ab of 6 μ L, 6 mg/mL
2two anti-compound solution of incubating drip and are applied on electrode surface, are placed in after 4 DEG C of refrigerators dry, ultrapure water, obtained prostate specific antigen sandwich type immunosensor.
embodiment 4au-NH
2the preparation of-GS Nanocomposite solution
(1) preparation of solution of gold nanoparticles
Be the HAuCl of 0.01% by 100 mL, massfraction
4solution is heated to boil, and drip 2.0 mL, massfraction is the sodium citrate aqueous solution of 0.5%, solution colour becomes rufous, is cooled to room temperature;
(3) amination Graphene NH
2the preparation of-GS
0.05 g graphene dispersion in the ethanol of 5 mL, ultrasonic agitation 1 h, add 0.1 mL, massfraction be 99% 3-aminopropyl triethoxysilane, under 70 DEG C of conditions, add thermal agitation 1.0 h; Add 0.1 mL, massfraction is the hydrazine hydrate of 80%, 95 DEG C of heating 1 h; Centrifugal 20 min under the rotating speed of 9000 rpm; Dry 8 h under vacuum, obtained amination Graphene NH
2-GS
;
(3) Au-NH
2the preparation of-GS Nanocomposite solution
Take the amination Graphene NH of 10 mg
2-GS is placed in small beaker, adds 10 mL gold nano solution, stirs 10 h, after centrifugal at vacuum condition 35 DEG C dry 12 h, obtained Au-NH
2-GS nano composite material, is mixed with the Au-NH of 0.5 mg/mL with water
2-GS Nanocomposite solution.
embodiment 5au-NH
2the preparation of-GS Nanocomposite solution
(1) preparation of solution of gold nanoparticles
Be the HAuCl of 0.015% by 100mL, massfraction
4solution is heated to boil, and drip 2.5 mL, massfraction is the sodium citrate aqueous solution of 1.0%, solution colour becomes rufous, is cooled to room temperature;
(4) amination Graphene NH
2the preparation of-GS
0.1 g graphene dispersion in the ethanol of 10 mL, ultrasonic agitation 1 h, add 0.2 mL, massfraction be 99% 3-aminopropyl triethoxysilane, under 70 DEG C of conditions, add thermal agitation 1.5 h; Add 0.1 mL, massfraction is the hydrazine hydrate of 80%, 95 DEG C of heating 1 h; Centrifugal 30 min under the rotating speed of 9000 rpm; Dry 10 h under vacuum, obtained amination Graphene NH
2-GS
;
(3) Au-NH
2the preparation of-GS Nanocomposite solution
Take the amination Graphene NH of 15 mg
2-GS is placed in small beaker, adds 15 mL gold nano solution, stirs 12 h, after centrifugal at vacuum condition 40 DEG C dry 12 h, obtained Au-NH
2-GS nano composite material, is mixed with the Au-NH of 1.0 mg/mL with water
2-GS Nanocomposite solution.
embodiment 6au-NH
2the preparation of-GS Nanocomposite solution
(1) preparation of solution of gold nanoparticles
Be the HAuCl of 0.02% by 100 mL, massfraction
4solution is heated to boil, and drip 3.0 mL, massfraction is the sodium citrate aqueous solution of 1.5%, solution colour becomes rufous, is cooled to room temperature;
(5) amination Graphene NH
2the preparation of-GS
0.2 g graphene dispersion in the ethanol of 15 mL, ultrasonic agitation 1 h, add 0.3 mL, massfraction be 99% 3-aminopropyl triethoxysilane, under 70 DEG C of conditions, add thermal agitation 2.0 h; Add 0.1 mL, massfraction is the hydrazine hydrate of 80%, 95 DEG C of heating 1 h; Centrifugal 40 min under the rotating speed of 9000 rpm; Dry 12 h under vacuum, obtained amination Graphene NH
2-GS
;
(3) Au-NH
2the preparation of-GS Nanocomposite solution
Take the amination Graphene NH of 20 mg
2-GS is placed in small beaker, adds 20 mL gold nano solution, stirs 14 h, after centrifugal at vacuum condition 45 DEG C dry 12 h, obtained Au-NH
2-GS nano composite material, is mixed with the Au-NH of 2 mg/mL with water
2-GS Nanocomposite solution.
embodiment 7gS-CNT-Pt/Ab
2two anti-preparations of incubating compound solution
(1) preparation of graphene oxide
By the graphite of 0.3 g and the mixing of 1.5 g potassium permanganate, put into the there-necked flask of 500 mL with magneton, add sulfuric acid and phosphoric acid mixed liquor that volume ratio is 8: 1, there-necked flask is put into oil bath pan, is heated to 50 DEG C, react 10 h; Sample is poured into 40 mL on ice, under magnetic agitation, drip the hydrogen peroxide of 0.5 mL, solution becomes khaki, centrifugal 0.5 h under the rotating speed of 8000 rpm, use the absolute ethyl alcohol centrifuge washing 3 times of the hydrochloric acid of 30 mL, 0.2 mol/L and 30 mL successively, abandoning supernatant, with ether centrifuge washing 1 time, abandoning supernatant, the solid sample obtained is placed in dry 12 h of vacuum drying chamber of 30oC, obtained graphene oxide yellow-brown solid powder;
(2) preparation of the GS-CNT nano composite material of three-dimensional structure
The carbon nano-tube CNT of 20 mg is joined 30 mL, 1 mg/mL graphene oxide uniform dispersion in, ultrasonic 30 min, the potpourri obtained is put into the autoclave of 50 mL, be heated to 160 DEG C, and keep 12 h, be cooled to room temperature, by the potpourri freeze drying obtained;
(3) preparation of the GS-CNT-Pt nano-complex of three-dimensional structure
Under the condition of vigorous stirring, by the K of 1 mL, 5 mmol/L
2ptCl
4the GS-CNT nano composite material of the three-dimensional structure of solution and 5 mg joins in the ultrapure water of 10 mL, ice bath 30 min, centrifuging 5 min under 7000 rpm, the GS-CNT-Pt nano-complex of obtained three-dimensional structure, wash with water, centrifuging 3 times, vacuum drying 12 h at 35 DEG C;
(4) GS-CNT-Pt/Ab
2two anti-preparations of incubating compound solution
The GS-CNT-Pt nano-complex of the three-dimensional structure of 3mg is joined in the phosphate buffered solution of 3 mL, pH=7.4, adds the anti-Ab of prostate cancer marker two of 150 μ L, 10 μ g/mL
2solution, vibrate in 4 DEG C of constant-temperature shaking incubators hatching 24 h, and centrifugal 10 min under the rotating speed of 3000 rpm, add 1 mL, pH=7.4 phosphate buffered solution, obtained GS-CNT-Pt/Ab
2two anti-incubate compound solution, save backup at 4 DEG C.
embodiment 8gS-CNT-Pt/Ab
2two anti-preparations of incubating compound solution
(1) preparation of graphene oxide
By the graphite of 0.3 g and the mixing of 1.8 g potassium permanganate, put into the there-necked flask of 500 mL with magneton, add sulfuric acid and phosphoric acid mixed liquor that volume ratio is 9: 1, there-necked flask is put into oil bath pan, is heated to 50 DEG C, react 11 h; Sample is poured into 40 mL on ice, under magnetic agitation, drip the hydrogen peroxide of 0.8 mL, solution becomes khaki, centrifugal 0.5 h under the rotating speed of 8000 rpm, use the absolute ethyl alcohol centrifuge washing 3 times of the hydrochloric acid of 30 mL, 0.2 mol/L and 30 mL successively, abandoning supernatant, with ether centrifuge washing 1 time, abandoning supernatant, the solid sample obtained is placed in dry 12 h of vacuum drying chamber of 35oC, obtained graphene oxide yellow-brown solid powder;
(2) preparation of the GS-CNT nano composite material of three-dimensional structure
The carbon nano-tube CNT of 30mg is joined 30 mL, 2 mg/mL graphene oxide uniform dispersion in, ultrasonic 30 min, the potpourri obtained is put into the autoclave of 50 mL, be heated to 180 DEG C, and keep 12 h, be cooled to room temperature, by the potpourri freeze drying obtained;
(3) preparation of the GS-CNT-Pt nano-complex of three-dimensional structure
Under the condition of vigorous stirring, by the K of 1 mL, 10 mmol/L
2ptCl
4the three-dimensional GS-CNT nano composite material of solution and 5 mg joins in the ultrapure water of 10 mL, ice bath 30 min, centrifuging 5 min under 7000 rpm, the GS-CNT-Pt nano-complex of obtained three-dimensional structure, wash with water, centrifuging 3 times, vacuum drying 12h at 40 DEG C;
(4) GS-CNT-Pt/Ab
2two anti-preparations of incubating compound solution
The GS-CNT-Pt nano-complex of the three-dimensional structure of 5 mg is joined in the phosphate buffered solution of 5 mL, pH=7.4, adds the anti-Ab of prostate cancer marker two of 200 μ L, 10 μ g/mL
2solution, vibrate in 4 DEG C of constant-temperature shaking incubators hatching 24 h, and centrifugal 10 min under the rotating speed of 3000 rpm, add 1 mL, pH=7.4 phosphate buffered solution, obtained GS-CNT-Pt/Ab
2two anti-incubate compound solution, save backup at 4 DEG C.
embodiment 9gS-CNT-Pt/Ab
2two anti-preparations of incubating compound solution
(1) preparation of graphene oxide
By the graphite of 0.3 g and the mixing of 2.0 g potassium permanganate, put into the there-necked flask of 500 mL with magneton, add sulfuric acid and phosphoric acid mixed liquor that volume ratio is 10: 1, there-necked flask is put into oil bath pan, is heated to 50 DEG C, react 12 h; Sample is poured into 40 mL on ice, under magnetic agitation, drip the hydrogen peroxide of 1.0 mL, solution becomes khaki, centrifugal 0.5 h under the rotating speed of 8000 rpm, use the absolute ethyl alcohol centrifuge washing 3 times of the hydrochloric acid of 30 mL, 0.2 mol/L and 30 mL successively, abandoning supernatant, with ether centrifuge washing 1 time, abandoning supernatant, the solid sample obtained is placed in dry 12 h of vacuum drying chamber of 40 DEG C, obtained graphene oxide yellow-brown solid powder;
(2) preparation of the GS-CNT nano composite material of three-dimensional structure
The carbon nano-tube CNT of 40 mg is joined 30 mL, 3 mg/mL graphene oxide uniform dispersion in, ultrasonic 30 min, the potpourri obtained is put into the autoclave of 50 mL, be heated to 190 DEG C, and keep 12 h, be cooled to room temperature, by the potpourri freeze drying obtained;
(3) preparation of the GS-CNT-Pt nano-complex of three-dimensional structure
Under the condition of vigorous stirring, by the K of 1 mL, 15 mmol/L
2ptCl
4the GS-CNT nano composite material of the three-dimensional structure of solution and 5 mg joins in the ultrapure water of 10 mL, ice bath 30 min, centrifuging 5 min under 7000 rpm, the GS-CNT-Pt nano-complex of obtained three-dimensional structure, wash with water, centrifuging 3 times, vacuum drying 12 h at 45 DEG C;
(4) GS-CNT-Pt/Ab
2two anti-preparations of incubating compound solution
The GS-CNT-Pt nano-complex of the three-dimensional structure of 6 mg is joined in the phosphate buffered solution of 6 mL, pH=7.4, adds the anti-Ab of prostate cancer marker two of 300 μ L, 10 μ g/mL
2solution, vibrate in 4 DEG C of constant-temperature shaking incubators hatching 24 h, and centrifugal 10 min under the rotating speed of 3000 rpm, add 1 mL, pH=7.4 phosphate buffered solution, obtained GS-CNT-Pt/Ab
2two anti-incubate compound solution, save backup at 4 DEG C.
embodiment 10the detection of prostate cancer marker
(1) electrochemical workstation is used to test with three-electrode system, saturated calomel electrode is contrast electrode, platinum electrode is auxiliary electrode, and prepared sensor is working electrode, tests in pH 7.4 phosphate buffered solution of 10 mL, 30 mmol/L;
(2) detect prostate specific antigen standard solution with chronoamperometry, input voltage is-0.4 V, sample interval 0.1 s, working time 300 s;
(3) after background current tends towards stability, inject the hydrogen peroxide solution of 10 μ L, 5 mol/L every 50 s in the pH=7.4 phosphate buffered solution of 10 mL, 30 mmol/L, record current changes, drawing curve;
(4) replace prostate specific antigen standard solution with blood serum sample solution, the method for drawing according to working curve detects.
embodiment 11the detection of prostate cancer marker
(1) electrochemical workstation is used to test with three-electrode system, saturated calomel electrode is contrast electrode, platinum electrode is auxiliary electrode, and prepared sensor is working electrode, tests in the pH8.0 phosphate buffered solution of 10 mL, 50 mmol/L;
(2) detect prostate specific antigen standard solution with chronoamperometry, input voltage is-0.4 V, sample interval 0.1 s, working time 300 s;
(3) after background current tends towards stability, inject the hydrogen peroxide solution of 10 μ L, 10 mol/L every 50 s in the pH=7.4 phosphate buffered solution of 10 mL, 50 mmol/L, record current changes, drawing curve;
(4) replace prostate specific antigen standard solution with blood serum sample solution, the method for drawing according to working curve detects, and the range of linearity is 0.50 pg/mL ~ 15 ng/mL, detects and is limited to 0.12 pg/mL.
Claims (4)
1. a prostate specific antigen sandwich type immunosensor preparation method, it is characterized in that, step is as follows:
(1) by diameter be the glass-carbon electrode of the 4 mm Al of 0.05 μm
2o
3burnishing powder is polished, and cleans up with ultrapure water; By the Au-NH of 4 ~ 6 μ L, 0.5 ~ 2 mg/mL
2-GS Nanocomposite solution is added drop-wise on the bare electrode handled well, places in 4 DEG C of refrigerators and uses ultrapure water again after drying, dry in 4 DEG C of refrigerators;
(2) by the prostate cancer marker primary antibodie Ab of 4 ~ 6 μ L, 8 ~ 12 μ g/mL
1solution is added drop-wise to electrode surface, places in 4 DEG C of refrigerators and uses ultrapure water again after drying, dry in 4 DEG C of refrigerators;
(3) be that the bovine serum albumin(BSA) BSA solution of 0.5 ~ 3.0 % is added drop-wise to electrode surface by 3 μ L, massfraction, with enclosed-electrode nonspecific activity site on the surface, place in 4 DEG C of refrigerators and use ultrapure water again after drying, dry in 4 DEG C of refrigerators;
(4) the prostate cancer marker antigen standard solution of a series of variable concentrations of dropping 4 ~ 6 μ L, 0.5 pg/mL ~ 15 ng/mL is to electrode surface, places in 4 DEG C of refrigerators and uses ultrapure water again after drying, dry in 4 DEG C of refrigerators;
(5) by the GS-CNT-Pt/Ab of 4 ~ 6 μ L, 3 ~ 6 mg/mL
2two anti-compound solution of incubating drip and are applied on electrode surface, are placed in after 4 DEG C of refrigerators dry, ultrapure water, obtained prostate specific antigen sandwich type immunosensor.
2. a kind of prostate specific antigen sandwich type immunosensor preparation method as claimed in claim 1, described Au-NH
2-GS Nanocomposite solution, is characterized in that, preparation process is as follows:
(1) preparation of solution of gold nanoparticles
Be the HAuCl of 0.01 ~ 0.02% by 100 mL, massfraction
4solution is heated to boil, and dropping 2.0 ~ 3.0 mL, massfraction are the sodium citrate aqueous solution of 0.5 ~ 1.5%, and solution colour becomes rufous, is cooled to room temperature;
Amination Graphene NH
2the preparation of-GS
0.05 ~ 0.2 g graphene dispersion in the ethanol of 5 ~ 15 mL, ultrasonic agitation 1 h, add 0.1 ~ 0.3 mL, massfraction be 99% 3-aminopropyl triethoxysilane, under 70 DEG C of conditions, add thermal agitation 1.0 ~ 2.0 h; Add 0.1mL, massfraction is the hydrazine hydrate of 80%, 95 DEG C of heating 1 h; Centrifugal 20 ~ 40 min under the rotating speed of 9000 rpm; Dry 8 ~ 12 h under vacuum, obtained amination Graphene NH
2-GS
;
(3) Au-NH
2the preparation of-GS Nanocomposite solution
Take the amination Graphene NH of 10 ~ 20 mg
2-GS is placed in small beaker, adds 10 ~ 20 mL gold nano solution, stirs 10 ~ 14 h, after centrifugal at vacuum condition 35 ~ 45 DEG C dry 12 h, obtained Au-NH
2-GS nano composite material, is mixed with the Au-NH of 0.5 ~ 2 mg/mL with water
2-GS Nanocomposite solution.
3. a kind of prostate specific antigen sandwich type immunosensor preparation method as claimed in claim 1, described GS-CNT-Pt/Ab
2two anti-incubate compound solution, and it is characterized in that, preparation process is as follows:
(1) preparation of graphene oxide
By the graphite of 0.3 g and the mixing of 1.5 ~ 2.0 g potassium permanganate, put into the there-necked flask of 500 mL with magneton, add sulfuric acid and phosphoric acid mixed liquor that volume ratio is 8 ~ 10: 1, there-necked flask is put into oil bath pan, is heated to 50 DEG C, reaction 10 ~ 12 h; Sample is poured into 40 mL on ice, under magnetic agitation, drip the hydrogen peroxide of 0.5 ~ 1.0 mL, solution becomes khaki, centrifugal 0.5 h under the rotating speed of 8000 rpm, use the absolute ethyl alcohol centrifuge washing 3 times of the hydrochloric acid of 30 mL, 0.2 mol/L and 30 mL successively, abandoning supernatant, with ether centrifuge washing 1 time, abandoning supernatant, the solid sample obtained is placed in dry 12 h of vacuum drying chamber of 30 ~ 40 DEG C, obtained graphene oxide yellow-brown solid powder;
(2) preparation of the GS-CNT nano composite material of three-dimensional structure
The carbon nano-tube CNT of 20 ~ 40 mg is joined 30 mL, 1 ~ 3 mg/mL graphene oxide uniform dispersion in, ultrasonic 30 min, the potpourri obtained is put into the autoclave of 50 mL, be heated to 160 ~ 190 DEG C, and keep 12 h, be cooled to room temperature, by the potpourri freeze drying obtained;
(3) preparation of the GS-CNT-Pt nano-complex of three-dimensional structure
Under the condition of vigorous stirring, by the K of 1 mL, 5 ~ 15 mmol/L
2ptCl
4the GS-CNT nano composite material of the three-dimensional structure of solution and 5 mg joins in the ultrapure water of 10 mL, ice bath 30 min, centrifuging 5 min under 7000 rpm, the GS-CNT-Pt nano-complex of obtained three-dimensional structure, wash with water, centrifuging 3 times, vacuum drying 12 h at 35 ~ 45 DEG C;
(4) GS-CNT-Pt/Ab
2two anti-preparations of incubating compound solution
The GS-CNT-Pt nano-complex of the three-dimensional structure of 3 ~ 6 mg is joined in the phosphate buffered solution of 3 ~ 6 mL, pH=7.4, adds the anti-Ab of prostate cancer marker two of 150 ~ 300 μ L, 10 μ g/mL
2solution, vibrate in 4 DEG C of constant-temperature shaking incubators hatching 24 h, and centrifugal 10 min under the rotating speed of 3000 rpm, add 1 mL, pH=7.4 phosphate buffered solution, obtained GS-CNT-Pt/Ab
2two anti-incubate compound solution, save backup at 4 DEG C.
4. a kind of prostate specific antigen sandwich type immunosensor of preparing of preparation method as claimed in claim 1, for the detection of prostate cancer marker, detecting step is as follows:
(1) electrochemical workstation is used to test with three-electrode system, saturated calomel electrode is contrast electrode, platinum electrode is auxiliary electrode, and prepared sensor is working electrode, tests in pH 5.9 ~ 8.0 phosphate buffered solution of 10 mL, 30 ~ 50 mmol/L;
(2) detect prostate specific antigen standard solution with chronoamperometry, input voltage is-0.4 V, sample interval 0.1 s, working time 300 s;
(3) after background current tends towards stability, inject the hydrogen peroxide solution of 10 μ L, 5 ~ 10 mol/L every 50 s in pH=5.9 ~ 8.0 phosphate buffered solution of 10 mL, 30 ~ 50 mmol/L, record current changes, drawing curve;
(4) replace prostate specific antigen standard solution with blood serum sample solution, the method for drawing according to working curve detects.
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