CN103454417B - Preparation method and application of biosensor for squamous cell carcinoma antigens - Google Patents
Preparation method and application of biosensor for squamous cell carcinoma antigens Download PDFInfo
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- CN103454417B CN103454417B CN201310396759.1A CN201310396759A CN103454417B CN 103454417 B CN103454417 B CN 103454417B CN 201310396759 A CN201310396759 A CN 201310396759A CN 103454417 B CN103454417 B CN 103454417B
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Abstract
The invention relates to a preparation method and an application of a biosensor for squamous cell carcinoma antigens, and belongs to the technical field of novel nanometer function materials and biosensing. The preparation method is particularly used for preparing a sandwich type electrochemical immunosensor for the squamous cell carcinoma antigens based on nitrogen-doped grapheme (N-GS) and Pd-Au/C supported nano-catalysts, and the sandwich type electrochemical immunosensor is used for detecting the squamous cell carcinoma antigens in serum. The preparation method is characterized by comprising the following steps of (1) preparing the nitrogen-doped grapheme; (2) preparing secondary antibody compounds of Pd-Au/C-squamous cell carcinoma antigens; (3) preparing the electrochemical immunosensor. The Pd-Au/C has very strong catalytic capacity to H2O2 and is beneficial to fixing multiple secondary antibodies and maintain biological activities of matters. The biosensor prepared by utilizing the preparation method has the advantages of high sensitivity, good specificity, easiness in operation and low detection limit.
Description
Technical field
The present invention relates to a kind of preparation method and application of squamous cell carcinoma-related antigen biology sensor.Specifically based on nitrogen-doped graphene (N-GS) and Pd-Au/C carrier nanometer catalyst (Pd-Au/C), the sandwich type electrochemical immunosensor of preparing a kind of squamous cell carcinoma-related antigen, for detection of the squamous cell carcinoma-related antigen in serum, belong to Nano-function thin films and biosensor technique field.
Background technology
Squamous cell carcinoma-related antigen (SCCA) is the hypotype of a kind of TA-4, and the ovalbumin family that belongs to serpin, extensively be present in the epithelial cell of Different Organs normal structure (content is atomic) and malignant change, be a kind of tumor markers of diagnosing squama cancer, the present invention is based on nitrogen-doped graphene (N-GS) and Pd-Au/C and prepare squamous cell carcinoma-related antigen sandwich type immunosensor.
N-GS is a kind of by chemical method, and the part carbon atom on Graphene is replaced with to a kind of material after nitrogen-atoms.So-called " doping ", refers to a kind of state that nitrogen-atoms and carbon atom coexist on Graphene upper skeleton, has high reduced level, and in multi-solvents, has good dispersiveness.N-GS is transparent tulle shape structure, and has bigger serface and good electric conductivity.Therefore,, because N-GS has good signal amplification, make N-GS be widely used in making the electrochemical immunosensor of the transmission medium of electronics.
Pd-Au/C carrier nanometer catalyst is a kind of activated carbon loaded Pd-Au bimetallic alloy catalyzer, and the present invention utilizes Pd-Au/C carrier nanometer catalyst as two anti-labels, is conducive to fixing more two anti-, maintenance substance bioactivities, and to H
2o
2there is very strong catalytic capability.The response current of the biology sensor that the present invention makes and SCCA concentration keep good linear relationship within the scope of 0.0050 ~ 2.0 ng/mL, detect and are limited to 1.6 pg/mL.
That sensor prepared by the present invention has is highly sensitive, specificity good, testing cost is low, can fast detecting squamous cell carcinoma-related antigen etc. advantage, effectively overcome the deficiency of current squamous cell carcinoma-related antigen detection method.
Summary of the invention
One of object of the present invention has been to provide a kind of preparation method who quick and precisely detects the electrochemical immunosensor of squamous cell carcinoma-related antigen.
Two of object of the present invention is this electrochemical immunosensor to be applied to the detection of squamous cell carcinoma-related antigen.
To achieve these goals, the present invention realizes by following measures.
A preparation method for squamous cell carcinoma-related antigen biology sensor, is characterized in that, comprises the following steps:
(1) graphene oxide is synthetic
At 0.1 ~ 0.6 g graphite and 0.6 ~ 4.0g KMnO
4in add 40 mL, H
2sO
4-H
3pO
4mixed solution, H
2sO
4and H
3pO
4volume ratio 9: 1, is heated to 40 ~ 60 ℃, continues 12h, stops reacting cool to room temperature, adds the H that 0.1 ~ 0.5 mL, volume fraction are 30% in ice-water bath
2o
2, magnetic agitation 30 ~ 60 min, potpourri is centrifugal, use respectively 0.2 molL
-1hCl, ethanol, ether washs, and washs 2 times;
(2) nitrogen-doped graphene (N-GS) is synthetic
Graphene oxide is scattered in to N, in dinethylformamide (DMF), be made into 0.5 ~ 2.0 mg/mL solution, ultrasonic 30 ~ 60 min, by mixed liquor under 120 ~ 160 ℃ of conditions, stirring and refluxing 1 ~ 2 h, cool to room temperature, centrifugal under the rotating speed of 9000 r/min, 50 ~ 60 ℃ of vacuum drying;
(3) palladium-Jin/carbon (Pd-Au/C) is synthetic
30 ~ 100 mg acticarbon, 1.0 ~ 5.0 mL, 0.50 molL
-1pdCl
2solution, the HAuCl that 0.5 ~ 1.0 mL, massfraction are 1%
4solution, adds 5mL water and 5mL tetrahydrofuran successively, and ultrasonic 0.5 ~ 2 h mixes, and magnetic agitation 12 h slowly add 10 ~ 30 mL to contain 0.1 molL
-1naBH
4with 0.05 molL
-1na
2cO
3mixed solution, magnetic agitation 1 h, the solid obtaining successively water and ethanol washs, after having washed, vacuum drying at 50 ~ 60 ℃, makes palladium-Jin/carbon nanomaterial (Pd-Au/C);
(4) anti-(Ab of Pd-Au/C-squamous cell carcinoma-related antigen two
2) preparation
Prepare respectively Pd-Au/C solution and Ab
2solution, by Pd-Au/C solution and Ab
2solution mixes by 1: 1 ~ 2 volume ratio, hatching concussion 12 ~ 24 h in the illumination box of 10 ℃, the Pd-Au/C-Ab making
2, by pH=7.4 phosphate buffered solution, cleaning, the refrigerator that is kept at 4 ℃ is interior standby.
The concentration of described Pd-Au/C solution is 5 ~ 15 mgmL
-1, described Ab
2the concentration of solution is 5 ~ 15 μ gmL
-1.
(5) preparation method of electrochemical immunosensor
1) glass-carbon electrode of diameter 4 mm is used successively to the alundum (Al2O3) burnishing powder polishing of 1.0,0.3 and 0.05 mm, ethanol ultrasonic cleaning, then rinse well with ultrapure water, then electrode is placed in to 5 ~ 10 mmolL
-1in potassium ferricyanide solution, under-0.2 ~ 0.6 V current potential, scan, make poor 90 mV that are less than of spike potential;
2) by 6 μ L, 0.4 ~ 2.0 mgmL
-1n-GS solution drip and be applied to electrode surface, drip 3 μ L, 2.5% glutaraldehyde solution in electrode surface, drying at room temperature;
3) by 6 μ L, 5 ~ 15 μ gmL
-1squamous cell carcinoma-related antigen primary antibodie (Ab
1) drip be coated onto step 2) electrode surface that N-GS modifies, be placed under 4 ℃ of wet condition and dry, washing;
4) by concentration, be 3 μ L, 100 μ gmL
-1bovine serum albumin(BSA) drips and is coated onto step 3) Ab
1the electrode surface of modifying, dries under wet condition under 4oC;
5) squamous cell carcinoma-related antigen of 6 μ L (SCCA) is dripped respectively to the electrode surface that is coated in step 4) Bovine Serum Albumin Modified, under wet condition, dry at 4 ℃, washing;
6) by the Pd-Au/C-Ab of 6 μ L
2drip and to be coated in the electrode surface that step 5) SCCA modifies, dry at 4 ℃ under wet condition, prepared by electrochemical immunosensor.
(6) electrochemical immunosensor of described preparation is for the detection of SCCA, and step is as follows:
1) adopt 0.066 molL
-1, the phosphate buffered solution of pH=7.4, is formulated in 5pgmL
-1~ 2ngmL
-1the SCCA solution of variable concentrations in scope, the SCCA solution of 6 μ L variable concentrations is dripped respectively and is applied to above-mentioned steps 4) electrode surface of Bovine Serum Albumin Modified, after drying, according to step 6) modified electrode, be connected in electrochemical workstation, respectively electrode be placed in to 10 mL and contain 3 ~ 6 mmolmL
-1h
2o
2the PBS buffer solution of pH=6.8 in measure its curent change;
2) linear according to gained electric current difference and SCCA concentration, drawing curve;
3) method for drafting according to working curve carries out sample detection, and testing result checks in from working curve.
useful achievement of the present invention
(1) squamous cell carcinoma-related antigen biology sensor, is incorporated into N-GS in the middle of the preparation of squamous cell carcinoma-related antigen biology, utilizes the electronics transmission capacity that N-GS is good, has significantly improved the performance of electrode.
(2) by Pd-Au/C and the anti-directly hatching of tumor markers two, utilize the biocompatibility of porpezite excellence and high catalytic performance, in two anti-labels, needn't use enzyme, avoided because of the inactivation of enzyme and leaked the detection error causing, simplify the making step of two anti-labels, significantly improved reappearance and the stability of electrochemical immunosensor.
(3) the prepared electrochemical immunosensor of the present invention, simple to operate, detection speed is fast, can realize at short notice the mensuration of batch sample.
Embodiment
embodiment 1synthesizing of graphene oxide
At 0.1 ~ 0.6 g graphite and 0.6 ~ 4.0g KMnO
4in add 40 mL, H
2sO
4-H
3pO
4mixed solution, H
2sO
4and H
3pO
4volume ratio 9: 1, is heated to 40 ~ 60 ℃, continues 12h, stops reacting cool to room temperature, adds the H that 0.1 ~ 0.5 mL, volume fraction are 30% in ice-water bath
2o
2, magnetic agitation 30 ~ 60 min, potpourri is centrifugal, use respectively 0.2 molL
-1hCl, ethanol, ether washs, and washs 2 times.
embodiment 2synthesizing of nitrogen-doped graphene (N-GS)
Graphene oxide is scattered in DMF (DMF), is made into 0.5 mg/mL solution, ultrasonic 30 min, by mixed liquor under 120 ℃ of conditions, stirring and refluxing 1 h, cool to room temperature, centrifugal under the rotating speed of 9000 r/min, 50 ℃ of vacuum drying.
embodiment 3synthesizing of nitrogen-doped graphene (N-GS)
Graphene oxide is scattered in DMF (DMF), is made into 2.0 mg/mL solution, ultrasonic 60 min, by mixed liquor under 160 ℃ of conditions, stirring and refluxing 2 h, cool to room temperature, centrifugal under the rotating speed of 9000 r/min, 60 ℃ of vacuum drying.
embodiment 4synthesizing of nitrogen-doped graphene (N-GS)
Graphene oxide is scattered in DMF (DMF), is made into 1.0 mg/mL solution, ultrasonic 50 min, by mixed liquor under 140 ℃ of conditions, stirring and refluxing 1.5h, cool to room temperature, centrifugal under the rotating speed of 9000 r/min, 55 ℃ of vacuum drying.
embodiment 5synthesizing of palladium-Jin/carbon (Pd-Au/C)
30 mg acticarbon, 1.0 mL, 0.50 molL
-1pdCl
2solution, the HAuCl that 0.5 mL, massfraction are 1%
4solution, adds 5mL water and 5mL tetrahydrofuran successively, and ultrasonic 0.5 h mixes, and magnetic agitation 12 h slowly add 10mL to contain 0.1 molL
-1naBH
4with 0.05 molL
-1na
2cO
3mixed solution, magnetic agitation 1 h, the solid obtaining successively water and ethanol washs, and has washed vacuum drying at latter 50 ℃, makes palladium-Jin/carbon nanomaterial (Pd-Au/C).
embodiment 6synthesizing of palladium-Jin/carbon (Pd-Au/C)
100 mg acticarbon, 5.0 mL, 0.50 molL
-1pdCl
2solution, the HAuCl that 1.0 mL, massfraction are 1%
4solution, adds 5mL water and 5mL tetrahydrofuran successively, and ultrasonic 2 h mix, and magnetic agitation 12 h slowly add 30 mL to contain 0.1 molL
-1naBH
4with 0.05 molL
-1na
2cO
3mixed solution, magnetic agitation 1 h, the solid obtaining successively water and ethanol washs, and has washed vacuum drying at latter 60 ℃, makes palladium-Jin/carbon nanomaterial (Pd-Au/C).
embodiment 7synthesizing of palladium-Jin/carbon (Pd-Au/C)
60 mg acticarbon, 3.0 mL, 0.50 molL
-1pdCl
2solution, the HAuCl that 0.7 mL, massfraction are 1%
4solution, adds 5mL water and 5mL tetrahydrofuran successively, and ultrasonic 1.0 h mix, and magnetic agitation 12 h slowly add 20mL to contain 0.1 molL
-1naBH
4with 0.05 molL
-1na
2cO
3mixed solution, magnetic agitation 1 h, the solid obtaining successively water and ethanol washs, and has washed vacuum drying at latter 55 ℃, makes palladium-Jin/carbon nanomaterial (Pd-Au/C).
embodiment 8anti-(the Ab of Pd-Au/C-squamous cell carcinoma-related antigen two
2) preparation
Prepare respectively Pd-Au/C solution and Ab
2solution, by Pd-Au/C solution and Ab
2solution mixes by the volume ratio of 1: 1, hatching concussion 12 h in the illumination box of 10 ℃, the Pd-Au/C-Ab making
2, by pH=7.4 phosphate buffered solution, cleaning, the refrigerator that is kept at 4 ℃ is interior standby.
embodiment 9anti-(the Ab of Pd-Au/C-squamous cell carcinoma-related antigen two
2) preparation
Prepare respectively Pd-Au/C solution and Ab
2solution, by Pd-Au/C solution and Ab
2solution mixes by the volume ratio of 1: 2, hatching concussion 24 h in the illumination box of 10 ℃, the Pd-Au/C-Ab making
2, by pH=7.4 phosphate buffered solution, cleaning, the refrigerator that is kept at 4 ℃ is interior standby.
embodiment 10anti-(the Ab of Pd-Au/C-squamous cell carcinoma-related antigen two
2) preparation
Prepare respectively Pd-Au/C solution and Ab
2solution, by Pd-Au/C solution and Ab
2solution mixes by the volume ratio of 1: 1.5, hatching concussion 18 h in the illumination box of 10 ℃, the Pd-Au/C-Ab making
2, by pH=7.4 phosphate buffered solution, cleaning, the refrigerator that is kept at 4 ℃ is interior standby.
embodiment 11the preparation of electrochemical immunosensor
(1) glass-carbon electrode of diameter 4 mm is used successively to the alundum (Al2O3) burnishing powder polishing of 1.0,0.3 and 0.05 mm, ethanol ultrasonic cleaning, then rinse well with ultrapure water, then electrode is placed in to 5 ~ 10 mmolL
-1in potassium ferricyanide solution, under-0.2 ~ 0.6 V current potential, scan, make poor 80 mV that are less than of spike potential;
(2) by 6 μ L, 0.4 mgmL
-1n-GS solution drip and be applied to electrode surface, dry under infrared lamp after, drip 3 μ L, 2.5% glutaraldehyde solution in electrode surface, drying at room temperature;
(3) by 6 μ L, 5 μ gmL
-1squamous cell carcinoma-related antigen primary antibodie (Ab
1) drip the electrode surface that is coated onto step (2) N-GS modification, be placed under 4 ℃ of wet condition and dry, washing;
(4) by concentration, be 3 μ L, 100 μ gmL
-1bovine serum albumin(BSA) drips and is coated onto step (3) Ab
1the electrode surface of modifying, dries under wet condition at 4o ℃;
(5) squamous cell carcinoma-related antigen of 6 μ L (SCCA) is dripped respectively to the electrode surface that is coated in step (4) Bovine Serum Albumin Modified, under wet condition, dry at 4 ℃, washing;
(6) by the Pd-Au/C-Ab of 6 μ L
2drip and be coated in the electrode surface that step (5) SCCA modifies, under 4oC, under wet condition, dry, prepared by electrochemical immunosensor.
embodiment 12the preparation of electrochemical immunosensor
(1) glass-carbon electrode of diameter 4 mm is used successively to the alundum (Al2O3) burnishing powder polishing of 1.0,0.3 and 0.05 mm, ethanol ultrasonic cleaning, then rinse well with ultrapure water, then electrode is placed in to 5 ~ 10 mmolL
-1in potassium ferricyanide solution, under-0.2 ~ 0.6 V current potential, scan, make the poor 80mV of being less than of spike potential;
(2) by 6 μ L, 2.0 mgmL
-1n-GS solution drip and be applied to electrode surface, dry under infrared lamp after, drip 3 μ L, 2.5% glutaraldehyde solution in electrode surface, drying at room temperature;
(3) by 6 μ L, 15 μ gmL
-1squamous cell carcinoma-related antigen primary antibodie (Ab
1) drip the electrode surface that is coated onto step (2) N-GS modification, be placed under 4 ℃ of wet condition and dry, washing;
(4) by concentration, be 3 μ L, 100 μ gmL
-1bovine serum albumin(BSA) drips and is coated onto step (3) Ab
1the electrode surface of modifying, dries under wet condition at 4 ℃;
(5) squamous cell carcinoma-related antigen of 6 μ L (SCCA) is dripped respectively to the electrode surface that is coated in step (4) Bovine Serum Albumin Modified, under wet condition, dry at 4 ℃, washing;
(6) by the Pd-Au/C-Ab of 6 μ L
2drip and to be coated in the electrode surface that step (5) SCCA modifies, dry at 4 ℃ under wet condition, prepared by electrochemical immunosensor.
embodiment 13the detection of SCCA
(1) adopt 0.066 molL
-1, the phosphate buffered solution of pH=7.4, is formulated in 5pgmL
-1~ 2ngmL
-1the SCCA solution of variable concentrations in scope, the SCCA solution of 6 μ L variable concentrations is dripped respectively to the electrode surface that is applied to step in embodiment 12 (4) Bovine Serum Albumin Modified, after drying, according to step (6) modified electrode, be connected in electrochemical workstation, respectively electrode be placed in to 10 mL and contain 3 ~ 6 mmolmL
-1h
2o
2the PBS buffer solution of pH=6.8 in measure its curent change;
(2) linear according to gained electric current difference and SCCA concentration, drawing curve;
(3) method for drafting according to working curve carries out sample detection, and testing result checks in from working curve.
Claims (3)
1. a preparation method for squamous cell carcinoma-related antigen biology sensor, is characterized in that, comprises the following steps:
(1) graphene oxide is synthetic, and step is as follows:
At 0.1 ~ 0.6 g graphite and 0.6 ~ 4.0 g KMnO
4in add 40 mL, H
2sO
4-H
3pO
4mixed solution, H
2sO
4and H
3pO
4volume ratio 9: 1, is heated to 40 ~ 60 ℃, continues 12h, stops reacting cool to room temperature, adds the H that 0.1 ~ 0.5 mL, volume fraction are 30% in ice-water bath
2o
2, magnetic agitation 30 ~ 60 min, potpourri is centrifugal, use respectively 0.2 molL
-1hCl, ethanol, ether washs, and washs 2 times;
(2) nitrogen-doped graphene (N-GS) is synthetic
Graphene oxide is scattered in to N, in dinethylformamide (DMF), be made into 0.5 ~ 2.0 mg/mL solution, ultrasonic 30 ~ 60 min, by mixed liquor under 120 ~ 160 ℃ of conditions, stirring and refluxing 1 ~ 2 h, cool to room temperature, centrifugal under the rotating speed of 9000 r/min, 50 ~ 60 ℃ of vacuum drying;
(3) palladium-Jin/carbon (Pd-Au/C) is synthetic
30 ~ 100 mg acticarbon, 1.0 ~ 5.0 mL, 0.50 molL
-1pdCl
2solution, the HAuCl that 0.5 ~ 1.0 mL, massfraction are 1%
4solution, adds 5 mL water and 5 mL tetrahydrofurans successively, and ultrasonic 0.5 ~ 2 h mixes, and magnetic agitation 12 h slowly add 10 ~ 30 mL to contain 0.1 molL
-1naBH
4with 0.05 molL
-1na
2cO
3mixed solution, magnetic agitation 1 h, the solid obtaining successively water and ethanol washs, after having washed, vacuum drying at 50 ~ 60 ℃, makes palladium-Jin/carbon nanomaterial (Pd-Au/C);
(4) anti-(Ab of Pd-Au/C-squamous cell carcinoma-related antigen two
2) preparation
(5) preparation of electrochemical immunosensor
The glass-carbon electrode of diameter 4 mm is used successively to the alundum (Al2O3) burnishing powder polishing of 1.0,0.3 and 0.05 mm, ethanol ultrasonic cleaning, then rinse well with ultrapure water, then electrode is placed in to 5 ~ 10 mmolL
-1in potassium ferricyanide solution, under-0.2 ~ 0.6 V current potential, scan, make poor 90 mV that are less than of spike potential; By 6 μ L, 0.4 ~ 2.0 mgmL
-1n-GS solution drip and be applied to electrode surface, dry under infrared lamp after, drip 3 μ L, 2.5% glutaraldehyde solution in electrode surface, drying at room temperature; By 6 μ L, 5 ~ 15 μ gmL
-1squamous cell carcinoma-related antigen primary antibodie (Ab
1) drip the electrode surface that is coated onto N-GS modification, be placed under 4 ℃ of wet condition and dry, washing; By concentration, be 3 μ L, 100 μ gmL
-1bovine serum albumin(BSA) drips and is coated onto Ab
1the electrode surface of modifying, dries under wet condition at 4 ℃; The squamous cell carcinoma-related antigen of 6 μ L (SCCA) is dripped respectively to the electrode surface that is coated in Bovine Serum Albumin Modified, under wet condition, dry at 4 ℃, washing; By the Pd-Au/C-Ab of 6 μ L
2drip and to be coated in the electrode surface that SCCA modifies, dry at 4 ℃ under wet condition, prepared by electrochemical immunosensor.
2. the preparation method of a kind of squamous cell carcinoma-related antigen biology sensor as claimed in claim 1, is characterized in that, described Pd-Au/C-Ab
2preparation, step is as follows:
Prepare respectively Pd-Au/C solution and Ab
2solution, by Pd-Au/C solution and Ab
2solution mixes by 1: 1 ~ 2 volume ratio, hatching concussion 12 ~ 24 h in the illumination box of 10 ℃, the Pd-Au/C-Ab making
2, by pH=7.4 phosphate buffered solution, cleaning, the refrigerator that is kept at 4 ℃ is interior standby.
3. the preparation method of a kind of squamous cell carcinoma-related antigen biology sensor as claimed in claim 2, is characterized in that, the concentration of described Pd-Au/C solution is 5 ~ 15 mgmL
-1, described Ab
2the concentration of solution is 5 ~ 15 μ gmL
-1.
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| CN104297480B (en) * | 2014-09-26 | 2015-08-19 | 济南大学 | A kind of prostate specific antigen sandwich type immunosensor preparation method and application |
| CN109158121B (en) * | 2018-08-15 | 2022-01-07 | 临沂大学 | Preparation method of dumbbell-shaped nanogold with excellent catalytic hydrogen peroxide performance |
| CN109201101B (en) * | 2018-08-15 | 2022-01-07 | 临沂大学 | Preparation method of stone-like nanogold with excellent catalytic hydrogen peroxide performance |
| CN110702758B (en) * | 2019-09-27 | 2021-04-30 | 广西师范大学 | Method for enhancing luminous intensity of squamous cell carcinoma antigen in electrochemical luminescence detection |
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| CN102980925A (en) * | 2012-11-26 | 2013-03-20 | 济南大学 | Sandwich type electrochemical immunosensor, preparation method and application thereof |
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