CN103868971A - Preparation method of glutathione transferase antigen biosensor and application thereof - Google Patents
Preparation method of glutathione transferase antigen biosensor and application thereof Download PDFInfo
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- CN103868971A CN103868971A CN201410036028.0A CN201410036028A CN103868971A CN 103868971 A CN103868971 A CN 103868971A CN 201410036028 A CN201410036028 A CN 201410036028A CN 103868971 A CN103868971 A CN 103868971A
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Abstract
The invention relates to a preparation method of a glutathione transferase antigen biosensor and an application thereof, belongs to the technical fields of a novel nano functional material and biosensing, and particularly relates to a sandwich-type electrochemical immunosensor for detecting glutathione transferase prepared on the basis of sulfated grapheme (HSO3-GS) and double metal nanometer porous alloy material-platinum and ferrum (PtFe) and used for detecting the glutathione transferase in serum. The preparation method is characterized by comprising the following steps: (1) preparing sulfated grapheme; (2) preparing a double metal porous material-PtFe-horse radish peroxidase-glutathione transferase second antibody compound; and (3) preparing the electrochemical immunosensor. The PtFe has the strong catalytic ability on H2O2, meanwhile, the fixation of a relatively large quantity of secondary antibodies and the keeping of the biological activity of a substance are facilitated, an electronic media body reddish black A is added to a base solution, the problem of leakage after the electronic media body reddish black A is modified on an electrode is avoided, and the prepared sensor is high in sensitivity, good in specificity, easy to operate and low in detection limit.
Description
Technical field
The present invention relates to a kind of preparation method and application of glutathione transferase antigen biology sensor.Specifically based on sulfonated Graphene (HSO
3-GS) and bimetal nano multi-porous alloy material platinum iron (PtFe), prepare the sandwich type electrochemical immunosensor of glutathione transferase, for detection of the glutathione transferase in serum.Belong to Nano-function thin films and biosensor technique field.
Background technology
Glutathione transferase (GST) is mainly a kind of biology enzyme that participates in bio-transformation second-phase reaction in liver, it can detect again in tissue in serum, and other many tumor markerses can only detect in tissue, and GST in detection serum to body without aggressive, can greatly facilitate clinical application, therefore the development of glutathione transferase immunosensor has great importance.
The present invention is based on the sulfonated Graphene (HSO of nano materials
3-GS) and bimetal nano porosint platinum iron (PtFe) prepare the research of glutathione transferase sandwich type immunosensor, given prominence to electrode modified material biology sensor increased response, improve detection sensitivity, increase the effects such as the range of linearity.HSO
3-GS have specific surface area large, can strengthen the advantages such as electronic conduction, utilize chemical bonding effect directly to connect with glutathione transferase primary antibodie, without the immobilization of the traditional crosslinking chemical glutaraldehyde of employing.Adopt PtFe to be conducive to fixing more two anti-, maintenance substance bioactivities, and to H
2o
2there is very strong catalytic capability.Both utilize simple drop-coating to modify can the electrochemical response of intensifier electrode in course of reaction on glass-carbon electrode.Reddish black electron mediator A added in end liquid to the leakage problem after having avoided modifying on electrode.
The response current of the biology sensor that the present invention makes and GST concentration keep good linear relationship within the scope of 0.01 ~ 12 ng/mL, detect and are limited to 2.1 pg/mL.
The present invention utilizes immunoreactive high specific, in conjunction with HSO
3-GS and PtFe, prepared a kind of glutathione transferase sandwich type immunosensor.
That the present invention has is highly sensitive, specificity good, testing cost is low, can fast detecting glutathione transferase etc. advantage, and preparation process of the present invention is simple, operating process is easy, has effectively overcome the deficiency of current glutathione transferase detection method.
Summary of the invention
One of object of the present invention is to avoid instrument and equipment complexity, the operating process of traditional detection method loaded down with trivial details, testing staff requires the shortcomings such as high, and the preparation method of the electrochemical immunosensor of a kind of highly sensitive, high specificity, favorable reproducibility, fast detecting glutathione transferase easy and simple to handle is provided.
Two of object of the present invention is the detections that this electrochemical immunosensor are applied to glutathione transferase.
To achieve these goals, the present invention realizes by following measures.
A preparation method for glutathione transferase antigen biology sensor, is characterized in that, comprises the following steps:
(1) preparation of PtFe
PtFeAl alloy sheet is put into 0.5 ~ 1.0 molL is housed
-1in the small beaker of NaOH solution, be placed in water bath with thermostatic control, under 40 ~ 50 ℃ of conditions, place 24 ~ 48 h, the product obtaining is washed repeatedly with intermediate water, under room temperature, dry, prepare required PtFe.
(2) the anti-compound (PtFe/HRP/Ab of PtFe/ horseradish peroxidase/glutathione transferase two
2) preparation
PtFe suspending liquid will be housed ultrasonic, pipette PtFe suspending liquid, HRP solution and Ab
2solution is in a centrifuge tube, and volume ratio is PtFe: HRP: Ab
2=1: 0.5 ~ 2: 0.5 ~ 2 mixes, under 4 ℃ of conditions, the hatching of vibrating, after hatching 12 ~ 24 h, is placed in 4 ℃ of refrigerators and preserves stand-by.
(3) 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 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) HSO shitosan being disperseed
3-GS modifies in glass-carbon electrode surface, drying at room temperature;
3) by glutathione transferase primary antibodie (Ab
1) drip be coated onto step 2) HSO
3the electrode surface that-GS modifies, is placed under 4 ℃ of wet condition and dries;
4) by concentration be 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) GST is dripped to the electrode surface that is coated in step 4) Bovine Serum Albumin Modified, under wet condition, dry at 4 ℃;
6) by PtFe/HRP/Ab
2drip and be coated in the electrode surface that step 5) GST modifies, dry at 4 ℃ under wet condition, prepared by electrochemical immunosensor.
Described HSO
3the concentration of-GS is 0.5 ~ 2.5 mgmL
-1, the concentration of described PtFe suspending liquid is 10 mgmL
-1, the concentration of described HRP is 1.0 mgmL
-1, described Ab
2concentration be 1.0 mgmL
-1.
(4) application of electrochemical immunosensor
For the detection of glutathione transferase, step is as follows:
1) adopt 0.10 molmL
-1, the phosphate buffered solution of pH=7.4, is formulated in 0.01 ~ 12 ngmL
-1the GST solution of variable concentrations in scope, the GST 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 above-mentioned steps 6) modified electrode, be connected in electrochemical workstation, electrode be placed in and contain 2.0 ~ 3.0 mmolL respectively
-1reddish black A and 1.0 ~ 5.0 mmolL
-1h
2o
2the PBS buffer solution of pH=7.4 in measure its curent change;
2) linear according to gained electric current difference and GST concentration, drawing curve;
3) carry out the detection of GST in sample according to the method for drafting of working curve, testing result checks in from working curve.
useful achievement of the present invention
(1) glutathione transferase antigen biology sensor, HSO
3-GS is incorporated in the middle of the preparation of glutathione transferase antigen electrochemical immunosensor, utilizes HSO
3the electronics transmission capacity that-GS is good, has significantly improved the performance of electrode.
(2) by PtFe and the anti-directly hatching of tumor markers two, utilize the biocompatibility of platinum iron excellence and high catalytic performance, significantly improved reappearance and the stability of electrochemical immunosensor.
(3) PtFe and horseradish peroxidase compound token two are anti-, have the collaborative sensitization to signal.
(4) 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 1the preparation of PtFe
PtFeAl alloy sheet is put into 0.5 molL is housed
-1in the small beaker of NaOH solution, be placed in water bath with thermostatic control, under 40 ℃ of conditions, place 24 h, by the product obtaining intermediate water cyclic washing, under room temperature, dry, prepare required PtFe.
embodiment 2the preparation of PtFe
PtFeAl alloy sheet is put into 1.0 molL are housed
-1in the small beaker of NaOH solution, be placed in water bath with thermostatic control, under 50 ℃ of conditions, place 48 h, by the product obtaining intermediate water cyclic washing, under room temperature, dry, prepare required PtFe.
embodiment 3the preparation of PtFe
PtFeAl alloy sheet is put into 0.7 molL is housed
-1in the small beaker of NaOH solution, be placed in water bath with thermostatic control, under 45 ℃ of conditions, place 35 h, by the product obtaining intermediate water cyclic washing, under room temperature, dry, prepare required PtFe.
embodiment 4ptFe/HRP/Ab
2preparation
PtFe suspending liquid will be housed ultrasonic, pipette PtFe suspending liquid, HRP solution and Ab
2solution is in a centrifuge tube, and volume ratio is PtFe: HRP: Ab
2=1: mix at 0.5: 0.5, under 4 ℃ of conditions, the hatching of vibrating, after hatching 12 h, is placed in 4 ℃ of refrigerators and preserves stand-by.
embodiment 5ptFe/HRP/Ab
2preparation
PtFe suspending liquid will be housed ultrasonic, pipette PtFe suspending liquid, HRP solution and Ab
2solution is in a centrifuge tube, and volume ratio is PtFe: HRP: Ab
2=1: mix at 1: 1, under 4 ℃ of conditions, the hatching of vibrating, after hatching 24h, is placed in 4 ℃ of refrigerators and preserves stand-by.
embodiment 6ptFe/HRP/Ab
2preparation
PtFe suspending liquid will be housed ultrasonic, pipette PtFe suspending liquid, HRP solution and Ab
2solution is in a centrifuge tube, and volume ratio is PtFe: HRP: Ab
2=1: mix at 2: 2, under 4 ℃ of conditions, the hatching of vibrating, after hatching 18h, is placed in 4 ℃ of refrigerators and preserves stand-by.
embodiment 7the 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 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) HSO shitosan being disperseed
3-GS modifies in glass-carbon electrode surface, drying at room temperature;
(3) by glutathione transferase primary antibodie (Ab
1) drip and be coated onto step (2) HSO
3the electrode surface that-GS modifies, is placed under 4 ℃ of wet condition and dries;
(4) by concentration be 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) GST is dripped to the electrode surface that is coated in step (4) Bovine Serum Albumin Modified, under wet condition, dry at 4 ℃;
(6) by PtFe/HRP/Ab
2drip and be coated in the electrode surface that step (5) GST modifies, dry at 4 ℃ under wet condition, prepared by electrochemical immunosensor.
embodiment 8the application of electrochemical immunosensor
For the detection of glutathione transferase, step is as follows:
(1) adopt 0.10 molmL
-1, the phosphate buffered solution of pH=7.4, is formulated in 0.01 ngmL
-1the GST solution of variable concentrations in scope, the GST solution of 6 μ L variable concentrations is dripped respectively to the electrode surface that is applied to step in embodiment 7 (4) Bovine Serum Albumin Modified, after drying, according to step (6) modified electrode in embodiment 7, be connected in electrochemical workstation, electrode be placed in and contain 2.0 mmolL respectively
-1reddish black A and 1.0 mmolL
-1h
2o
2the PBS buffer solution of pH=7.4 in measure its curent change;
(2) linear according to gained electric current difference and GST concentration, drawing curve;
(3) carry out the detection of GST in sample according to the method for drafting of working curve, testing result checks in from working curve.
embodiment 9the application of electrochemical immunosensor
For the detection of glutathione transferase, step is as follows:
(1) adopt 0.10 molmL
-1, the phosphate buffered solution of pH=7.4, is formulated in 12 ngmL
-1the GST solution of variable concentrations in scope, the GST solution of 6 μ L variable concentrations is dripped respectively to the electrode surface that is applied to step in embodiment 7 (4) Bovine Serum Albumin Modified, after drying, according to step (6) modified electrode in embodiment 7, be connected in electrochemical workstation, electrode be placed in and contain 3.0 mmolL respectively
-1reddish black A and 5.0 mmolL
-1h
2o
2the PBS buffer solution of pH=7.4 in measure its curent change;
(2) linear according to gained electric current difference and GST concentration, drawing curve;
(3) carry out the detection of GST in sample according to the method for drafting of working curve, testing result checks in from working curve.
embodiment 10the application of electrochemical immunosensor
For the detection of glutathione transferase, step is as follows:
(1) adopt 0.10 molmL
-1, the phosphate buffered solution of pH=7.4, is formulated in 5.0 ngmL
-1the GST solution of variable concentrations in scope, the GST solution of 6 μ L variable concentrations is dripped respectively to the electrode surface that is applied to step in embodiment 7 (4) Bovine Serum Albumin Modified, after drying, according to step (6) modified electrode in embodiment 7, be connected in electrochemical workstation, electrode be placed in and contain 2.5 mmolL respectively
-1reddish black A and 3.0 mmolL
-1h
2o
2the PBS buffer solution of pH=7.4 in measure its curent change;
(2) linear according to gained electric current difference and GST concentration, drawing curve;
(3) carry out the detection of GST in sample according to the method for drafting of working curve, testing result checks in from working curve.
Claims (4)
1. a preparation method for glutathione transferase antigen biology sensor, is characterized in that, comprises the following steps:
(1) preparation of bimetal nano porosint platinum iron (PtFe)
PtFeAl alloy sheet is put into 0.5 ~ 1.0 molL is housed
-1in the small beaker of NaOH solution, be placed in water bath with thermostatic control, under 40 ~ 50 ℃ of conditions, place 24 ~ 48 h, by the product obtaining intermediate water cyclic washing, under room temperature, dry, prepare required PtFe;
(2) the anti-compound (PtFe/HRP/Ab of PtFe/ horseradish peroxidase/glutathione transferase two
2) preparation
By ultrasonic PtFe suspending liquid, pipette suspending liquid, HRP solution and Ab
2solution is in a centrifuge tube, and volume ratio is PtFe:HRP: Ab
2=1: 0.5 ~ 2: 0.5 ~ 2 mixes, under 4 ℃ of conditions, the hatching of vibrating, after hatching 12 ~ 24 h, is placed in 4 ℃ of refrigerators and preserves stand-by;
(3) preparation of electrochemical immunosensor.
2. the preparation method of a kind of glutathione transferase antigen biology sensor as claimed in claim 1, is characterized in that, the preparation of described electrochemical immunosensor, and step is as follows:
(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 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) HSO shitosan being disperseed
3-GS modifies in glass-carbon electrode surface, drying at room temperature;
(3) by glutathione transferase primary antibodie (Ab
1) drip and be coated onto step (2) HSO
3the electrode surface that-GS modifies, is placed under 4 ℃ of wet condition and dries;
(4) by concentration be 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) glutathione transferase antigen (GST) is dripped and is coated in the electrode surface of step (4) Bovine Serum Albumin Modified, under wet condition, dry at 4 ℃;
(6) by PtFe/HRP/Ab
2drip and be coated in the electrode surface that step (5) GST modifies, dry at 4 ℃ under wet condition, prepared by electrochemical immunosensor.
3. the preparation method of a kind of glutathione transferase antigen biology sensor as claimed in claim 1, is characterized in that, described HSO
3the concentration of-GS is 0.5 ~ 2.5 mgmL
-1, the concentration of described PtFe suspending liquid is 10 mgmL
-1, the concentration of described HRP is 1.0 mgmL
-1, described Ab
2concentration be 1.0 mgmL
-1.
4. the detection method of the sensor that prepared by the preparation method as described in one of claim 1 ~ 3, is characterized in that, comprises the steps:
(1) adopt 0.10 molmL
-1, the phosphate buffered solution of pH=7.4, is formulated in 0.01 ~ 12 ngmL
-1the GST solution of variable concentrations in scope, the GST solution of 6 μ L variable concentrations is dripped respectively to the electrode surface that is applied to step in claim 2 (4) Bovine Serum Albumin Modified, after drying, according to step in claim 2 (6) modified electrode, be connected in electrochemical workstation, electrode be placed in and contain 2.0 ~ 3.0 mmolL respectively
-1reddish black A and 1.0 ~ 5.0 mmolL
-1h
2o
2the PBS buffer solution of pH=7.4 in measure its curent change;
(2) linear according to gained electric current difference and GST concentration, drawing curve;
(3) carry out the detection of GST in sample according to the method for drafting of working curve, testing result checks in from working curve.
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| CN104569427A (en) * | 2014-12-30 | 2015-04-29 | 山东理工大学 | Preparation method and application of immunosensor established based on manganese dioxide loaded silver nanoparticle multiwalled carbon nanotube |
| CN104714027A (en) * | 2015-02-06 | 2015-06-17 | 济南大学 | Preparation method and application of myohemoglobin biosensor constructed based on palladium tripod |
| CN109342525A (en) * | 2018-11-15 | 2019-02-15 | 江苏科技大学 | Redox graphene and gather reddish black A duplicature and the preparation method and application thereof |
| CN110609067A (en) * | 2018-06-16 | 2019-12-24 | 浙江晨阳新材料有限公司 | Alcohol sensor and preparation method thereof |
| CN112684020A (en) * | 2020-12-01 | 2021-04-20 | 哈尔滨医科大学 | Biomarker for evaluating individual zinc nutrition state and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104569427A (en) * | 2014-12-30 | 2015-04-29 | 山东理工大学 | Preparation method and application of immunosensor established based on manganese dioxide loaded silver nanoparticle multiwalled carbon nanotube |
| CN104714027A (en) * | 2015-02-06 | 2015-06-17 | 济南大学 | Preparation method and application of myohemoglobin biosensor constructed based on palladium tripod |
| CN104714027B (en) * | 2015-02-06 | 2016-01-20 | 济南大学 | A kind of preparation method of the myoglobins biology sensor based on palladium tripod structure and application |
| CN110609067A (en) * | 2018-06-16 | 2019-12-24 | 浙江晨阳新材料有限公司 | Alcohol sensor and preparation method thereof |
| CN110609067B (en) * | 2018-06-16 | 2022-08-23 | 浙江晨阳新材料有限公司 | Alcohol sensor and preparation method thereof |
| CN109342525A (en) * | 2018-11-15 | 2019-02-15 | 江苏科技大学 | Redox graphene and gather reddish black A duplicature and the preparation method and application thereof |
| CN112684020A (en) * | 2020-12-01 | 2021-04-20 | 哈尔滨医科大学 | Biomarker for evaluating individual zinc nutrition state and application thereof |
| CN112684020B (en) * | 2020-12-01 | 2023-12-29 | 哈尔滨医科大学 | Biomarker for evaluating zinc nutrition status of individual and application thereof |
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| CN103868971B (en) | 2014-12-17 |
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