CN103868971B - 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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- CN103868971B CN103868971B CN201410036028.0A CN201410036028A CN103868971B CN 103868971 B CN103868971 B CN 103868971B CN 201410036028 A CN201410036028 A CN 201410036028A CN 103868971 B CN103868971 B CN 103868971B
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- glutathione transferase
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- gst
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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 detecting the glutathione transferase in serum.Belong to Nano-function thin films and biosensor technique field.
Background technology
Glutathione transferase (GST) mainly participates in the biological a kind of biology enzyme transforming second-phase reaction in liver, it can detect again in the tissue in serum, and other many tumor markers can only detect in the tissue, and the 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, highlight electrode modified material and biology sensor increased to response, improve detection sensitivity, increase the effects such as the range of linearity.HSO
3-GS have specific surface area large, can the advantages such as electronic conduction be strengthened, utilize chemical bonding effect directly to connect with glutathione transferase primary antibodie, without the need to adopting the immobilization of conventional cross-linking agent glutaraldehyde.PtFe is adopted 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 for electron mediator A is added in end liquid, avoids and modify the leakage problem after to electrode.
Response current and the GST concentration of the biology sensor that the present invention obtains 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, has prepared a kind of glutathione transferase sandwich type immunosensor.
The present invention have highly sensitive, specificity good, testing cost is low, can detect the advantages such as glutathione transferase fast, and preparation process of the present invention is simple, and operating process is easy, effectively overcomes the deficiency of current glutathione transferase detection method.
Summary of the invention
An object of the present invention is to avoid that the instrument and equipment of traditional detection method is complicated, operating process is loaded down with trivial details, testing staff requires the shortcomings such as high, provides the preparation method of electrochemical immunosensor of a kind of highly sensitive, high specificity, favorable reproducibility, quick detection glutathione transferase easy and simple to handle.
Two of object of the present invention is the detections this electrochemical immunosensor being applied to glutathione transferase.
To achieve these goals, the present invention is realized by following measures.
A preparation method for glutathione transferase antigen biology sensor, is characterized in that, comprise 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 DEG C of conditions, place 24 ~ 48 h, the product intermediate water obtained is washed repeatedly, dries under room temperature, namely 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 mix, and under 4 DEG C of conditions, carry out vibration hatching, after hatching 12 ~ 24 h, are placed in 4 DEG C of refrigerators and preserve stand-by.
(3) preparation of electrochemical immunosensor
1) glass-carbon electrode of diameter 4 mm is used successively the alundum (Al2O3) burnishing powder polishing of 1.0,0.3 and 0.05 mm, EtOH Sonicate cleans, cleaner with ultrapure water, then electrode is placed in 5 mmolL
-1in potassium ferricyanide solution, scan under-0.2 ~ 0.6 V current potential, make spike potential difference be less than 90 mV;
2) by the HSO of shitosan dispersion
3-GS modifies in glassy 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, dries under being placed in 4 DEG C of wet condition;
4) by concentration be 100 μ gmL
-1bovine serum albumin(BSA) drips and is coated onto step 3) Ab
1the electrode surface modified, dries under wet condition at 4 DEG C;
5) GST is dripped the electrode surface being coated in step 4) Bovine Serum Albumin Modified, dry under wet condition at 4 DEG C;
6) by PtFe/HRP/Ab
2drip the electrode surface being coated in step 5) GST and modifying, dry under wet condition at 4 DEG C, 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) 0.10 molmL is adopted
-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, respectively electrode be placed in containing 2.0 ~ 3.0 mmolL
-1reddish black A and 1.0 ~ 5.0 mmolL
-1h
2o
2pH=7.4 PBS buffer solution in measure its curent change;
2) according to gained current differential and GST concentration linear, drawing curve;
3) method for drafting of foundation working curve carries out the detection of GST in sample, and 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 electron transmission ability that-GS is good, significantly improves the performance of electrode.
(2) by PtFe and tumor markers two is anti-directly hatches, utilize the biocompatibility of platinum iron excellence and high catalytic performance, significantly improve reappearance and the stability of electrochemical immunosensor.
(3) PtFe and horseradish peroxidase compound token two resist, and have the synergistic sensitization to signal.
(4) electrochemical immunosensor prepared by the present invention, simple to operate, detection speed is fast, can realize the mensuration of batch sample at short notice.
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 DEG C of conditions, place 24 h, by the product intermediate water cyclic washing obtained, dry under room temperature, namely 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 DEG C of conditions, place 48 h, by the product intermediate water cyclic washing obtained, dry under room temperature, namely 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 DEG C of conditions, place 35 h, by the product intermediate water cyclic washing obtained, dry under room temperature, namely 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
2mix at=1: 0.5: 0.5, under 4 DEG C of conditions, carries out vibration hatching, after hatching 12 h, be placed in 4 DEG C of refrigerators and preserve 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
2mix at=1: 1: 1, under 4 DEG C of conditions, carries out vibration hatching, after hatching 24h, be placed in 4 DEG C of refrigerators and preserve 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
2mix at=1: 2: 2, under 4 DEG C of conditions, carries out vibration hatching, after hatching 18h, be placed in 4 DEG C of refrigerators and preserve stand-by.
embodiment 7the preparation of electrochemical immunosensor
(1) glass-carbon electrode of diameter 4 mm is used successively the alundum (Al2O3) burnishing powder polishing of 1.0,0.3 and 0.05 mm, EtOH Sonicate cleans, cleaner with ultrapure water, then electrode is placed in 5 mmolL
-1in potassium ferricyanide solution, scan under-0.2 ~ 0.6 V current potential, make spike potential difference be less than 90 mV;
(2) by the HSO of shitosan dispersion
3-GS modifies in glassy 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, dries under being placed in 4 DEG C of wet condition;
(4) by concentration be 100 μ gmL
-1bovine serum albumin(BSA) drips and is coated onto step (3) Ab
1the electrode surface modified, dries under wet condition at 4 DEG C;
(5) GST is dripped the electrode surface being coated in step (4) Bovine Serum Albumin Modified, dry under wet condition at 4 DEG C;
(6) by PtFe/HRP/Ab
2drip the electrode surface being coated in step (5) GST and modifying, dry under wet condition at 4 DEG C, prepared by electrochemical immunosensor.
embodiment 8the application of electrochemical immunosensor
For the detection of glutathione transferase, step is as follows:
(1) 0.10 molmL is adopted
-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 the electrode surface being applied to step (4) Bovine Serum Albumin Modified in embodiment 7 respectively, after drying, according to step (6) modified electrode in embodiment 7, be connected in electrochemical workstation, respectively electrode be placed in containing 2.0 mmolL
-1reddish black A and 1.0 mmolL
-1h
2o
2pH=7.4 PBS buffer solution in measure its curent change;
(2) according to gained current differential and GST concentration linear, drawing curve;
(3) method for drafting of foundation working curve carries out the detection of GST in sample, and testing result checks in from working curve.
embodiment 9the application of electrochemical immunosensor
For the detection of glutathione transferase, step is as follows:
(1) 0.10 molmL is adopted
-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 the electrode surface being applied to step (4) Bovine Serum Albumin Modified in embodiment 7 respectively, after drying, according to step (6) modified electrode in embodiment 7, be connected in electrochemical workstation, respectively electrode be placed in containing 3.0 mmolL
-1reddish black A and 5.0 mmolL
-1h
2o
2pH=7.4 PBS buffer solution in measure its curent change;
(2) according to gained current differential and GST concentration linear, drawing curve;
(3) method for drafting of foundation working curve carries out the detection of GST in sample, and testing result checks in from working curve.
embodiment 10the application of electrochemical immunosensor
For the detection of glutathione transferase, step is as follows:
(1) 0.10 molmL is adopted
-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 the electrode surface being applied to step (4) Bovine Serum Albumin Modified in embodiment 7 respectively, after drying, according to step (6) modified electrode in embodiment 7, be connected in electrochemical workstation, respectively electrode be placed in containing 2.5 mmolL
-1reddish black A and 3.0 mmolL
-1h
2o
2pH=7.4 PBS buffer solution in measure its curent change;
(2) according to gained current differential and GST concentration linear, drawing curve;
(3) method for drafting of foundation working curve carries out the detection of GST in sample, and testing result checks in from working curve.
Claims (3)
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 DEG C of conditions, place 24 ~ 48 h, by the product intermediate water cyclic washing obtained, dry under room temperature, namely prepare required PtFe;
(2) the anti-compound (PtFe/HRP/Ab of PtFe/ horseradish peroxidase/glutathione transferase two
2) preparation
By ultrasonic for 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 mix, and under 4 DEG C of conditions, carry out vibration hatching, after hatching 12 ~ 24 h, are placed in 4 DEG C of refrigerators and preserve stand-by;
(3) preparation of electrochemical immunosensor, step is as follows:
1) glass-carbon electrode of diameter 4 mm is used successively the alundum (Al2O3) burnishing powder polishing of 1.0,0.3 and 0.05 mm, EtOH Sonicate cleans, cleaner with ultrapure water, then electrode is placed in 5 mmolL
-1in potassium ferricyanide solution, scan under-0.2 ~ 0.6 V current potential, make spike potential difference be less than 80 mV;
2) by the HSO of shitosan dispersion
3-GS modifies in glassy 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, dries under being placed in 4 DEG C of wet condition;
4) by concentration be 100 μ gmL
-1bovine serum albumin(BSA) drips and is coated onto step 3) Ab
1the electrode surface modified, dries under wet condition at 4 DEG C;
5) glutathione transferase antigen (GST) is dripped the electrode surface being coated in step 4) Bovine Serum Albumin Modified, dry under wet condition at 4 DEG C;
6) by PtFe/HRP/Ab
2drip the electrode surface being coated in step 5) GST and modifying, dry under wet condition at 4 DEG C, prepared by electrochemical immunosensor.
2. 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.
3. the detection method of sensor prepared by the preparation method as described in one of claim 1 ~ 2, is characterized in that, comprise the steps:
(1) 0.10 molmL is adopted
-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 the electrode surface being applied to step in claim 2 (4) Bovine Serum Albumin Modified respectively, after drying, according to step in claim 2 (6) modified electrode, be connected in electrochemical workstation, respectively electrode be placed in containing 2.0 ~ 3.0 mmolL
-1reddish black A and 1.0 ~ 5.0 mmolL
-1h
2o
2pH=7.4 PBS buffer solution in measure its curent change;
(2) according to gained current differential and GST concentration linear, drawing curve;
(3) method for drafting of foundation working curve carries out the detection of GST in sample, and testing result checks in from working curve.
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| CN104569427B (en) * | 2014-12-30 | 2016-06-22 | 山东理工大学 | The preparation method of a kind of immunosensor based on manganese dioxide load Nano silver grain multi-walled carbon nano-tubes structure and application |
| 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 |
| 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 |
| CN109342525B (en) * | 2018-11-15 | 2021-01-26 | 江苏科技大学 | Reduced graphene oxide and polyaspartic acid A double-layer film and preparation method 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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| US20060240492A1 (en) * | 2004-11-12 | 2006-10-26 | Rusling James F | Carbon nanotube based immunosensors and methods of making and using |
| CN101963592B (en) * | 2010-08-13 | 2013-04-17 | 上海师范大学 | Method for preparing electrochemical biosensor based on horse radish peroxidase-porous zirconium phytate nanoparticle modified glassy carbon electrode |
| CN102507953B (en) * | 2011-10-20 | 2013-04-17 | 济南大学 | Preparation method of electrochemistry immunosensor for determining alpha fetoprotein |
| CN102707049B (en) * | 2012-05-14 | 2014-12-10 | 宁波大学 | Preparation method and application of magnetic sandwich nano immunosensor |
| CN102914648A (en) * | 2012-10-09 | 2013-02-06 | 重庆医科大学 | Electrochemical immunosensor for detecting toxoplasma gondii IgM antibody and preparation method thereof |
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