CN104897757A - Production method and application of PdNi alloy/nitrogen doped graphene nanoribbon dual-amplification immunosensor - Google Patents
Production method and application of PdNi alloy/nitrogen doped graphene nanoribbon dual-amplification immunosensor Download PDFInfo
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- CN104897757A CN104897757A CN201510211215.2A CN201510211215A CN104897757A CN 104897757 A CN104897757 A CN 104897757A CN 201510211215 A CN201510211215 A CN 201510211215A CN 104897757 A CN104897757 A CN 104897757A
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Abstract
The invention relates to a production method and an application of a PdNi alloy/nitrogen doped graphene nanoribbon dual-amplification immunosensor, and belongs to the technical field of novel functional materials and biologic sensing detection. The sandwich electrochemical immunosensor is produced based on a PdNi alloy hybrid nitrogen doped graphene nanoribbon composite material. PdNi alloy and graphene nanoribbon respectively have a good catalysis performance on hydrogen peroxide, so the immunosensor has a dual-amplification effect. The above hybrid material has good biocompatibility and a high catalysis efficiency, and can greatly improve the sensitivity and the stability of the immunosensor, and the immunosensor is very important for early stage diagnosis and prognosis determination of breast cancer tumors.
Description
Technical field
The present invention relates to the preparations and applicatio of the immunosensor of a kind of PdNi alloy/dual amplification of nitrogen-doped graphene nanobelt.Specifically adopt nitrogen-doped graphene nanobelt and the PdNi alloy with better catalytic effect, prepare a kind of electrochemical immunosensor detecting the dual amplification of markers for breast cancer, belong to new function material and bio-sensing detection technique field.
Background technology
Current carbon nano-tube, due to performances such as the structure of its uniqueness and excellent electricity, mechanics and optics, receives much concern at physics, chemistry, material and even biological field.Carbon nano-tube has biocompatibility and can be used as biology sensor, timbering material, pharmaceutical carrier etc., has become the study hotspot in nanobiology field.The multi-walled carbon nano-tubes of N doping is except having above-mentioned character, and it also has good catalytic performance to hydrogen peroxide.But the multi-walled carbon nano-tubes due to N doping is water-soluble poor, therefore carried out microwave stripping to it, the multi-walled carbon nano-tubes after stripping not only maintains good catalytic performance, and has water-soluble preferably.With its carrier as PdNi alloy, reach the effect of the dual amplification of signal, reduce the detectability analyzing thing, thus improve the sensitivity detected.
Summary of the invention
An object of the present invention is the dual amplification electrochemical immunosensor built based on PdNi alloy/nitrogen-doped graphene nanobelt.
Two of object of the present invention is the detections this electrochemical immunosensor being applied to breast cancer tumour mark.
technical scheme of the present invention
1. the preparation of the immunosensor of PdNi alloy/dual amplification of nitrogen-doped graphene nanobelt
(1) by diameter be the alundum (Al2O3) burnishing powder polishing that the glass-carbon electrode of 4 mm uses 1.0,0.3,0.05 μm successively, clean up with ultrapure water, then electrode is placed in 5 mmol/L potassium ferricyanide solutions, scans under-0.2 ~ 0.6 V current potential, make spike potential difference be less than 110 mV;
(2) 6 μ L, 0.5 ~ 3 mg/mL is got
βthe solution of-cyclodextrin Graphene is added drop-wise to electrode surface, dries under room temperature;
(3) the capture antibody Ab of markers for breast cancer of adamantanecarboxylic acid functionalization of 6 μ L, 5 ~ 10 μ g/mL is dripped
1, dry at 4 DEG C, ultrapure water;
(4) continue dropping 3 μ L, massfraction be the bovine serum albumin solution of 1% to electrode surface, dry at 4 DEG C, ultrapure water;
(5) the markers for breast cancer solution continuing a series of variable concentrations of dropping 6 μ L, to electrode surface, dries at 4 DEG C, ultrapure water;
(6) continue dropping 4 ~ 6 μ L and detect antibody hatching thing-PdNi alloy/nitrogen-doped graphene nanobelt-Ab
2solution, to electrode surface, is placed in 4 DEG C of refrigerators and hatches 1 h, after cleaning, dry, the immunosensor of obtained a kind of PdNi alloy/dual amplification of nitrogen-doped graphene nanobelt.
2. the preparation of beta-schardinger dextrin-Graphene
The graphene oxide of 6 ~ 12 mg is dissolved in the ultrapure water of 20 mL, and the beta-schardinger dextrin-of 60 ~ 100 mg is dissolved in the water of 20 mL, and two solution mixing, add ammonia spirit and 10 ~ 40 μ L hydrazine hydrates of 200 ~ 600 μ L; Stir 10 min, be heated to 60 DEG C and keep 3.5 h, gained potpourri through washing, centrifuging, vacuum drying at 45 DEG C, i.e. obtained beta-schardinger dextrin-Graphene;
3. detect antibody hatching thing-PdNi alloy/nitrogen-doped graphene nanobelt-Ab
2the preparation of solution
(1) preparation of nitrogen-doped graphene nanobelt
By the H of the multi-walled carbon nano-tubes of the N doping of 50 mg, 18 mL
2sO
4, 2 mL H
3pO
4mixing, at 50 ~ 140 DEG C, microwave reaction 4 ~ 30 min, is cooled to room temperature; Add the KMnO of 0.15 ~ 0.5 g
4, microwave reaction 2 ~ 10 min at 60 ~ 70 DEG C, potpourri through washing, centrifuging, vacuum drying at 35 ~ 45 DEG C, obtained nitrogen-doped graphene nanobelt;
(2) preparation of antibody labeling thing-PdNi alloy/nitrogen-doped graphene nanobelt is detected
Respectively by the Na of 15 ~ 25 mg
2pdCl
4, the NiCl of 20 ~ 30 mg
26H
2the glutamic acid of O and 50 ~ 60 mg is placed in the ethylene glycol of 40 mL, regulates the pH of solution to be 11, adds the nitrogen-doped graphene nanobelt of 25 ~ 35 mg, stir 1 ~ 3 h by the NaOH solution of 1mol/L, moves in reactor, reacts 2 ~ 8 h at 100 ~ 200 DEG C; Potpourri through washing, centrifuging, vacuum drying at 35 DEG C, obtained PdNi alloy/nitrogen-doped graphene nanobelt;
(4) antibody hatching thing-PdNi alloy/nitrogen-doped graphene nanobelt-Ab is detected
2the preparation of solution
The PdNi of 1 ~ 3 mg alloy/nitrogen-doped graphene nanobelt is scattered in 1 mL ultrapure water, add the pH=7.4 phosphate buffered solution of 100 μ L, the detection antibody-solutions of 8 ~ 12 μ g/mL and 900 μ L, 1/15 mol/L, vibrate hatching 12 h at 4 DEG C, centrifuging, lower sediment be added in the pH=7.4 phosphate buffered solution of 1 mL, 1/15 mol/L, the obtained antibody that detects hatches thing-PdNi alloy/nitrogen-doped graphene nanobelt-Ab
2solution, saves backup at 4 DEG C.
4. the immunosensor of PdNi alloy/dual amplification of nitrogen-doped graphene nanobelt is used for a detection for markers for breast cancer, and step is as follows:
(1) three-electrode system is adopted to measure, using the immunosensor prepared by claim 1 as working electrode, be connected in electrochemical workstation, respectively electrode is placed in the phosphate buffered solution of pH=7.0, during employing, the method for m-electric current scans, input voltage is-0.4 V, working time 400 s, record current change;
(2) after background current tends towards stability, inject the hydrogen peroxide solution of 10 μ L, 5 mol/L every 50 s in the phosphate buffered solution of 10 mL, pH=7.0, record current changes, drawing curve;
6. the preparation of the immunosensor of a kind of PdNi alloy/dual amplification of nitrogen-doped graphene nanobelt of the present invention, described markers for breast cancer is selected from one of following: CA153, CA125, CEA.
useful achievement of the present invention
(1) cyclodextrin Graphene has larger surface area and good electric conductivity, and good biocompatibility and stability, in addition, it can the capture antibody of specific recognition adamantanecarboxylic acid functionalization, improve antibody in the charge capacity on its surface and the dispersiveness in water, add transducer sensitivity and stability.
(2) by the carrier of the graphene nanobelt of N doping as PdNi alloy, PdNi alloy can be dispersed on nitrogen-doped graphene nanobelt, improves the sensitivity of sensor.And the graphene nanobelt of N doping has good catalytic performance, thus reach the effect of dual amplification.
(3) electrochemical immunosensor prepared of the present invention is for the detection of markers for breast cancer, response time is short, and detectability is low, and the range of linearity is wide, simple, quick, highly sensitive and specific detection can be realized, 0.22 pg/mL can be reached to the detectability of markers for breast cancer.
Embodiment
embodiment 1the preparation of markers for breast cancer immunosensor
(1) by diameter be the alundum (Al2O3) burnishing powder polishing that the glass-carbon electrode of 4 mm uses 1.0,0.3,0.05 μm successively, clean up with ultrapure water, then electrode is placed in 5 mmol/L potassium ferricyanide solutions, scans under-0.2 ~ 0.6 V current potential, make spike potential difference be less than 110 mV;
(2) 6 μ L, 0.5 mg/mL is got
βthe solution of-cyclodextrin Graphene is added drop-wise to electrode surface, dries under room temperature;
(3) the capture antibody Ab of markers for breast cancer of adamantanecarboxylic acid functionalization of 6 μ L, 5 μ g/mL is dripped
1, dry at 4 DEG C, ultrapure water;
(4) continue dropping 3 μ L, massfraction be the bovine serum albumin solution of 1% to electrode surface, dry at 4 DEG C, ultrapure water;
(5) the markers for breast cancer solution continuing a series of variable concentrations of dropping 6 μ L, to electrode surface, dries at 4 DEG C, ultrapure water;
(6) continue dropping 4 μ L and detect antibody hatching thing-PdNi alloy/nitrogen-doped graphene nanobelt-Ab
2solution, to electrode surface, is placed in 4 DEG C of refrigerators and hatches 1 h, after cleaning, dry, the immunosensor of obtained a kind of PdNi alloy/dual amplification of nitrogen-doped graphene nanobelt.
embodiment 2the preparation of markers for breast cancer immunosensor
(1) by diameter be the alundum (Al2O3) burnishing powder polishing that the glass-carbon electrode of 4 mm uses 1.0,0.3,0.05 μm successively, clean up with ultrapure water, then electrode is placed in 5 mmol/L potassium ferricyanide solutions, scans under-0.2 ~ 0.6 V current potential, make spike potential difference be less than 110 mV;
(2) 6 μ L, 1.5 mg/mL are got
βthe solution of-cyclodextrin Graphene is added drop-wise to electrode surface, dries under room temperature;
(3) the capture antibody Ab of markers for breast cancer of adamantanecarboxylic acid functionalization of 6 μ L, 7 μ g/mL is dripped
1, dry at 4 DEG C, ultrapure water;
(4) continue dropping 3 μ L, massfraction be the bovine serum albumin solution of 1% to electrode surface, dry at 4 DEG C, ultrapure water;
(5) the markers for breast cancer solution continuing a series of variable concentrations of dropping 6 μ L, to electrode surface, dries at 4 DEG C, ultrapure water;
(6) continue dropping 5 μ L and detect antibody hatching thing-PdNi alloy/nitrogen-doped graphene nanobelt-Ab
2solution, to electrode surface, is placed in 4 DEG C of refrigerators and hatches 1 h, after cleaning, dry, the immunosensor of obtained a kind of PdNi alloy/dual amplification of nitrogen-doped graphene nanobelt.
embodiment 3the preparation of markers for breast cancer immunosensor
(1) by diameter be the alundum (Al2O3) burnishing powder polishing that the glass-carbon electrode of 4 mm uses 1.0,0.3,0.05 μm successively, clean up with ultrapure water, then electrode is placed in 5 mmol/L potassium ferricyanide solutions, scans under-0.2 ~ 0.6 V current potential, make spike potential difference be less than 110 mV;
(2) 6 μ L, 3 mg/mL are got
βthe solution of-cyclodextrin Graphene is added drop-wise to electrode surface, dries under room temperature;
(3) the capture antibody Ab of markers for breast cancer of adamantanecarboxylic acid functionalization of 6 μ L, 10 μ g/mL is dripped
1, dry at 4 DEG C, ultrapure water;
(4) continue dropping 3 μ L, massfraction be the bovine serum albumin solution of 1% to electrode surface, dry at 4 DEG C, ultrapure water;
(5) the markers for breast cancer solution continuing a series of variable concentrations of dropping 6 μ L, to electrode surface, dries at 4 DEG C, ultrapure water;
(6) continue dropping 6 μ L and detect antibody hatching thing-PdNi alloy/nitrogen-doped graphene nanobelt-Ab
2solution, to electrode surface, is placed in 4 DEG C of refrigerators and hatches 1 h, after cleaning, dry, the immunosensor of obtained a kind of PdNi alloy/dual amplification of nitrogen-doped graphene nanobelt.
embodiment 4the preparation of beta-schardinger dextrin-Graphene
The graphene oxide of 6 mg is dissolved in the ultrapure water of 20 mL, and the beta-schardinger dextrin-of 60 mg is dissolved in the water of 20 mL, and two solution mixing, add ammonia spirit and the 10 μ L hydrazine hydrates of 200 μ L; Stir 10 min, be heated to 60 DEG C and keep 3.5 h, gained potpourri through washing, centrifuging, vacuum drying at 45 DEG C, i.e. obtained beta-schardinger dextrin-Graphene;
embodiment 5the preparation of beta-schardinger dextrin-Graphene
The graphene oxide of 8 mg is dissolved in the ultrapure water of 20 mL, and the beta-schardinger dextrin-of 80 mg is dissolved in the water of 20 mL, and two solution mixing, add ammonia spirit and the 20 μ L hydrazine hydrates of 400 μ L; Stir 10 min, be heated to 60 DEG C and keep 3.5 h, gained potpourri through washing, centrifuging, vacuum drying at 45 DEG C, i.e. obtained beta-schardinger dextrin-Graphene;
embodiment 6the preparation of beta-schardinger dextrin-Graphene
The graphene oxide of 12 mg is dissolved in the ultrapure water of 20 mL, and the beta-schardinger dextrin-of 100 mg is dissolved in the water of 20 mL, and two solution mixing, add ammonia spirit and the 40 μ L hydrazine hydrates of 600 μ L; Stir 10 min, be heated to 60 DEG C and keep 3.5 h, gained potpourri through washing, centrifuging, vacuum drying at 45 DEG C, i.e. obtained beta-schardinger dextrin-Graphene;
embodiment 7detect antibody hatching thing alloy PdNi/ nitrogen-doped graphene nanobelt-Ab
2the preparation of solution
(1) preparation of nitrogen-doped graphene nanobelt
By the H of the multi-walled carbon nano-tubes of the N doping of 50 mg, 18 mL
2sO
4, 2 mL H
3pO
4mixing, at 50 DEG C, microwave reaction 4 min, is cooled to room temperature; Add the KMnO of 0.15g
4, microwave reaction 2 min at 60 DEG C, potpourri through washing, centrifuging, vacuum drying at 35 DEG C, obtained nitrogen-doped graphene nanobelt;
(2) preparation of antibody labeling thing-PdNi alloy/nitrogen-doped graphene nanobelt is detected
Respectively by the Na of 15 mg
2pdCl
4, the NiCl of 20 mg
26H
2the glutamic acid of O and 50 mg is placed in the ethylene glycol of 40 mL, regulates the pH of solution to be 11, adds the nitrogen-doped graphene nanobelt of 25 mg, stir 1 h, move in reactor, react 2 h at 100 DEG C by the NaOH solution of 1 mol/L; Potpourri through washing, centrifuging, vacuum drying at 35 DEG C, obtained PdNi alloy/nitrogen-doped graphene nanobelt;
(4) antibody hatching thing-PdNi alloy/nitrogen-doped graphene nanobelt-Ab is detected
2the preparation of solution
The PdNi of 1 mg alloy/nitrogen-doped graphene nanobelt is scattered in 1 mL ultrapure water, add the pH=7.4 phosphate buffered solution of 100 μ L, the detection antibody-solutions of 8 μ g/mL and 900 μ L, 1/15 mol/L, vibrate hatching 12 h at 4 DEG C, centrifuging, lower sediment be added in the pH=7.4 phosphate buffered solution of 1 mL, 1/15 mol/L, the obtained antibody that detects hatches thing-PdNi alloy/nitrogen-doped graphene nanobelt-Ab
2solution, saves backup at 4 DEG C.
embodiment 8detect antibody hatching thing alloy PdNi/ nitrogen-doped graphene nanobelt-Ab
2the preparation of solution
(1) preparation of nitrogen-doped graphene nanobelt
By the H of the multi-walled carbon nano-tubes of the N doping of 50 mg, 18 mL
2sO
4, 2 mL H
3pO
4mixing, at 100 DEG C, microwave reaction 20 min, is cooled to room temperature; Add the KMnO of 0.2 g
4, microwave reaction 8 min at 65 DEG C, potpourri through washing, centrifuging, vacuum drying at 40 DEG C, obtained nitrogen-doped graphene nanobelt;
(2) preparation of antibody labeling thing-PdNi alloy/nitrogen-doped graphene nanobelt is detected
Respectively by the Na of 20 mg
2pdCl
4, the NiCl of 25 mg
26H
2the glutamic acid of O and 55 mg is placed in the ethylene glycol of 40 mL, regulates the pH of solution to be 11, adds the nitrogen-doped graphene nanobelt of 30 mg, stir 2 h, move in reactor, react 6 h at 150 DEG C by the NaOH solution of 1 mol/L; Potpourri through washing, centrifuging, vacuum drying at 35 DEG C, obtained PdNi alloy/nitrogen-doped graphene nanobelt;
(4) antibody hatching thing-PdNi alloy/nitrogen-doped graphene nanobelt-Ab is detected
2the preparation of solution
The PdNi of 2 mg alloy/nitrogen-doped graphene nanobelt is scattered in 1 mL ultrapure water, add the pH=7.4 phosphate buffered solution of 100 μ L, the detection antibody-solutions of 10 μ g/mL and 900 μ L, 1/15 mol/L, vibrate hatching 12 h at 4 DEG C, centrifuging, lower sediment be added in the pH=7.4 phosphate buffered solution of 1 mL, 1/15 mol/L, the obtained antibody that detects hatches thing-PdNi alloy/nitrogen-doped graphene nanobelt-Ab
2solution, saves backup at 4 DEG C.
embodiment 9detect antibody hatching thing alloy PdNi/ nitrogen-doped graphene nanobelt-Ab
2the preparation of solution
(1) preparation of nitrogen-doped graphene nanobelt
By the H of the multi-walled carbon nano-tubes of the N doping of 50 mg, 18 mL
2sO
4, 2 mL H
3pO
4mixing, at 140 DEG C, microwave reaction 30 min, is cooled to room temperature; Add the KMnO of 0.5g
4, microwave reaction 10 min at 70 DEG C, potpourri through washing, centrifuging, vacuum drying at 45 DEG C, obtained nitrogen-doped graphene nanobelt;
(2) preparation of antibody labeling thing-PdNi alloy/nitrogen-doped graphene nanobelt is detected
Respectively by the Na of 25 mg
2pdCl
4, the NiCl of 30 mg
26H
2the glutamic acid of O and 60 mg is placed in the ethylene glycol of 40 mL, regulates the pH of solution to be 11, adds the nitrogen-doped graphene nanobelt of 35 mg, stir 3 h, move in reactor, react 8 h at 200 DEG C by the NaOH solution of 1 mol/L; Potpourri through washing, centrifuging, vacuum drying at 35 DEG C, obtained PdNi alloy/nitrogen-doped graphene nanobelt;
(4) antibody hatching thing-PdNi alloy/nitrogen-doped graphene nanobelt-Ab is detected
2the preparation of solution
The PdNi of 3 mg alloy/nitrogen-doped graphene nanobelt is scattered in 1 mL ultrapure water, add the pH=7.4 phosphate buffered solution of 100 μ L, the detection antibody-solutions of 12 μ g/mL and 900 μ L, 1/15 mol/L, vibrate hatching 12 h at 4 DEG C, centrifuging, lower sediment be added in the pH=7.4 phosphate buffered solution of 1 mL, 1/15 mol/L, the obtained antibody that detects hatches thing-PdNi alloy/nitrogen-doped graphene nanobelt-Ab
2solution, saves backup at 4 DEG C.
embodiment 10the detection of markers for breast cancer CA153
(1) three-electrode system is adopted to measure, using the immunosensor prepared by claim 1 as working electrode, be connected in electrochemical workstation, respectively electrode is placed in the phosphate buffered solution of pH=7.0, during employing, the method for m-electric current scans, input voltage is-0.4 V, working time 400 s, record current change;
(2) after background current tends towards stability, inject the hydrogen peroxide solution of 10 μ L, 5 mol/L every 50 s in the phosphate buffered solution of 10 mL, pH=7.0, record current changes;
(3) according to the linear relationship between strength of current and breast cancer tumour mark CA153, drawing curve, recording the range of linearity is 1.0 pg/mL ~ 16 ng/mL, detects and is limited to 0.30 pg/mL.
embodiment 11the detection of markers for breast cancer CA125
Drawing curve step, with embodiment 10, carries out the sample analysis of CA125 according to the method for drawing curve, recording the range of linearity is 1.0 pg/mL ~ 16 ng/mL, detects and is limited to 0.22 pg/mL.
embodiment 12the detection of markers for breast cancer CEA
Drawing curve step, with embodiment 10, carries out the sample analysis of CEA according to the method for drawing curve, recording the range of linearity is 1.2 pg/mL ~ 20 ng/mL, detects and is limited to 0.31 pg/mL.
Claims (4)
1. a preparation for the immunosensor of PdNi alloy/dual amplification of nitrogen-doped graphene nanobelt, is characterized in that, comprise the following steps:
(1) by diameter be the alundum (Al2O3) burnishing powder polishing that the glass-carbon electrode of 4 mm uses 1.0,0.3,0.05 μm successively, clean up with ultrapure water, then electrode is placed in 5 mmol/L potassium ferricyanide solutions, scans under-0.2 ~ 0.6 V current potential, make spike potential difference be less than 110 mV;
(2) 6 μ L, 0.5 ~ 3 mg/mL is got
βthe solution of-cyclodextrin Graphene is added drop-wise to electrode surface, dries under room temperature;
(3) the capture antibody Ab of markers for breast cancer of adamantanecarboxylic acid functionalization of 6 μ L, 5 ~ 10 μ g/mL is dripped
1, dry at 4 DEG C, ultrapure water;
(4) continue dropping 3 μ L, massfraction be the bovine serum albumin solution of 1% to electrode surface, dry at 4 DEG C, ultrapure water;
(5) the markers for breast cancer solution continuing a series of variable concentrations of dropping 6 μ L, to electrode surface, dries at 4 DEG C, ultrapure water;
(6) continue dropping 4 ~ 6 μ L and detect antibody hatching thing-PdNi alloy/nitrogen-doped graphene nanobelt-Ab
2solution, to electrode surface, is placed in 4 DEG C of refrigerators and hatches 1 h, after cleaning, dry, the immunosensor of obtained a kind of PdNi alloy/dual amplification of nitrogen-doped graphene nanobelt.
2. the preparation of the immunosensor of a kind of PdNi alloy/dual amplification of nitrogen-doped graphene nanobelt as claimed in claim 1, is characterized in that, described detection antibody hatching thing-PdNi alloy/nitrogen-doped graphene nanobelt-Ab
2the preparation of solution, comprises the following steps:
(1) preparation of nitrogen-doped graphene nanobelt
By the H of the multi-walled carbon nano-tubes of the N doping of 50 mg, 18 mL
2sO
4, 2 mL H
3pO
4mixing, at 50 ~ 140 DEG C, microwave reaction 4 ~ 30 min, is cooled to room temperature; Add the KMnO of 0.15 ~ 0.5g
4, microwave reaction 2 ~ 10 min at 60 ~ 70 DEG C, potpourri through washing, centrifuging, vacuum drying at 35 ~ 45 DEG C, obtained nitrogen-doped graphene nanobelt;
(2) preparation of antibody labeling thing-PdNi alloy/nitrogen-doped graphene nanobelt is detected
Respectively by the Na of 15 ~ 25 mg
2pdCl
4, the NiCl of 20 ~ 30 mg
26H
2the glutamic acid of O and 50 ~ 60 mg is placed in the ethylene glycol of 40 mL, regulates the pH of solution to be 11, adds the nitrogen-doped graphene nanobelt of 25 ~ 35 mg, stir 1 ~ 3 h by the NaOH solution of 1 mol/L, moves in reactor, reacts 2 ~ 8 h at 100 ~ 200 DEG C; Potpourri through washing, centrifuging, vacuum drying at 35 DEG C, obtained PdNi alloy/nitrogen-doped graphene nanobelt;
(4) antibody hatching thing-PdNi alloy/nitrogen-doped graphene nanobelt-Ab is detected
2the preparation of solution
The PdNi of 1 ~ 3 mg alloy/nitrogen-doped graphene nanobelt is scattered in 1 mL ultrapure water, add the pH=7.4 phosphate buffered solution of 100 μ L, the detection antibody-solutions of 8 ~ 12 μ g/mL and 900 μ L, 1/15 mol/L, vibrate hatching 12 h at 4 DEG C, centrifuging, lower sediment be added in the pH=7.4 phosphate buffered solution of 1 mL, 1/15 mol/L, the obtained antibody that detects hatches thing-PdNi alloy/nitrogen-doped graphene nanobelt-Ab
2solution, saves backup at 4 DEG C.
3. the immunosensor prepared of preparation method as claimed in claim 1 is for the detection of markers for breast cancer, it is characterized in that, comprises following analytical procedure:
(1) three-electrode system is adopted to measure, using the immunosensor prepared by claim 1 as working electrode, be connected in electrochemical workstation, respectively electrode is placed in the phosphate buffered solution of pH=7.0, during employing, the method for m-electric current scans, input voltage is-0.4 V, working time 400 s, record current change;
(2) after background current tends towards stability, inject the hydrogen peroxide solution of 10 μ L, 5 mol/L every 50 s in the phosphate buffered solution of 10 mL, pH=7.0, record current changes, drawing curve.
4. the preparation of the immunosensor of a kind of PdNi alloy/dual amplification of nitrogen-doped graphene nanobelt as claimed in claim 1, described markers for breast cancer is selected from one of following: CA153, CA125, CEA.
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