CN106124586B - A kind of preparation method and application of sensor that is while detecting two kinds of hepatitis b virus marker HBs/HBe - Google Patents
A kind of preparation method and application of sensor that is while detecting two kinds of hepatitis b virus marker HBs/HBe Download PDFInfo
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- CN106124586B CN106124586B CN201610438126.6A CN201610438126A CN106124586B CN 106124586 B CN106124586 B CN 106124586B CN 201610438126 A CN201610438126 A CN 201610438126A CN 106124586 B CN106124586 B CN 106124586B
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
- G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
- G01N27/3278—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction involving nanosized elements, e.g. nanogaps or nanoparticles
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/576—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis
- G01N33/5761—Hepatitis B
Abstract
The invention belongs to nano-functional material, immunoassay and biosensor technique fields, provide the preparation method and application of sensor that is a kind of while detecting two kinds of hepatitis b virus marker HBs/HBe.SnO is respectively adopted2‑GS@Au@Pt NPs、SnO2GS@Ag cys Au nano materials are as detection antibody marker, the interlayer type electrochemical immunosensor of preparation is realized to being detected while hepatitis b virus marker HBs and HBe, with high specificity, the advantages such as high sensitivity and detection limit are low have important scientific meaning and clinical value to the detection of hepatitis b virus marker HBs/HBe.
Description
Technical field
The invention belongs to nano-functional material, immunoassay and biosensor technique fields, provide a kind of while examining
Survey the preparation method and application of the sensor of two kinds of hepatitis b virus marker HBs/HBe.
Background technology
Virus B hepatitis be caused by hepatitis B, based on liver inflammatory lesion, and multiple organ can be caused to damage
A kind of harmful disease.Hepatitis B is widely current in countries in the world, and main to invade children and person between twenty and fifty, it is hard that small number of patients can be converted into liver
Change or liver cancer.Therefore, it have become the worldwide disease for seriously threatening human health and China's current popular the most extensively,
A kind of disease of harmfulness most serious.Virus B hepatitis can fall ill throughout the year without certain epizootic modeling, but more categories dissipate
Hair.Hepatitis B incidence is in the trend obviously increased in recent years.Therefore the accurate detection of hepatitis b virus marker, to hepatitis B
Treatment can play an important role.Interlayer type electrochemical immunosensor combines the immuno analytical method and Gao Ling of high specific
Quick electrochemical analysis techniques, have many advantages, such as high sensitivity, prepare simple, detection is quick, at low cost, led in clinical examination
Domain has important application value.And the key for building electrochemical immunosensor has at 2 points：One is using it is simple, quickly,
Effective method is by biomolecule fixations such as antigen-antibodies in electrode surface；The second is the signal amplification technique of exploitation sensor.
Invention content
The present invention provides it is a kind of and meanwhile detect two kinds of hepatitis b virus marker HBs/HBe sensor preparation method and
Using realizing the highly sensitive of hepatitis b virus marker HBs and HBe while detecting.
An object of the present invention is to provide sensor that is a kind of while detecting two kinds of hepatitis b virus marker HBs/HBe
Preparation method.
The second object of the present invention is to will be prepared while detect two kinds of hepatitis b virus marker HBs/HBe sensing
Device, for being detected while hepatitis b virus marker HBs and HBe.
Technical scheme of the present invention includes the following steps.
1. the preparation method of sensor that is a kind of while detecting two kinds of hepatitis b virus marker HBs/HBe, steps are as follows：
（1）By the glass-carbon electrode Al of a diameter of 3 ~ 5 mm2O3Polishing powder is polished, and ultra-pure water cleans up；
（2）Above-mentioned electrode is immersed into 10 mL, mass fraction is 1% HAuCl4In solution, under -0.2 V voltages, plating
30 s, dry at room temperature, with ultrapure water electrode surface, dry；
（3）Continue above-mentioned electrode immersing hepatitis b virus marker capture antibody A nti-HBs and Anti-HBe concentration difference
In mixed solution for 8 ~ 12 μ g/mL, hatch 12 h in 4 DEG C of refrigerators, takes out in 4 DEG C of refrigerators and dry；
（4）Continue the BSA solution of 3 μ L, 1 ~ 3 mg/mL being added drop-wise to electrode surface, to non-on enclosed-electrode surface
Activity specific site, ultrapure water electrode surface dry in 4 DEG C of refrigerators；
（5）HBs the and HBe antigens that above-mentioned electrode is immersed to a series of various concentrations of the ng/mL of 0.1 pg/mL ~ 50 again are mixed
Close 37 DEG C of 50 min of hatching in solution；It dries at room temperature, ultrapure water, it is dry in 4 DEG C of refrigerators；
（6）Finally by above-mentioned steps（5）The electrode of modification immerses the HBs detections of the graphene-supported golden hydridization platinum of stannic oxide
Antibody incubation content solution S nO2-GS@Au@Pt NPs-HBs-Ab2It is anti-with the HBe detections of the graphene-supported silver-colored hydridization gold of stannic oxide
Body incubation content solution S nO2-GS@ Ag-cys-Au NPs-HBe-Ab2Concentration be respectively 1 ~ 3 mg/mL mixed liquor in, room
Temperature is lower to hatch 50 min, is placed in drying in 4 DEG C of refrigerators, is made a kind of while two kinds of hepatitis b virus marker HBs/HBe of detection
Sensor.
2. the detection antibody incubation content solution S nO2-GS@Au@Pt NPs-HBs-Ab2Preparation, steps are as follows：
(1) prepared by Au@Pt NPs
It is 1% HAuCl by 1 ~ 3 mL, mass fraction4Solution is added in 100 mL ultra-pure waters, is rapidly joined under stirring
3 ~ 6mL, the sodium citrate solution that mass fraction is 1%, are slowly added to NaBH4Become rufous, stirring to solution from faint yellow
Overnight；
By 3 ~ 7 mL, the H that mass fraction is 1%2PtCl6The above-mentioned HAuCl boiled is added in solution4In solution, 2 are added
The ascorbic acid solution of ~ 4 mL, 0.1moL/L heats 30 min, and the solution of Au@Pt NPs is made；
(2) SnO2The preparation of-GS@Au@Pt NPs
0.1 ~ 0.3 mL、 0.5 mg/mL SnO2The above-mentioned preparation Au@Pt NPs solution of-GS and 6 mL is mixed 12
H is centrifuged, and washs drying, and product is dispersed in the SnO that 1 ~ 3 mg/mL is made in ultra-pure water again2-GS@ Au@Pt NPs
Solution；
(3) detection antibody incubation content solution S nO2-GS@ Au@Pt NPs HBs-Ab2Preparation
By the HBs detection antibody As b of 1 ~ 3 mL, 10 μ g/mL2With the SnO of 1 mL, 1 ~ 3 mg/mL2-GS@ Au@Pt
NPs mixes 4 DEG C of concussion incubated overnights, and SnO is made in centrifuge washing2-GS@Au@Pt NPs-HBs-Ab2Detect antibody incubation content；With
The PBS buffer solutions of pH 7.4 disperse again, and the detection antibody incubation content solution S nO of 1 ~ 3 mg/mL is made2-GS@ Au@Pt
NPs HBs-Ab2It is spare.
3. the detection antibody incubation content solution S nO2-GS@ Ag-cys-Au NPs-HBe-Ab2Preparation, steps are as follows：
(1) preparation of Ag-cys-Au nanospheres
1) preparation of L-cys@Au NPs
20 ~ 30 mL, 2 mmoL/L L-cysteine L-cys solution in, sequentially add while stirring 250 μ L,
The HAuCl of 0.05moL/L4Solution, 350 μ L, 0.01 moL/L NaBH4, 2 h are stirred at 300k, are centrifuged, and are surpassed
Pure water is dried in vacuo 12h at room temperature, and L-cys@Au NPs are made, spare；
2) preparation of Ag-cys-Au nanospheres
10 ~ 15 mL, 0.1 mg/mL L-cys@Au NPs in, sequentially add 1 mL, mass fraction is 1% polyethylene
The ascorbic acid of pyrrolidones PVP, 1 mL, 0.1 moL/L, 1 mL, the AgNO of 0.1 moL/L3Solution, at room temperature stir 30 ~
40 min are centrifuged, and ultra-pure water cleaning is dried in vacuo Ag-cys-Au nanospheres are made at room temperature；
(2) SnO2The preparation of-GS@Ag-cys-Au NPs
0.1 ~ 0.3 mL、0.5 mg/mL SnO2The Ag-cys-Au nanosphere solution of-GS and 6 mL, 1 ~ 3 mg/mL
12 h are mixed, centrifuge, wash drying, product is dispersed in the SnO that 1 ~ 3 mg/mL is made in ultra-pure water again2-
GS@Ag-cys-Au NPs solution；
(3) detection antibody incubation content solution S nO2-GS@ Ag-cys-Au NPs-HBe-Ab2Preparation
By the HBe detection antibody As b of 1 ~ 3 mL, 10 μ g/mL2With the SnO of 1 mL, 1 ~ 3 mg/mL2-GS@ Ag-
Cys-Au NPs mix 4 DEG C of concussion incubated overnights, centrifuge washing；SnO is made2-GS@ Ag-cys-Au NPs-HBe-Ab2Capture
Antibody incubation content；Disperseed again with the PBS buffer solutions of pH 7.4, the SnO of 1 ~ 3 mg/mL is made2-GS@ Ag-cys-Au
NPs-HBe-Ab2Detect antibody incubation content solution for standby.
4. being detected while hepatitis b virus marker HBs/HBe, steps are as follows：
（1）It is tested with three-electrode system using electrochemical workstation, saturated calomel electrode is reference electrode, platinum filament electricity
Extremely auxiliary electrode, prepared sensor are working electrode, slow in 5.1 ~ 8.6 phosphate of pH of 10 mL, 50 mmol/L
It rushes in solution and is tested；
（2）Hepatitis b virus marker HBs/HBe is detected with chronoamperometry, input voltage is -0.4 V, sampling
It is spaced 0.1 s, 400 s of run time；
（3）After background current tends towards stability, the phosphate-buffereds of pH=7.4 every 50 s to 10 mL, 50 mmol/L are molten
The hydrogen peroxide solution of 10 μ L, 5 mol/L, record current variation are injected in liquid.
Raw materials of the present invention can be bought in chemical reagents corporation or biopharmaceutical company.
The useful achievement of the present invention
（1）Present invention uses electroplating golds as base material, and gold utensil has good electric conductivity and excellent biofacies
Capacitive can be good at immobilized capture antibody, and can accelerate the transmission of electronics, for realizing the highly sensitive and low of sensor
Detection limit is of great significance.
（2）SnO is respectively adopted2-GS@ Au@Pt NPs、SnO2- GS@Ag-cys-Au NPs are as detection antibody label
Object, SnO2There is-GS big specific surface area, Pt, Au and Ag to have good conductive capability and have catalysis to make hydrogen peroxide
With the collaboration for realizing signal is amplified, therefore improves the sensitivity of sensor, reduces detection limit；
（3）Two kinds of hepatitis b virus markers of sensor pair that are a kind of while detecting two kinds of hepatitis b virus marker HBs/HBe
The detection of HBs/HBe, the range of linearity reach the ng/mL of 0.1 pg/mL~50, and detection is limited up to 0.033 pg/mL.
Specific implementation mode
Now the present invention is further illustrated by specific implementation mode, but not limited to this.
Embodiment 1 is a kind of while detecting the preparation method of the sensor of two kinds of hepatitis b virus marker HBs/HBe, and step is such as
Under：
（1）By the glass-carbon electrode Al of a diameter of 3 mm2O3Polishing powder is polished, and ultra-pure water cleans up；
（2）Above-mentioned electrode is immersed into 10 mL, mass fraction is 1% HAuCl4In solution, under -0.2 V voltages, plating
30 s, dry at room temperature, with ultrapure water electrode surface, dry；
（3）Continue above-mentioned electrode immersing hepatitis b virus marker capture antibody A nti-HBs and Anti-HBe concentration difference
In mixed solution for 8 μ g/mL, hatch 12 h in 4 DEG C of refrigerators, takes out in 4 DEG C of refrigerators and dry；
（4）Continue the BSA solution of 3 μ L, 1 mg/mL being added drop-wise to electrode surface, to non-specific on enclosed-electrode surface
Property active site, ultrapure water electrode surface dry in 4 DEG C of refrigerators；
（5）HBs the and HBe antigens that above-mentioned electrode is immersed to a series of various concentrations of the ng/mL of 0.1 pg/mL ~ 50 again are mixed
Close 37 DEG C of 50 min of hatching in solution；It dries at room temperature, ultrapure water, it is dry in 4 DEG C of refrigerators；
（6）Finally by above-mentioned steps（5）The electrode of modification immerses the HBs detections of the graphene-supported golden hydridization platinum of stannic oxide
Antibody incubation content solution S nO2-GS@Au@Pt NPs-HBs-Ab2It is anti-with the HBe detections of the graphene-supported silver-colored hydridization gold of stannic oxide
Body incubation content solution S nO2-GS@ Ag-cys-Au NPs-HBe-Ab2Concentration be respectively 1 mg/mL mixed liquor in, at room temperature
Hatch 50 min, is placed in 4 DEG C of refrigerators dry, sensing that is obtained a kind of while detecting two kinds of hepatitis b virus marker HBs/HBe
Device.
Embodiment 2 is a kind of while detecting the preparation method of the sensor of two kinds of hepatitis b virus marker HBs/HBe, and step is such as
Under：
（1）By the glass-carbon electrode Al of a diameter of 4 mm2O3Polishing powder is polished, and ultra-pure water cleans up；
（2）Above-mentioned electrode is immersed into 10 mL, mass fraction is 1% HAuCl4In solution, under -0.2 V voltages, plating
30 s, dry at room temperature, with ultrapure water electrode surface, dry；
（3）Continue above-mentioned electrode immersing hepatitis b virus marker capture antibody A nti-HBs and Anti-HBe concentration difference
In mixed solution for 10 μ g/mL, hatch 12 h in 4 DEG C of refrigerators, takes out in 4 DEG C of refrigerators and dry；
（4）Continue the BSA solution of 3 μ L, 2 mg/mL being added drop-wise to electrode surface, to non-specific on enclosed-electrode surface
Property active site, ultrapure water electrode surface dry in 4 DEG C of refrigerators；
（5）HBs the and HBe antigens that above-mentioned electrode is immersed to a series of various concentrations of the ng/mL of 0.1 pg/mL ~ 50 again are mixed
Close 37 DEG C of 50 min of hatching in solution；It dries at room temperature, ultrapure water, it is dry in 4 DEG C of refrigerators；
（6）Finally by above-mentioned steps（5）The electrode of modification immerses the HBs detections of the graphene-supported golden hydridization platinum of stannic oxide
Antibody incubation content solution S nO2-GS@Au@Pt NPs-HBs-Ab2It is anti-with the HBe detections of the graphene-supported silver-colored hydridization gold of stannic oxide
Body incubation content solution S nO2-GS@ Ag-cys-Au NPs-HBe-Ab2Concentration be respectively 2 mg/mL mixed liquor in, at room temperature
Hatch 50 min, is placed in 4 DEG C of refrigerators dry, sensing that is obtained a kind of while detecting two kinds of hepatitis b virus marker HBs/HBe
Device.
Embodiment 3 is a kind of while detecting the preparation method of the sensor of two kinds of hepatitis b virus marker HBs/HBe, and step is such as
Under：
（1）By the glass-carbon electrode Al of a diameter of 5 mm2O3Polishing powder is polished, and ultra-pure water cleans up；
（2）Above-mentioned electrode is immersed into 10 mL, mass fraction is 1% HAuCl4In solution, under -0.2 V voltages, plating
30 s, dry at room temperature, with ultrapure water electrode surface, dry；
（3）Continue above-mentioned electrode immersing hepatitis b virus marker capture antibody A nti-HBs and Anti-HBe concentration difference
In mixed solution for 12 μ g/mL, hatch 12 h in 4 DEG C of refrigerators, takes out in 4 DEG C of refrigerators and dry；
（4）Continue the BSA solution of 3 μ L, 3 mg/mL being added drop-wise to electrode surface, to non-specific on enclosed-electrode surface
Property active site, ultrapure water electrode surface dry in 4 DEG C of refrigerators；
（5）HBs the and HBe antigens that above-mentioned electrode is immersed to a series of various concentrations of the ng/mL of 0.1 pg/mL ~ 50 again are mixed
Close 37 DEG C of 50 min of hatching in solution；It dries at room temperature, ultrapure water, it is dry in 4 DEG C of refrigerators；
（6）Finally by above-mentioned steps（5）The electrode of modification immerses the HBs detections of the graphene-supported golden hydridization platinum of stannic oxide
Antibody incubation content solution S nO2-GS@Au@Pt NPs-HBs-Ab2It is anti-with the HBe detections of the graphene-supported silver-colored hydridization gold of stannic oxide
Body incubation content solution S nO2-GS@ Ag-cys-Au NPs-HBe-Ab2Concentration be respectively 3 mg/mL mixed liquor in, at room temperature
Hatch 50 min, is placed in 4 DEG C of refrigerators dry, sensing that is obtained a kind of while detecting two kinds of hepatitis b virus marker HBs/HBe
Device.
Antibody incubation content solution S nO is detected described in embodiment 42-GS@Au@Pt NPs-HBs-Ab2Preparation, steps are as follows：
(1) prepared by Au@Pt NPs
It is 1% HAuCl by 1 mL, mass fraction4Solution is added in 100 mL ultra-pure waters, and 3 are rapidly joined under stirring
ML, the sodium citrate solution that mass fraction is 1%, are slowly added to NaBH4Become rufous to solution from faint yellow, is stirred overnight；
By 3 mL, the H that mass fraction is 1%2PtCl6The above-mentioned HAuCl boiled is added in solution4In solution, 2 are added
The ascorbic acid solution of mL, 0.1moL/L heat 30 min, and the solution of Au@Pt NPs is made；
(2) SnO2The preparation of-GS@Au@Pt NPs
0.1 mL、 0.5 mg/mL SnO212 h, centrifugation is mixed in the above-mentioned preparation Au@Pt NPs solution of-GS and 6 mL
Separation, washs drying, and product is dispersed in the SnO that 1 mg/mL is made in ultra-pure water again2- GS@Au@Pt NPs solution；
(3) detection antibody incubation content solution S nO2-GS@ Au@Pt NPs HBs-Ab2Preparation
By the HBs detection antibody As b of 1 mL, 10 μ g/mL2With the SnO of 1 mL, 1 mg/mL2- GS@Au@Pt NPs mixing
SnO is made in 4 DEG C of concussion incubated overnights, centrifuge washing2-GS@Au@Pt NPs-HBs-Ab2Detect antibody incubation content；With pH 7.4
PBS buffer solutions disperse again, the detection antibody incubation content solution S nO of 1 mg/mL is made2-GS@ Au@Pt NPs HBs-
Ab2It is spare.
Antibody incubation content solution S nO is detected described in embodiment 52-GS@Au@Pt NPs-HBs-Ab2Preparation, steps are as follows：
(1) prepared by Au@Pt NPs
It is 1% HAuCl by 2 mL, mass fraction4Solution is added in 100 mL ultra-pure waters, and 4.5 are rapidly joined under stirring
ML, the sodium citrate solution that mass fraction is 1%, are slowly added to NaBH4Become rufous to solution from faint yellow, is stirred overnight；
By 5 mL, the H that mass fraction is 1%2PtCl6The above-mentioned HAuCl boiled is added in solution4In solution, 3 are added
The ascorbic acid solution of mL, 0.1moL/L heat 30 min, and the solution of Au@Pt NPs is made；
(2) SnO2The preparation of-GS@Au@Pt NPs
0.2 mL、 0.5 mg/mL SnO212 h, centrifugation is mixed in the above-mentioned preparation Au@Pt NPs solution of-GS and 6 mL
Separation, washs drying, and product is dispersed in the SnO that 2 mg/mL are made in ultra-pure water again2- GS@Au@Pt NPs solution；
(3) detection antibody incubation content solution S nO2-GS@ Au@Pt NPs HBs-Ab2Preparation
By the HBs detection antibody As b of 2 mL, 10 μ g/mL2With the SnO of 1 mL, 2 mg/mL2- GS@Au@Pt NPs mixing
SnO is made in 4 DEG C of concussion incubated overnights, centrifuge washing2-GS@Au@Pt NPs-HBs-Ab2Detect antibody incubation content；With pH 7.4
PBS buffer solutions disperse again, the detection antibody incubation content solution S nO of 2 mg/mL is made2-GS@ Au@Pt NPs HBs-
Ab2It is spare.
Antibody incubation content solution S nO is detected described in embodiment 62-GS@Au@Pt NPs-HBs-Ab2Preparation, step is such as
Under：
(1) prepared by Au@Pt NPs
It is 1% HAuCl by 3 mL, mass fraction4Solution is added in 100 mL ultra-pure waters, rapidly joined under stirring 6mL,
The sodium citrate solution that mass fraction is 1%, is slowly added to NaBH4Become rufous to solution from faint yellow, is stirred overnight；
By 7 mL, the H that mass fraction is 1%2PtCl6The above-mentioned HAuCl boiled is added in solution4In solution, 4 are added
The ascorbic acid solution of mL, 0.1moL/L heat 30 min, and the solution of Au@Pt NPs is made；
(2) SnO2The preparation of-GS@Au@Pt NPs
0.3 mL、 0.5 mg/mL SnO212 h, centrifugation is mixed in the above-mentioned preparation Au@Pt NPs solution of-GS and 6 mL
Separation, washs drying, and product is dispersed in the SnO that 3 mg/mL are made in ultra-pure water again2- GS@Au@Pt NPs solution；
(3) detection antibody incubation content solution S nO2-GS@ Au@Pt NPs HBs-Ab2Preparation
By the HBs detection antibody As b of 3 mL, 10 μ g/mL2With the SnO of 1 mL, 3 mg/mL2- GS@Au@Pt NPs mixing
SnO is made in 4 DEG C of concussion incubated overnights, centrifuge washing2-GS@Au@Pt NPs-HBs-Ab2Detect antibody incubation content；With pH 7.4
PBS buffer solutions disperse again, the detection antibody incubation content solution S nO of 3 mg/mL is made2-GS@ Au@Pt NPs HBs-
Ab2It is spare.
Antibody incubation content solution S nO is detected described in embodiment 72-GS@ Ag-cys-Au NPs-HBe-Ab2Preparation, step
It is as follows：
(1) preparation of Ag-cys-Au nanospheres
1) preparation of L-cys@Au NPs
20 mL, 2 mmoL/L L-cysteine L-cys solution in, sequentially add while stirring 250 μ L,
The HAuCl of 0.05moL/L4Solution, 350 μ L, 0.01 moL/L NaBH4, 2 h are stirred at 300k, are centrifuged, and are surpassed
Pure water is dried in vacuo 12h at room temperature, and L-cys@Au NPs are made, spare；
2) preparation of Ag-cys-Au nanospheres
10 mL, 0.1 mg/mL L-cys@Au NPs in, sequentially add 1 mL, mass fraction is 1% polyvinyl pyrrole
The ascorbic acid of alkanone PVP, 1 mL, 0.1 moL/L, 1 mL, the AgNO of 0.1 moL/L3Solution stirs 30 min at room temperature,
It centrifuges, ultra-pure water cleaning is dried in vacuo Ag-cys-Au nanospheres are made at room temperature；
(2) SnO2The preparation of-GS@Ag-cys-Au NPs
0.1 mL、0.5 mg/mL SnO2The Ag-cys-Au nanosphere solution mixing of-GS and 6 mL, 1 mg/mL
12 h are centrifuged, and wash drying, and product is dispersed in the SnO that 1 ~ 3 mg/mL is made in ultra-pure water again2-GS@ Ag-
Cys-Au NPs solution；
(3) detection antibody incubation content solution S nO2-GS@ Ag-cys-Au NPs-HBe-Ab2Preparation
By the HBe detection antibody As b of 1 mL, 10 μ g/mL2With the SnO of 1 mL, 1 mg/mL2-GS@ Ag-cys-Au NPs
4 DEG C of concussion incubated overnights of mixing, centrifuge washing；SnO is made2-GS@ Ag-cys-Au NPs-HBe-Ab2Capture antibody incubation content；
Disperseed again with the PBS buffer solutions of pH 7.4, the SnO of 1 mg/mL is made2-GS@ Ag-cys-Au NPs-HBe-Ab2Detection
Antibody incubation content solution for standby.
Antibody incubation content solution S nO is detected described in embodiment 82-GS@ Ag-cys-Au NPs-HBe-Ab2Preparation, step
It is as follows：
(1) preparation of Ag-cys-Au nanospheres
1) preparation of L-cys@Au NPs
25 mL, 2 mmoL/L L-cysteine L-cys solution in, sequentially add while stirring 250 μ L,
The HAuCl of 0.05moL/L4Solution, 350 μ L, 0.01 moL/L NaBH4, 2 h are stirred at 300k, are centrifuged, and are surpassed
Pure water is dried in vacuo 12h at room temperature, and L-cys@Au NPs are made, spare；
2) preparation of Ag-cys-Au nanospheres
12.5 mL, 0.1 mg/mL L-cys@Au NPs in, sequentially add 1 mL, mass fraction is 1% polyethylene pyrrole
The ascorbic acid of pyrrolidone PVP, 1 mL, 0.1 moL/L, 1 mL, the AgNO of 0.1 moL/L3Solution stirs 35 at room temperature
Min is centrifuged, and ultra-pure water cleaning is dried in vacuo Ag-cys-Au nanospheres are made at room temperature；
(2) SnO2The preparation of-GS@Ag-cys-Au NPs
0.2 mL、0.5 mg/mL SnO2The Ag-cys-Au nanosphere solution mixing of-GS and 6 mL, 2 mg/mL
12 h are centrifuged, and wash drying, and product is dispersed in the SnO that 2 mg/mL are made in ultra-pure water again2-GS@ Ag-cys-Au
NPs solution；
(3) detection antibody incubation content solution S nO2-GS@ Ag-cys-Au NPs-HBe-Ab2Preparation
By the HBe detection antibody As b of 2 mL, 10 μ g/mL2With the SnO of 1 mL, 2 mg/mL2-GS@ Ag-cys-Au NPs
4 DEG C of concussion incubated overnights of mixing, centrifuge washing；SnO is made2-GS@ Ag-cys-Au NPs-HBe-Ab2Capture antibody incubation content；
Disperseed again with the PBS buffer solutions of pH 7.4, the SnO of 2 mg/mL is made2-GS@ Ag-cys-Au NPs-HBe-Ab2Detection
Antibody incubation content solution for standby.
Antibody incubation content solution S nO is detected described in embodiment 92-GS@ Ag-cys-Au NPs-HBe-Ab2Preparation, step
It is as follows：
(1) preparation of Ag-cys-Au nanospheres
1) preparation of L-cys@Au NPs
30 mL, 2 mmoL/L L-cysteine L-cys solution in, sequentially add while stirring 250 μ L,
The HAuCl of 0.05moL/L4Solution, 350 μ L, 0.01 moL/L NaBH4, 2 h are stirred at 300k, are centrifuged, and are surpassed
Pure water is dried in vacuo 12h at room temperature, and L-cys@Au NPs are made, spare；
2) preparation of Ag-cys-Au nanospheres
15 mL, 0.1 mg/mL L-cys@Au NPs in, sequentially add 1 mL, mass fraction is 1% polyvinyl pyrrole
The ascorbic acid of alkanone PVP, 1 mL, 0.1 moL/L, 1 mL, the AgNO of 0.1 moL/L3Solution stirs 40 min at room temperature,
It centrifuges, ultra-pure water cleaning is dried in vacuo Ag-cys-Au nanospheres are made at room temperature；
(2) SnO2The preparation of-GS@Ag-cys-Au NPs
0.3 mL、0.5 mg/mL SnO2The Ag-cys-Au nanosphere solution mixing of-GS and 6 mL, 3 mg/mL
12 h are centrifuged, and wash drying, and product is dispersed in the SnO that 3 mg/mL are made in ultra-pure water again2-GS@ Ag-cys-Au
NPs solution；
(3) detection antibody incubation content solution S nO2-GS@ Ag-cys-Au NPs-HBe-Ab2Preparation
By the HBe detection antibody As b of 3 mL, 10 μ g/mL2With the SnO of 1 mL, 3 mg/mL2-GS@ Ag-cys-Au NPs
4 DEG C of concussion incubated overnights of mixing, centrifuge washing；SnO is made2-GS@ Ag-cys-Au NPs-HBe-Ab2Capture antibody incubation content；
Disperseed again with the PBS buffer solutions of pH 7.4, the SnO of 3 mg/mL is made2-GS@ Ag-cys-Au NPs-HBe-Ab2Detection
Antibody incubation content solution for standby.
It is detected while 10 hepatitis b virus marker HBs/HBe of embodiment, steps are as follows：
（1）It is tested with three-electrode system using electrochemical workstation, saturated calomel electrode is reference electrode, platinum filament electricity
Extremely auxiliary electrode, prepared sensor are working electrode, slow in 5.1 ~ 8.6 phosphate of pH of 10 mL, 50 mmol/L
It rushes in solution and is tested；
（2）Hepatitis b virus marker HBs/HBe is detected with chronoamperometry, input voltage is -0.4 V, sampling
It is spaced 0.1 s, 400 s of run time；
（3）After background current tends towards stability, the phosphate-buffereds of pH=7.4 every 50 s to 10 mL, 50 mmol/L are molten
The hydrogen peroxide solution of 10 μ L, 5 mol/L, record current variation are injected in liquid；
（4）Using calibration curve method, the range of linearity for measuring this method detection hepatitis b virus marker HBs and HBe is
The ng/mL of 0.1pg/mL ~ 50, detection are limited to 0.033pg/mL.
Claims (2)
1. the preparation method of sensor that is a kind of while detecting two kinds of hepatitis b virus marker HBs/HBe, which is characterized in that step
It is as follows：
（1）By the glass-carbon electrode Al of a diameter of 3 ~ 5 mm2O3Polishing powder is polished, and ultra-pure water cleans up；
（2）By above-mentioned steps（1）The electrode of modification immerses 10 mL, mass fraction is 1% HAuCl4In solution, in -0.2 V voltages
Under, 30 s are electroplated, dries, with ultrapure water electrode surface, dries at room temperature；
（3）Continue above-mentioned steps（2）The electrode of modification immerses hepatitis b virus marker capture antibody A nti-HBs and Anti-
HBe concentration is respectively to hatch 12 h in 4 DEG C of refrigerators in the mixed solution of 8 ~ 12 μ g/mL, takes out in 4 DEG C of refrigerators and dries；
（4）Continue the BSA solution of 3 μ L, 1 ~ 3 mg/mL being added drop-wise to above-mentioned steps（3）The electrode surface of modification, to seal
Close nonspecific activity site on electrode surface, ultrapure water electrode surface dries in 4 DEG C of refrigerators；
（5）Again by above-mentioned steps（4）The electrode of modification immerse a series of various concentrations of the ng/mL of 0.1 pg/mL ~ 50 HBs and
37 DEG C of 50 min of hatching in HBe antigen mixed solutions；It dries at room temperature, ultrapure water, it is dry in 4 DEG C of refrigerators；
（6）Finally by above-mentioned steps（5）The electrode of modification, which immerses the stannic oxide graphene that concentration is respectively 1 ~ 3 mg/mL, to be born
Carry the HBs detection antibody incubation content solution Ss nO of golden hydridization platinum2-GS@Au@Pt NPs-HBs-Ab2It is graphene-supported with stannic oxide
The HBe detection antibody incubation content solution Ss nO of silver-colored hydridization gold2-GS@ Ag-cys-Au NPs-HBe-Ab2Mixed liquor in, at room temperature
Hatch 50 min, is placed in 4 DEG C of refrigerators dry, sensing that is obtained a kind of while detecting two kinds of hepatitis b virus marker HBs/HBe
Device；
The detection antibody incubation content solution S nO2-GS@Au@Pt NPs-HBs-Ab2Preparation, which is characterized in that steps are as follows：
(1) prepared by Au@Pt NPs
It is 1% HAuCl by 1 ~ 3 mL, mass fraction4Solution is added in 100 mL ultra-pure waters, rapidly join 3 under stirring ~
6mL, the sodium citrate solution that mass fraction is 1%, are slowly added to NaBH4Become rufous to solution from faint yellow, it is stirred
Night；
By 3 ~ 7 mL, the H that mass fraction is 1%2PtCl6The above-mentioned HAuCl boiled is added in solution4In solution, 2 ~ 4 are added
The ascorbic acid solution of mL, 0.1moL/L heat 30 min, and the solution of Au@Pt NPs is made；
(2) SnO2The preparation of-GS@Au@Pt NPs
0.1 ~ 0.3 mL、 0.5 mg/mL SnO212 h are mixed in-GS and the Au@Pt NPs solution of the above-mentioned preparations of 6 mL,
It centrifuges, washs drying, product is dispersed in the SnO that 1 ~ 3 mg/mL is made in ultra-pure water again2- GS@Au@Pt NPs are molten
Liquid；
(3) detection antibody incubation content solution S nO2-GS@ Au@Pt NPs HBs-Ab2Preparation
By the HBs detection antibody As b of 1 ~ 3 mL, 10 μ g/mL2With the SnO of 1 mL, 1 ~ 3 mg/mL2-GS@ Au@Pt NPs
SnO is made in 4 DEG C of concussion incubated overnights of mixing, centrifuge washing2-GS@Au@Pt NPs-HBs-Ab2Detect antibody incubation content；Use pH
7.4 PBS buffer solutions disperse again, and the detection antibody incubation content solution S nO of 1 ~ 3 mg/mL is made2-GS@ Au@Pt
NPs HBs-Ab2It is spare；
The detection antibody incubation content solution S nO2-GS@ Ag-cys-Au NPs-HBe-Ab2Preparation, which is characterized in that step
It is as follows：
(1) preparation of Ag-cys-Au nanospheres
1) preparation of L-cys@Au NPs
20 ~ 30 mL, 2 mmoL/L L-cysteine L-cys solution in, sequentially add while stirring 250 μ L,
The HAuCl of 0.05moL/L4Solution, 350 μ L, 0.01 moL/L NaBH4, 2 h are stirred at 300k, are centrifuged, and are surpassed
Pure water is dried in vacuo 12h at room temperature, and L-cys@Au NPs are made, spare；
2) preparation of Ag-cys-Au nanospheres
10 ~ 15 mL, 0.1 mg/mL L-cys@Au NPs in, sequentially add 1 mL, mass fraction is 1% polyvinyl pyrrole
The ascorbic acid of alkanone PVP, 1 mL, 0.1 moL/L, 1 mL, the AgNO of 0.1 moL/L3Solution stirs 30 ~ 40 at room temperature
Min is centrifuged, and ultra-pure water cleaning is dried in vacuo Ag-cys-Au nanospheres are made at room temperature；
(2) SnO2The preparation of-GS@Ag-cys-Au NPs
0.1 ~ 0.3 mL、0.5 mg/mL SnO2- GS is mixed with the Ag-cys-Au nanosphere solution of 6 mL, 1 ~ 3 mg/mL
12 h are stirred, are centrifuged, drying is washed, product is dispersed in the SnO that 1 ~ 3 mg/mL is made in ultra-pure water again2-GS@
Ag-cys-Au NPs solution；
(3) detection antibody incubation content solution S nO2-GS@ Ag-cys-Au NPs-HBe-Ab2Preparation
By the HBe detection antibody As b of 1 ~ 3 mL, 10 μ g/mL2With the SnO of 1 mL, 1 ~ 3 mg/mL2-GS@ Ag-cys-Au
NPs mixes 4 DEG C of concussion incubated overnights, centrifuge washing；SnO is made2-GS@ Ag-cys-Au NPs-HBe-Ab2Capture antibody hatching
Object；Disperseed again with the PBS buffer solutions of pH 7.4, the SnO of 1 ~ 3 mg/mL is made2-GS@ Ag-cys-Au NPs-HBe-
Ab2Detect antibody incubation content solution for standby.
2. the preparation side of sensor that is as described in claim 1 a kind of while detecting two kinds of hepatitis b virus marker HBs/HBe
Immunosensor prepared by method, for being detected while hepatitis b virus marker HBs/HBe, detecting step is as follows：
（1）It is tested with three-electrode system using electrochemical workstation, saturated calomel electrode is reference electrode, and platinum electrode is
Auxiliary electrode, prepared sensor is working electrode, molten in 5.1 ~ 8.6 phosphate-buffereds of pH of 10 mL, 50 mmol/L
It is tested in liquid；
（2）Hepatitis b virus marker HBs/HBe is detected with chronoamperometry, input voltage is -0.4 V, sampling interval
0.1 s, 400 s of run time；
（3）After background current tends towards stability, every 50 s to 10 mL, 50 mmol/L the phosphate buffer solutions of pH=7.4 in
Inject the hydrogen peroxide solution of 10 μ L, 5 mol/L, record current variation.
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| CN106596942B (en) * | 2016-12-21 | 2018-08-28 | 山东理工大学 | A kind of construction method of interlayer type hepatitis b virus marker immunosensor and application |
| CN106770529B (en) * | 2017-02-13 | 2019-02-15 | 山东理工大学 | A kind of preparation method and application for the interlayer type immunosensor loading vulcanization copper nano-wire based on silver |
| CN107179342B (en) * | 2017-05-18 | 2019-04-02 | 山东理工大学 | A kind of preparation method and application of the Serum Markers of Hepatitis B Virus immunosensor based on GQDs-CuO@3D-rGO |
| CN107677716B (en) * | 2017-09-26 | 2019-08-06 | 山东理工大学 | A kind of preparation method and application of the immunosensor based on AuPdCu/N-GQDs@PS |
| CN108051491B (en) * | 2017-12-18 | 2019-10-18 | 重庆医科大学 | It is a kind of for detecting the electrochemical immunosensor of LAG-3 albumen |
| CN109342745A (en) * | 2018-12-21 | 2019-02-15 | 山东理工大学 | A kind of construction method of the interlayer type electrochemical immunosensor based on PdCu@GO and application |
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