CN106596942A - Construction method and application of sandwich-type hepatitis B virus marker immunosensor - Google Patents
Construction method and application of sandwich-type hepatitis B virus marker immunosensor Download PDFInfo
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Abstract
The invention belongs to the technical field of novel nano composite materials, immunoassay and biological sensing, and relates to a construction method and application of a sandwich-type hepatitis B virus marker immunosensor. An electrochemical immunosensor constructed from a platinum nanowire@SBA-15 microsphere composite material capable of adsorbing thionine is used for quantitatively detecting a hepatitis B virus marker, has the advantages of high specificity, high sensitivity and low detection limit, and has important scientific significance and application values for early diagnosis of hepatitis B virus infection.
Description
Technical field
The invention belongs to novel nanocomposite materials, immunoassay and biosensor technique field, there is provided a kind of sandwich
The construction method of type hepatitis b virus marker immunosensor, is applied to the detection of hepatitis b virus marker.
Background technology
Hepatitis b virus infected is one of health problem of global most serious, and it can cause chronic hepatitiss, liver cirrhosis etc.
Disease, or even cause the hepatocarcinoma for occupying global cancer mortality second place.In blood, the concentration of Markers of HBV is
The standard of diagnosis of hepatitis b virus infection, the accurate detection of hepatitis b virus marker concentration is to hepatitis b virus infected
Early diagnosiss it is significant.Therefore, develop the quantitative detecting method of high-sensitive hepatitis b virus marker particularly
Urgently.
In recent years, developing rapidly with clinical diagnosis technology, the electrochemical immunosensor of superior performance are shown one's talent,
And be widely used in the detection of virus marker thing or tumor markerses.Interlayer type electrochemical immunosensor is based on antigen
A kind of analysis method combined with antibody specificity, with detection it is rapid, detection limit is low, sensitivity is high, simple to operate and prepare
The advantage of low cost, the detection to trace level virus and tumor markerses have important value.
The important component of base material and catalyst material as electrochemical immunosensor, to improving immunosensor
Sensitivity has important function.In recent years, nano material and its composite be widely used in immunosensor structure work as
In.The present invention utilizes layer-by-layer, the double layer gold built with deposited Au thin film and golden nanometer particle as substrate, with suction
The Pt nanowires@SBA-15 microspheres of attached sulfur a beautiful gem build a kind of sandwich type hepatitis b virus marker as detection antibody label
Immunosensor, has the advantages that detection range is wide, Monitoring lower-cut is low, simple to operate, detection speed is fast, the morning to hepatitis B
Phase, diagnosis was with important using value.
The content of the invention
The invention provides a kind of construction method of sandwich type hepatitis b virus marker immunosensor and application, real
The super sensitivity detection to hepatitis b virus marker is showed.
An object of the present invention is to provide a kind of structure side of sandwich type hepatitis b virus marker immunosensor
Method.
The second object of the present invention is to use a kind of prepared sandwich type hepatitis b virus marker immunosensor
In the detection of hepatitis b virus marker.
Technical scheme, comprises the following steps.
1. a kind of construction method of sandwich type hepatitis b virus marker immunosensor, step are as follows：
（1）By the glass-carbon electrode Al of a diameter of 3.0 ~ 5.0 mm2O3Polishing powder is polished to minute surface, the ultrasound in dehydrated alcohol
Clean up；
（2）Using chronoamperometry, under -0.2 V, polished glass-carbon electrode is placed in into 5.0 mL, 1.0 wt% ~ 2.0
In the chlorauric acid solution of wt%, 25 ~ 35 s are electroplated, deposited Au thin film is formed in electrode surface, with ultrapure water electrode table
Dry under face, room temperature；
（3）By above-mentioned electrode immerse 30 mL, 1.0 mmol/L 4- aminothiophenol solution, soak 2.0 h, ultrapure water,
Dry under room temperature；
（4）Continue electrode is immersed the golden nanometer particle dispersion liquid of 50 mL, 0.04 ~ 0.05 mg/mL, soak 2.0 h, it is ultrapure
Water is rinsed, and is dried under room temperature；
（5）By hepatitis b virus marker capture antibody A b of 6.0 L, 5.0 ~ 10.0 g/mL1It is added drop-wise to electrode table
Face, dries in 4.0 °C of refrigerators；
（6）The bovine serum albumen solution of 3.0 L, 0.5 wt% ~ 1.0 wt% is added drop-wise to electrode surface by continuation, to close
Electrode is rinsed in nonspecific activity site on electrode surface, pH=7.0 phosphate buffers, is dried in 4.0 °C of refrigerators；
（7）Continue 6.0 L of Deca, a series of hepatitis b virus marker of variable concentrations of 0.00001 ~ 100 ng/mL
Antigenic solution, dries in 4.0 °C of refrigerators；
（8）By 6.0 L, Pt nanowires@SBA-15 microspheres/detection antibody Ab of the absorption sulfur a beautiful gem of 1.0 ~ 3.0 mg/mL2Incubate
Compound dispersant liquid drop is applied to electrode surface, is placed in 4.0 °C of refrigerators, stands 40 min, is rushed with the phosphate buffer of pH=7.0
Wash, dry in 4.0 °C of refrigerators, a kind of sandwich type hepatitis b virus marker immunosensor is obtained.
2. a kind of construction method of sandwich type hepatitis b virus marker immunosensor, the system of the associated materials
Standby, step is as follows：
（1）The preparation of golden nanometer particle dispersion liquid
The chlorauric acid solution of 1.0 ~ 2.0 mL, 1.0 wt% is added in 99.0 mL ultra-pure waters, boiling is heated to, is added
The sodium citrate solution of 1.5 ~ 3.5 mL, 1.0 wt%, continue backflow 10 ~ 20 min, after be cooled to room temperature, obtain Jenner
Rice corpuscles dispersion liquid；
（2）Pt nanowires@SBA-15 microspheres/detection antibody HBs-Ab of absorption sulfur a beautiful gem2The preparation of incubation content dispersion liquid
1. the preparation of Pt nanowires@SBA-15 microspheres
Take 1.0 ~ 1.5 mL, the chloroplatinous acid potassium solution of 12 wt% is placed in 100 mL beakers, inserts 0.4 ~ 0.6 g's
SBA-15 powder, stirs, and after 15 ~ 20 min of ultrasound, is placed in 25 °C of vacuum drying ovens and is dried 24 h, continuously add
After 2.0 ~ 3.0 mL, the ascorbic acid solution of 1.0 mol/mL, 1.0 h of reduction in 25 °C of vacuum drying ovens are placed in, continue to add
Enter 5.0 ~ 7.0 mL, the hydrofluoric acid solution of 10 wt%, after persistently stirring 20 ~ 40 min, centrifugation, with milli-Q water, is incited somebody to action
Resulting solid is placed in 30 °C of vacuum drying ovens and is dried 24 h, obtains Pt nanowires@SBA-15 microspheres；
2. adsorb the preparation of the Pt nanowires@SBA-15 microspheres of sulfur a beautiful gem
Weigh the sulfur a beautiful gem solution that 40 ~ 50 mg Pt nanowires@SBA-15 microspheres are immersed in 10 mL, 1.0 ~ 1.5 mol/mL
In, ultrasonic disperse, and in an oscillator after 6.0 h of persistent oscillation, centrifugation, with milli-Q water, resulting solid is placed in
24 h are dried in 30 °C of vacuum drying ovens, obtain adsorbing the Pt nanowires@SBA-15 microspheres of sulfur a beautiful gem；
3. adsorb Pt nanowires@SBA-15 microspheres/detection antibody HBs-Ab of sulfur a beautiful gem2The preparation of incubation content dispersion liquid
Take 2.0 ~ 6.0 mg absorption sulfur a beautiful gem Pt nanowires@SBA-15 microspheres be distributed in 1.0 mL ultra-pure waters, add 1.0 ~
Hepatitis b virus marker detection antibody Ab of 2.0 mL, 20 ~ 30 μ g/mL2Dispersion liquid, is placed in 4.0 °C of constant temperature oscillations
In incubator after 6.0 ~ 8.0 h of vibration hatching, 5.0 ~ 10.0 min are centrifuged under 5000 rpm rotating speeds, lower sediment are taken and is added
Enter 1.0 mL, pH=7.0 phosphate buffered solution centrifuge washing 1 time, take lower sediment and continuously add 1.0 mL, pH=7.0 phosphoric acid
Salt buffer solution, is uniformly dispersed, and obtains adsorbing Pt nanowires@SBA-15 microspheres/detection antibody Ab of sulfur a beautiful gem2Incubation content is disperseed
Liquid, preserves under 4.0 °C.
3. a kind of sandwich type hepatitis b virus marker immunosensor, for hepatitis b virus marker antigen
Detection, step is as follows：
（1）Using electrochemical workstation, tested under three-electrode system, with saturated calomel electrode as reference electrode, with platinum
Silk electrode is, to electrode, with prepared immunosensor as working electrode, to include 5.0 mmol/L hydrogen peroxide in 10 mL molten
Tested in liquid, 5.3 ~ 8.0 phosphate buffered solution of pH of 2.0 mmol/L o-phenylenediamine solutions；
（2）Hepatitis b virus marker antigen is detected using differential pulse voltammetry, sweep limitss are 0.0 ~ 0.6
V, pulse amplitude be 50 mV, pulse width be 50 ms, the pulse period be 50 ms, record current peak value；
（3）The current peak corresponding to hepatitis b virus marker antigen under record variable concentrations；
（4）Using working curve method, the concentration of hepatitis b virus marker antigen in testing sample is obtained.
Raw material used by the present invention can be bought in chemical reagents corporation or biopharmaceutical company.
The useful achievement of the present invention
（1）The present invention utilizes double layer gold that deposited Au thin film and golden nanometer particle build be effectively increased the electricity of electrode surface
Sub- transmission efficiency, also, as golden nanometer particle has good biocompatibility, enable double layer gold stable bonds of structure
Substantial amounts of active capture antibody is so as to increase the stability of immunosensor, sensitive for raising immunosensor
Degree is with important function；
（2）The present invention is using the Pt nanowires@SBA-15 microspheres of the absorption sulfur a beautiful gem of function admirable by as detection antibody label
For in the structure of immunosensor.After the splendid Pt nanowires of catalytic performance and electric conductivity are mounted to the inside of SBA-15,
The electric conductivity of SBA-15 is effectively enhanced, and ordered mesopore structure flourishing in SBA-15 can provide substantial amounts of catalytic active site
Point, and the time of contact of reactant and Pt nanowires@SBA-15 microspheres can be effectively increased, it is mutual by this synergism and advantage
Benefit is acted on, and increases the sensitivity of immunosensor.The absorption of sulfur a beautiful gem makes the electric conductivity of SBA-15 microspheres further increase.Additionally,
There is in sulfur a beautiful gem amido functional group, make the Pt nanowires@SBA-15 microspheres of absorption sulfur a beautiful gem that there is more preferable biocompatibility, improve
The load capacity of detection antibody.Therefore, effectively put using the Pt nanowires@SBA-15 microspheres that adsorb sulfur a beautiful gem as detection antibody label
The big signal of telecommunication, improves the sensitivity of immunosensor, reduces the Monitoring lower-cut of immunosensor；
（3）A kind of detection of sandwich type hepatitis b virus marker immunosensor to different hepatitis b virus markers,
Its linear detection range to hepatitis B virus surface antigen HBs-Ag is 0.00001 ng/mL ~ 100 ng/mL, minimum
Monitoring lower-cut is 3.3 fg/mL；To the linear detection range of hepatitis B virus e antigen HBe-Ag be 0.0005 ng/mL ~
50 ng/mL, lowest detection lower limit are 0.167 pg/mL；Linear detection range to hepatitis B virus core antigen HBc-Ag
It is 0.00003 ng/mL ~ 60 ng/mL, lowest detection lower limit is 10 fg/mL；Show a kind of sandwich type hepatitis B viruss
Marker immunosensor can reach the purpose that accurate quantification detects hepatitis b virus marker.
Specific embodiment
Now the present invention is further illustrated by specific embodiment, but not limited to this.
A kind of construction method of 1 sandwich type hepatitis b virus marker immunosensor of embodiment
（1）By the glass-carbon electrode Al of a diameter of 4.0 mm2O3Polishing powder is polished to minute surface, is cleaned by ultrasonic dry in dehydrated alcohol
Only；
（2）Using chronoamperometry, under -0.2 V, polished glass-carbon electrode is placed in into the gold chloride of 5.0 mL, 1.0 wt%
In solution, 25 s are electroplated, deposited Au thin film is formed in electrode surface, use and dry under ultrapure water electrode surface, room temperature；
（3）By above-mentioned electrode immerse 30 mL, 1.0 mmol/L 4- aminothiophenol solution, soak 2.0 h, ultrapure water,
Dry under room temperature；
（4）Continue electrode is immersed the golden nanometer particle dispersion liquid of 50 mL, 0.04 mg/mL, 2.0 h of immersion, ultrapure water,
Dry under room temperature；
（5）By hepatitis b virus marker capture antibody A b of 6.0 L, 5.0 g/mL1It is added drop-wise to electrode surface, 4.0 °C
Dry in refrigerator；
（6）The bovine serum albumen solution of 3.0 L, 0.5 wt% is added drop-wise to electrode surface by continuation, on enclosed-electrode surface
Electric face is rinsed in nonspecific activity site, pH=7.0 phosphate buffers, dries in 4.0 °C of refrigerators；
（7）Continue 6.0 L of Deca, a series of hepatitis b virus marker of variable concentrations of 0.00001 ~ 100 ng/mL
Antigenic solution, dries in 4 °C of refrigerators；
（8）By 6.0 L, Pt nanowires@SBA-15 microspheres/detection antibody Ab of the absorption sulfur a beautiful gem of 1.0 mg/mL2Incubation content point
Dispersion liquid drop coating is placed in 4.0 °C of refrigerators in electrode surface, stands 40 min, is rinsed with the phosphate buffer of pH=7.0,4.0
Dry in °C refrigerator, a kind of sandwich type hepatitis b virus marker immunosensor is obtained.
A kind of construction method of 2 sandwich type hepatitis b virus marker immunosensor of embodiment
（1）By the glass-carbon electrode Al of a diameter of 4.0 mm2O3Polishing powder is polished to minute surface, is cleaned by ultrasonic dry in dehydrated alcohol
Only；
（2）Using chronoamperometry, under -0.2 V, polished glass-carbon electrode is placed in into the gold chloride of 5.0 mL, 1.5 wt%
In solution, 30 s are electroplated, deposited Au thin film is formed in electrode surface, use and dry under ultrapure water electrode surface, room temperature；
（3）By above-mentioned electrode immerse 30 mL, 1.0 mmol/L 4- aminothiophenol solution, soak 2.0 h, ultrapure water,
Dry under room temperature；
（4）Continue electrode is immersed the golden nanometer particle dispersion liquid of 50 mL, 0.05 mg/mL, 2.0 h of immersion, ultrapure water,
Dry under room temperature；
（5）By hepatitis b virus marker capture antibody A b of 6.0 L, 7.5 g/mL1It is added drop-wise to electrode surface, 4.0 °C
Dry in refrigerator；
（6）The bovine serum albumen solution of 3.0 L, 0.75 wt% is added drop-wise to electrode surface by continuation, on enclosed-electrode surface
Electrode is rinsed in nonspecific activity site, pH=7.0 phosphate buffers, is dried in 4.0 °C of refrigerators；
（7）Continue 6.0 L of Deca, a series of hepatitis b virus marker of variable concentrations of 0.00001 ~ 100 ng/mL
Antigenic solution, dries in 4 °C of refrigerators；
（8）By 6.0 L, Pt nanowires@SBA-15 microspheres/detection antibody Ab of the absorption sulfur a beautiful gem of 2.0 mg/mL2Incubation content point
Dispersion liquid drop coating is placed in 4.0 °C of refrigerators in electrode surface, stands 40 min, is rinsed with the phosphate buffer of pH=7.0,4.0
Dry in °C refrigerator, a kind of sandwich type hepatitis b virus marker immunosensor is obtained.
A kind of construction method of 3 sandwich type hepatitis b virus marker immunosensor of embodiment
（1）By the glass-carbon electrode Al of a diameter of 4.0 mm2O3Polishing powder is polished to minute surface, is cleaned by ultrasonic dry in dehydrated alcohol
Only；
（2）Using chronoamperometry, under -0.2 V, polished glass-carbon electrode is placed in into the gold chloride of 5.0 mL, 2.0 wt%
In solution, 35 s are electroplated, deposited Au thin film is formed in electrode surface, use and dry under ultrapure water electrode surface, room temperature；
（3）By above-mentioned electrode immerse 30 mL, 1.0 mmol/L 4- aminothiophenol solution, soak 2.0 h, ultrapure water,
Dry under room temperature；
（4）Continue electrode is immersed the golden nanometer particle dispersion liquid of 50 mL, 0.05 mg/mL, 2.0 h of immersion, ultrapure water,
Dry under room temperature；
（5）By hepatitis b virus marker capture antibody A b of 6.0 L, 10.0 g/mL1It is added drop-wise to electrode surface, 4.0 °
Dry in C refrigerators；
（6）The bovine serum albumen solution of 3.0 L, 1.0 wt% is added drop-wise to electrode surface by continuation, on enclosed-electrode surface
Electrode is rinsed in nonspecific activity site, pH=7.0 phosphate buffers, is dried in 4.0 °C of refrigerators；
（7）Continue 6.0 L of Deca, a series of hepatitis b virus marker of variable concentrations of 0.00001 ~ 100 ng/mL
Antigenic solution, dries in 4 °C of refrigerators；
（8）By 6.0 L, Pt nanowires@SBA-15 microspheres/detection antibody Ab of the absorption sulfur a beautiful gem of 3.0 mg/mL2Incubation content point
Dispersion liquid drop coating is placed in 4.0 °C of refrigerators in electrode surface, stands 40 min, is rinsed with the phosphate buffer of pH=7.0,4.0
Dry in °C refrigerator, a kind of sandwich type hepatitis b virus marker immunosensor is obtained.
The preparation of golden nanometer particle dispersion liquid described in embodiment 4
The chlorauric acid solution of 1.0 mL, 1.0 wt% is added in 99.0 mL ultra-pure waters, is heated to seething with excitement, 1.5 mL of addition,
The sodium citrate solution of 1.0 wt%, continue backflow 10 min, after be cooled to room temperature, obtain golden nanometer particle dispersion liquid.
The preparation of golden nanometer particle dispersion liquid described in embodiment 5
The chlorauric acid solution of 1.5 mL, 1.0 wt% is added in 99.0 mL ultra-pure waters, is heated to seething with excitement, 2.5 mL of addition,
The sodium citrate solution of 1.0 wt%, continue backflow 15 min, after be cooled to room temperature, obtain golden nanometer particle dispersion liquid.
The preparation of golden nanometer particle dispersion liquid described in embodiment 6
The chlorauric acid solution of 2.0 mL, 1.0 wt% is added in 99.0 mL ultra-pure waters, is heated to seething with excitement, 3.5 mL of addition,
The sodium citrate solution of 1.0 wt%, continue backflow 20 min, after be cooled to room temperature, obtain golden nanometer particle dispersion liquid.
Adsorb Pt nanowires@SBA-15 microspheres/detection antibody Ab of sulfur a beautiful gem described in embodiment 72The system of incubation content dispersion liquid
It is standby
1. the preparation of Pt nanowires@SBA-15 microspheres
Take 1.0 mL, the chloroplatinous acid potassium solution of 12 wt% is placed in 100 mL beakers, insert the SBA-15 powder of 0.4 g, stir
Mix uniform, after ultrasonic 15 min, be placed in 25 °C of vacuum drying ovens and be dried 24 h, continuously add 2.0 mL, 1.0 mol/mL
After ascorbic acid solution, be placed in 1.0 h of reduction in 25 °C of vacuum drying ovens, continuously add 5.0 mL, 10 wt% Fluohydric acid. it is molten
Liquid, after persistently stirring 20 min, centrifugation, with milli-Q water, resulting solid is placed in 30 °C of vacuum drying ovens and is dried 24
H, obtains Pt nanowires@SBA-15 microspheres；
2. adsorb the preparation of the Pt nanowires@SBA-15 microspheres of sulfur a beautiful gem
Weigh 40 mg Pt nanowires@SBA-15 microspheres to be immersed in the sulfur a beautiful gem solution of 10 mL, 1.0 mol/mL, ultrasonic disperse,
And in an oscillator after 6.0 h of persistent oscillation, resulting solid, with milli-Q water, is placed in 30 °C of vacuum drying by centrifugation
24 h are dried in case, obtain adsorbing the Pt nanowires@SBA-15 microspheres of sulfur a beautiful gem；
3. adsorb Pt nanowires@SBA-15 microspheres/detection antibody Ab of sulfur a beautiful gem2The preparation of incubation content dispersion liquid
The Pt nanowires@SBA-15 microspheres for taking 2.0 mg absorption sulfur a beautiful gems are distributed in 1.0 mL ultra-pure waters, add 1.0 mL, 20 μ
Hepatitis b virus marker detection antibody Ab of g/mL2Dispersion liquid, is placed in vibration hatching in 4.0 °C of constant-temperature shaking incubators
After 6.0 h, 5.0 min are centrifuged under 5000 rpm rotating speeds, take lower sediment and add 1.0 mL, pH=7.0 phosphate buffered solution
Centrifuge washing 1 time, takes lower sediment, continuously adds 1.0 mL, pH=7.0 phosphate buffered solution, is uniformly dispersed, is adsorbed
Pt nanowires@SBA-15 microspheres/detection antibody Ab of sulfur a beautiful gem2Incubation content dispersion liquid, preserves under 4.0 °C.
Adsorb Pt nanowires@SBA-15 microspheres/detection antibody Ab of sulfur a beautiful gem described in embodiment 82The system of incubation content dispersion liquid
It is standby
1. the preparation of Pt nanowires@SBA-15 microspheres
Take 1.5 mL, the chloroplatinous acid potassium solution of 12 wt% is placed in 100 mL beakers, insert the SBA-15 powder of 0.5 g, stir
Mix uniform, after ultrasonic 20 min, be placed in 25 °C of vacuum drying ovens and be dried 24 h, continuously add 2.5 mL, 1.0 mol/mL
After ascorbic acid solution, be placed in 1.0 h of reduction in 25 °C of vacuum drying ovens, continuously add 6.0 mL, 10 wt% Fluohydric acid. it is molten
Liquid, after persistently stirring 30 min, centrifugation, with milli-Q water, resulting solid is placed in 30 °C of vacuum drying ovens and is dried 24
H, obtains Pt nanowires@SBA-15 microspheres；
2. adsorb the preparation of the Pt nanowires@SBA-15 microspheres of sulfur a beautiful gem
Weigh 45 mg Pt nanowires@SBA-15 microspheres to be immersed in the sulfur a beautiful gem solution of 10 mL, 1.5 mol/mL, ultrasonic disperse,
And in an oscillator after 6.0 h of persistent oscillation, resulting solid, with milli-Q water, is placed in 30 °C of vacuum drying by centrifugation
24 h are dried in case, obtain adsorbing the Pt nanowires@SBA-15 microspheres of sulfur a beautiful gem；
3. adsorb Pt nanowires@SBA-15 microspheres/detection antibody Ab of sulfur a beautiful gem2The preparation of incubation content dispersion liquid
The Pt nanowires@SBA-15 microspheres for taking 4.0 mg absorption sulfur a beautiful gems are distributed in 1.0 mL ultra-pure waters, add 1.5 mL, 25 μ
Hepatitis b virus marker detection antibody Ab of g/mL2Dispersion liquid, is placed in vibration hatching in 4.0 °C of constant-temperature shaking incubators
After 7.0 h, 5.0 min are centrifuged under 5000 rpm rotating speeds, obtain lower sediment, add 1.0 mL, pH=7.0 phosphate-buffered
Solution centrifugal is washed 1 time, obtains lower sediment, is added 1.0 mL, pH=7.0 phosphate buffered solution, is uniformly dispersed, is adsorbed
Pt nanowires@SBA-15 microspheres/detection antibody Ab of sulfur a beautiful gem2Incubation content dispersion liquid, preserves under 4.0 °C.
Adsorb Pt nanowires@SBA-15 microspheres/detection antibody Ab of sulfur a beautiful gem described in embodiment 92The system of incubation content dispersion liquid
It is standby
1. the preparation of Pt nanowires@SBA-15 microspheres
Take 1.5 mL, the chloroplatinous acid potassium solution of 12 wt% is placed in 100 mL beakers, insert the SBA-15 powder of 0.6 g, stir
Mix uniform, after ultrasonic 20 min, be placed in 25 °C of vacuum drying ovens and be dried 24 h, continuously add 3.0 mL, 1.0 mol/mL
After ascorbic acid solution, be placed in 1.0 h of reduction in 25 °C of vacuum drying ovens, continuously add 7.0 mL, 10 wt% Fluohydric acid. it is molten
Liquid, after persistently stirring 40 min, centrifugation, with milli-Q water, resulting solid is placed in 30 °C of vacuum drying ovens and is dried
24 h, obtain Pt nanowires@SBA-15 microspheres；
2. adsorb the preparation of the Pt nanowires@SBA-15 microspheres of sulfur a beautiful gem
Weigh 50 mg Pt nanowires@SBA-15 microspheres to be immersed in the sulfur a beautiful gem solution of 10 mL, 1.5 mol/mL, ultrasonic disperse,
And in an oscillator after 6.0 h of persistent oscillation, resulting solid, with milli-Q water, is placed in 30 °C of vacuum drying by centrifugation
24 h are dried in case, obtain adsorbing the Pt nanowires@SBA-15 microspheres of sulfur a beautiful gem；
3. adsorb Pt nanowires@SBA-15 microspheres/detection antibody Ab of sulfur a beautiful gem2The preparation of incubation content dispersion liquid
The Pt nanowires@SBA-15 microspheres for taking 6.0 mg absorption sulfur a beautiful gems are distributed in 1.0 mL ultra-pure waters, add 2.0 mL, 30 μ
Hepatitis b virus marker detection antibody Ab of g/mL2Dispersion liquid, is placed in vibration hatching in 4.0 °C of constant-temperature shaking incubators
After 8.0 h, 10 min are centrifuged under 5000 rpm rotating speeds, take lower sediment and add 1.0 mL, pH=7.0 phosphate buffered solution
Centrifuge washing 1 time, takes lower sediment, continuously adds 1.0 mL, pH=7.0 phosphate buffered solution, is uniformly dispersed, is adsorbed
Pt nanowires@SBA-15 microspheres/detection antibody Ab of sulfur a beautiful gem2Incubation content dispersion liquid, preserves under 4.0 °C.
Described in embodiment 10, sandwich type hepatitis b virus marker immunosensor is to hepatitis B virus surface antigen
The detection of HBs-Ag
（1）Using electrochemical workstation, tested under three-electrode system, with saturated calomel electrode as reference electrode, with platinum
Silk electrode is, to electrode, with prepared immunosensor as working electrode, to include 5.0 mmol/L hydrogen peroxide in 10 mL molten
Tested in liquid, pH=7.0 phosphate buffered solution of 2.0 mmol/L o-phenylenediamine solutions；
（2）Analyte is detected using differential pulse voltammetry, sweep limitss are 0.0 ~ 0.6 V, and pulse amplitude is 50
MV, pulse width be 50 ms, the pulse period be 50 ms, record current peak value；
（3）The current peak corresponding to hepatitis b virus marker antigen under record variable concentrations；
（4）Using working curve method, linear inspection of the immunosensor to hepatitis B virus surface antigen HBs-Ag is obtained
It is 0.00001 ng/mL ~ 100 ng/mL to survey scope, and lowest detection lower limit is 3.3 fg/mL.
Described in embodiment 11, sandwich type hepatitis b virus marker immunosensor is to hepatitis B virus e antigen
The detection of HBe-Ag
Method according to embodiment 10 detects that to hepatitis B virus e antigen HBe-Ag its linear detection range is
0.0005 ng/mL ~ 50 ng/mL, lowest detection lower limit are 0.167 pg/mL.
Described in embodiment 12, sandwich type hepatitis b virus marker immunosensor is to hepatitis B virus core antigen
The detection of HBc-Ag
Method according to embodiment 10 detects that to hepatitis B virus core antigen HBc-Ag its linear detection range is
0.00003 ng/mL ~ 60 ng/mL, lowest detection lower limit are 10 fg/mL.
Claims (4)
1. a kind of construction method of sandwich type hepatitis b virus marker immunosensor, it is characterised in that including following step
Suddenly：
（1）By the glass-carbon electrode Al of a diameter of 3.0 ~ 5.0 mm2O3Polishing powder is polished to minute surface, the ultrasound in dehydrated alcohol
Clean up；
（2）Using chronoamperometry, under -0.2 V, polished glass-carbon electrode is placed in into 5.0 mL, 1.0 wt% ~ 2.0
In the chlorauric acid solution of wt%, 25 ~ 35 s are electroplated, deposited Au thin film is formed in electrode surface, with ultrapure water electrode table
Dry under face, room temperature；
（3）By above-mentioned electrode immerse 30 mL, 1.0 mmol/L 4- aminothiophenol solution, soak 2.0 h, ultrapure water,
Dry under room temperature；
（4）Continue electrode is immersed the golden nanometer particle dispersion liquid of 50 mL, 0.04 ~ 0.05 mg/mL, soak 2.0 h, it is ultrapure
Water is rinsed, and is dried under room temperature；
（5）By hepatitis b virus marker capture antibody A b of 6.0 L, 5.0 ~ 10.0 g/mL1It is added drop-wise to electrode surface,
Dry in 4.0 °C of refrigerators；
（6）The bovine serum albumen solution of 3.0 L, 0.5 wt% ~ 1.0 wt% is added drop-wise to electrode surface by continuation, to close
Electrode is rinsed in nonspecific activity site on electrode surface, pH=7.0 phosphate buffers, is dried in 4.0 °C of refrigerators；
（7）Continue 6.0 L of Deca, a series of hepatitis b virus marker of variable concentrations of 0.00001 ~ 100 ng/mL
Antigenic solution, dries in 4 °C of refrigerators；
（8）By 6.0 L, Pt nanowires@SBA-15 microspheres/detection antibody Ab of the absorption sulfur a beautiful gem of 1.0 ~ 3.0 mg/mL2Hatching
Thing dispersant liquid drop is applied to electrode surface, is placed in 4.0 °C of refrigerators, stands 40 min, is rushed with the phosphate buffer of pH=7.0
Wash, dry in 4.0 °C of refrigerators, a kind of sandwich type hepatitis b virus marker immunosensor is obtained.
2. a kind of construction method of sandwich type hepatitis b virus marker immunosensor as claimed in claim 1, described
The preparation of associated materials, including following step：
（1）The preparation of golden nanometer particle dispersion liquid
The chlorauric acid solution of 1.0 ~ 2.0 mL, 1.0 wt% is added in 99.0 mL ultra-pure waters, boiling is heated to, is added
The sodium citrate solution of 1.5 ~ 3.5 mL, 1.0 wt%, continue backflow 10 ~ 20 min, after be cooled to room temperature, obtain Jenner
Rice corpuscles dispersion liquid；
（2）Pt nanowires@SBA-15 microspheres/detection antibody HBs-Ab of absorption sulfur a beautiful gem2The preparation of incubation content dispersion liquid
1. the preparation of Pt nanowires@SBA-15 microspheres
Take 1.0 ~ 1.5 mL, the chloroplatinous acid potassium solution of 12 wt% is placed in 100 mL beakers, inserts 0.4 ~ 0.6 g's
SBA-15 powder, stirs, and after 15 ~ 20 min of ultrasound, is placed in 25 °C of vacuum drying ovens and is dried 24 h, continuously add
After 2.0 ~ 3.0 mL, the ascorbic acid solution of 1.0 mol/mL, 1.0 h of reduction in 25 °C of vacuum drying ovens are placed in, continue to add
Enter 5.0 ~ 7.0 mL, the hydrofluoric acid solution of 10 wt%, after persistently stirring 20 ~ 40 min, centrifugation, with milli-Q water, is incited somebody to action
Resulting solid is placed in 30 °C of vacuum drying ovens and is dried 24 h, obtains Pt nanowires@SBA-15 microspheres；
2. adsorb the preparation of the Pt nanowires@SBA-15 microspheres of sulfur a beautiful gem
Weigh the sulfur a beautiful gem solution that 40 ~ 50 mg Pt nanowires@SBA-15 microspheres are immersed in 10 mL, 1.0 ~ 1.5 mol/mL
In, ultrasonic disperse, and in an oscillator after 6.0 h of persistent oscillation, centrifugation, with milli-Q water, resulting solid is placed in
24 h are dried in 30 °C of vacuum drying ovens, obtain adsorbing the Pt nanowires@SBA-15 microspheres of sulfur a beautiful gem；
3. adsorb Pt nanowires@SBA-15 microspheres/detection antibody HBs-Ab of sulfur a beautiful gem2The preparation of incubation content dispersion liquid
Take 2.0 ~ 6.0 mg absorption sulfur a beautiful gem Pt nanowires@SBA-15 microspheres be distributed in 1.0 mL ultra-pure waters, add 1.0 ~
Hepatitis b virus marker detection antibody Ab of 2.0 mL, 20 ~ 30 μ g/mL2Dispersion liquid, is placed in 4.0 °C of constant temperature oscillations
In incubator after 6.0 ~ 8.0 h of vibration hatching, 5.0 ~ 10.0 min are centrifuged under 5000 rpm rotating speeds, lower sediment are taken and is added
Enter 1.0 mL, pH=7.0 phosphate buffered solution centrifuge washing 1 time, take lower sediment and continuously add 1.0 mL, pH=7.0 phosphoric acid
Salt buffer solution, is uniformly dispersed, and obtains adsorbing Pt nanowires@SBA-15 microspheres/detection antibody Ab of sulfur a beautiful gem2Incubation content is disperseed
Liquid, preserves under 4.0 °C.
3. a kind of sandwich type hepatitis b virus marker immunosensor that construction method as claimed in claim 1 builds,
For the detection of hepatitis b virus marker antigen, step is as follows：
（1）Using electrochemical workstation, tested under three-electrode system, with saturated calomel electrode as reference electrode, with platinum
Silk electrode is, to electrode, with prepared immunosensor as working electrode, to include 5.0 mmol/L hydrogen peroxide in 10 mL molten
Tested in liquid, 5.3 ~ 8.0 phosphate buffered solution of pH of 2.0 mmol/L o-phenylenediamine solutions；
（2）Hepatitis b virus marker antigen is detected using differential pulse voltammetry, sweep limitss are 0.0 ~ 0.6
V, pulse amplitude be 50 mV, pulse width be 50 ms, the pulse period be 50 ms, record current peak value；
（3）The current peak corresponding to hepatitis b virus marker antigen under record variable concentrations；
（4）Using working curve method, the concentration of hepatitis b virus marker antigen in testing sample is obtained.
4. a kind of construction method of sandwich type hepatitis b virus marker immunosensor as claimed in claim 1 builds
Immunosensor, for the measure of hepatitis b virus marker antigen, it is characterised in that the virus marker thing is selected from following
One of：Hepatitis B virus surface antigen HBs-Ag, hepatitis B virus e antigen HBe-Ag, hepatitis B virus core antigen
HBc-Ag。
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