CN106596942B - A kind of construction method of interlayer type hepatitis b virus marker immunosensor and application - Google Patents
A kind of construction method of interlayer type hepatitis b virus marker immunosensor and application Download PDFInfo
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Abstract
The invention belongs to novel nanocomposite materials, immunoassay and biosensor technique fields, the present invention relates to a kind of construction method of interlayer type hepatitis b virus marker immunosensor and applications, the electrochemical immunosensor of 15 microballoon composite materials of Pt nanowires@SBA structure based on absorption sulphur a beautiful gem, for quantitatively detecting hepatitis b virus marker, with high specificity, high sensitivity, detection limits low advantage, has important scientific meaning and application value to hepatitis b virus infected early diagnosis.
Description
Technical field
The invention belongs to novel nanocomposite materials, immunoassay and biosensor technique fields, provide 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 the health problem of global most serious, it can cause chronic hepatitis, hepatic sclerosis etc.
Disease, or even cause the liver cancer for occupying global cancer mortality second place.The concentration of Markers of HBV is in blood
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 diagnosis be of great significance.Therefore, develop the quantitative detecting method of highly sensitive hepatitis b virus marker particularly
Urgently.
In recent years, with the rapid development of clinical diagnosis technology, the electrochemical immunosensor of superior performance is shown one's talent,
And it is widely used in the detection of virus marker object or tumor markers.Interlayer type electrochemical immunosensor is to be based on antigen
A kind of analysis method combined with antibody specificity, has that detection is rapid, detection limit is low, high sensitivity, easy to operate and prepare
Advantage at low cost has important value to the detection of trace level virus and tumor markers.
The important component of base material and catalyst material as electrochemical immunosensor, to improving immunosensor
Sensitivity plays an important roll.In recent years, nano material and its composite material are widely used in the structure of immunosensor and work as
In.The present invention utilizes layer-by-layer, and the double layer gold built using deposited Au film and gold nanoparticle is substrates, to inhale
The Pt nanowires@SBA-15 microballoons of attached sulphur a beautiful gem build a kind of interlayer type hepatitis b virus marker as detection antibody marker
Immunosensor has many advantages, such as that detection range is wide, Monitoring lower-cut is low, easy to operate, detection speed is fast, to the morning of hepatitis B
Phase diagnosis has important application value.
Invention content
The present invention provides a kind of construction method of interlayer type hepatitis b virus marker immunosensor and applications, 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 interlayer type hepatitis b virus marker immunosensor
Method.
The second object of the present invention is to use a kind of prepared interlayer type hepatitis b virus marker immunosensor
In the detection of hepatitis b virus marker.
Technical scheme of the present invention includes the following steps.
1. a kind of construction method of interlayer type hepatitis b virus marker immunosensor, steps 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, in absolute ethyl alcohol
It is cleaned by ultrasonic clean；
（2）Using chronoamperometry, at -0.2 V, by polished glass-carbon electrode be placed in 5.0 mL, 1.0 wt% ~
In the chlorauric acid solution of 2.0 wt%, 25 ~ 35 s are electroplated, deposited Au film is formed in electrode surface, with ultrapure water electricity
Pole surface is dried at room temperature；
（3）Above-mentioned electrode is immersed to the 4- aminothiophenol solution of 30 mL, 1.0 mmol/L, impregnates 2.0 h, ultra-pure water
It rinses, dries at room temperature；
（4）Continue, by the golden nanometer particle dispersion liquid of 50 mL of electrode immersion, 0.04 ~ 0.05 mg/mL, to impregnate 2.0 h,
Ultrapure water dries at room temperature；
（5）By the hepatitis b virus marker capture antibody A b of 6.0 μ L, 5.0 ~ 10.0 μ g/mL1It is added drop-wise to electrode
Surface is dried in 4.0 °C of refrigerators；
（6）Continue the bovine serum albumen solution of 3.0 μ L, the wt% of 0.5 wt% ~ 1.0 being added drop-wise to electrode surface, to
Nonspecific activity site on enclosed-electrode surface, the phosphate buffers of pH=7.0 rinse electrode, are dried in 4.0 °C of refrigerators；
（7）Continue a series of hepatitis type B virus mark that various concentrations of 6.0 μ L, 0.00001 ~ 100 ng/mL are added dropwise
Will object antigenic solution dries in 4.0 °C of refrigerators；
（8）By Pt nanowires@SBA-15 microballoons/detection antibody of the absorption sulphur a beautiful gem of 6.0 μ L, 1.0 ~ 3.0 mg/mL
Ab2Incubation content dispersant liquid drop is applied to electrode surface, is placed in 4.0 °C of refrigerators, stands 40 min, slow with the phosphate of pH=7.0
Fliud flushing is rinsed, and is dried in 4.0 °C of refrigerators, and a kind of interlayer type hepatitis b virus marker immunosensor is made.
2. a kind of construction method of interlayer type hepatitis b virus marker immunosensor, the system of the associated materials
Standby, steps are as follows：
（1）The preparation of golden nanometer particle dispersion liquid
The chlorauric acid solution of 1.0 ~ 2.0 mL, 1.0 wt% are added in 99.0 mL ultra-pure waters, is heated to boiling, add
The sodium citrate solution for entering 1.5 ~ 3.5 mL, 1.0 wt%, continues 10 ~ 20 min of reflux, and postcooling to room temperature obtains gold
Nanoparticle dispersion liquid；
（2）Adsorb Pt nanowires@SBA-15 microballoons/detection antibody HBs-Ab of sulphur a beautiful gem2The preparation of incubation content dispersion liquid
1. the preparation of Pt nanowires@SBA-15 microballoons
Take 1.0 ~ 1.5 mL, the chloroplatinous acid potassium solution of 12 wt% is placed in 100 mL beakers, merging 0.4 ~ 0.6
The SBA-15 powder of g, stirs evenly, and after 15 ~ 20 min of ultrasound, is placed in 25 °C of vacuum drying chambers dry 24 h, continues to add
It after the ascorbic acid solution for entering 2.0 ~ 3.0 mL, 1.0 mol/mL, is placed in 25 °C of vacuum drying chambers and restores 1.0 h, continue
The hydrofluoric acid solution of 5.0 ~ 7.0 mL, 10 wt% is added, after persistently stirring 20 ~ 40 min, centrifugation, with milli-Q water,
Obtained solid is placed in 30 °C of vacuum drying chambers dry 24 h, obtains Pt nanowires@SBA-15 microballoons；
2. adsorbing the preparation of the Pt nanowires@SBA-15 microballoons of sulphur a beautiful gem
Weighing 40 ~ 50 mg Pt nanowires@SBA-15 microballoons, to be immersed in 10 mL, the sulphur a beautiful gem of 1.0 ~ 1.5 mol/mL molten
In liquid, ultrasonic disperse, and in an oscillator after 6.0 h of persistent oscillation, with milli-Q water, obtained solid is set for centrifugation
In dry 24 h in 30 °C of vacuum drying chambers, the Pt nanowires@SBA-15 microballoons of absorption sulphur a beautiful gem are obtained；
3. adsorbing Pt nanowires@SBA-15 microballoons/detection antibody HBs-Ab of sulphur a beautiful gem2The preparation of incubation content dispersion liquid
It takes the Pt nanowires@SBA-15 microballoons of 2.0 ~ 6.0 mg absorption sulphur a beautiful gems to be distributed in 1.0 mL ultra-pure waters, is added
The hepatitis b virus marker detection antibody A b of 1.0 ~ 2.0 mL, 20 ~ 30 μ g/mL2Dispersion liquid is placed in 4.0 °C of perseverances
In warm shaken cultivation case after 6.0 ~ 8.0 h of oscillation hatching, 5.0 ~ 10.0 min are centrifuged under 5000 rpm rotating speeds, remove layer
The phosphate buffer solutions of 1.0 mL, pH=7.0 centrifuge washings is added 1 time in precipitation, take lower sediment continuously add 1.0 mL, pH=
7.0 phosphate buffer solutions, are uniformly dispersed, and obtain Pt nanowires@SBA-15 microballoons/detection antibody A b of absorption sulphur a beautiful gem2Hatching
Object dispersion liquid preserves under 4.0 °C.
3. a kind of interlayer type hepatitis b virus marker immunosensor is used for hepatitis b virus marker antigen
Detection, steps are as follows：
（1）Using electrochemical workstation, tested under three-electrode system, using saturated calomel electrode as reference electrode,
It is to electrode with platinum electrode, includes 5.0 mmol/L peroxidating in 10 mL using prepared immunosensor as working electrode
Hydrogen solution, 2.0 mmol/L o-phenylenediamine solutions 5.3 ~ 8.0 phosphate buffer solutions of pH in tested；
（2）Hepatitis b virus marker antigen is detected using differential pulse voltammetry, scanning range be 0.0 ~
0.6 V, pulse amplitude are 50 mV, and pulse width is 50 ms, and the pulse period is 50 ms, record current peak value；
（3）Record the current peak corresponding to the hepatitis b virus marker antigen under various concentration；
（4）Using working curve method, the concentration of hepatitis b virus marker antigen in sample to be tested is obtained.
Raw material used in the present invention can be bought in chemical reagents corporation or biopharmaceutical company.
The useful achievement of the present invention
（1）The present invention can be effectively increased electrode surface using double layer gold that deposited Au film and gold nanoparticle are built
Electron transmission efficiency, also, due to gold nanoparticle have good biocompatibility, so that double layer gold of structure is stablized
In conjunction with a large amount of active capture antibody, to increase the stability of immunosensor, for improving immunosensor
Sensitivity plays an important roll；
（2）The present invention is using the Pt nanowires@SBA-15 microballoons of the absorption sulphur a beautiful gem of function admirable by as detection antibody mark
Remember object in the structure of immunosensor.Catalytic performance and the splendid Pt nanowires of electric conductivity are mounted to the inside of SBA-15
Afterwards, the electric conductivity of SBA-15 is effectively enhanced, and ordered mesopore structure flourishing in SBA-15 is capable of providing a large amount of catalysis and lives
Property site, and the time of contact of reactant and Pt nanowires@SBA-15 microballoons can be effectively increased, by this synergistic effect and excellent
Gesture complementation increases the sensitivity of immunosensor.The absorption of sulphur a beautiful gem makes the electric conductivity of SBA-15 microballoons further increase.This
Outside, there is amido functional group in sulphur a beautiful gem, make the Pt nanowires@SBA-15 microballoons of absorption sulphur a beautiful gem that there is better biocompatibility,
Improve the load capacity of detection antibody.Therefore, have using the Pt nanowires@SBA-15 microballoons for adsorbing sulphur a beautiful gem as detection antibody marker
Effect amplification electric signal, improves the sensitivity of immunosensor, reduces the Monitoring lower-cut of immunosensor；
（3）A kind of inspection of interlayer type hepatitis b virus marker immunosensor to different hepatitis b virus markers
It surveys, the linear detection range to hepatitis b virus s antigen HBs-Ag is the ng/mL of 0.00001 ng/mL ~ 100, most
Low-detection lower limit is 3.3 fg/mL；Linear detection range to hepatitis B virus e antigen HBe-Ag be 0.0005 ng/mL ~
50 ng/mL, lowest detection lower limit are 0.167 pg/mL；To the linear detection range of hepatitis B virus core antigen HBc-Ag
It is the ng/mL of 0.00003 ng/mL ~ 60, lowest detection lower limit is 10 fg/mL；Show a kind of interlayer type hepatitis type B virus
Marker immunosensor can achieve the purpose that accurate quantification detects hepatitis b virus marker.
Specific implementation mode
Now the present invention is further illustrated by specific implementation mode, but not limited to this.
A kind of construction method of 1 interlayer 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, and ultrasound is clear in absolute ethyl alcohol
Wash clean；
（2）Using chronoamperometry, at -0.2 V, polished glass-carbon electrode is placed in the chlorine of 5.0 mL, 1.0 wt%
In auric acid solution, 25 s are electroplated, form deposited Au film in electrode surface is dried in the air at room temperature with ultrapure water electrode surface
It is dry；
（3）Above-mentioned electrode is immersed to the 4- aminothiophenol solution of 30 mL, 1.0 mmol/L, impregnates 2.0 h, ultra-pure water
It rinses, dries at room temperature；
（4）Continue, by the golden nanometer particle dispersion liquid of 50 mL of electrode immersion, 0.04 mg/mL, to impregnate 2.0 h, ultra-pure water
It rinses, dries at room temperature；
（5）By the hepatitis b virus marker capture antibody A b of 6.0 μ L, 5.0 μ g/mL1Electrode surface is added drop-wise to,
It is dried in 4.0 °C of refrigerators；
（6）Continue the bovine serum albumen solution of 3.0 μ L, 0.5 wt% being added drop-wise to electrode surface, to enclosed-electrode table
Nonspecific activity site on face, the phosphate buffers of pH=7.0 rinse electric face, are dried in 4.0 °C of refrigerators；
（7）Continue a series of hepatitis type B virus mark that various concentrations of 6.0 μ L, 0.00001 ~ 100 ng/mL are added dropwise
Will object antigenic solution dries in 4 °C of refrigerators；
（8）By Pt nanowires@SBA-15 microballoons/detection antibody A b of the absorption sulphur a beautiful gem of 6.0 μ L, 1.0 mg/mL2Hatching
Object 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
It washes, is dried in 4.0 °C of refrigerators, a kind of interlayer type hepatitis b virus marker immunosensor is made.
A kind of construction method of 2 interlayer 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, and ultrasound is clear in absolute ethyl alcohol
Wash clean；
（2）Using chronoamperometry, at -0.2 V, polished glass-carbon electrode is placed in the chlorine of 5.0 mL, 1.5 wt%
In auric acid solution, 30 s are electroplated, form deposited Au film in electrode surface is dried in the air at room temperature with ultrapure water electrode surface
It is dry；
（3）Above-mentioned electrode is immersed to the 4- aminothiophenol solution of 30 mL, 1.0 mmol/L, impregnates 2.0 h, ultra-pure water
It rinses, dries at room temperature；
（4）Continue, by the golden nanometer particle dispersion liquid of 50 mL of electrode immersion, 0.05 mg/mL, to impregnate 2.0 h, ultra-pure water
It rinses, dries at room temperature；
（5）By the hepatitis b virus marker capture antibody A b of 6.0 μ L, 7.5 μ g/mL1Electrode surface is added drop-wise to,
It is dried in 4.0 °C of refrigerators；
（6）Continue the bovine serum albumen solution of 3.0 μ L, 0.75 wt% being added drop-wise to electrode surface, to enclosed-electrode table
Nonspecific activity site on face, the phosphate buffers of pH=7.0 rinse electrode, are dried in 4.0 °C of refrigerators；
（7）Continue a series of hepatitis type B virus mark that various concentrations of 6.0 μ L, 0.00001 ~ 100 ng/mL are added dropwise
Will object antigenic solution dries in 4 °C of refrigerators；
（8）By Pt nanowires@SBA-15 microballoons/detection antibody A b of the absorption sulphur a beautiful gem of 6.0 μ L, 2.0 mg/mL2Hatching
Object 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
It washes, is dried in 4.0 °C of refrigerators, a kind of interlayer type hepatitis b virus marker immunosensor is made.
A kind of construction method of 3 interlayer 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, and ultrasound is clear in absolute ethyl alcohol
Wash clean；
（2）Using chronoamperometry, at -0.2 V, polished glass-carbon electrode is placed in the chlorine of 5.0 mL, 2.0 wt%
In auric acid solution, 35 s are electroplated, form deposited Au film in electrode surface is dried in the air at room temperature with ultrapure water electrode surface
It is dry；
（3）Above-mentioned electrode is immersed to the 4- aminothiophenol solution of 30 mL, 1.0 mmol/L, impregnates 2.0 h, ultra-pure water
It rinses, dries at room temperature；
（4）Continue, by the golden nanometer particle dispersion liquid of 50 mL of electrode immersion, 0.05 mg/mL, to impregnate 2.0 h, ultra-pure water
It rinses, dries at room temperature；
（5）By the hepatitis b virus marker capture antibody A b of 6.0 μ L, 10.0 μ g/mL1Electrode surface is added drop-wise to,
It is dried in 4.0 °C of refrigerators；
（6）Continue the bovine serum albumen solution of 3.0 μ L, 1.0 wt% being added drop-wise to electrode surface, to enclosed-electrode table
Nonspecific activity site on face, the phosphate buffers of pH=7.0 rinse electrode, are dried in 4.0 °C of refrigerators；
（7）Continue a series of hepatitis type B virus mark that various concentrations of 6.0 μ L, 0.00001 ~ 100 ng/mL are added dropwise
Will object antigenic solution dries in 4 °C of refrigerators；
（8）By Pt nanowires@SBA-15 microballoons/detection antibody A b of the absorption sulphur a beautiful gem of 6.0 μ L, 3.0 mg/mL2Hatching
Object 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
It washes, is dried in 4.0 °C of refrigerators, a kind of interlayer type hepatitis b virus marker immunosensor is made.
The preparation of golden nanometer particle dispersion liquid described in embodiment 4
The chlorauric acid solution of 1.0 mL, 1.0 wt% are added in 99.0 mL ultra-pure waters, are heated to boiling, is added 1.5
The sodium citrate solution of mL, 1.0 wt%, continue 10 min that flow back, and postcooling to room temperature obtains 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% are added in 99.0 mL ultra-pure waters, are heated to boiling, is added 2.5
The sodium citrate solution of mL, 1.0 wt%, continue 15 min that flow back, and postcooling to room temperature obtains 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% are added in 99.0 mL ultra-pure waters, are heated to boiling, is added 3.5
The sodium citrate solution of mL, 1.0 wt%, continue 20 min that flow back, and postcooling to room temperature obtains golden nanometer particle dispersion liquid.
Pt nanowires@SBA-15 microballoons/detection antibody A b of sulphur a beautiful gem is adsorbed described in embodiment 72The system of incubation content dispersion liquid
It is standby
1. the preparation of Pt nanowires@SBA-15 microballoons
Take 1.0 mL, the chloroplatinous acid potassium solution of 12 wt% is placed in 100 mL beakers, the SBA-15 powder of 0.4 g of merging
End stirs evenly, and after 15 min of ultrasound, is placed in 25 °C of vacuum drying chambers dry 24 h, continuously adds 2.0 mL, 1.0
It after the ascorbic acid solution of mol/mL, is placed in 25 °C of vacuum drying chambers and restores 1.0 h, continuously add 5.0 mL, 10 wt%
Hydrofluoric acid solution, after persistently stirring 20 min, acquired solid with milli-Q water, is placed in 30 °C of vacuum drying chambers by centrifugation
24 h of interior drying obtains Pt nanowires@SBA-15 microballoons；
2. adsorbing the preparation of the Pt nanowires@SBA-15 microballoons of sulphur a beautiful gem
It weighs 40 mg Pt nanowires@SBA-15 microballoons to be immersed in the sulphur a beautiful gem solution of 10 mL, 1.0 mol/mL, ultrasound point
It dissipates, and in an oscillator after 6.0 h of persistent oscillation, obtained solid with milli-Q water, is placed in 30 °C of vacuum by centrifugation
Dry 24 h, obtain the Pt nanowires@SBA-15 microballoons of absorption sulphur a beautiful gem in drying box；
3. adsorbing Pt nanowires@SBA-15 microballoons/detection antibody A b of sulphur a beautiful gem2The preparation of incubation content dispersion liquid
The Pt nanowires@SBA-15 microballoons of 2.0 mg absorption sulphur a beautiful gems are taken to be distributed in 1.0 mL ultra-pure waters, 1.0 mL of addition,
The hepatitis b virus marker detection antibody A b of 20 μ g/mL2Dispersion liquid is placed in 4.0 °C of constant-temperature shaking incubators and vibrates
After hatching 6.0 h, 5.0 min are centrifuged under 5000 rpm rotating speeds, take lower sediment that the phosphate-buffereds of 1.0 mL, pH=7.0 are added
Solution centrifuge washing 1 time, takes lower sediment, continuously adds the phosphate buffer solutions of 1.0 mL, pH=7.0, is uniformly dispersed, obtains
Adsorb Pt nanowires@SBA-15 microballoons/detection antibody A b of sulphur a beautiful gem2Incubation content dispersion liquid preserves under 4.0 °C.
Pt nanowires@SBA-15 microballoons/detection antibody A b of sulphur a beautiful gem is adsorbed described in embodiment 82The system of incubation content dispersion liquid
It is standby
1. the preparation of Pt nanowires@SBA-15 microballoons
Take 1.5 mL, the chloroplatinous acid potassium solution of 12 wt% is placed in 100 mL beakers, the SBA-15 powder of 0.5 g of merging
End stirs evenly, and after 20 min of ultrasound, is placed in 25 °C of vacuum drying chambers dry 24 h, continuously adds 2.5 mL, 1.0
It after the ascorbic acid solution of mol/mL, is placed in 25 °C of vacuum drying chambers and restores 1.0 h, continuously add 6.0 mL, 10 wt%
Hydrofluoric acid solution, after persistently stirring 30 min, acquired solid with milli-Q water, is placed in 30 °C of vacuum drying chambers by centrifugation
24 h of interior drying obtains Pt nanowires@SBA-15 microballoons；
2. adsorbing the preparation of the Pt nanowires@SBA-15 microballoons of sulphur a beautiful gem
It weighs 45 mg Pt nanowires@SBA-15 microballoons to be immersed in the sulphur a beautiful gem solution of 10 mL, 1.5 mol/mL, ultrasound point
It dissipates, and in an oscillator after 6.0 h of persistent oscillation, obtained solid with milli-Q water, is placed in 30 °C of vacuum by centrifugation
Dry 24 h, obtain the Pt nanowires@SBA-15 microballoons of absorption sulphur a beautiful gem in drying box；
3. adsorbing Pt nanowires@SBA-15 microballoons/detection antibody A b of sulphur a beautiful gem2The preparation of incubation content dispersion liquid
The Pt nanowires@SBA-15 microballoons of 4.0 mg absorption sulphur a beautiful gems are taken to be distributed in 1.0 mL ultra-pure waters, 1.5 mL of addition,
The hepatitis b virus marker detection antibody A b of 25 μ g/mL2Dispersion liquid is placed in 4.0 °C of constant-temperature shaking incubators and vibrates
After hatching 7.0 h, 5.0 min are centrifuged under 5000 rpm rotating speeds, obtain lower sediment, the phosphate of 1.0 mL, pH=7.0 is added
Buffer solution centrifuge washing 1 time obtains lower sediment, and the phosphate buffer solutions of 1.0 mL, pH=7.0 are added, is uniformly dispersed, obtains
Adsorb Pt nanowires@SBA-15 microballoons/detection antibody A b of sulphur a beautiful gem2Incubation content dispersion liquid preserves under 4.0 °C.
Pt nanowires@SBA-15 microballoons/detection antibody A b of sulphur a beautiful gem is adsorbed described in embodiment 92The system of incubation content dispersion liquid
It is standby
1. the preparation of Pt nanowires@SBA-15 microballoons
Take 1.5 mL, the chloroplatinous acid potassium solution of 12 wt% is placed in 100 mL beakers, the SBA-15 powder of 0.6 g of merging
End stirs evenly, and after 20 min of ultrasound, is placed in 25 °C of vacuum drying chambers dry 24 h, continuously adds 3.0 mL, 1.0
It after the ascorbic acid solution of mol/mL, is placed in 25 °C of vacuum drying chambers and restores 1.0 h, continuously add 7.0 mL, 10 wt%
Hydrofluoric acid solution, after persistently stirring 40 min, obtained solid with milli-Q water, is placed in 30 °C of vacuum drying by centrifugation
Dry 24 h, obtain Pt nanowires@SBA-15 microballoons in case；
2. adsorbing the preparation of the Pt nanowires@SBA-15 microballoons of sulphur a beautiful gem
It weighs 50 mg Pt nanowires@SBA-15 microballoons to be immersed in the sulphur a beautiful gem solution of 10 mL, 1.5 mol/mL, ultrasound point
It dissipates, and in an oscillator after 6.0 h of persistent oscillation, obtained solid with milli-Q water, is placed in 30 °C of vacuum by centrifugation
Dry 24 h, obtain the Pt nanowires@SBA-15 microballoons of absorption sulphur a beautiful gem in drying box；
3. adsorbing Pt nanowires@SBA-15 microballoons/detection antibody A b of sulphur a beautiful gem2The preparation of incubation content dispersion liquid
The Pt nanowires@SBA-15 microballoons of 6.0 mg absorption sulphur a beautiful gems are taken to be distributed in 1.0 mL ultra-pure waters, 2.0 mL of addition,
The hepatitis b virus marker detection antibody A b of 30 μ g/mL2Dispersion liquid is placed in 4.0 °C of constant-temperature shaking incubators and vibrates
After hatching 8.0 h, 10 min are centrifuged under 5000 rpm rotating speeds, take lower sediment that the phosphate-buffereds of 1.0 mL, pH=7.0 are added
Solution centrifuge washing 1 time, takes lower sediment, continuously adds the phosphate buffer solutions of 1.0 mL, pH=7.0, is uniformly dispersed, obtains
Adsorb Pt nanowires@SBA-15 microballoons/detection antibody A b of sulphur a beautiful gem2Incubation content dispersion liquid preserves under 4.0 °C.
Interlayer type hepatitis b virus marker immunosensor is to hepatitis b virus s antigen described in embodiment 10
The detection of HBs-Ag
（1）Using electrochemical workstation, tested under three-electrode system, using saturated calomel electrode as reference electrode,
It is to electrode with platinum electrode, includes 5.0 mmol/L peroxidating in 10 mL using prepared immunosensor as working electrode
Hydrogen solution, 2.0 mmol/L o-phenylenediamine solutions the phosphate buffer solutions of pH=7.0 in tested；
（2）Analyte is detected using differential pulse voltammetry, scanning range is 0.0 ~ 0.6 V, pulse amplitude
For 50 mV, pulse width is 50 ms, and the pulse period is 50 ms, record current peak value；
（3）Record the current peak corresponding to the hepatitis b virus marker antigen under various concentration；
（4）Using working curve method, line of the immunosensor to hepatitis b virus s antigen HBs-Ag is obtained
Property detection range be the ng/mL of 0.00001 ng/mL ~ 100, lowest detection lower limit be 3.3 fg/mL.
Interlayer type hepatitis b virus marker immunosensor is to hepatitis B virus e antigen described in embodiment 11
The detection of HBe-Ag
Hepatitis B virus e antigen HBe-Ag is detected according to the method for embodiment 10, linear detection range is
The ng/mL of 0.0005 ng/mL ~ 50, lowest detection lower limit are 0.167 pg/mL.
Interlayer type hepatitis b virus marker immunosensor is to hepatitis B virus core antigen described in embodiment 12
The detection of HBc-Ag
Hepatitis B virus core antigen HBc-Ag is detected according to the method for embodiment 10, linear detection range
It is the ng/mL of 0.00003 ng/mL ~ 60, lowest detection lower limit is 10 fg/mL.
Claims (4)
1. a kind of construction method of interlayer type hepatitis b virus marker immunosensor, which is characterized 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, ultrasonic in absolute ethyl alcohol
It cleans up；
（2）Using chronoamperometry, at -0.2 V, polished glass-carbon electrode is placed in 5.0 mL, 1.0 wt% ~ 2.0
In the chlorauric acid solution of wt%, 25 ~ 35 s are electroplated, deposited Au film are formed in electrode surface, with ultrapure water electrode table
Face is dried at room temperature；
（3）Above-mentioned electrode surface is immersed to the 4- aminothiophenol solution of 30 mL, 1.0 mmol/L, impregnates 2.0 h, ultra-pure water
It rinses, dries at room temperature；
（4）Continue, by the golden nanometer particle dispersion liquid of 50 mL of electrode immersion, 0.04 ~ 0.05 mg/mL, to impregnate 2.0 h, it is ultrapure
Water rinses, and dries at room temperature；
（5）By the hepatitis b virus marker capture antibody A b of 6.0 μ L, 5.0 ~ 10.0 μ g/mL1Electrode surface is added drop-wise to,
It is dry in 4.0 °C of refrigerators；
（6）Continue the bovine serum albumen solution of 3.0 μ L, the wt% of 0.5 wt% ~ 1.0 being added drop-wise to electrode surface, to close
Nonspecific activity site on electrode surface, the phosphate buffers of pH=7.0 rinse electrode surface, are dried in 4.0 °C of refrigerators；
（7）Continue a series of hepatitis b virus marker that various concentrations of 6.0 μ L, 0.00001 ~ 100 ng/mL are added dropwise
Antigenic solution, it is dry in 4 °C of refrigerators；
（8）By Pt nanowires@SBA-15 microballoons/detection antibody A b of the absorption sulphur a beautiful gem of 6.0 μ L, 1.0 ~ 3.0 mg/mL2Hatching
Object 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
It washes, it is dry in 4.0 °C of refrigerators, a kind of interlayer type hepatitis b virus marker immunosensor is made.
2. a kind of construction method of interlayer type hepatitis b virus marker immunosensor as described in claim 1, described
Pt nanowires@SBA-15 microballoons/detection antibody HBs-Ab of golden nanometer particle dispersion liquid and absorption sulphur a beautiful gem2Incubation content dispersion liquid
It prepares, including following steps：
（1）The preparation of golden nanometer particle dispersion liquid
The chlorauric acid solution of 1.0 ~ 2.0 mL, 1.0 wt% are added in 99.0 mL ultra-pure waters, are heated to boiling, is added
The sodium citrate solution of 1.5 ~ 3.5 mL, 1.0 wt%, continue 10 ~ 20 min of reflux, and postcooling to room temperature obtains Jenner
Rice corpuscles dispersion liquid；
（2）Adsorb Pt nanowires@SBA-15 microballoons/detection antibody HBs-Ab of sulphur a beautiful gem2The preparation of incubation content dispersion liquid
1. the preparation of Pt nanowires@SBA-15 microballoons
Take 1.0 ~ 1.5 mL, the chloroplatinous acid potassium solution of 12 wt% is placed in 100 mL beakers, 0.4 ~ 0.6 g of merging
SBA-15 powder, stirs evenly, and after 15 ~ 20 min of ultrasound, is placed in 25 °C of vacuum drying chambers dry 24 h, continuously adds
2.0 ~ 3.0 mL, 1.0 mol/mL ascorbic acid solution after, be placed in 25 °C of vacuum drying chambers and restore 1.0 h, continue plus
Enter the hydrofluoric acid solution of 5.0 ~ 7.0 mL, 10 wt%, after persistently stirring 20 ~ 40 min, centrifugation will with milli-Q water
Obtained solid is placed in 30 °C of vacuum drying chambers dry 24 h, obtains Pt nanowires@SBA-15 microballoons；
2. adsorbing the preparation of the Pt nanowires@SBA-15 microballoons of sulphur a beautiful gem
Weigh the sulphur a beautiful gem solution that 40 ~ 50 mg Pt nanowires@SBA-15 microballoons 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, with milli-Q water, obtained solid is placed in for centrifugation
Dry 24 h, obtain the Pt nanowires@SBA-15 microballoons of absorption sulphur a beautiful gem in 30 °C of vacuum drying chambers；
3. adsorbing Pt nanowires@SBA-15 microballoons/detection antibody HBs-Ab of sulphur a beautiful gem2The preparation of incubation content dispersion liquid
The Pt nanowires@SBA-15 microballoons of 2.0 ~ 6.0 mg absorption sulphur a beautiful gems are taken to be distributed in 1.0 mL ultra-pure waters, addition 1.0 ~
The hepatitis b virus marker detection antibody A b 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 oscillation hatching, 5.0 ~ 10.0 min are centrifuged under 5000 rpm rotating speeds, it is heavy to obtain lower layer
It forms sediment, the phosphate buffer solutions of 1.0 mL, pH=7.0 centrifuge washing is added 1 time, obtain lower sediment, the phosphorus of 1.0 mL, pH=7.0 is added
Hydrochlorate buffer solution, is uniformly dispersed, and obtains Pt nanowires@SBA-15 microballoons/detection antibody A b of absorption sulphur a beautiful gem2Incubation content is disperseed
Liquid preserves under 4.0 °C.
3. a kind of interlayer type hepatitis b virus marker immunosensor of construction method structure as described in claim 1,
For the detection of hepatitis b virus marker antigen, steps are as follows：
（1）It using electrochemical workstation, is tested under three-electrode system, using saturated calomel electrode as reference electrode, with platinum
Silk electrode is to electrode, includes that 5.0 mmol/L hydrogen peroxide are molten in 10 mL using prepared immunosensor as working electrode
Liquid, 2.0 mmol/L o-phenylenediamine solutions 5.3 ~ 8.0 phosphate buffer solutions of pH in tested；
（2）Hepatitis b virus marker antigen is detected using differential pulse voltammetry, scanning range is 0.0 ~ 0.6
V, pulse amplitude are 50 mV, and pulse width is 50 ms, and the pulse period is 50 ms, record current peak value；
（3）Record the current peak corresponding to the hepatitis b virus marker antigen under various concentration；
（4）Using working curve method, the concentration of hepatitis b virus marker antigen in sample to be tested is obtained.
4. a kind of construction method structure of interlayer type hepatitis b virus marker immunosensor as described in claim 1
Immunosensor is used for the measurement of hepatitis b virus marker antigen, which is characterized in that the virus marker object is selected from following
One of：Hepatitis b virus s antigen HBs-Ag, hepatitis B virus e antigen HBe-Ag, hepatitis B virus core antigen
HBc-Ag。
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