CN107525835B - A kind of preparation method and application of the immunosensor of the phenolic resin micropore carbon ball of the functionalization based on Au AgNPs - Google Patents

A kind of preparation method and application of the immunosensor of the phenolic resin micropore carbon ball of the functionalization based on Au AgNPs Download PDF

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CN107525835B
CN107525835B CN201710673361.6A CN201710673361A CN107525835B CN 107525835 B CN107525835 B CN 107525835B CN 201710673361 A CN201710673361 A CN 201710673361A CN 107525835 B CN107525835 B CN 107525835B
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agnps
phenolic resin
solution
functionalization
carbon ball
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CN107525835A (en
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李月云
张晓波
吕慧
王平
董云会
刘青
孔玲
陈志伟
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Shandong University of Technology
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Shandong University of Technology
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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
    • 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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54386—Analytical elements
    • 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
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites

Abstract

The invention belongs to immunoassays and biosensor technique field, provide a kind of preparation method and application of the immunosensor of the phenolic resin micropore carbon ball of functionalization based on Au AgNPs.Specifically using the phenolic resin micropore carbon ball of the functionalization of load Au AgNPs as marker, it is prepared for a kind of electrochemical immunosensor for detecting tumor markers antigen, realize the detection of common hepatic carcinoma marker AFP, CEA, with high specificity, high sensitivity, detection limit is low, has important scientific meaning and application value.

Description

A kind of immune sensing of the phenolic resin micropore carbon ball of the functionalization based on Au AgNPs The preparation method and application of device
Technical field
The invention belongs to new function materials and bio-sensing detection technique field, provide a kind of based on Au@AgNPs's The preparation method and application of the immunosensor of the phenolic resin micropore carbon ball of functionalization.Specifically use the function of Au@AgNPs The phenolic resin micropore carbon ball of change as detection antibody marker, exempt from by the electrochemistry for being prepared for a kind of detection tumor markers antigen Epidemic disease sensor.
Background technique
Liver cancer is the death rate in a kind of disease and all cancers that harm to the human body is very big, the most fast evil of death rate One of property tumour.The Sensitive Detection of liver cancer tumor markers AFP, CEA, clinically for the early detection of tumour, tumour The screening of people at highest risk, the antidiastole of benign and malignant tumour, the judgement of tumor development degree, the sight of the therapeutic effect of tumour It examines and evaluates and the prediction of tumor recurrence and prognosis generates strong influence, cause the extensive concern of people.
Electrochemical immunosensor is widely used for the detection of tumor markers, interlayer type electrochemistry immuno-sensing at present Device combine high specific immuno analytical method and highly sensitive electrochemical analysis techniques, have high sensitivity, preparation it is simple, The advantages that quick, at low cost is detected, is had in fields such as clinical examination, environmental monitoring, food safety control, biological monitorings important Application value.
The present invention successfully constructs a kind of immune sensing of the phenolic resin micropore carbon ball of functionalization based on Au AgNPs Device.The phenolic resin hard carbon microballoon of poly-dopamine functionalization is three-dimensional micropore chondritic, and porous structure can promote electric work Property substance absorption on the surface of the material, shorten transmission range of the ion between aperture, reduce the charge transfer resistance of material, mention High electric conductivity.Carbon ball after functionalization can form stable chemical bond with noble metal nano particles, can effectively adsorb your gold Belong to nanoparticle, while the reuniting effect of nanoparticle can also be reduced.Gold and silver core-shell nano is relative to monometallic nanoparticle Son, possesses more catalysis points, shows concerted catalysis effect to the reduction reaction of hydrogen peroxide, substantially increases material Electrocatalysis characteristic.
Summary of the invention
The present invention provides a kind of immunosensors of the phenolic resin micropore carbon ball of functionalization based on Au AgNPs Preparation method and application realizes the Sensitive Detection to tumor markers.
An object of the present invention is to provide a kind of exempting from for the phenolic resin micropore carbon ball of functionalization based on Au AgNPs The preparation method of epidemic disease sensor.
The second object of the present invention is to by the phenolic resin micropore carbon ball of the prepared functionalization based on Au AgNPs Immunosensor is applied to highly sensitive, the specific detection of tumor markers.
Technical solution of the present invention includes the following steps.
1. a kind of preparation method of the immunosensor of the phenolic resin micropore carbon ball of functionalization based on Au AgNPs, Steps are as follows:
(1) the glass-carbon electrode Al for being 4 mm by diameter2O3Polishing powder polishing, ultrapure water clean up；
(2) above-mentioned glass-carbon electrode is placed in the HAuCl of 0.1 %4In solution, using i-t method, at -0.2 V, operation 30 ~ 35 s；
(3) continue the tumor markers capture antibody of 6 μ L, 8 ~ 12 μ g/mL being added drop-wise to electrode surface, ultrapure water punching It washes, it is dry in 4 °C of refrigerators；
(4) continue the bovine serum albumin(BSA) BSA solution of 3 μ L, 0.8 ~ 1.5mg/mL being added drop-wise to electrode surface, it is ultrapure Water rinses electrode surface, dries in 4 °C of refrigerators；
(5) the tumor markers antigenic solution of the various concentration of 6 μ L, the ng/mL of 20 fg/mL ~ 100, ultrapure water is added dropwise Rinse electrode surface, drying in 4 °C of refrigerators；
(6) the phenolic resin micropore carbon ball of the functionalization of the load Au AgNPs of 6 μ L, 1 ~ 3 mg/mL is detected into antibody Incubation content solution, drop coating are dried on electrode surface, being placed in 4 °C of refrigerators, and a kind of functionalization based on Au@AgNPs is made The immunosensor of phenolic resin micropore carbon ball.
2. Au@AgNPs described in, preparation step are as follows:
By the HAuCl of 64 mL, 1 ~ 3 mmol/L4Three neck round bottom flask is added in solution, and 115 °C of oil bath are heated at reflux 10 The sodium citrate of 12.5 ~ 13.5 mL, 38.8 mmol/L is added in ~ 15 min, until to have yellow to become wine red for solution colour AuNPs solution is made in color；
The AuNPs solution of the above-mentioned preparation of 5 ~ 7 mL is taken, 400 ~ 600 μ L, 7.1 mmol/L that will newly configure are added Silver nitrate aqueous solution concussion, rufous is become from claret to solution, three times with ultrapure water centrifuge washing, ultrasonic disperse to 10 Au@AgNPs solution is made in the ultrapure water of mL, it is spare.
3. the phenolic resin micropore carbon ball of the functionalization of the load Au AgNPs detects antibody incubation content solution, preparation step It is rapid as follows:
(1) preparation of phenolic resin micropore carbon ball
20 mL ultrapure water water, 8 mL dehydrated alcohols are taken, 0.1 mL, the ammonium hydroxide that mass fraction is 25 % are added in beaker, stir Mix 10 ~ 20 min；It sequentially adds 0.2 ~ 0.4 g phenol and 0.25 ~ 0.35 mL, mass fraction is molten for 37% formaldehyde Liquid is stirred to react 24 h under 30 °C of waters bath with thermostatic control；100 °C of lower 24 h of aging in polytetrafluoroethylene (PTFE) autoclave are transferred to, Centrifuge washing, obtained solid dry 16 ~ 24 h in 40 °C of vacuum ovens；In mass ratio by dried solid and KOH 4:1 mixing, grinds 1h, is activated, after be put into tube type resistance furnace and be carbonized under the protection of nitrogen；Carbonization is completed Afterwards, it is taken out out of resistance furnace, with 1 h of salt acid soak of 10 %, centrifuge washing is dry, spare；
Specific step is as follows for the carbonization: being warming up to 350 °C with the speed of 1 °C/min, constant temperature keeps 1 h；Then with 2 °C/min is warming up to 700 °C, constant temperature keeps 2 h；After be cooled to room temperature；
(2) preparation of the phenolic resin micropore carbon ball of the functionalization of Au AgNPs is loaded
Three hydroxyls by the phenolic resin micropore carbon ball ultrasonic disperse of 30 ~ 50 mg in pH=8.5 of 15 mL, 10 mmol/L In aminomethane buffer, the Dopamine hydrochloride of 10 ~ 15 mg is added, shakes 24 h at room temperature；Addition 5 ~ The Au@AgNPs of the above-mentioned preparation of 7 mL continues to shake 1 h；Three times with ultrapure water centrifuge washing, it is done in 40 °C of vacuum ovens The phenolic resin micropore carbon ball solid powder of the functionalization of load Au AgNPs is made in dry 16 ~ 20 h；
(3) preparation of the phenolic resin micropore carbon ball detection antibody incubation content solution of the functionalization of Au AgNPs is loaded
By 2 ~ 6 mg load Au AgNPs functionalization phenolic resin micropore carbon ball ultrasonic disperse in 1 mL pH= In 7.0 phosphate buffer solution, add 100 μ L, 80 ~ 120 μ g/mL tumor-marker analyte detection antibody-solutions and The phosphate buffer solution of pH=7.0 of 900 μ L, 50 mmol/L vibrates in 4 °C of constant-temperature shaking incubators, hatches 12 h, system The phenolic resin micropore carbon ball detection antibody incubation content solution of the functionalization of Au AgNPs must be loaded, is saved backup under 4 °C.
4. the detection of tumor markers, steps are as follows:
(1) it is tested using electrochemical workstation with three-electrode system, saturated calomel electrode is reference electrode, platinum filament electricity Extremely auxiliary electrode, prepared sensor are working electrode, slow in pH=5.6 ~ 8.0 phosphate of 10 mL, 50 mmol/L It rushes in solution and is tested；
(2) used time m- current method detects analyte, and input voltage is -0.4 V, 0.1 s of sampling interval, operation 400 s of time；
(3) after background current tends towards stability, every 50 s to the phosphate-buffered of pH=7.0 of 10 mL, 50 mmol/L The hydrogen peroxide solution of 10 μ L, 5 mol/L, record current variation are injected in solution；
(4) working curve method is utilized, the concentration of tumor markers antigen in sample to be tested is obtained.
Tumor markers described above are selected from AFP or CEA.
Raw materials of the present invention can be bought in chemical reagents corporation or biopharmaceutical company.
Beneficial achievement of the invention
(1) using the phenolic resin micropore carbon ball of the poly-dopamine functionalization of load Au AgNPs as antibody marker. The phenolic resin hard carbon microballoon of poly-dopamine functionalization is three-dimensional micropore chondritic, and porous structure can promote electric active matter The absorption on the surface of the material of matter shortens transmission range of the ion between aperture, reduces the charge transfer resistance of material, improve Electric conductivity.Carbon ball has big specific surface area, and the carbon ball after functionalization can form stable chemistry with noble metal nano particles Key can effectively adsorb noble metal nano particles, while can also reduce the reuniting effect of nanoparticle.Gold and silver core-shell nano phase For monometallic nanoparticle, possess more catalysis points, concerted catalysis effect shown to the reduction reaction of hydrogen peroxide, Substantially increase the electrocatalysis characteristic of material.
(2) a kind of immunosensor of the phenolic resin micropore carbon ball of functionalization based on Au AgNPs is to tumor markers Detection, to AFP its range of linearity ng/mL of 20 fg/mL ~ 100, detection is limited to 6.7 fg/mL；To its range of linearity of CEA The ng/mL of 30 fg/mL ~ 100, detection are limited to 10 fg/mL；Show a kind of phenolic resin of functionalization based on Au@AgNPs The immunosensor of micropore carbon ball can achieve the purpose of Accurate Determining.
Specific embodiment
Now the present invention is further illustrated by specific embodiment, but not limited to this.
A kind of preparation side of the immunosensor of the phenolic resin micropore carbon ball of the functionalization based on Au AgNPs of embodiment 1 Method, steps are as follows:
(1) the glass-carbon electrode Al for being 4 mm by diameter2O3Polishing powder polishing, ultrapure water clean up；
(2) above-mentioned glass-carbon electrode is placed in the HAuCl of 0.1 %4In solution, using i-t method, at -0.2 V, operation 30 s；
(3) continue for the tumor markers capture antibody of 6 μ L, 8 μ g/mL to be added drop-wise to electrode surface, ultrapure water, 4 It is dry in °C refrigerator；
(4) continue the bovine serum albumin(BSA) BSA solution of 3 μ L, 0.8 mg/mL being added drop-wise to electrode surface, ultrapure water Electrode surface dries in 4 °C of refrigerators；
(5) the tumor markers antigenic solution of the various concentration of 6 μ L, the ng/mL of 20 fg/mL ~ 100, ultrapure water is added dropwise Rinse electrode surface, drying in 4 °C of refrigerators；
(6) by the phenolic resin micropore carbon ball detection antibody hatching of the functionalization of the load Au AgNPs of 6 μ L, 1 mg/mL Object solution, drop coating are dried on electrode surface, being placed in 4 °C of refrigerators, and a kind of phenolic aldehyde of functionalization based on Au@AgNPs is made The immunosensor of resin micropore carbon ball.
A kind of preparation side of the immunosensor of the phenolic resin micropore carbon ball of the functionalization based on Au AgNPs of embodiment 2 Method, steps are as follows:
(1) the glass-carbon electrode Al for being 4 mm by diameter2O3Polishing powder polishing, ultrapure water clean up；
(2) above-mentioned glass-carbon electrode is placed in the HAuCl of 0.1 %4In solution, using i-t method, at -0.2 V, operation 32 s；
(3) continue for the tumor markers capture antibody of 6 μ L, 10 μ g/mL to be added drop-wise to electrode surface, ultrapure water, 4 It is dry in °C refrigerator；
(4) continue the bovine serum albumin(BSA) BSA solution of 3 μ L, 1.2 mg/mL being added drop-wise to electrode surface, ultrapure water Electrode surface dries in 4 °C of refrigerators；
(5) the tumor markers antigenic solution of the various concentration of 6 μ L, the ng/mL of 20 fg/mL ~ 100, ultrapure water is added dropwise Rinse electrode surface, drying in 4 °C of refrigerators；
(6) the phenolic resin micropore carbon ball detection antibody of the functionalization of the load Au Ag NPs of 6 μ L, 2 mg/mL is incubated Compound solution, drop coating are dried on electrode surface, being placed in 4 °C of refrigerators, and a kind of phenol of functionalization based on Au@AgNPs is made The immunosensor of urea formaldehyde micropore carbon ball.
A kind of preparation side of the immunosensor of the phenolic resin micropore carbon ball of the functionalization based on Au AgNPs of embodiment 3 Method, steps are as follows:
(1) the glass-carbon electrode Al for being 4 mm by diameter2O3Polishing powder polishing, ultrapure water clean up；
(2) above-mentioned glass-carbon electrode is placed in the HAuCl of 0.1 %4In solution, using i-t method, at -0.2 V, operation 35 s；
(3) continue for the tumor markers capture antibody of 6 μ L, 12 μ g/mL to be added drop-wise to electrode surface, ultrapure water, 4 It is dry in °C refrigerator；
(4) continue the bovine serum albumin(BSA) BSA solution of 3 μ L, 1.5mg/mL being added drop-wise to electrode surface, ultrapure water Electrode surface dries in 4 °C of refrigerators；
(5) the tumor markers antigenic solution of the various concentration of 6 μ L, the ng/mL of 20 fg/mL ~ 100, ultrapure water is added dropwise Rinse electrode surface, drying in 4 °C of refrigerators；
(6) by the phenolic resin micropore carbon ball detection antibody hatching of the functionalization of the load Au AgNPs of 6 μ L, 3 mg/mL Object solution, drop coating are dried on electrode surface, being placed in 4 °C of refrigerators, and a kind of phenolic aldehyde of functionalization based on Au@AgNPs is made The immunosensor of resin micropore carbon ball.
Au@AgNPs as described in example 4, preparation step are as follows:
By the HAuCl of 64 mL, 1 mmol/L4Three neck round bottom flask is added in solution, and 115 °C of oil bath are heated at reflux 10 The sodium citrate of 12.5 mL, 38.8 mmol/L is added in min, until solution colour has yellow to become claret, it is molten that AuNPs is made Liquid；
The AuNPs solution of the above-mentioned preparation of 5 mL is taken, the silver nitrate water of 400 μ L, 7.1 mmol/L that will newly configure is added Solution concussion, becomes rufous from claret to solution, three times with ultrapure water centrifuge washing, ultrasonic disperse is ultrapure to 10 mL's Au@AgNPs solution is made in water, it is spare.
Au@AgNPs, preparation step described in embodiment 5 are as follows:
By the HAuCl of 64 mL, 2 mmol/L4Three neck round bottom flask is added in solution, and 115 °C of oil bath are heated at reflux 12 The sodium citrate of 13.0 mL, 38.8 mmol/L is added in min, until solution colour has yellow to become claret, it is molten that AuNPs is made Liquid；
The AuNPs solution of the above-mentioned preparation of 6 mL is taken, the silver nitrate water of 500 μ L, 7.1 mmol/L that will newly configure is added Solution concussion, becomes rufous from claret to solution, three times with ultrapure water centrifuge washing, ultrasonic disperse is ultrapure to 10 mL's Au@AgNPs solution is made in water, it is spare.
Au@Ag NPs, preparation step described in embodiment 6 are as follows:
By the HAuCl of 64 mL, 3 mmol/L4Three neck round bottom flask is added in solution, and 115 °C of oil bath are heated at reflux 15 The sodium citrate of 13.5 mL, 38.8 mmol/L is added in min, until solution colour has yellow to become claret, it is molten that AuNPs is made Liquid；
The AuNPs solution of the above-mentioned preparation of 7 mL is taken, the silver nitrate water of 600 μ L, 7.1 mmol/L that will newly configure is added Solution concussion, becomes rufous from claret to solution, three times with ultrapure water centrifuge washing, ultrasonic disperse is ultrapure to 10 mL's Au@AgNPs solution is made in water, it is spare.
The phenolic resin micropore carbon ball that the functionalization of Au AgNPs is loaded described in embodiment 7 detects antibody incubation content solution, Preparation step is as follows:
(1) preparation of phenolic resin micropore carbon ball
20 mL ultrapure water water, 8 mL dehydrated alcohols are taken, 0.1 mL, the ammonium hydroxide that mass fraction is 25 % are added in beaker, stir Mix 10 min；0.2 g phenol and 0.25 mL, the formalin that mass fraction is 37% are sequentially added, under 30 °C of waters bath with thermostatic control It is stirred to react 24 h；100 °C of lower 24 h of aging in polytetrafluoroethylene (PTFE) autoclave are transferred to, centrifuge washing, obtained solid is 40 Dry 16 h in °C vacuum oven；Dried solid is mixed with KOH 4:1 in mass ratio, 1h is ground, carries out at activation Reason, after be put into tube type resistance furnace and be carbonized under the protection of nitrogen；It after the completion of carbonization, is taken out out of resistance furnace, with 10 % 1 h of salt acid soak, centrifuge washing is dry, spare；
Specific step is as follows for the carbonization: being warming up to 350 °C with the speed of 1 °C/min, constant temperature keeps 1 h；Then with 2 °C/min is warming up to 700 °C, constant temperature keeps 2 h；After be cooled to room temperature；
(2) preparation of the phenolic resin micropore carbon ball of the functionalization of Au AgNPs is loaded
Trihydroxy methyl by the phenolic resin micropore carbon ball ultrasonic disperse of 30 mg in pH=8.5 of 15 mL, 10 mmol/L In aminomethane buffer solution, the Dopamine hydrochloride of 10 mg is added, shakes 24 h at room temperature；The above-mentioned system of 5 mL is added Standby Au@AgNPs continues to shake 1 h；Three times with ultrapure water centrifuge washing, dry 16 h in 40 °C of vacuum ovens, are made negative Carry the phenolic resin micropore carbon ball solid powder of the functionalization of Au AgNPs；
(3) preparation of the phenolic resin micropore carbon ball detection antibody incubation content solution of the functionalization of Au AgNPs is loaded
By the phenolic resin micropore carbon ball ultrasonic disperse of the functionalization of the load Au AgNPs of 2 mg in pH=7.0 of 1 mL Phosphate buffer solution in, add the tumor-marker analyte detection antibody-solutions and 900 μ L, 50 of 100 μ L, 80 μ g/mL The phosphate buffer solution of pH=7.0 of mmol/L vibrates in 4 °C of constant-temperature shaking incubators, hatches 12 h, and load Au@is made The phenolic resin micropore carbon ball of the functionalization of AgNPs detects antibody incubation content solution, saves backup under 4 °C.
The phenolic resin micropore carbon ball that the functionalization of Au AgNPs is loaded described in embodiment 8 detects antibody incubation content solution, Preparation step is as follows:
(1) preparation of phenolic resin micropore carbon ball
20 mL ultrapure water water, 8 mL dehydrated alcohols are taken, 0.1 mL, the ammonium hydroxide that mass fraction is 25 % are added in beaker, stir Mix 15 min；0.3 g phenol and 0.30 mL, the formalin that mass fraction is 37% are sequentially added, under 30 °C of waters bath with thermostatic control It is stirred to react 24 h；100 °C of lower 24 h of aging in polytetrafluoroethylene (PTFE) autoclave are transferred to, centrifuge washing, obtained solid is 40 Dry 20 h in °C vacuum oven；Dried solid is mixed with KOH 4:1 in mass ratio, 1h is ground, carries out at activation Reason, after be put into tube type resistance furnace and be carbonized under the protection of nitrogen；It after the completion of carbonization, is taken out out of resistance furnace, with 10 % 1 h of salt acid soak, centrifuge washing is dry, spare；
Specific step is as follows for the carbonization: being warming up to 350 °C with the speed of 1 °C/min, constant temperature keeps 1 h；Then with 2 °C/min is warming up to 700 °C, constant temperature keeps 2 h；After be cooled to room temperature；
(2) preparation of the phenolic resin micropore carbon ball of the functionalization of Au AgNPs is loaded
Trihydroxy methyl by the phenolic resin micropore carbon ball ultrasonic disperse of 40 mg in pH=8.5 of 15 mL, 10 mmol/L In aminomethane buffer solution, the Dopamine hydrochloride of 12.5 mg is added, shakes 24 h at room temperature；The above-mentioned of 6 mL is added The Au@AgNPs of preparation continues to shake 1 h；Three times with ultrapure water centrifuge washing, dry 18 h in 40 °C of vacuum ovens, are made Load the phenolic resin micropore carbon ball solid powder of the functionalization of Au AgNPs；
(3) preparation of the phenolic resin micropore carbon ball detection antibody incubation content solution of the functionalization of Au Ag NPs is loaded
By the phenolic resin micropore carbon ball ultrasonic disperse of the functionalization of the load Au AgNPs of 4 mg in pH=7.0 of 1 mL Phosphate buffer solution in, add the tumor-marker analyte detection antibody-solutions and 900 μ L, 50 of 100 μ L, 100 μ g/mL The phosphate buffer solution of pH=7.0 of mmol/L vibrates in 4 °C of constant-temperature shaking incubators, hatches 12 h, and load Au@is made The phenolic resin micropore carbon ball of the functionalization of AgNPs detects antibody incubation content solution, saves backup under 4 °C.
The phenolic resin micropore carbon ball that the functionalization of Au AgNPs is loaded described in embodiment 9 detects antibody incubation content solution, Preparation step is as follows:
(1) preparation of phenolic resin micropore carbon ball
20 mL ultrapure water water, 8 mL dehydrated alcohols are taken, 0.1 mL, the ammonium hydroxide that mass fraction is 25 % are added in beaker, stir Mix 20 min；0.4 g phenol and 0.35 mL, the formalin that mass fraction is 37% are sequentially added, under 30 °C of waters bath with thermostatic control It is stirred to react 24 h；100 °C of lower 24 h of aging in polytetrafluoroethylene (PTFE) autoclave are transferred to, centrifuge washing, obtained solid is 40 Dry 24 h in °C vacuum oven；Dried solid is mixed with KOH 4:1 in mass ratio, 1h is ground, carries out at activation Reason, after be put into tube type resistance furnace and be carbonized under the protection of nitrogen；It after the completion of carbonization, is taken out out of resistance furnace, with 10 % 1 h of salt acid soak, centrifuge washing is dry, spare；
Specific step is as follows for the carbonization: being warming up to 350 °C with the speed of 1 °C/min, constant temperature keeps 1 h；Then with 2 °C/min is warming up to 700 °C, constant temperature keeps 2 h；After be cooled to room temperature；
(2) preparation of the phenolic resin micropore carbon ball of the functionalization of Au AgNPs is loaded
Trihydroxy methyl by the phenolic resin micropore carbon ball ultrasonic disperse of 50 mg in pH=8.5 of 15 mL, 10 mmol/L In aminomethane buffer solution, the Dopamine hydrochloride of 15 mg is added, shakes 24 h at room temperature；The above-mentioned system of 7 mL is added Standby Au@AgNPs continues to shake 1 h；Three times with ultrapure water centrifuge washing, dry 20 h in 40 °C of vacuum ovens, are made negative Carry the phenolic resin micropore carbon ball solid powder of the functionalization of Au AgNPs；
(3) preparation of the phenolic resin micropore carbon ball detection antibody incubation content solution of the functionalization of Au AgNPs is loaded
By the phenolic resin micropore carbon ball ultrasonic disperse of the functionalization of the load Au AgNPs of 6 mg in pH=7.0 of 1 mL Phosphate buffer solution in, add the tumor-marker analyte detection antibody-solutions and 900 μ L, 50 of 100 μ L, 120 μ g/mL The phosphate buffer solution of pH=7.0 of mmol/L vibrates in 4 °C of constant-temperature shaking incubators, hatches 12 h, and load Au@is made The phenolic resin micropore carbon ball of the functionalization of AgNPs detects antibody incubation content solution, saves backup under 4 °C.
The detection of 10 tumor markers AFP of embodiment, steps are as follows:
(1) it is tested using electrochemical workstation with three-electrode system, saturated calomel electrode is reference electrode, platinum filament electricity Extremely auxiliary electrode, prepared sensor are working electrode, slow in pH=5.6 ~ 8.0 phosphate of 10 mL, 50 mmol/L It rushes in solution and is tested；
(2) used time m- current method detects analyte, and input voltage is -0.4 V, 0.1 s of sampling interval, operation 400 s of time；
(3) after background current tends towards stability, every 50 s to the phosphate-buffered of pH=7.0 of 10 mL, 50 mmol/L The hydrogen peroxide solution of 10 μ L, 5 mol/L, record current variation are injected in solution；
(4) working curve method is utilized, the range of linearity for measuring AFP in sample is the ng/mL of 20 fg/mL ~ 100, detection It is limited to 6.7 fg/mL.
The detection of 11 tumor markers CEA of embodiment
CEA in sample is detected according to the method for embodiment 10, the range of linearity ng/mL of 30 fg/mL ~ 100, Detection is limited to 10 fg/mL.

Claims (5)

1. a kind of preparation method of the immunosensor of the phenolic resin micropore carbon ball of functionalization based on Au AgNPs, feature It is, steps are as follows:
(1) the glass-carbon electrode Al for being 4 mm by diameter2O3Polishing powder polishing, ultrapure water clean up；
(2) above-mentioned glass-carbon electrode is placed in the HAuCl of 0.1 %4In solution, using i-t method, at -0.2 V, operation 30 ~ 35 s；
(3) continue for the tumor markers capture antibody of 6 μ L, 8 ~ 12 μ g/mL to be added drop-wise to electrode surface, ultrapure water, 4 It is dry in °C refrigerator；
(4) continue for the bovine serum albumin(BSA) BSA solution of 3 μ L, 0.8 ~ 1.5mg/mL to be added drop-wise to electrode surface, ultrapure water punching Electrode surface is washed, is dried in 4 °C of refrigerators；
(5) the tumor markers antigenic solution of the various concentration of 6 μ L, the ng/mL of 20 fg/mL ~ 100, ultrapure water is added dropwise Electrode surface, it is dry in 4 °C of refrigerators；
(6) by the phenolic resin micropore carbon ball detection antibody hatching of the functionalization of the load Au AgNPs of 6 μ L, 1 ~ 3 mg/mL Object solution, drop coating are dried on electrode surface, being placed in 4 °C of refrigerators, and a kind of phenolic aldehyde of functionalization based on Au@AgNPs is made The immunosensor of resin micropore carbon ball；
The phenolic resin micropore carbon ball of the functionalization of the load Au AgNPs detects antibody incubation content solution, which is characterized in that Preparation step is as follows:
(1) preparation of phenolic resin micropore carbon ball
20 mL ultrapure waters, 8 mL dehydrated alcohols are taken, 0.1 mL, the ammonium hydroxide that mass fraction is 25 % are added in beaker, stirring 10 ~ 20 min；0.2 ~ 0.4 g phenol and 0.25 ~ 0.35 mL, the formalin that mass fraction is 37% are sequentially added, at 30 ° 24 h are stirred to react under C water bath with thermostatic control；Transfer to 100 °C of lower 24 h of aging in polytetrafluoroethylene (PTFE) autoclave, centrifuge washing, Obtained solid dry 16 ~ 24 h in 40 °C of vacuum ovens；Dried solid is mixed with KOH 4:1 in mass ratio, Grind 1h, be activated, after be put into tube type resistance furnace and be carbonized under the protection of nitrogen；After the completion of carbonization, from electricity It hinders in furnace and takes out, with 1 h of salt acid soak of 10 %, centrifuge washing is dry, spare；
Specific step is as follows for the carbonization: being warming up to 350 °C with the speed of 1 °C/min, constant temperature keeps 1 h；Then with 2 °C/ Min is warming up to 700 °C, and constant temperature keeps 2 h；After be cooled to room temperature；
(2) preparation of the phenolic resin micropore carbon ball of the functionalization of Au AgNPs is loaded
Trihydroxy methyl by the phenolic resin micropore carbon ball ultrasonic disperse of 30 ~ 50 mg in pH=8.5 of 15 mL, 10 mmol/L In aminomethane buffer solution, the Dopamine hydrochloride of 10 ~ 15 mg is added, shakes 24 h at room temperature；5 ~ 7 mL are added Above-mentioned preparation Au@AgNPs continue shake 1 h；Three times with ultrapure water centrifuge washing, dry 16 in 40 °C of vacuum ovens The phenolic resin micropore carbon ball solid powder of the functionalization of load Au AgNPs is made in ~ 20 h；
(3) preparation of the phenolic resin micropore carbon ball detection antibody incubation content solution of the functionalization of Au AgNPs is loaded
By the phenolic resin micropore carbon ball ultrasonic disperse of the functionalization of the load Au AgNPs of 2 ~ 6 mg in pH=7.0 of 1 mL Phosphate buffer solution in, add the tumor-marker analyte detection antibody-solutions and 900 μ of 100 μ L, 80 ~ 120 μ g/mL L, the phosphate buffer solution of pH=7.0 of 50 mmol/L vibrates in 4 °C of constant-temperature shaking incubators, hatches 12 h, is made negative The phenolic resin micropore carbon ball for carrying the functionalization of Au AgNPs detects antibody incubation content solution, saves backup under 4 °C.
2. a kind of immunosensor of the phenolic resin micropore carbon ball of the functionalization based on Au AgNPs as described in claim 1 Preparation method, the Au@AgNPs, which is characterized in that preparation step is as follows:
By the HAuCl of 64 mL, 1 ~ 3 mmol/L4Three neck round bottom flask is added in solution, and 115 °C of oil bath are heated at reflux 10 ~ 15 The sodium citrate of 12.5 ~ 13.5 mL, 38.8 mmol/L is added in min, until solution colour has yellow to become claret, is made AuNPs solution；
The AuNPs solution of the above-mentioned preparation of 5 ~ 7 mL is taken, the nitre of 400 ~ 600 μ L, 7.1 mmol/L that will newly configure is added Sour silver aqueous solution concussion, becomes rufous from claret to solution, three times with ultrapure water centrifuge washing, ultrasonic disperse to 10 mL Ultrapure water in be made Au@AgNPs solution, it is spare.
3. a kind of immunosensor of the phenolic resin micropore carbon ball of the functionalization based on Au AgNPs as described in claim 1 Preparation method, which is characterized in that the tumor markers be selected from AFP or CEA.
4. a kind of phenolic resin microporous carbon of functionalization based on Au@AgNPs of preparation method preparation as described in claim 1 The immunosensor of ball, for the detection of tumor markers, detecting step is as follows:
(1) it is tested using electrochemical workstation with three-electrode system, saturated calomel electrode is reference electrode, and platinum electrode is Auxiliary electrode, prepared sensor is working electrode, molten in pH=5.6 ~ 8.0 phosphate-buffereds of 10 mL, 50 mmol/L It is tested in liquid；
(2) used time m- current method detects analyte, and input voltage is -0.4 V, 0.1 s of sampling interval, runing time 400 s；
(3) after background current tends towards stability, every 50 s to the phosphate buffer solution of pH=7.0 of 10 mL, 50 mmol/L The hydrogen peroxide solution of 10 μ L of middle injection, 5 mol/L, record current variation.
5. a kind of immune sensing of the phenolic resin micropore carbon ball of the functionalization based on Au AgNPs as claimed in claim 4 Device, which is characterized in that the tumor markers are selected from AFP or CEA.
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