CN105572356B - A kind of preparation method and application of breast cancer tumour marker immunosensor - Google Patents

A kind of preparation method and application of breast cancer tumour marker immunosensor Download PDF

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CN105572356B
CN105572356B CN201610104965.4A CN201610104965A CN105572356B CN 105572356 B CN105572356 B CN 105572356B CN 201610104965 A CN201610104965 A CN 201610104965A CN 105572356 B CN105572356 B CN 105572356B
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carbon nanotube
benzenethiol
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李月云
李明党
董云会
王平
刘青
刘会
陈磊
张道鹏
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Shandong University of Technology
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    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
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Abstract

The invention belongs to immunoassay and biosensor technique field, there is provided a kind of preparation method and application of breast cancer tumour marker immunosensor；The electrochemical immunosensor marked to benzenethiol functionalization Sulfonated carbon nanotube using Nano silver grain@, the detection of common breast cancer tumour mark is realized, with high specificity, sensitivity is high, test limit is low, has important scientific meaning and application value to the detection of breast cancer.

Description

A kind of preparation method and application of breast cancer tumour marker immunosensor
Technical field
The invention belongs to novel function nanometer material, immunoassay and biosensor technique field, there is provided a kind of mammary gland The preparation method and application of tumor marker immunosensor.
Background technology
If cancer can be early diagnosed, treated in time, 1/3rd cancer can be healing；It is various swollen in serum Early diagnosis of the sensitive determination of tumor markers to cancer plays an important role；Hence set up quick, sensitive and accurate detection blood The analysis method of tumor markers becomes current study hotspot in clear, causes everybody extensive concern.
Breast cancer is one of most common malignant tumour of women, and according to relevant statistics, the incidence of disease of breast cancer accounts for whole body The 10% of various malignant tumours, is a kind of common cancer of the physically and mentally healthy even threat to life for having a strong impact on women；Cancer embryo Antigen（CEA）, the common tumor markers such as CA125 (CA125), CA15-3 (CA15-3), for mammary gland The diagnosis of cancer can play a role.
Interlayer type electrochemical immunosensor combines the immuno analytical method of high specific and highly sensitive electrochemical credit Analysis technology, with sensitivity it is high, prepare that simple, detection is quick, low cost and other advantages, in clinical analysis, environmental monitoring, food peace There is important application value in the fields such as full control, biological monitoring.
With the development of nanometer technology, various nano-particles are widely used in the building process of sensor.Graphene There is substantial amounts of carboxyl functional group on surface, and with big specific surface area, good electron transmission ability and catalytic performance, can be effective Adsorb immobilized antibody；The Graphene of load gold nano particle has more preferable electric conductivity；Nano silver grain has catalysis well Performance, the premium properties not only to benzenethiol functionalization Sulfonated carbon nanotube with CNT, and by sulfonation, functionalization Even more considerably increase the ability of immobilized antibody, thus Nano silver grain@to benzenethiol functionalization Sulfonated carbon nanotube as inspection Survey antibody labeling thing preferably can realize amplifying to determining signal, improve the sensitivity of tumor-marker analyte detection.
The content of the invention
The invention provides a kind of preparation method and application of breast cancer tumour marker immunosensor, realize to breast The super sensitivity detection of adenocarcinoma tumor mark.
An object of the present invention is to provide a kind of preparation method of breast cancer tumour marker immunosensor.
The second object of the present invention is by a kind of prepared breast cancer tumour marker immunosensor, for breast cancer The detection of tumor markers.
Technical scheme, comprises the following steps
1. a kind of preparation method of breast cancer tumour marker immunosensor, step is as follows：
（1）By the glass-carbon electrode Al of a diameter of 3 ~ 5mm2O3Polishing powder is polished, and ultra-pure water is cleaned up；
（2）Take 6 μ L, the load gold nano particle Graphene of 0.5 ~ 2.0mg/mL is added drop-wise to electrode surface, dry in the air at room temperature It is dry, ultrapure water electrode surface is used, dry；
（3）Continue the tumor markers capture antibody A b of 6 μ L, 5 ~ 15 μ g/mL1It is added drop-wise to electrode surface, ultra-pure water Rinse, dried in 4 DEG C of refrigerators；
（4）The BSA solution of 3 μ L, 1 ~ 2 mg/mL is added drop-wise to electrode surface by continuation, to non-on enclosed-electrode surface Activity specific site, ultrapure water electrode surface dries in 4 DEG C of refrigerators；6 μ L, 0.001 ~ 30 ng/mL is added dropwise A series of tumor markers antigen A g solution of various concentrations, ultrapure water electrode surface is dried in 4 DEG C of refrigerators；
（5）By 6 μ L, 1 ~ 3 mg/mL Nano silver grain@to benzenethiol functionalization Sulfonated carbon nanotube detection antibody Ab2Incubation content solution, drop coating is placed in 4 DEG C of refrigerators and dries in electrode surface, and a kind of immune biography of breast cancer tumour mark is obtained Sensor.
2. a kind of preparation method of breast cancer tumour marker immunosensor, the preparation of the associated materials, including with Under several steps：
（1）The preparation of load gold nano particle Graphene
1. the preparation of amination graphene
Take 40 ~ 60mg graphene oxides to be dissolved in the dry toluene of 10mL, add the ethoxy of 0.2mL 3- aminopropyls three Base silane, oil bath heating to 60 ~ 70˚C, magnetic agitation 1.5h, after 110˚Drying obtains amination graphene under C；
2. the preparation of solution of gold nanoparticles
By the HAuCl that 1 ~ 2mL, mass fraction are 1%4Be added in 99mL ultra-pure waters, be heated to boiling add 1.5 ~ 3.5mL, mass fraction are 1% sodium citrate solution, flow back 15 ~ 30min, after be cooled to room temperature, obtain the Jenner of claret Rice corpuscles solution；
3. the preparation of the Graphene of load gold nano particle
Take 20 ~ 30mg amination graphenes and be added to above-mentioned steps 2. in obtained solution of gold nanoparticles, vibrate 12h, Load gold nano particle Graphene is obtained；
（2）Nano silver grain@is to benzenethiol functionalization Sulfonated carbon nanotube detection antibody Ab2The preparation of incubation content solution
1. the preparation of Sulfonated carbon nanotube
The multi-walled carbon nano-tubes for weighing 0.5 ~ 1.5g is placed in 250mL there-necked flasks, sequentially add the 66mL concentrated sulfuric acids and 22mL concentrated nitric acids, 120˚30 ~ 50min is reacted under C oil baths, after being cooled to room temperature, gained suspension 500mL beakers is moved into, Plus the dilution of 300mL ultra-pure waters, suction filtration, with milli-Q water to filtrate into neutrality, gained solid is in 75 ~ 85˚C vacuum drying chambers Interior 10 ~ 14h of drying, obtains Sulfonated carbon nanotube；
2. to the preparation of benzenethiol functionalization Sulfonated carbon nanotube
9 ~ 15mg Sulfonated carbon nanotubes are sequentially added in 250mL round-bottomed flasks, the hydrochloric acid solution of 50mL, 1mol/L surpasses Sound disperses 1h, adds 1.0g 4,4 '-diaminourea diphenyl disulfide, ice bath to be cooled to 0˚C, is added slowly with stirring 10mL sub- Sodium nitrate aqueous solution, reacts 1h, is warming up to 40˚C, continues to stir 1.5 ~ 2.5 h of lower reaction, with 0.45 μm of poly tetrafluoroethylene Suction filtration, milli-Q water 3 times, absolute ethanol washing 3 times in the solid ultrasonic disperse for obtaining to 50mL ethanol, is sequentially added 50mL acetonitriles and 520mg triphenyl phosphorus, stir 1h, with 0.45 μm of poly tetrafluoroethylene suction filtration, with absolute ethanol washing 5 times, Gained solid 80˚12h is dried in C vacuum drying chambers, is obtained to benzenethiol functionalization Sulfonated carbon nanotube；
Described sodium nitrite in aqueous solution is that 280mg solid sodium nitrites are dissolved in into 10mL ultra-pure waters to be obtained；
3. the preparation of silver nano-particle solution
The silver nitrate and 1mL of 1mL, 50mmol/L, 5% sodium citrate are added in 48mL ultra-pure waters under agitation, 45 ~ 55mg sodium borohydrides are subsequently adding, 10min is reacted under agitation, solution is changed into brown color, lasting stirring, to solution colour No longer change, be cooled to room temperature, 5min is centrifuged under rotating speed 5000rpm, the supernatant of gained is silver nano-particle solution；
4. Nano silver grain@is to benzenethiol functionalization Sulfonated carbon nanotube detection antibody Ab2The preparation of incubation content solution
5 ~ 10 mg are taken to be added in 20 ~ 40mL silver nano-particle solutions benzenethiol functionalization Sulfonated carbon nanotube, 10 ~ 14h, centrifugation, 80 are vibrated under 130rpm rotating speeds˚Dried in C vacuum drying chambers, Nano silver grain@is obtained to benzenethiol function Change Sulfonated carbon nanotube；
The Nano silver grain@of 2 ~ 6 mg is distributed in 1mL ultra-pure waters to benzenethiol functionalization Sulfonated carbon nanotube, is added The tumor markers detection antibody Ab of 100 μ L, 80 ~ 120 μ g/mL2The phosphate of pH 7.4 of solution and 900 μ L, 50mmol/L delays Rush solution, 4˚12 h of hatching are vibrated in C constant-temperature shaking incubators；4˚Under C, 15 min are centrifuged under 6000 rpm rotating speeds, obtain Lower sediment, the pH=7.4 PBSs centrifuge washing of addition 1mL, 50mmol/L 1 time, obtains lower sediment, finally adds Enter the pH=7.4 PBSs of 1mL, 50mmol/L, Nano silver grain@is obtained to benzenethiol functionalization sulfonation carbon nanometer Pipe detection antibody Ab2Incubation content solution, 4˚Saved backup under C.
A kind of breast cancer tumour marker immunosensor, for the detection of breast cancer tumour mark, step is as follows：
（1）Tested with three-electrode system using electrochemical workstation, saturated calomel electrode is reference electrode, platinum filament electricity Extremely auxiliary electrode, prepared sensor is working electrode, in 10 mL, the phosphate of pH 5.10 ~ 7.98 of 50 mmol/L Tested in cushioning liquid；
（2）Used time, m- current method was detected to analyte, and input voltage is -0.4 V, the s of sampling interval 0.1, operation The s of time 400；
（3）It is molten to the pH=7.4 phosphate-buffereds of 10 mL, 50 mmol/L every 50 s after background current tends towards stability 10 μ L, the hydrogen peroxide solution of 5 mol/L, record current change are injected in liquid.
Tumor markers described above is selected from one of following：Carcinomebryonic antigen, CA125, CA15-3.
Raw materials of the present invention can be bought in chemical reagents corporation or biopharmaceutical company.
Useful achievement of the invention
（1）Present invention uses load gold nano particle Graphene as substrate, Graphene has big specific surface area, has Substantial amounts of carboxyl is contained on the binding site of substantial amounts of antibody, surface, and carboxyl can effectively be combined with the amino on antibody, realizes anti- Body it is immobilized, golden nanometer particle has good electric conductivity, and the big immobilized antibody of specific surface area, can speed up electronics biography Pass, play an important roll for improving transducer sensitivity.
（2）Using Nano silver grain@to benzenethiol functionalization Sulfonated carbon nanotube as detection antibody label, carbon nanometer Pipe has high intensity and good electric conductivity, and has catalytic action to hydrogen peroxide, and the carbon to benzenethiol functionalization sulfonation is received Mitron has preferably dispersiveness and biocompatibility, and the group of functionalization preferably antibody can be combined, Nano silver grain pair Hydrogen peroxide also has catalytic action, therefore, Nano silver grain@is urged hydrogen peroxide benzenethiol functionalization Sulfonated carbon nanotube Change effect is in multiple amplification, so as to improve the sensitivity of sensor, reduces test limit.
（3）A kind of detection of breast cancer tumour marker immunosensor to different breast cancer tumour marks, it is to cancer The range of linearity of embryonal antigen is 0.5 pg/mL ~ 25 ng/mL, and detection is limited to 0.12 pg/mL；To the linear model of CA125 It is 0.5pg/mL ~ 20ng/mL to enclose, and detection is limited to 0.12 pg/mL；The range of linearity to CA15-3 is 1 pg/mL ~ 20 Ng/mL, detection is limited to 0.21 pg/mL；Show that a kind of breast cancer tumour marker immunosensor can reach Accurate Determining Purpose.
Specific embodiment
Now the present invention is further illustrated by specific embodiment, but not limited to this.
A kind of preparation of breast cancer tumour marker immunosensor of embodiment 1
（1）By the glass-carbon electrode Al of a diameter of 3mm2O3Polishing powder is polished, and ultra-pure water is cleaned up；
（2）Take 6 μ L, the load gold nano particle Graphene of 0.5 mg/mL is added drop-wise to electrode surface, dry at room temperature, use Ultrapure water electrode surface, dries；
（3）Continue the tumor markers capture antibody A b of 6 μ L, 5 μ g/mL1It is added drop-wise to electrode surface, ultra-pure water punching Wash, dried in 4 DEG C of refrigerators；
（4）The BSA solution of 3 μ L, 1 mg/mL is added drop-wise to electrode surface by continuation, to non-specific on enclosed-electrode surface Property avtive spot, ultrapure water electrode surface dries in 4 DEG C of refrigerators；6 μ L are added dropwise, the one of 0.001 ~ 30 ng/mL are The tumor markers antigen A g solution of row various concentrations, ultrapure water electrode surface is dried in 4 DEG C of refrigerators；
（5）By 6 μ L, 1 mg/mL Nano silver grain@to benzenethiol functionalization Sulfonated carbon nanotube detection antibody Ab2Incubate Compound solution, drop coating is placed in 4 DEG C of refrigerators and dries in electrode surface, and a kind of breast cancer tumour marker immunosensor is obtained.
A kind of preparation of breast cancer tumour marker immunosensor of embodiment 2
（1）By the glass-carbon electrode Al of a diameter of 4mm2O3Polishing powder is polished, and ultra-pure water is cleaned up；
（2）The load gold nano particle Graphene for taking 6 μ L, 1.0mg/mL is added drop-wise to electrode surface, dries at room temperature, with super Pure water rinsing electrode surface, dries；
（3）Continue the tumor markers capture antibody A b of 6 μ L, 10 μ g/mL1It is added drop-wise to electrode surface, ultra-pure water punching Wash, dried in 4 DEG C of refrigerators；
（4）The BSA solution of 3 μ L, 1.5 mg/mL is added drop-wise to electrode surface by continuation, to non-spy on enclosed-electrode surface Specific activities site, ultrapure water electrode surface dries in 4 DEG C of refrigerators；6 μ L, the one of 0.001 ~ 30 ng/mL is added dropwise The tumor markers antigen A g solution of serial various concentrations, ultrapure water electrode surface is dried in 4 DEG C of refrigerators；
（5）By 6 μ L, 2 mg/mL Nano silver grain@to benzenethiol functionalization Sulfonated carbon nanotube detection antibody Ab2Incubate Compound solution, drop coating is placed in 4 DEG C of refrigerators and dries in electrode surface, and a kind of breast cancer tumour marker immunosensor is obtained.
A kind of preparation of breast cancer tumour marker immunosensor of embodiment 3
（1）By the glass-carbon electrode Al of a diameter of 5 mm2O3Polishing powder is polished, and ultra-pure water is cleaned up；
（2）Take 6 μ L, the load gold nano particle Graphene of 2.0 mg/mL is added drop-wise to electrode surface, dry at room temperature, use Ultrapure water electrode surface, dries；
（3）Continue the tumor markers capture antibody A b of 6 μ L, 15 μ g/mL1It is added drop-wise to electrode surface, ultra-pure water punching Wash, dried in 4 DEG C of refrigerators；
（4）The BSA solution of 3 μ L, 2 mg/mL is added drop-wise to electrode surface by continuation, to non-specific on enclosed-electrode surface Property avtive spot, ultrapure water electrode surface dries in 4 DEG C of refrigerators；6 μ L are added dropwise, the one of 0.001 ~ 30 ng/mL are The tumor markers antigen A g solution of row various concentrations, ultrapure water electrode surface is dried in 4 DEG C of refrigerators；
（5）By 6 μ L, 3 mg/mL Nano silver grain@to benzenethiol functionalization Sulfonated carbon nanotube detection antibody Ab2Incubate Compound solution, drop coating is placed in 4 DEG C of refrigerators and dries in electrode surface, and a kind of breast cancer tumour marker immunosensor is obtained.
A kind of preparation method of breast cancer tumour marker immunosensor of embodiment 4, the preparation of the associated materials, bag Include following steps：
（1）The preparation of load gold nano particle Graphene
1. the preparation of amination graphene
Take 40 mg graphene oxides to be dissolved in the dry toluene of 10mL, add 0.2mL 3- aminopropyl-triethoxy silicon Alkane, oil bath heating to 60˚C, magnetic agitation 1.5h, after 110˚Drying obtains amination graphene under C；
2. the preparation of solution of gold nanoparticles
By the HAuCl that 1mL, mass fraction are 1%4It is added in 99mL ultra-pure waters, is heated to boiling and adds 1.5mL, matter Amount fraction be 1% sodium citrate solution, flow back 15 min, after be cooled to room temperature, obtain the solution of gold nanoparticles of claret；
3. the preparation of the Graphene of load gold nano particle
Take 20mg amination graphenes and be added to above-mentioned steps 2. in obtained solution of gold nanoparticles, vibrate 12h, be obtained Load gold nano particle Graphene；
（2）Nano silver grain@is to benzenethiol functionalization Sulfonated carbon nanotube detection antibody Ab2The preparation of incubation content solution
1. the preparation of Sulfonated carbon nanotube
The multi-walled carbon nano-tubes for weighing 0.5g is placed in 250mL there-necked flasks, sequentially adds the 66mL concentrated sulfuric acids and 22mL is dense Nitric acid, 120˚30min is reacted under C oil baths, it is after being cooled to room temperature, gained suspension immigration 500mL beakers, plus 300mL is ultrapure Water dilutes, suction filtration, and with milli-Q water to filtrate into neutrality, gained solid is in 75˚10h is dried in C vacuum drying chambers, sulphur is obtained Carbon nano tube；
2. to the preparation of benzenethiol functionalization Sulfonated carbon nanotube
9mg Sulfonated carbon nanotubes, the hydrochloric acid solution of 50mL, 1mol/L, ultrasound are sequentially added in 250mL round-bottomed flasks Dispersion 1h, adds 1.0g 4,4 '-diaminourea diphenyl disulfide, ice bath to be cooled to 0˚C, is added slowly with stirring 10mL nitrous Acid sodium aqueous solution, reacts 1h, is warming up to 40˚C, continues to stir 1.5 h of lower reaction, with 0.45 μm of poly tetrafluoroethylene suction filtration, Milli-Q water 3 times, absolute ethanol washing 3 times in the solid ultrasonic disperse for obtaining to 50mL ethanol, sequentially adds 50mL acetonitriles With 520mg triphenyl phosphorus, 1h is stirred, with 0.45 μm of poly tetrafluoroethylene suction filtration, with absolute ethanol washing 5 times, gained solid 80˚12h is dried in C vacuum drying chambers, is obtained to benzenethiol functionalization Sulfonated carbon nanotube；
Described sodium nitrite in aqueous solution is that 280mg solid sodium nitrites are dissolved in into 10mL ultra-pure waters to be obtained；
3. the preparation of silver nano-particle solution
The silver nitrate and 1mL of 1mL, 50mmol/L, 5% sodium citrate are added in 48mL ultra-pure waters under agitation, 45mg sodium borohydrides are subsequently adding, 10min is reacted under agitation, solution is changed into brown color, lasting stirring, to solution colour no longer Change, is cooled to room temperature, and 5min is centrifuged under rotating speed 5000rpm, and the supernatant of gained is silver nano-particle solution；
4. Nano silver grain@is to benzenethiol functionalization Sulfonated carbon nanotube detection antibody Ab2The preparation of incubation content solution
Take 5 mg to be added in 20mL silver nano-particle solutions benzenethiol functionalization Sulfonated carbon nanotube, 130rpm turns Speed 10 h of lower vibration, centrifugation, 80˚Dried in C vacuum drying chambers, Nano silver grain@is obtained benzenethiol functionalization sulfonation carbon is received Mitron；
The Nano silver grain@of 2 mg is distributed in 1mL ultra-pure waters to benzenethiol functionalization Sulfonated carbon nanotube, is added The tumor markers detection antibody Ab of 100 μ L, 80 μ g/mL2The phosphate-buffereds of pH 7.4 of solution and 900 μ L, 50mmol/L are molten Liquid, 4˚12 h of hatching are vibrated in C constant-temperature shaking incubators；4˚Under C, 15 min are centrifuged under 6000 rpm rotating speeds, obtain lower floor Precipitation, the pH=7.4 PBSs centrifuge washing of addition 1mL, 50mmol/L 1 time, obtains lower sediment, is eventually adding The pH=7.4 PBSs of 1mL, 50mmol/L, are obtained Nano silver grain@to benzenethiol functionalization Sulfonated carbon nanotube Detection antibody Ab2Incubation content solution, 4˚Saved backup under C.
A kind of preparation method of breast cancer tumour marker immunosensor of embodiment 5, the preparation of the associated materials, bag Include following steps：
（1）The preparation of load gold nano particle Graphene
1. the preparation of amination graphene
Take 50mg graphene oxides to be dissolved in the dry toluene of 10mL, add 0.2mL 3- aminopropyl-triethoxy silicon Alkane, oil bath heating to 65˚C, magnetic agitation 1.5h, after 110˚Drying obtains amination graphene under C；
2. the preparation of solution of gold nanoparticles
By the HAuCl that 1.5mL, mass fraction are 1%4Be added in 99mL ultra-pure waters, be heated to boiling add 2.5mL, Mass fraction is 1% sodium citrate solution, flow back 20min, after be cooled to room temperature, obtain the solution of gold nanoparticles of claret；
3. the preparation of the Graphene of load gold nano particle
Take 25mg amination graphenes and be added to above-mentioned steps 2. in obtained solution of gold nanoparticles, vibrate 12h, be obtained Load gold nano particle Graphene；
（2）Nano silver grain@is to benzenethiol functionalization Sulfonated carbon nanotube detection antibody Ab2The preparation of incubation content solution
1. the preparation of Sulfonated carbon nanotube
The multi-walled carbon nano-tubes for weighing 1.0g is placed in 250mL there-necked flasks, sequentially adds the 66mL concentrated sulfuric acids and 22mL is dense Nitric acid, 120˚40min is reacted under C oil baths, it is after being cooled to room temperature, gained suspension immigration 500mL beakers, plus 300mL is ultrapure Water dilutes, suction filtration, and with milli-Q water to filtrate into neutrality, gained solid is in 80˚12h is dried in C vacuum drying chambers, sulphur is obtained Carbon nano tube；
2. to the preparation of benzenethiol functionalization Sulfonated carbon nanotube
12mg Sulfonated carbon nanotubes, the hydrochloric acid solution of 50mL, 1mol/L, ultrasound are sequentially added in 250mL round-bottomed flasks Dispersion 1h, adds 1.0g 4,4 '-diaminourea diphenyl disulfide, ice bath to be cooled to 0˚C, is added slowly with stirring 10mL nitrous Acid sodium aqueous solution, reacts 1h, is warming up to 40˚C, continues to stir 2.0 h of lower reaction, with 0.45 μm of poly tetrafluoroethylene suction filtration, Milli-Q water 3 times, absolute ethanol washing 3 times in the solid ultrasonic disperse for obtaining to 50mL ethanol, sequentially adds 50mL acetonitriles With 520mg triphenyl phosphorus, 1h is stirred, with 0.45 μm of poly tetrafluoroethylene suction filtration, with absolute ethanol washing 5 times, gained solid 80˚12h is dried in C vacuum drying chambers, is obtained to benzenethiol functionalization Sulfonated carbon nanotube；
Described sodium nitrite in aqueous solution is that 280mg solid sodium nitrites are dissolved in into 10mL ultra-pure waters to be obtained；
3. the preparation of silver nano-particle solution
The silver nitrate and 1mL of 1mL, 50mmol/L, 5% sodium citrate are added in 48mL ultra-pure waters under agitation, 50mg sodium borohydrides are subsequently adding, 10min is reacted under agitation, solution is changed into brown color, lasting stirring, to solution colour no longer Change, is cooled to room temperature, and 5min is centrifuged under rotating speed 5000rpm, and the supernatant of gained is silver nano-particle solution；
4. Nano silver grain@is to benzenethiol functionalization Sulfonated carbon nanotube detection antibody Ab2The preparation of incubation content solution
Take 7.5 mg to be added in 30mL silver nano-particle solutions benzenethiol functionalization Sulfonated carbon nanotube, 130rpm 12h is vibrated under rotating speed, is centrifuged, 80˚Dried in C vacuum drying chambers, Nano silver grain@is obtained benzenethiol functionalization sulfonation carbon is received Mitron；
The Nano silver grain@of 4 mg is distributed in 1mL ultra-pure waters to benzenethiol functionalization Sulfonated carbon nanotube, is added The tumor markers detection antibody Ab of 100 μ L, 100 μ g/mL2The phosphate-buffereds of pH 7.4 of solution and 900 μ L, 50mmol/L Solution, 4˚12 h of hatching are vibrated in C constant-temperature shaking incubators；4˚Under C, 15 min are centrifuged under 6000 rpm rotating speeds, obtain down Layer precipitation, the pH=7.4 PBSs centrifuge washing of addition 1mL, 50mmol/L 1 time, obtains lower sediment, is eventually adding The pH=7.4 PBSs of 1mL, 50mmol/L, are obtained Nano silver grain@to benzenethiol functionalization Sulfonated carbon nanotube Detection antibody Ab2Incubation content solution, 4˚Saved backup under C.
A kind of preparation method of breast cancer tumour marker immunosensor of embodiment 6, the preparation of the associated materials, bag Include following steps：
（1）The preparation of load gold nano particle Graphene
1. the preparation of amination graphene
Take 60mg graphene oxides to be dissolved in the dry toluene of 10mL, add 0.2mL 3- aminopropyl-triethoxy silicon Alkane, oil bath heating to 70˚C, magnetic agitation 1.5h, after 110˚Drying obtains amination graphene under C；
2. the preparation of solution of gold nanoparticles
By the HAuCl that 2mL, mass fraction are 1%4It is added in 99mL ultra-pure waters, is heated to boiling and adds 3.5mL, matter Amount fraction be 1% sodium citrate solution, flow back 30min, after be cooled to room temperature, obtain the solution of gold nanoparticles of claret；
3. the preparation of the Graphene of load gold nano particle
Take 30mg amination graphenes and be added to above-mentioned steps 2. in obtained solution of gold nanoparticles, vibrate 12h, be obtained Load gold nano particle Graphene；
（2）Nano silver grain@is to benzenethiol functionalization Sulfonated carbon nanotube detection antibody Ab2The preparation of incubation content solution
1. the preparation of Sulfonated carbon nanotube
The multi-walled carbon nano-tubes for weighing 1.5g is placed in 250mL there-necked flasks, sequentially adds the 66mL concentrated sulfuric acids and 22mL is dense Nitric acid, 120˚50min is reacted under C oil baths, it is after being cooled to room temperature, gained suspension immigration 500mL beakers, plus 300mL is ultrapure Water dilutes, suction filtration, and with milli-Q water to filtrate into neutrality, gained solid is in 85˚14h is dried in C vacuum drying chambers, sulphur is obtained Carbon nano tube；
2. to the preparation of benzenethiol functionalization Sulfonated carbon nanotube
15mg Sulfonated carbon nanotubes, the hydrochloric acid solution of 50mL, 1mol/L, ultrasound are sequentially added in 250mL round-bottomed flasks Dispersion 1h, adds 1.0g 4,4 '-diaminourea diphenyl disulfide, ice bath to be cooled to 0˚C, is added slowly with stirring 10mL nitrous Acid sodium aqueous solution, reacts 1h, is warming up to 40˚C, continues to stir 2.5 h of lower reaction, with 0.45 μm of poly tetrafluoroethylene suction filtration, Milli-Q water 3 times, absolute ethanol washing 3 times in the solid ultrasonic disperse for obtaining to 50mL ethanol, sequentially adds 50mL acetonitriles With 520mg triphenyl phosphorus, 1h is stirred, with 0.45 μm of poly tetrafluoroethylene suction filtration, with absolute ethanol washing 5 times, gained solid 80˚12h is dried in C vacuum drying chambers, is obtained to benzenethiol functionalization Sulfonated carbon nanotube；
Described sodium nitrite in aqueous solution is that 280mg solid sodium nitrites are dissolved in into 10mL ultra-pure waters to be obtained；
3. the preparation of silver nano-particle solution
The silver nitrate and 1mL of 1mL, 50mmol/L, 5% sodium citrate are added in 48mL ultra-pure waters under agitation, 55mg sodium borohydrides are subsequently adding, 10min is reacted under agitation, solution is changed into brown color, lasting stirring, to solution colour no longer Change, is cooled to room temperature, and 5min is centrifuged under rotating speed 5000rpm, and the supernatant of gained is silver nano-particle solution；
4. Nano silver grain@is to benzenethiol functionalization Sulfonated carbon nanotube detection antibody Ab2The preparation of incubation content solution
Take 10 mg to be added in 40mL silver nano-particle solutions benzenethiol functionalization Sulfonated carbon nanotube, 130rpm turns The lower vibration 14h of speed, centrifugation, 80˚Dried in C vacuum drying chambers, Nano silver grain@is obtained to benzenethiol functionalization sulfonation carbon nanometer Pipe；
The Nano silver grain@of 6 mg is distributed in 1mL ultra-pure waters to benzenethiol functionalization Sulfonated carbon nanotube, is added The tumor markers detection antibody Ab of 100 μ L, 120 μ g/mL2The phosphate-buffereds of pH 7.4 of solution and 900 μ L, 50mmol/L Solution, 4˚12 h of hatching are vibrated in C constant-temperature shaking incubators；4˚Under C, 15 min are centrifuged under 6000 rpm rotating speeds, obtain down Layer precipitation, the pH=7.4 PBSs centrifuge washing of addition 1mL, 50mmol/L 1 time, obtains lower sediment, is eventually adding The pH=7.4 PBSs of 1mL, 50mmol/L, are obtained Nano silver grain@to benzenethiol functionalization Sulfonated carbon nanotube Detection antibody Ab2Incubation content solution, 4˚Saved backup under C.
The detection of the breast cancer tumour mark carcinomebryonic antigen of embodiment 7
（1）Tested with three-electrode system using electrochemical workstation, saturated calomel electrode is reference electrode, platinum filament electricity Extremely auxiliary electrode, prepared sensor is working electrode, in 10 mL, the phosphate of pH 5.10 ~ 7.98 of 50 mmol/L Tested in cushioning liquid；
（2）Used time, m- current method was detected to analyte, and input voltage is -0.4 V, the s of sampling interval 0.1, operation The s of time 400；
（3）It is molten to the pH=7.4 phosphate-buffereds of 10 mL, 50 mmol/L every 50 s after background current tends towards stability 10 μ L, the hydrogen peroxide solution of 5 mol/L, record current change are injected in liquid；
（4）The range of linearity of carcinomebryonic antigen is 0.5 pg/mL ~ 25 ng/mL in determination sample, and detection is limited to 0.12 pg/ mL。
The detection of the breast cancer cancerous swelling tumor markers CA125 of embodiment 8
Method according to embodiment 7 detects to CA125 in sample, its range of linearity be 0.5pg/mL ~ 20ng/mL, detection is limited to 0.12 pg/mL.
The detection of the breast cancer tumour mark CA15-3 of embodiment 9
Method according to embodiment 7 detects that its range of linearity is 1 pg/mL ~ 20 to CA15-3 in sample Ng/mL, detection is limited to 0.21 pg/mL.

Claims (3)

1. a kind of preparation method of breast cancer tumour marker immunosensor, it is characterised in that comprise the following steps：
（1）By the glass-carbon electrode Al of a diameter of 3 ~ 5mm2O3Polishing powder is polished, and ultra-pure water is cleaned up；
（2）Take 6 μ L, the load gold nano particle Graphene of 0.5 ~ 2.0mg/mL is added drop-wise to electrode surface, dry at room temperature, use Ultrapure water electrode surface, dries；
（3）Continue the tumor markers capture antibody A b of 6 μ L, 5 ~ 15 μ g/mL1It is added drop-wise to electrode surface, ultra-pure water punching Wash, dried in 4 DEG C of refrigerators；
（4）The BSA solution of 3 μ L, 1 ~ 2 mg/mL is added drop-wise to electrode surface by continuation, to non-specific on enclosed-electrode surface Property avtive spot, ultrapure water electrode surface dries in 4 DEG C of refrigerators；6 μ L are added dropwise, the one of 0.001 ~ 30 ng/mL are The tumor markers antigen A g solution of row various concentrations, ultrapure water electrode surface is dried in 4 DEG C of refrigerators；
（5）By 6 μ L, 1 ~ 3 mg/mL Nano silver grain@to benzenethiol functionalization Sulfonated carbon nanotube detection antibody Ab2Hatching Thing solution, drop coating is placed in 4 DEG C of refrigerators and dries in electrode surface, and a kind of breast cancer tumour marker immunosensor is obtained；
The preparation process of wherein load gold nano particle Graphene is as follows：1. the preparation of amination graphene takes 40 ~ 60mg oxygen Graphite alkene is dissolved in the dry toluene of 10mL, addition 0.2mL 3- aminopropyl triethoxysilanes, and oil bath heating to 60 ~ 70˚C, magnetic agitation 1.5h, after 110˚Drying obtains amination graphene under C；2. the preparation of solution of gold nanoparticles by 1 ~ 2mL, mass fraction are 1% HAuCl4It is added in 99mL ultra-pure waters, is heated to boiling and adds 1.5 ~ 3.5mL, mass fraction Be 1% sodium citrate solution, flow back 15 ~ 30min, after be cooled to room temperature, obtain the solution of gold nanoparticles of claret；③ The preparation of the Graphene of load gold nano particle takes 20 ~ 30mg amination graphenes and is added to above-mentioned steps 2. obtained Jenner In rice corpuscles solution, 12h is vibrated, load gold nano particle Graphene is obtained；
Nano silver grain@is to benzenethiol functionalization Sulfonated carbon nanotube detection antibody Ab2The preparation process of incubation content solution is as follows： 1. the preparation of Sulfonated carbon nanotube weighs the multi-walled carbon nano-tubes of 0.5 ~ 1.5g and is placed in 250mL there-necked flasks, sequentially adds The 66mL concentrated sulfuric acids and 22mL concentrated nitric acids, 120˚30 ~ 50min is reacted under C oil baths, after being cooled to room temperature, gained suspension is moved into 500mL beakers, plus the dilution of 300mL ultra-pure waters, suction filtration, with milli-Q water to filtrate into neutrality, gained solid is in 75 ~ 85˚C 10 ~ 14h is dried in vacuum drying chamber, Sulfonated carbon nanotube is obtained；2. the preparation to benzenethiol functionalization Sulfonated carbon nanotube exists Sequentially add 9 ~ 15mg Sulfonated carbon nanotubes in 250mL round-bottomed flasks, the hydrochloric acid solution of 50mL, 1mol/L, ultrasonic disperse 1h, plus Enter 1.0g 4,4 '-diaminourea diphenyl disulfide, ice bath is cooled to 0˚C, is added slowly with stirring 10mL natrium nitrosums water-soluble Liquid, reacts 1h, is warming up to 40˚C, 1.5 ~ 2.5 h of reaction, ultrapure with 0.45 μm of poly tetrafluoroethylene suction filtration under continuing to stir Water washing 3 times, absolute ethanol washing 3 times, in the solid ultrasonic disperse for obtaining to 50mL ethanol, sequentially add 50mL acetonitriles and 520mg triphenyl phosphorus, stir 1h, with 0.45 μm of poly tetrafluoroethylene suction filtration, with absolute ethanol washing 5 times, gained solid 80˚ 12h is dried in C vacuum drying chambers, is obtained to benzenethiol functionalization Sulfonated carbon nanotube；Described sodium nitrite in aqueous solution be by 280mg solid sodium nitrites are dissolved in 10mL ultra-pure waters and are obtained；3. the preparation of silver nano-particle solution is by the nitre of 1mL, 50mmol/L Sour silver and 1mL, 5% sodium citrate, are added in 48mL ultra-pure waters under agitation, are subsequently adding 45 ~ 55mg sodium borohydrides, 10min is reacted under agitation, solution is changed into brown color, lasting stirring no longer changes to solution colour, is cooled to room temperature, is turning 5min is centrifuged under fast 5000rpm, the supernatant of gained is silver nano-particle solution；4. Nano silver grain@is to benzenethiol functionalization Sulfonated carbon nanotube detection antibody Ab2The preparation of incubation content solution takes 5 ~ 10 mg and benzenethiol functionalization Sulfonated carbon nanotube is added Enter in 20 ~ 40mL silver nano-particle solutions, 10 ~ 14h, centrifugation, 80 are vibrated under 130rpm rotating speeds˚Done in C vacuum drying chambers It is dry, Nano silver grain@is obtained to benzenethiol functionalization Sulfonated carbon nanotube；By the Nano silver grain@of 2 ~ 6 mg to benzenethiol work( Sulfonated carbon nanotube can be changed to be distributed in 1mL ultra-pure waters, 100 μ L, the tumor markers detection antibody of 80 ~ 120 μ g/mL is added Ab2The PBSs of pH 7.4 of solution and 900 μ L, 50mmol/L, 4˚Hatching 12 is vibrated in C constant-temperature shaking incubators h；4˚Under C, 15 min are centrifuged under 6000 rpm rotating speeds, obtain lower sediment, add the pH=7.4 phosphorus of 1mL, 50mmol/L Hydrochlorate cushioning liquid centrifuge washing 1 time, obtains lower sediment, and the pH=7.4 phosphate-buffereds for being eventually adding 1mL, 50mmol/L are molten Liquid, is obtained Nano silver grain@to benzenethiol functionalization Sulfonated carbon nanotube detection antibody Ab2Incubation content solution, 4˚Preserve standby under C With.
2. a kind of breast cancer tumour marker immunosensor that prepared by preparation method as claimed in claim 1, for mammary gland The detection of cancerous swelling tumor markers, step is as follows：
（1）Tested with three-electrode system using electrochemical workstation, saturated calomel electrode is reference electrode, platinum electrode is Auxiliary electrode, prepared sensor is working electrode, in 10 mL, the phosphate-buffereds of pH 5.10 ~ 7.98 of 50 mmol/L Tested in solution；
（2）Used time, m- current method was detected to analyte, and input voltage is -0.4 V, the s of sampling interval 0.1, run time 400 s；
（3）After background current tends towards stability, every 50 s in 10 mL, the pH=7.4 PBSs of 50 mmol/L Inject 10 μ L, the hydrogen peroxide solution of 5 mol/L, record current change.
3. the sensor that prepared by a kind of preparation method of breast cancer tumour marker immunosensor as claimed in claim 1, For the measure of breast cancer tumour mark, it is characterised in that the tumor markers is selected from one of following：Carcinomebryonic antigen, sugar Class antigen 125, CA15-3.
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