CN102778561A - Preparation and application of tumor marker immunosensor built by putamen nanometer materials - Google Patents
Preparation and application of tumor marker immunosensor built by putamen nanometer materials Download PDFInfo
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- CN102778561A CN102778561A CN201210261102XA CN201210261102A CN102778561A CN 102778561 A CN102778561 A CN 102778561A CN 201210261102X A CN201210261102X A CN 201210261102XA CN 201210261102 A CN201210261102 A CN 201210261102A CN 102778561 A CN102778561 A CN 102778561A
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Abstract
The invention relates to preparation and application of a tumor marker immunosensor built by putamen nanometer materials and belongs to the technical fields of novel nanometer functional materials and biosensors. The preparation and the application are characterized by comprising (1) preparation of nanometer multihole gold film; (2) preparation of a secondary antibody composite of an Au@Pd@Pt putamen nanometer material-tumor marker; and (3) building of an electrochemistry immunosensor. The preparation and application have the advantages of being high in sensitivity, good in specificity, easy to operate, low in limit of detection and capable of achieving high sensitivity, specificity and rapid and accurate detection of a plurality of tumor markers.
Description
Technical field
The present invention relates to the preparation and the application of the tumor markers immunosensor of shell nuclear nano material structure.Specifically be based on nano-porous gold film and AuPdPt shell nuclear nano material, the preparation method and the application of the sandwich type electrochemical immunosensor of the detection kinds of tumors mark of structure are used for detecting the tumor markers of serum.Belong to novel nano functional material and biosensor technique field.
Background technology
Tumor markers has important practical value in the early diagnosis of cancer.Tumor markers is a kind of in the generation and breeding of tumour cell, is produced, is perhaps produced by the reaction of body to tumour cell by tumour cell itself, one type of material that the reaction tumour exists and grows.Comprise protein, hormone, enzyme, polyamines and oncoprotein etc.
Tumor markers is widely used in clinical, the clinical stage of the screening for cancer in normal population, the cancer auxiliary diagnosis that the symptom is arranged, cancer by stages, the prognosis evaluation therapeutic scheme of Cancerous disease process, judge cancer plays an important role in whether recurring.
The Clinical detection method of present existing tumor markers is a lot, and radioimmunoassay is arranged, enzymoimmunoassay, chemiluminescent immunoassay etc.But these detection methods exist following not enough.
(1) radioimmunoassay: radiommunoassay has been widely used in biomedical every field since coming out.It has easy and simple to handle, low cost and other advantages.But this method radioactive contamination is serious, and sensitivity is low, detects limit for height, has therefore limited its application.
(2) EIA enzyme immunoassay determination method: enzyme immunoassay (EIA) is simple because of the label preparation, and the term of validity is long, and characteristics such as environmentally safe have obtained popularizing rapidly and development.But the easy inactivation of the enzyme in the EIA enzyme immunoassay determination method causes its signal time short, has reduced the sensitivity and the reappearance of method.
(3) chemiluminscence immunoassay method: this method have sensitivity, fast, stable, selectivity is strong, favorable reproducibility, easy operating, the advantage that flexible method is various.But the factor of testing result that influences chemiluminescence analysis is more, so its less stable, and after chemical reaction took place, the luminous of sample can't be reproduced.
Can find out by above analysis, invent that a kind of detectability is low, highly sensitive, the tumor markers sensor of high specificity, favorable reproducibility is extremely urgent.
Nanometer-material-modified electrochemical immunosensor is because it is simple, with low cost, high specificity, sensitivity advantage fast; Enjoyed people to pay close attention in recent years, studied a series of electrochemical immunosensors that make up based on nano material [referring to (a) Lai G.S.; Yan F.; Wu J.; Leng C.; Ju H.X. Anal. Chem., 2011,83,2726-2732. (b) Du D.; Wang L.M.; Shao Y.Y.; Wang J.; Engelhard M.H.; Lin Y.H. Anal. Chem., 2011,83,746-752.].
The present invention examines the structure that nano material is used for electrochemical immunosensor with shell, gives electrochemical immunosensor more outstanding characteristic.At first adopt nano-porous gold film fixing anti-; Because the nano-porous gold film has three-dimensional open-celled structure and big specific surface area continuously, help fixing more one and resist, and nano-porous gold film excellent biological compatibility; Can anti-directly combine with one, avoid the use of crosslinking chemical; Adopt AuPdPt shell nuclear nano material mark two anti-then; AuPdPt had both kept the advantage of Au, Pd, Pt three kinds of noble metal nanometer materials; Embody excellent collaborative effect of enhanced sensitivity again; Thereby improved the electron transport efficient of its electrode surface, reduced detectability, increased the detection sensitivity of sensor.
Under no enzyme situation, directly adopt AuPdPt shell nuclear nano material as two anti-labels, simplified operation steps, strengthened the stability of sensor, prepared a kind of sandwich type electrochemical immunosensor that detects the kinds of tumors mark that is applicable to.
Retrieval through existing tumor markers being detected patented technology finds that CN200910212772.0 discloses a kind of electrochemiluminescence immunosensor that is used to detect alpha-fetoprotein at present, and the detectability of this sensor reaches 0.035 ngmL
-1, the range of linearity is 0.01 ~ 20 ngmL
-1
CN201110199112.0 discloses a kind of electrochemical immunosensor that detects phosphatization albumen, and its detection is limited to 0.01 ngmL
-1, the range of linearity is 0.02 ~ 20 ngmL
-1
CN03113053.4 discloses a kind of detection CA-125 does not have the reagent ampere immunity sensor.
The present invention adopts nano-porous gold film and AuPdPt shell nuclear nano material as electrode modification and two anti-marker materials respectively, has reduced the detection limit of sensor, to the detection limit of kinds of tumors label at 0.91 ~ 1.5 pgmL
-1Between.
This shows that the sensitivity of its method is significantly improved, sensitivity all is superior to above three kinds of methods, accurately detection by quantitative kinds of tumors mark.
The present invention utilizes immunoreactive high specific, and combining nano porous gold film and AuPdPt shell have been examined nano material preparation a kind of sandwich type electrochemical immunosensor also is used for detecting the kinds of tumors mark.
That the present invention has is highly sensitive, specificity good, detect cost low, can fast detecting kinds of tumors mark etc. advantages, and the present invention to prepare process simple, operating process is easy, has effectively overcome the deficiency of present tumor-marker object detecting method.
Summary of the invention
One of the object of the invention is to avoid that the instrument and equipment of traditional detection method is complicated, operating process is loaded down with trivial details, the testing staff requires high shortcoming, and the preparation method of the electrochemical immunosensor of a kind of highly sensitive, high specificity, favorable reproducibility, fast detecting kinds of tumors label easy and simple to handle is provided.
Two of the object of the invention is the detections that this tumor markers electrochemical immunosensor are applied to the kinds of tumors mark.
To achieve these goals, the present invention realizes through following measure.
1. the preparation and the application of the tumor markers immunosensor of shell nuclear nano material structure is characterized in that, may further comprise the steps:
(1) preparation of nano-porous gold film;
(2) preparation of AuPdPt shell nuclear nano material-tumor marker two anti-compounds;
(3) structure of electrochemical immunosensor.
The preparation of the nano-porous gold film (1) specifically may further comprise the steps:
1) silver-colored billon film is swum in 14 ~ 16 molL
-10.5 ~ 5 min erodes silver on the nitric acid liquid level, processes the nano-porous gold film;
2) the nano-porous gold film is washed to its pH=7.0 with ultrapure water.
The preparation of the AuPdPt shell nuclear nano material-tumor marker two anti-compounds (2) specifically may further comprise the steps:
1) with AuPdPt shell nuclear nano material and tumor marker two anti-being mixed to join in the PBS of pH=7.4, making AuPdPt shell nuclear nano material and tumor marker two anti-mass concentration ratios is 500 ~ 1000:1, is mixed with mixed liquor A;
2) with concussion hatching 12 ~ 24 h under the above-mentioned mixed liquor A room temperature; Centrifugal under 10000 ~ 12000 rpm; Clean with pH=7.4 PBSs, be scattered in again in the PBS of pH=7.4 then, be mixed with mixed liquid B and be stored under 4 oC subsequent use.
The structure of the electrochemical immunosensor (3) specifically may further comprise the steps:
1) glass-carbon electrode of diameter 4 mm is used successively the alundum (Al burnishing powder polishing of 1.0,0.3 and 0.05 mm, the ethanol ultrasonic cleaning is rinsed well with ultrapure water again, then electrode is placed 5 mmolL
-1In the potassium ferricyanide solution, scanning makes the spike potential difference less than 110 mV under-0.2 ~ 0.6 V current potential;
2) nano-porous gold is film modified in glass-carbon electrode surface, drying at room temperature;
3) tumor markers one anti-dripping is coated onto step 2) the film modified electrode surface of nano-porous gold, place under the 4 oC wet condition and dry;
4) be 100 μ gmL with concentration
-1Bovine serum albumin(BSA) drips and is coated onto the step 3) tumor markers one anti-electrode surface of modifying, and dries under the wet condition under the 4oC;
5) tumor markers antigen is dripped the electrode surface that is coated in the modification of step 4) bovine serum albumin(BSA), dry under the wet condition under 4 oC;
6) solution B is dripped the electrode surface that is coated in the modification of step 5) tumor markers antigen, dry under the wet condition under 4 oC, the electrochemical immunosensor preparation is accomplished.
2. the shell of preparation of the present invention is examined the tumor markers immunosensor that nano material makes up, and it is characterized in that be used for the detection of tumor markers, step is following:
(1) drafting of working curve;
(2) detection of tumor markers.
The drafting of the working curve (1), step is following:
1) with contrast electrode-saturated calomel electrode, correctly is connected on the electrochemical workstation to electrode-platinum electrode and working electrode;
2) in pH=7 ~ 8 phosphate buffers, detect of the response of the good working electrode of modification to hydrogen peroxide through chronoamperometry;
3) according to the relation of gained current-responsive and tumor markers antigen concentration of standard solution, the drawing curve.
3. the tumor markers immunosensor that shell nuclear nano material of the present invention makes up is characterized in that described tumor marker; Be selected from one of high tumor markers of the following present incidence of disease: carcinomebryonic antigen (CEA), alpha-fetoprotein (AFP), alpha-L-fucosidase (AFU); PSA (PSA), CA-125, CA-199; CA-724, CA-242, human chorionic gonadotrophin (HCG).
Useful achievement of the present invention
(1) preparation method of tumor markers electrochemical immunosensor; The nano-porous gold film is incorporated in the middle of the preparation of tumor markers electrochemical immunosensor; Utilize nano-porous gold film good electron transmission capacity with and excellent biocompatibility, significantly improved the performance of electrode.
(2) with AuPdPt shell nuclear nano material and tumor markers two anti-directly hatchings; Utilize noble metal excellent biocompatibility and high catalytic performance; In the label that two resist, needn't use enzyme; Avoided inactivation and the detection error that leakage causes, simplified the making step of two anti-labels, significantly improved the reappearance and the stability of electrochemical immunosensor because of enzyme.
(3) use identical nano material and method of modifying; Utilize antigen to combine with the specificity of antibody; Only need to change highly sensitive, the specific detection that the tumor-marker species can realize the kinds of tumors mark, the method is simple, economical, helps the commercialization of tumor markers sensor.
(4) the prepared electrochemical immunosensor of the present invention, simple to operate, detection speed is fast, and 30 s can accomplish sample determination, can realize the mensuration of sample in batches at short notice.
(5) the present invention detects the method for tumor markers, and testing result all has instrument to accomplish automatically and record, has avoided the influence of subjective factor, and good reappearance is arranged, and is convenient to on-the-spot the detection.
Description of drawings
Below in conjunction with description of drawings and specific embodiment the present invention is described in further detail.
Fig. 1 is the transmission electron microscope picture of AuPdPt shell nuclear nano material.
Fig. 2 is a kind of building process of tumor markers electrochemical immunosensor of shell nuclear nano material structure.
Embodiment
Embodiment 1
The preparation of the tumor markers immunosensor that a kind of shell nuclear nano material makes up may further comprise the steps.
(1) silver-colored billon film is swum in 15 molL
-10.5 min on the nitric acid liquid level utilizes red fuming nitric acid (RFNA) that silver is eroded, and processes the nano-porous gold film.The nano-porous gold film is washed to its pH=7.0 with ultrapure water.
(2) in flask, add 20 mmolL
-1Gold chloride 2.5 mL; 20 mmolL
-1Tetrachloro-palladium acid sodium 4.0 mL; 20 mmolL
-1Platinous Potassium Chloride 4.0 mL; Blocked polyethers F-127 0.1 g adds 0.4 molL immediately after mixing
-1Ascorbic acid 1.0 mL.Stir 1 h under the room temperature.AuPdPt shell nuclear nano material is promptly processed in centrifugal washing 3 times after the drying, the pattern of its nano material is seen Fig. 1; Can be found out that by Fig. 1 transmission electron microscope picture AuPdPt is a core-shell structure, its particle diameter is 20 nm; Dispersion of materials property is good, uniform particles, is suitable as the manufacturing materials of electrochemical immunosensor.
(3) AuPdPt shell nuclear nano material and tumor marker two anti-being mixed to join in pH=7.4 PBSs are processed mixed liquor; Making AuPdPt shell nuclear nano material and tumor marker two anti-mass concentration ratios is 500: 1, with concussion hatching 12 h under the above-mentioned mixed liquor room temperature.Centrifugal under 10000 rpm, clean with pH=7.4 PBSs, be scattered in again then in pH=7.4 PBSs, obtain AuPdPt shell nuclear nano material-tumor marker two anti-complex solutions.
(4) glass-carbon electrode of diameter 4 mm is used successively the alundum (Al burnishing powder polishing of 1.0,0.3 and 0.05 mm, the ethanol ultrasonic cleaning is rinsed well with ultrapure water again, then electrode is placed 5 mmolL
-1In the potassium ferricyanide solution, scanning makes the spike potential difference less than 110 mV under-0.2 ~ 0.6 V current potential.
The building process of the tumor markers electrochemical immunosensor that (5) makes up according to a kind of shell of Fig. 2 nuclear nano material prepares.Nano-porous gold is film modified in glass-carbon electrode surface, drying at room temperature.With 10 μ gmL
-1Tumor markers one anti-6 μ L drip and are coated onto the film modified electrode surface of nano-porous gold, place under the 4 oC wet condition and dry.With concentration is 100 μ gmL
-1Bovine serum albumin(BSA) 3 μ L drip and are coated onto the tumor markers one anti-electrode surface of modifying, and dry under the wet condition under 4 oC.Tumor markers antigen 6 μ L are dripped the electrode surface that is coated in the bovine serum albumin(BSA) modification, dry under the wet condition under 4 oC.AuPdPt shell nuclear nano material-tumor marker two anti-complex solution 6 μ L of step (2) preparation are dripped the electrode surface that is applied to the modification of tumor markers antigen, dry under the wet condition under 4 oC.
Embodiment 2
The preparation of the tumor markers immunosensor that a kind of shell nuclear nano material makes up may further comprise the steps.
(1) silver-colored billon film is swum in 15 molL
-13 min on the nitric acid liquid level utilize red fuming nitric acid (RFNA) that silver is eroded, and process the nano-porous gold film.The nano-porous gold film is washed to its pH=7.0 with ultrapure water.
(2) in flask, add 20 mmolL
-1Gold chloride 2.5 mL; 20 mmolL
-1Tetrachloro-palladium acid sodium 4.0 mL; 20 mmolL
-1Platinous Potassium Chloride 4.0 mL; Blocked polyethers F-127 0.1g adds 0.4 molL immediately after mixing
-1Ascorbic acid 1.0 mL.Stir 1 h under the room temperature.AuPdPt shell nuclear nano material is promptly processed in centrifugal washing 3 times after the drying, its transmission electron microscope pattern is seen Fig. 1.
(3) AuPdPt shell nuclear nano material and tumor marker two anti-being mixed to join in pH=7.4 PBSs are processed mixed liquor; Making AuPdPt shell nuclear nano material and tumor marker two anti-mass concentration ratios is 750: 1, with concussion hatching 12 h under the above-mentioned mixed liquor room temperature.Centrifugal under 10000 rpm, clean with pH=7.4 PBSs, be scattered in again then in pH=7.4 PBSs.
(4) glass-carbon electrode of diameter 4 mm is used successively the alundum (Al burnishing powder polishing of 1.0,0.3 and 0.05 mm, the ethanol ultrasonic cleaning is rinsed well with ultrapure water again, then electrode is placed 5 mmolL
-1In the potassium ferricyanide solution, scanning makes the spike potential difference less than 110 mV under-0.2 ~ 0.6 V current potential.
The building process of the tumor markers electrochemical immunosensor that (5) makes up according to a kind of shell of Fig. 2 nuclear nano material prepares.Nano-porous gold is film modified in glass-carbon electrode surface, drying at room temperature.With 10 μ gmL
-1Tumor markers one anti-6 μ L drip and are coated onto the film modified electrode surface of nano-porous gold, place under the 4 oC wet condition and dry.With concentration is 100 μ gmL
-1Bovine serum albumin(BSA) 3 μ L drip and are coated onto the tumor markers one anti-electrode surface of modifying, and dry under the wet condition under 4 oC.Tumor markers antigen 6 μ L are dripped the electrode surface that is coated in the bovine serum albumin(BSA) modification, dry under the wet condition under 4 oC.AuPdPt shell nuclear nano material-tumor marker two anti-complex solution 6 μ L of step (2) preparation are dripped the electrode surface that is applied to the modification of tumor markers antigen, dry under the wet condition under 4 oC.
Embodiment 3
The preparation of the tumor markers immunosensor that a kind of shell nuclear nano material makes up may further comprise the steps.
(1) silver-colored billon film is swum in 15 molL
-15 min on the nitric acid liquid level utilize red fuming nitric acid (RFNA) that silver is eroded, and process the nano-porous gold film.The nano-porous gold film is washed to its pH=7.0 with ultrapure water.
(2) in flask, add 20 mmolL
-1Gold chloride 2.5 mL; 20 mmolL
-1Tetrachloro-palladium acid sodium 4.0 mL; 20 mmolL
-1Platinous Potassium Chloride 4.0 mL; Blocked polyethers F-127 0.1 g adds 0.4 molL immediately after mixing
-1Ascorbic acid 1.0 mL.Stir 1 h under the room temperature.AuPdPt shell nuclear nano material is promptly processed in centrifugal washing 3 times after the drying, its transmission electron microscope pattern is seen Fig. 1.
(3) AuPdPt shell nuclear nano material and tumor marker two anti-being mixed to join in pH=7.4 PBSs are processed mixed liquor; Making AuPdPt shell nuclear nano material and tumor marker two anti-mass concentration ratios is 1000: 1; With concussion hatching 12 h under the above-mentioned mixed liquor room temperature; Centrifugal under 10000 rpm, clean with pH=7.4 PBSs, be scattered in again then in pH=7.4 PBSs.
(4) glass-carbon electrode of diameter 4 mm is used successively the alundum (Al burnishing powder polishing of 1.0,0.3 and 0.05 mm, the ethanol ultrasonic cleaning is rinsed well with ultrapure water again, then electrode is placed 5 mmolL
-1In the potassium ferricyanide solution, scanning makes the spike potential difference less than 110 mV under-0.2 ~ 0.6 V current potential.
The building process of the tumor markers electrochemical immunosensor that (5) makes up according to a kind of shell of Fig. 2 nuclear nano material prepares.Nano-porous gold is film modified in glass-carbon electrode surface, drying at room temperature.With 10 μ g.mL
-1Tumor markers one anti-6 μ L drip and are coated onto the film modified electrode surface of nano-porous gold, place under the 4 oC wet condition and dry.With concentration is 100 μ gmL
-1Bovine serum albumin(BSA) 3 μ L drip and are coated onto the tumor markers one anti-electrode surface of modifying, and dry under the wet condition under 4 oC.Tumor markers antigen 6 μ L are dripped the electrode surface that is coated in the bovine serum albumin(BSA) modification, dry under the wet condition under 4 oC.AuPdPt shell nuclear nano material-tumor marker two anti-complex solution 6 μ L of step (2) preparation are dripped the electrode surface that is applied to the modification of tumor markers antigen, dry under the wet condition under 4 oC.
Embodiment 4
The tumor markers electrochemical immunosensor of embodiment 1 ~ 3 preparation is respectively applied for tumor markers and detects, and may further comprise the steps.
(1) drafting of working curve, step is following:
1) with contrast electrode-saturated calomel electrode, electrode-platinum electrode and working electrode correctly are connected on the CHI760D electrochemical workstation;
2) in the pH=7.4 phosphate buffer, detect of the response of the good working electrode of modification to hydrogen peroxide through chronoamperometry;
3) according to the concentration relationship of gained current-responsive and tumor markers antigen standard solution, the drawing curve.
(2) detection of tumor markers.
Embodiment 5Breast cancer tumour mark: CA-125, CEA or HCG
A kind of preparation and application of breast cancer tumour mark immunosensor may further comprise the steps:
(1) selects the breast cancer tumour mark, make up electrochemical immunosensor according to embodiment 1 described step;
(2) detect according to embodiment 4 described steps, the detection technique index of breast cancer tumour mark is seen table 1.
The detection technique index of table 1 breast cancer tumour mark
| Tumor markers | The range of linearity, ngmL -1 | Detectability, pgmL -1 |
| CA-125 | 0.004?~20 | 1.3 |
| CEA | 0.004?~30 | 1.2 |
| HCG | 0.005~20 | 1.5 |
The result shows by table 1 detection technique index, and this electrochemical immunosensor is used for the detection of breast cancer tumour mark, and its range of linearity is wide, and detectability is low, and method is highly sensitive.
Embodiment 6Liver cancer tumor markers: AFP or AFU
A kind of preparation and application of liver cancer tumor markers immunosensor may further comprise the steps:
(1) selects the liver cancer tumor markers, make up electrochemical immunosensor according to embodiment 2 described steps;
(2) detect according to embodiment 4 described steps, the detection technique index of liver cancer tumor markers is seen table 2.
The detection technique index of table 2 liver cancer tumor markers
| Tumor markers | The range of linearity, ngmL -1 | Detectability, pgmL -1 |
| AFP | 0.003~?20 | 1.3 |
| AFU | 0.003~20 | 0.91 |
The result shows by table 2 detection technique index, and this electrochemical immunosensor is used for the detection of liver cancer tumor markers, and its range of linearity is wide, and detectability is low, and method is highly sensitive.
Embodiment 7Cancer of the stomach tumor markers: CA-724, CA-199 or CA-242
A kind of preparation and application of cancer of the stomach tumor markers immunosensor may further comprise the steps:
(1) selects the cancer of the stomach tumor markers, make up electrochemical immunosensor according to embodiment 3 described steps;
(2) detect according to embodiment 4 described steps, the detection technique index of cancer of the stomach tumor markers is seen table 3.
The detection technique index of table 3 cancer of the stomach tumor markers
| Tumor markers | The range of linearity, ngmL -1 | Detectability, pgmL -1 |
| CA-724 | 0.005?~?20 | 1.2 |
| CA-199 | 0.005~20 | 1.3 |
| CA-242 | 0.003~20 | 1.3 |
The result shows by table 3 detection technique index, and this electrochemical immunosensor is used for the detection of cancer of the stomach tumor markers, and its range of linearity is wide, and detectability is low, and method is highly sensitive.
Embodiment 8The detection of tumor markers in the human serum
Accurately pipette the human serum sample, detect according to embodiment 4 described steps, testing result is seen table 4.
The testing result of kinds of tumors mark in table 4 human serum
Can be known that by table 4 testing result result's relative standard deviation (RSD) is less than 3.0%, average recovery rate is 95.0 ~ 102%, shows that the present invention is used for the detection of human serum kinds of tumors mark, and the precision of method is high, and the result accurately and reliably.
Claims (7)
1. the preparation of the tumor markers immunosensor of shell nuclear nano material structure is characterized in that, may further comprise the steps:
(1) preparation of nano-porous gold film;
(2) preparation of AuPdPt shell nuclear nano material-tumor marker two anti-compounds;
(3) structure of electrochemical immunosensor.
2. the preparation of the tumor markers immunosensor of the nuclear of the shell described in claim 1 nano material structure is characterized in that, the preparation of described nano-porous gold film, and step is following:
(1) silver-colored billon film is swum in 14 ~ 16 molL
-10.5 ~ 5 min erodes silver on the nitric acid liquid level, processes the nano-porous gold film;
(2) the nano-porous gold film is washed to its pH=7.0 with ultrapure water.
3. the preparation of the tumor markers immunosensor of the nuclear of the shell described in claim 1 nano material structure is characterized in that, the preparation of described AuPdPt shell nuclear nano material-tumor marker two anti-compounds, and step is following:
(1) with AuPdPt shell nuclear nano material and tumor marker two anti-being mixed to join in pH=7.4 PBSs, making AuPdPt shell nuclear nano material and tumor marker two anti-mass concentration ratios is 500 ~ 1000:1, is mixed with mixed liquor A;
(2) with concussion hatching 12 ~ 24 h under the above-mentioned mixed liquor A room temperature; Centrifugal under 10000 ~ 12000 rpm; Clean with pH=7.4 PBSs, be scattered in again then in pH=7.4 PBSs and be mixed with mixed liquid B and be stored under the 4oC subsequent use.
4. the preparation of the tumor markers immunosensor of the nuclear of the shell described in claim 1 nano material structure is characterized in that, the structure of described electrochemical immunosensor, and step is following:
(1) glass-carbon electrode of diameter 4 mm is used successively the alundum (Al burnishing powder polishing of 1.0,0.3 and 0.05 mm, the ethanol ultrasonic cleaning is rinsed well with ultrapure water again, then electrode is placed 5 mmolL
-1In the potassium ferricyanide solution, scanning makes the spike potential difference less than 110 mV under-0.2 ~ 0.6 V current potential;
(2) nano-porous gold is film modified in glass-carbon electrode surface, drying at room temperature;
(3) tumor markers one anti-dripping is coated onto the film modified electrode surface of step (2) nano-porous gold, places under the 4oC wet condition and dry;
(4) be 100 μ gmL with concentration
-1Bovine serum albumin(BSA) drips and is coated onto step (3) the tumor markers one anti-electrode surface of modifying, and dries under the wet condition under the 4oC;
(5) tumor markers antigen is dripped the electrode surface that is coated in the modification of step (4) bovine serum albumin(BSA), dry under the wet condition under the 4oC;
(6) solution B is dripped the electrode surface that is coated in the modification of step (5) tumor markers antigen, dry under the wet condition under the 4oC, the electrochemical immunosensor preparation is accomplished.
5. examine the tumor markers immunosensor that nano material makes up like the shell of the described preparation of claim 1 ~ 4, it is characterized in that be used for the detection of tumor markers, method is following:
(1) drafting of working curve;
(2) detection of tumor markers.
6. the tumor markers immunosensor of the shell of the preparation described in claim 5 nuclear nano material structure is used for the detection of tumor markers, it is characterized in that, and the drafting of described working curve, step is following:
(1) with contrast electrode-saturated calomel electrode, correctly is connected on the electrochemical workstation to electrode-platinum electrode and working electrode;
(2) in the phosphate buffer of pH=7 ~ 8, detect of the response of the good working electrode of modification to hydrogen peroxide through chronoamperometry;
(3) according to the relation of gained current-responsive and tumor markers antigen concentration of standard solution, the drawing curve.
7. the tumor markers immunosensor that makes up like the described shell nuclear of claim 1 ~ 6 nano material is characterized in that described tumor marker; Be selected from one of high tumor markers of the following present incidence of disease: carcinomebryonic antigen (CEA), alpha-fetoprotein (AFP), alpha-L-fucosidase (AFU); PSA (PSA), CA-125, CA-199; CA-724, CA-242, human chorionic gonadotrophin (HCG).
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