CN102778561B - 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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- CN102778561B CN102778561B CN201210261102XA CN201210261102A CN102778561B CN 102778561 B CN102778561 B CN 102778561B CN 201210261102X A CN201210261102X A CN 201210261102XA CN 201210261102 A CN201210261102 A CN 201210261102A CN 102778561 B CN102778561 B CN 102778561B
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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 preparation and the application of the tumor markers immunosensor of shell core nano material structure.Specifically based on nano-porous gold film and Au@Pd@Pt shell core nano material, preparation method and the application of the sandwich type electrochemical immunosensor of the detection Diagnostic Value of Several Serum Tumor Markers of structure, for detection of the tumor markers in serum.Belong to Nano-function thin films and biosensor technique field.
Background technology
Tumor markers has important practical value in the early diagnosis of cancer.Tumor markers is a kind ofly produced or by the reaction of body to tumour cell, is produced by tumour cell itself in the generation and breeding of tumour cell, the reaction tumour exists and a class material of growth.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 aided Cancer 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 there is following deficiency in these detection methods.
(1) radioimmunoassay: radiommunoassay since coming out, has been widely used in biomedical every field.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 the characteristics such as environmentally safe have obtained universal and development rapidly.But the easy inactivation of the enzyme in the EIA enzyme immunoassay determination method, cause its signal time short, reduced sensitivity and the reappearance of method.
(3) chemiluminscence immunoassay method: this method have sensitivity, fast, stable, selectivity is strong, favorable reproducibility, easy operating, the flexile advantage of method.But affect the many factors of the testing result of chemiluminescence analysis, so its less stable, and after chemical reaction occurred, the luminous of sample can't be reproduced.
Can be found 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 because it is simple, with low cost, high specificity, sensitivity advantage fast, enjoy in recent years people to pay close attention to, studied a series of electrochemical immunosensors that build 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 is used for shell core nano material the structure of electrochemical immunosensor, gives electrochemical immunosensor more outstanding characteristic.At first adopt fixedly primary antibodie of nano-porous gold film, because the nano-porous gold film has three-dimensional open-celled structure and large specific surface area continuously, be conducive to fixing more primary antibodie, and the good biocompatibility of nano-porous gold film, can with the direct combination of primary antibodie, avoided the use of crosslinking chemical; Then adopt Au@Pd@Pt shell core nano material mark two anti-, Au@Pd@Pt had both kept the advantage of Au, Pd, three kinds of noble metal nanometer materials of Pt, embody again excellent collaborative effect of enhanced sensitivity, thereby improved the electronics transmission efficiency of its electrode surface, reduced detectability, increased the detection sensitivity of sensor.
In without the enzyme situation, directly adopt Au@Pd@Pt shell core nano material as two anti-labels, simplified operation steps, strengthened the stability of sensor, prepared a kind of sandwich type electrochemical immunosensor that is applicable to detect Diagnostic Value of Several Serum Tumor Markers.
Find through the retrieval that existing tumor markers is detected patented technology, CN200910212772.0 discloses a kind of immunosensor of electrochemiluminescence for detection of 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 without the reagent ampere immunity sensor.
The present invention adopts respectively nano-porous gold film and Au@Pd@Pt shell core nano material as electrode modification and two anti-marker materials, 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, the sensitivity of its method is significantly improved, and sensitivity all is better than above three kinds of methods, can detect Diagnostic Value of Several Serum Tumor Markers by accurate quantitative analysis.
The present invention utilizes immunoreactive high specific, and combining nano porous gold film and Au@Pd@Pt shell core nano material have prepared a kind of sandwich type electrochemical immunosensor and be used for detecting Diagnostic Value of Several Serum Tumor Markers.
Highly sensitive, the advantages such as specificity good, testing cost is low, energy fast detecting Diagnostic Value of Several Serum Tumor Markers that the present invention has, and preparation process of the present invention is simple, and operating process is easy, has effectively overcome the deficiency of present tumor-marker object detecting method.
Summary of the invention
One of purpose of the present 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 the shortcomings such as high, 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 purpose of the present invention is this tumor markers electrochemical immunosensor to be applied to the detection of Diagnostic Value of Several Serum Tumor Markers.
To achieve these goals, the present invention realizes by following measures.
1. preparation and the application of the tumor markers immunosensor of shell core nano material structure, is characterized in that, comprises the following steps:
(1) preparation of nano-porous gold film;
(2) preparation of Au@Pd@Pt shell core nano material-tumor marker two anti-compounds;
(3) structure of electrochemical immunosensor.
The preparation of the nano-porous gold film (1) specifically comprises the following steps:
1) with silver-colored billon film floating at 14 ~ 16 molL
-1On the nitric acid liquid level, 0.5 ~ 5 min, erode silver, makes 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 Au@Pd@Pt shell core nano material-tumor marker two anti-compounds (2) specifically comprises the following steps:
1) be mixed to join in the phosphate buffered solution of pH=7.4 Au@Pd@Pt shell core nano material and tumor marker two is anti-, making the anti-mass concentration ratio of Au Pd Pt shell core nano material and tumor marker two is 500 ~ 1000:1, is mixed with mixed liquor A;
2) with concussion hatching 12 ~ 24 h under above-mentioned mixed liquor A room temperature, centrifugal under 10000 ~ 12000 rpm, clean with pH=7.4 phosphate buffered solution, then again be scattered in the phosphate buffered solution of pH=7.4, be mixed with mixed liquid B and be stored under 4 oC standby.
The structure of the electrochemical immunosensor (3) specifically comprises the following steps:
1) glass-carbon electrode of diameter 4 mm is used successively the alundum (Al2O3) burnishing powder polishing of 1.0,0.3 and 0.05 mm, the ethanol ultrasonic cleaning, then with ultrapure water, rinse well, then electrode is placed in 5 mmolL
-1In potassium ferricyanide solution, scan under-0.2 ~ 0.6 V current potential, make spike potential poor less than 110 mV;
2) nano-porous gold is film modified in glass-carbon electrode surface, drying at room temperature;
3) the tumor markers primary antibodie is dripped and is coated onto step 2) the film modified electrode surface of nano-porous gold, be placed under 4 oC wet condition and dry;
4) with concentration be 100 μ gmL
-1Bovine serum albumin(BSA) drips and is coated onto the electrode surface that step 3) tumor markers primary antibodie is modified, and dries under wet condition under 4oC;
5) tumor markers antigen is dripped the electrode surface that is coated in the step 4) Bovine Serum Albumin Modified, dry under wet condition under 4 oC;
6) solution B is dripped the electrode surface that is coated in step 5) tumor markers Modified antigen, dry under wet condition under 4 oC, the electrochemical immunosensor preparation is completed.
2. the tumor markers immunosensor of the shell core nano material of preparation of the present invention structure, is characterized in that, is used for the detection of tumor markers, and step is as follows:
(1) drafting of working curve;
(2) detection of tumor markers.
The drafting of the working curve (1), step is as follows:
1) with contrast electrode-saturated calomel electrode, to electrode-platinum electrode and working electrode exact connect ion on electrochemical workstation;
2) in pH=7 ~ 8 phosphate buffers, detect the response to hydrogen peroxide of the working electrode modified by 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 builds of shell core nano material of the present invention, it is characterized in that described tumor marker is selected from one of high tumor markers of the following present incidence of disease: carcinomebryonic antigen (CEA), alpha-fetoprotein (AFP), alpha-L-fucosidase (AFU), prostate specific antigen (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 the good electronics transmission capacity of nano-porous gold film with and excellent biocompatibility, significantly improved the performance of electrode.
(2) with Au@Pd@Pt shell core nano material and the anti-directly hatching of tumor markers two, utilize the biocompatibility of noble metal excellence and high catalytic performance, needn't use enzyme in two labels that resist, avoided because of the inactivation of enzyme and leaked the detection error that causes, simplify the making step of two anti-labels, significantly improved reappearance and the stability of electrochemical immunosensor.
(3) use identical nano material and method of modifying, utilize the specific binding of antigen and antibody, only need to change the tumor-marker species and can realize highly sensitive, the specific detection of Diagnostic Value of Several Serum Tumor Markers, the method is simple, economical, is conducive to 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 complete sample determination, can realize at short notice the mensuration of batch sample.
(5) the present invention detects the method for tumor markers, and testing result all has instrument automatically to complete and record, has avoided the impact of subjective factor, and good reappearance is arranged, and is convenient to Site 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 Au@Pd@Pt shell core nano material.
Fig. 2 is a kind of building process of tumor markers electrochemical immunosensor of shell core nano material structure.
Embodiment
Embodiment 1
The preparation of the tumor markers immunosensor that a kind of shell core nano material builds, comprise the following steps.
(1) with silver-colored billon film floating at 15 molL
-10.5 min on the nitric acid liquid level, utilize red fuming nitric acid (RFNA) that silver is eroded, and makes the nano-porous gold film.The nano-porous gold film is washed to its pH=7.0 with ultrapure water.
(2) add 20 mmolL in flask
-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, add 0.4 molL immediately after mixing
-1Ascorbic acid 1.0 mL.Stir 1 h under room temperature.Centrifugal washing 3 times, namely make Au@Pd@Pt shell core nano material after drying, the pattern of its nano material is seen Fig. 1, can be found out by Fig. 1 transmission electron microscope picture, Au@Pd@Pt is core-shell structure, its particle diameter is 20 nm, and material scatter is good, uniform particles, is suitable as the making material of electrochemical immunosensor.
(3) Au@Pd@Pt shell core nano material and anti-being mixed to join in pH=7.4 phosphate buffered solution of tumor marker two are made mixed liquor, making the anti-mass concentration ratio of Au Pd Pt shell core nano material and tumor marker two is 500: 1, with concussion hatching 12 h under above-mentioned mixed liquor room temperature.Centrifugal under 10000 rpm, clean with pH=7.4 phosphate buffered solution, then again be scattered in pH=7.4 phosphate buffered solution, obtain Au@Pd@Pt shell core nano material-tumor marker two anti-complex solutions.
(4) glass-carbon electrode of diameter 4 mm is used successively the alundum (Al2O3) burnishing powder polishing of 1.0,0.3 and 0.05 mm, the ethanol ultrasonic cleaning, then with ultrapure water, rinse well, then electrode is placed in 5 mmolL
-1In potassium ferricyanide solution, scan under-0.2 ~ 0.6 V current potential, make spike potential poor less than 110 mV.
(5) building process a kind of according to Fig. 2 of tumor markers electrochemical immunosensor of shell core nano material structure is prepared.Nano-porous gold is film modified in glass-carbon electrode surface, drying at room temperature.With 10 μ gmL
-1Tumor markers primary antibodie 6 μ L drip and are coated onto the film modified electrode surface of nano-porous gold, are placed under 4 oC wet condition and dry.Be 100 μ gmL with concentration
-1Bovine serum albumin(BSA) 3 μ L drip and are coated onto the electrode surface that the tumor markers primary antibodie is modified, and dry under wet condition under 4 oC.Tumor markers antigen 6 μ L are dripped the electrode surface that is coated in Bovine Serum Albumin Modified, dry under wet condition under 4 oC.Au@Pd@Pt shell core 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 tumor markers Modified antigen, dry under wet condition under 4 oC.
Embodiment 2
The preparation of the tumor markers immunosensor that a kind of shell core nano material builds, comprise the following steps.
(1) with silver-colored billon film floating at 15 molL
-13 min on the nitric acid liquid level, utilize red fuming nitric acid (RFNA) that silver is eroded, and makes the nano-porous gold film.The nano-porous gold film is washed to its pH=7.0 with ultrapure water.
(2) add 20 mmolL in flask
-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, add 0.4 molL immediately after mixing
-1Ascorbic acid 1.0 mL.Stir 1 h under room temperature.Centrifugal washing 3 times, namely make Au Pd Pt shell core nano material after drying, its transmission electron microscope pattern is seen Fig. 1.
(3) Au@Pd@Pt shell core nano material and anti-being mixed to join in pH=7.4 phosphate buffered solution of tumor marker two are made mixed liquor, making the anti-mass concentration ratio of Au Pd Pt shell core nano material and tumor marker two is 750: 1, with concussion hatching 12 h under above-mentioned mixed liquor room temperature.Centrifugal under 10000 rpm, clean, then again be scattered in pH=7.4 phosphate buffered solution with pH=7.4 phosphate buffered solution.
(4) glass-carbon electrode of diameter 4 mm is used successively the alundum (Al2O3) burnishing powder polishing of 1.0,0.3 and 0.05 mm, the ethanol ultrasonic cleaning, then with ultrapure water, rinse well, then electrode is placed in 5 mmolL
-1In potassium ferricyanide solution, scan under-0.2 ~ 0.6 V current potential, make spike potential poor less than 110 mV.
(5) building process a kind of according to Fig. 2 of tumor markers electrochemical immunosensor of shell core nano material structure is prepared.Nano-porous gold is film modified in glass-carbon electrode surface, drying at room temperature.With 10 μ gmL
-1Tumor markers primary antibodie 6 μ L drip and are coated onto the film modified electrode surface of nano-porous gold, are placed under 4 oC wet condition and dry.Be 100 μ gmL with concentration
-1Bovine serum albumin(BSA) 3 μ L drip and are coated onto the electrode surface that the tumor markers primary antibodie is modified, and dry under wet condition under 4 oC.Tumor markers antigen 6 μ L are dripped the electrode surface that is coated in Bovine Serum Albumin Modified, dry under wet condition under 4 oC.Au@Pd@Pt shell core 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 tumor markers Modified antigen, dry under wet condition under 4 oC.
Embodiment 3
The preparation of the tumor markers immunosensor that a kind of shell core nano material builds, comprise the following steps.
(1) with silver-colored billon film floating at 15 molL
-15 min on the nitric acid liquid level, utilize red fuming nitric acid (RFNA) that silver is eroded, and makes the nano-porous gold film.The nano-porous gold film is washed to its pH=7.0 with ultrapure water.
(2) add 20 mmolL in flask
-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, add 0.4 molL immediately after mixing
-1Ascorbic acid 1.0 mL.Stir 1 h under room temperature.Centrifugal washing 3 times, namely make Au Pd Pt shell core nano material after drying, its transmission electron microscope pattern is seen Fig. 1.
(3) Au@Pd@Pt shell core nano material and anti-being mixed to join in pH=7.4 phosphate buffered solution of tumor marker two are made mixed liquor, making the anti-mass concentration ratio of Au Pd Pt shell core nano material and tumor marker two is 1000: 1, with concussion hatching 12 h under above-mentioned mixed liquor room temperature, centrifugal under 10000 rpm, clean, then again be scattered in pH=7.4 phosphate buffered solution with pH=7.4 phosphate buffered solution.
(4) glass-carbon electrode of diameter 4 mm is used successively the alundum (Al2O3) burnishing powder polishing of 1.0,0.3 and 0.05 mm, the ethanol ultrasonic cleaning, then with ultrapure water, rinse well, then electrode is placed in 5 mmolL
-1In potassium ferricyanide solution, scan under-0.2 ~ 0.6 V current potential, make spike potential poor less than 110 mV.
(5) building process a kind of according to Fig. 2 of tumor markers electrochemical immunosensor of shell core nano material structure is prepared.Nano-porous gold is film modified in glass-carbon electrode surface, drying at room temperature.With 10 μ g.mL
-1Tumor markers primary antibodie 6 μ L drip and are coated onto the film modified electrode surface of nano-porous gold, are placed under 4 oC wet condition and dry.Be 100 μ gmL with concentration
-1Bovine serum albumin(BSA) 3 μ L drip and are coated onto the electrode surface that the tumor markers primary antibodie is modified, and dry under wet condition under 4 oC.Tumor markers antigen 6 μ L are dripped the electrode surface that is coated in Bovine Serum Albumin Modified, dry under wet condition under 4 oC.Au@Pd@Pt shell core 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 tumor markers Modified antigen, dry under wet condition under 4 oC.
Embodiment 4
The tumor markers electrochemical immunosensor of embodiment 1 ~ 3 preparation, be respectively used to tumor markers and detect, and comprises the following steps.
(1) drafting of working curve, step is as follows:
1) with contrast electrode-saturated calomel electrode, to electrode-platinum electrode and working electrode exact connect ion on the CHI760D electrochemical workstation;
2) in the pH=7.4 phosphate buffer, detect the response to hydrogen peroxide of the working electrode modified by 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 comprise the following steps:
(1) select the breast cancer tumour mark, according to the described step of embodiment 1, build electrochemical immunosensor;
(2) detect according to the described step of embodiment 4, the detection technique index of breast cancer tumour mark is in 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 |
Shown by table 1 detection technique index result, 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 comprise the following steps:
(1) select the liver cancer tumor markers, according to the described step of embodiment 2, build electrochemical immunosensor;
(2) detect according to the described step of embodiment 4, the detection technique index of liver cancer tumor markers is in 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 |
Shown by table 2 detection technique index result, 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 7Stomach neoplasms tumor markers: CA-724, CA-199 or CA-242
A kind of preparation of stomach neoplasms tumor markers immunosensor and application comprise the following steps:
(1) select stomach neoplasms tumor markers, according to the described step of embodiment 3, build electrochemical immunosensor;
(2) detect according to the described step of embodiment 4, the detection technique index of stomach neoplasms tumor markers is in Table 3.
The detection technique index of table 3 stomach neoplasms 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 |
Shown by table 3 detection technique index result, this electrochemical immunosensor is used for the detection of stomach neoplasms 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 human serum
Accurately pipette the human serum sample, according to the described step of embodiment 4, detect, testing result is in Table 4.
The testing result of Diagnostic Value of Several Serum Tumor Markers in table 4 human serum
By table 4 testing result as can be known, the relative standard deviation of result (RSD) is less than 3.0%, and average recovery rate is 95.0 ~ 102%, shows that the present invention is used for the detection of human serum Diagnostic Value of Several Serum Tumor Markers, and the precision of method is high, and result accurately and reliably.
Claims (3)
1. the preparation method of the tumor markers immunosensor of a shell core nano material structure, is characterized in that, comprises the following steps:
(1) preparation of nano-porous gold film, step is as follows:
1) with silver-colored billon film floating at 14 ~ 16 molL
-1On the nitric acid liquid level, 0.5 ~ 5 min, erode silver, makes the nano-porous gold film;
2) the nano-porous gold film is washed to its pH=7.0 with ultrapure water;
(2) preparation of Au@Pd@Pt shell core nano material-tumor marker two anti-compounds, step is as follows:
1) be mixed to join in pH=7.4 phosphate buffered solution Au@Pd@Pt shell core nano material and tumor marker two is anti-, making the anti-mass concentration ratio of Au Pd Pt shell core nano material and tumor marker two is 500 ~ 1000: 1, is mixed with mixed liquor A;
2) with concussion hatching 12 ~ 24 h under above-mentioned mixed liquor A room temperature, centrifugal under 10000 ~ 12000 rpm, clean, then again be scattered in pH=7.4 phosphate buffered solution and be mixed with mixed liquid B and be stored under 4oC standby with pH=7.4 phosphate buffered solution;
(3) structure of electrochemical immunosensor.
2. the preparation method of the tumor markers immunosensor that builds of a kind of shell core nano material as described in claim 1, is characterized in that, the structure of described electrochemical immunosensor, and step is as follows:
(1) glass-carbon electrode of diameter 4 mm is used successively the alundum (Al2O3) burnishing powder polishing of 1.0,0.3 and 0.05 mm, the ethanol ultrasonic cleaning, then with ultrapure water, rinse well, then electrode is placed in 5 mmolL
-1In potassium ferricyanide solution, scan under-0.2 ~ 0.6 V current potential, make spike potential poor less than 110 mV;
(2) nano-porous gold is film modified in glass-carbon electrode surface, drying at room temperature;
(3) the tumor markers primary antibodie is dripped be coated onto the film modified electrode surface of step (2) nano-porous gold, be placed under the 4oC wet condition and dry;
(4) with concentration be 100 μ gmL
-1Bovine serum albumin(BSA) drips and is coated onto the electrode surface that step (3) tumor markers primary antibodie is modified, and dries under wet condition under 4oC;
(5) tumor markers antigen is dripped the electrode surface that is coated in step (4) Bovine Serum Albumin Modified, dry under wet condition under 4oC;
(6) solution B is dripped the electrode surface that is coated in step (5) tumor markers Modified antigen, dry under wet condition under 4oC, the electrochemical immunosensor preparation is completed.
3. the preparation method of the tumor markers immunosensor that builds of a kind of shell core nano material as described in claim 1 or 2, it is characterized in that described tumor marker is selected from one of high tumor markers of the following present incidence of disease: carcinomebryonic antigen (CEA), alpha-fetoprotein (AFP), alpha-L-fucosidase (AFU), prostate specific antigen (PSA), CA-125, CA-199, CA-724, CA-242, human chorionic gonadotrophin (HCG).
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| CN106198699B (en) * | 2016-07-20 | 2019-01-18 | 太原理工大学 | Prepare two kinds of secondary antibody conjugates and its at the same the method that detects alpha-fetoprotein and carcinomebryonic antigen |
| CN107543924A (en) * | 2017-09-06 | 2018-01-05 | 重庆医科大学 | Biology sensor detection myeloperoxidase based on copper palladium platinum mesh nanometer material |
| CN108982629B (en) * | 2018-07-20 | 2020-02-14 | 山东理工大学 | Preparation method and application of current type immunosensor mutually verified by double detection methods |
| CN110082403B (en) * | 2019-03-27 | 2021-11-02 | 宁波大学 | Histone acetyltransferase timing-current sensor based on gold-palladium nanoflower/graphene composite material and application thereof |
| CN110068601A (en) * | 2019-04-27 | 2019-07-30 | 山东理工大学 | A kind of preparation method and application of the electrochemical sensor based on the mesoporous stick label of mulberries shape Au@PtPd |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101706498A (en) * | 2009-11-09 | 2010-05-12 | 东南大学 | Preparing method of electrochemiluminescence immunosensor |
| CN102072954A (en) * | 2010-10-29 | 2011-05-25 | 济南大学 | Research and application of electrochemiluminescent immunosensor for detecting tumor markers |
| CN102253221A (en) * | 2011-07-15 | 2011-11-23 | 华中师范大学 | Electrochemical immune sensor for phosphating protein |
-
2012
- 2012-07-26 CN CN201210261102XA patent/CN102778561B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101706498A (en) * | 2009-11-09 | 2010-05-12 | 东南大学 | Preparing method of electrochemiluminescence immunosensor |
| CN102072954A (en) * | 2010-10-29 | 2011-05-25 | 济南大学 | Research and application of electrochemiluminescent immunosensor for detecting tumor markers |
| CN102253221A (en) * | 2011-07-15 | 2011-11-23 | 华中师范大学 | Electrochemical immune sensor for phosphating protein |
Non-Patent Citations (12)
| Title |
|---|
| Designing nanomaterial-enhanced electrochemical immunosensors for cancer biomarker proteins.;James F. Rusling et al.;《Bioelectrochemistry》;20090405;第76卷;第189－194页 * |
| Dumbbell-like Au-Fe3O4 nanoparticles as label for the preparation of electrochemical immunosensors.;Qin Wei et al.;《Biosensors and Bioelectronics》;20100712;第26卷;第628页第2.1节－第2.5节，图3－图5 * |
| Electrochemical immunosensors for cancer biomarker with signal amplification based on ferrocene functionalized iron oxide nanoparticles.;He Li et al.;《Biosensors and Bioelectronics》;20110216;第26卷;第3591－3592页，第2.1节－第2.4节 * |
| He Li et al..Electrochemical immunosensors for cancer biomarker with signal amplification based on ferrocene functionalized iron oxide nanoparticles..《Biosensors and Bioelectronics》.2011,第26卷 |
| James F. Rusling et al..Designing nanomaterial-enhanced electrochemical immunosensors for cancer biomarker proteins..《Bioelectrochemistry》.2009,第76卷 |
| Liang Wang, Yusuke Yamauchi.Autoprogrammed synthesis of triple-layered Au@Pd@Pt core-shell nanoparticle consisting of a Au@Pd bimetallic cor and nanoporous Pt shell..《J Am Chem Soc》.2010,第132卷 |
| Liang Wang, Yusuke Yamauchi.Autoprogrammed synthesis of triple-layered AuPdPt core-shell nanoparticle consisting of a AuPd bimetallic cor and nanoporous Pt shell..《J Am Chem Soc》.2010,第132卷 * |
| Nanoporous gold film based immunosensor for label-free detection of cancer biomarker.;Qin Wei et al.;《Biosensors and Bioelectronics》;20110221;第26卷;第3715页第2.2节，第2.3节，第3717页"结论"部分 * |
| Noble metal nanomaterials:controllable synthesis and application in fuel cells and analytical sensors.;Shaojun Guo, Erkang Wang;《Nano Today》;20110524;第6卷;第246页左侧栏最后一段，第247页左侧栏 * |
| Qin Wei et al..Dumbbell-like Au-Fe3O4 nanoparticles as label for the preparation of electrochemical immunosensors..《Biosensors and Bioelectronics》.2010,第26卷 |
| Qin Wei et al..Nanoporous gold film based immunosensor for label-free detection of cancer biomarker..《Biosensors and Bioelectronics》.2011,第26卷 |
| Shaojun Guo, Erkang Wang.Noble metal nanomaterials:controllable synthesis and application in fuel cells and analytical sensors..《Nano Today》.2011,第6卷 |
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