技术领域technical field
本发明涉及一种基于PPy-NH2GO-Ag2SeCdSe电致化学发光免疫传感器的制备方法及应用。具体涉及一种Ag2SeCdSe纳米大头针作为发光材料，PPy-NH2GO-Ag2SeCdSe作为捕获抗体基底的电致化学发光免疫传感器的制备与应用，属于电化学发光检测技术领域。The invention relates to a preparation method and application of an electrochemiluminescent immunosensor based on PPy-NH2 GO-Ag2 SeCdSe. It specifically relates to the preparation and application of an electrochemiluminescence immunosensor with Ag2 SeCdSe nano pin as a luminescent material and PPy-NH2 GO-Ag2 SeCdSe as a capture antibody substrate, belonging to the technical field of electrochemiluminescence detection.
背景技术Background technique
胃癌抗原CA724是检测胃癌和各种消化道癌症的化验标志，全称为相关性糖蛋白，属粘蛋白类癌胚胎抗原，存在于5%的乳腺组织和85%~95%的结肠、胰腺、胃、肺和卵巢的肿瘤中。因此，对于胰腺、卵巢、胃等部位的肿瘤有一定的诊断和鉴别诊断意义。Gastric cancer antigen CA724 is a laboratory marker for detecting gastric cancer and various digestive tract cancers. It is called related glycoprotein and belongs to mucin carcinoid embryonic antigen. , lung and ovarian tumors. Therefore, it has certain diagnostic and differential diagnosis significance for tumors in pancreas, ovary, stomach and other parts.
目前测定CA72-4的免疫分析方法主要有放射免疫分析法、酶联免疫分析法和试剂盒法。申请号为201110235595.5的专利中公开了糖类蛋白CA72-4定量测定试剂盒的制备方法及应用；申请号为201320494726.6的专利中公开了检测CA72-4试剂盒的制备方法及应用。但是试剂有效期短，操作周期长，步骤繁琐。为了克服以上色谱分析方法的缺点，本发明设计了一种特异性强，灵敏度高，无放射性污染，操作快速简便的电致化学发光免疫分析方法。而电致化学发光免疫传感器法检测糖类蛋白CA72-4未见公开的专利和文献报道。At present, the immunoassay methods for the determination of CA72-4 mainly include radioimmunoassay, enzyme-linked immunoassay and kit method. The patent application number 201110235595.5 discloses the preparation method and application of the glycoprotein CA72-4 quantitative assay kit; the patent application number 201320494726.6 discloses the preparation method and application of the CA72-4 detection kit. However, the validity period of the reagent is short, the operation cycle is long, and the steps are cumbersome. In order to overcome the shortcomings of the above chromatographic analysis method, the present invention designs an electrochemiluminescence immunoassay method with strong specificity, high sensitivity, no radioactive pollution, and quick and easy operation. However, there are no published patents and literature reports on the detection of glycoprotein CA72-4 by the electrochemiluminescence immunosensor method.
本发明利用Ag2SeCdSe纳米大头针为电致化学发光信号源，以Ag2SeCdSe纳米大头针为电致化学发光信号源，利用PPy-NH2GO大的比表面积将Ag2SeCdSe纳米大头针固载在PPy-NH2GO表面制得抗体捕获基底，PPy-NH2GO优良的导电性可以有效加强传感器的发光信号。在本发明中构建的致电化学发光免疫传感器具有操作简单，成本低，无放射性污染，灵敏度高，特异性强等优点，克服了放射免疫分析法、酶联免疫分析法和试剂盒法的一些不足。The present invention uses Ag2 SeCdSe nano pins as the electrochemiluminescence signal source, uses the Ag2 SeCdSe nano pins as the electrochemiluminescence signal source, and utilizes the large specific surface area of PPy-NH2 GO to immobilize the Ag2 SeCdSe nano pins on PPy -NH2 GO surface was used to prepare the antibody capture substrate, and the excellent conductivity of PPy-NH2 GO could effectively enhance the luminescent signal of the sensor. The electrochemiluminescence immunosensor constructed in the present invention has the advantages of simple operation, low cost, no radioactive pollution, high sensitivity and strong specificity, and overcomes some shortcomings of radioimmunoassay, enzyme-linked immunoassay and kit method .
发明内容Contents of the invention
本发明的目的是针对现有的糖类蛋白CA72-4检测方法的问题，提供一种简单、快速、无污染的PPy-NH2GO-Ag2SeCdSe电致化学发光免疫传感器的制备方法及应用，实现对CA72-4的快速灵敏检测。The purpose of the present invention is to provide a simple, rapid and pollution-free PPy-NH2 GO-Ag2 SeCdSe electrochemiluminescence immunosensor preparation method and application for the problems of the existing glycoprotein CA72-4 detection method , to achieve rapid and sensitive detection of CA72-4.
本发明的技术方案如下：Technical scheme of the present invention is as follows:
1.一种基于PPy-NH2GO-Ag2SeCdSe的胃癌抗原电致化学发光传感器的制备方法1. Preparation method of gastric cancer antigen electrochemiluminescence sensor based on PPy-NH2 GO-Ag2 SeCdSe
（1）将直径4 mm的玻碳电极依次用1.0 μm、0.3 μm、0.05 μm氧化铝抛光粉依次做抛光处理，用乙醇超声清洗，再用超纯水冲洗干净；(1) Polish the glassy carbon electrode with a diameter of 4 mm with 1.0 μm, 0.3 μm, and 0.05 μm alumina polishing powder in sequence, clean it ultrasonically with ethanol, and rinse it with ultrapure water;
（2）滴涂4 μL、0.5~2.0 mg mL-1的PPy-NH2GO-Ag2SeCdSe-Ab抗体孵化溶液于电极表面，4℃保存至干燥；(2) Drop-coat 4 μL, 0.5-2.0 mg mL-1 PPy-NH2 GO-Ag2 SeCdSe-Ab antibody incubation solution on the surface of the electrode, and store at 4°C until dry;
（3）滴加4 μL、质量分数为1~3%的牛血清白蛋白溶液，以封闭电极表面的非特异性活性位点，用pH 6.4~8.4的PBS缓冲溶液冲洗电极表面，4℃晾干；(3) Add 4 μL bovine serum albumin solution with a mass fraction of 1-3% dropwise to seal the non-specific active sites on the electrode surface, rinse the electrode surface with PBS buffer solution with pH 6.4-8.4, and dry at 4°C ;
（4）滴加4 μL的胃癌抗原CA72-4溶液，用pH 6.4~8.4的PBS缓冲溶液冲洗电极表面，放置在4 ℃的冰箱中孵化3~12 h晾干，制得一种基于PPy-NH2GO-Ag2SeCdSe的胃癌抗原电致化学发光传感器。(4) Add 4 μL of gastric cancer antigen CA72-4 solution dropwise, wash the electrode surface with PBS buffer solution of pH 6.4-8.4, place it in a refrigerator at 4 °C for 3-12 h and dry it to prepare a PPy-based NH2 GO-Ag2 SeCdSe Electrochemiluminescent Sensor for Gastric Cancer Antigen.
2. 上述所述的PPy-NH2GO-Ag2SeCdSe-Ab抗体孵化溶液的制备2. Preparation of PPy-NH2 GO-Ag2 SeCdSe-Ab antibody incubation solution as described above
0.05~0.15 mL、1 μg/mL CA72-4抗体Ab加入到PPy-NH2GO-Ag2SeCdSe纳米复合物溶液中，在4 ℃下振荡孵化12~24 h，离心除去过量的CA72-4抗体Ab，将产物分散到1 mL、pH 7.4的PBS中，制得PPy-NH2GO-Ag2SeCdSe-Ab抗体孵化溶液，储存在4 ℃下备用。Add 0.05~0.15 mL, 1 μg/mL CA72-4 antibody Ab to the PPy-NH2 GO-Ag2 SeCdSe nanocomposite solution, shake and incubate at 4 ℃ for 12~24 h, and centrifuge to remove excess CA72-4 antibody Ab, the product was dispersed into 1 mL of PBS with pH 7.4 to prepare PPy-NH2 GO-Ag2 SeCdSe-Ab antibody incubation solution, which was stored at 4°C for use.
3. 上述所述PPy-NH2GO-Ag2SeCdSe纳米复合物溶液的制备3. Preparation of the PPy-NH2 GO-Ag2 SeCdSe nanocomposite solution described above
（1）氨基化石墨烯NH2GO的制备(1) Preparation of aminated graphene NH2 GO
将90~110 mg 氧化石墨烯分散到30~50 mL乙二醇中，边超声边滴加0.8~1.2 mL 氨水，将得到的深褐色溶液转入聚四氟乙烯内衬高压反应釜中，170~190 ℃下反应8~12 h，过滤并用超纯水洗涤，50~70 ℃干燥12~36 h；Disperse 90-110 mg of graphene oxide into 30-50 mL of ethylene glycol, add 0.8-1.2 mL of ammonia water dropwise while ultrasonicating, and transfer the obtained dark brown solution into a polytetrafluoroethylene-lined high-pressure reactor, 170 React at ~190 °C for 8~12 h, filter and wash with ultrapure water, and dry at 50~70 °C for 12~36 h;
（2）聚吡咯插层氨基化石墨烯PPy-NH2GO的制备(2) Preparation of polypyrrole-intercalated aminated graphene PPy-NH2 GO
将氨基化石墨烯NH2GO分散到20~30 mL超纯水中，超声25~35 min，边超声边加入90~110 mL、0.13~0.15 g/mL的十二烷基苯磺酸钠，超声0.5~1.5 h后，将温度降至0 ℃，滴加200 μL吡咯，磁力搅拌20~40 min，边搅拌边加入20~40 mL、2~3 mol/L的三氯化铁溶液，搅拌反应2~3 h，过滤洗涤至中性，40~60 ℃干燥36~64 h；Disperse the aminated graphene NH2 GO into 20-30 mL ultrapure water, sonicate for 25-35 min, add 90-110 mL, 0.13-0.15 g/mL sodium dodecylbenzenesulfonate while sonicating, After ultrasonication for 0.5-1.5 h, lower the temperature to 0 °C, add 200 μL of pyrrole dropwise, stir magnetically for 20-40 min, add 20-40 mL, 2-3 mol/L ferric chloride solution while stirring, and stir React for 2-3 hours, filter and wash until neutral, and dry at 40-60 °C for 36-64 hours;
（3）Ag2SeCdSe纳米大头针溶液的制备(3) Preparation of Ag2 SeCdSe nano-pin solution
将20~26 mg二氯化镉，10~12 mg二氧化硒和0.1~0.3 g聚乙烯吡咯烷酮k-30溶入8~12 mL的二甲基甲酰胺中，边搅拌边加入4~6 mL油酸，0.1~0.3 mL、4 mmol/L的硝酸银水溶液，0.06~0.09 g的六偏磷酸钠和50 μL的巯基乙酸，将得到的混合溶液转入聚四氟乙烯内衬高压反应釜中，90~110 ℃下反应15 h, 离心分离并用异丙醇洗涤，将产物分散到异丙醇中，制得Ag2SeCdSe纳米大头针溶液；Dissolve 20~26 mg of cadmium dichloride, 10~12 mg of selenium dioxide and 0.1~0.3 g of polyvinylpyrrolidone K-30 into 8~12 mL of dimethylformamide, and add 4~6 mL of Oleic acid, 0.1-0.3 mL, 4 mmol/L silver nitrate aqueous solution, 0.06-0.09 g sodium hexametaphosphate and 50 μL thioglycolic acid, transfer the resulting mixed solution into a polytetrafluoroethylene-lined autoclave , reacted at 90~110 ℃ for 15 h, centrifuged and washed with isopropanol, dispersed the product in isopropanol to prepare Ag2 SeCdSe nano pin solution;
（4）PPy-NH2GO-Ag2SeCdSe纳米复合物溶液的制备(4) Preparation of PPy-NH2 GO-Ag2 SeCdSe nanocomposite solution
在Ag2SeCdSe纳米大头针溶液中加入1 mL、2 mg/mL的聚吡咯插层氨基化石墨烯PPy-NH2GO溶液，振荡孵化12~24 h，制得PPy-NH2GO-Ag2SeCdSe纳米复合物溶液。PPy-NH2 GO-Ag2 SeCdSe was prepared by adding 1 mL, 2 mg/mL polypyrrole-intercalated aminated graphene PPy-NH2 GO solution to the Ag2 SeCdSe nano-pin solution, shaking and incubating for 12-24 h Nanocomposite solution.
4. CA72-4的检测4. Detection of CA72-4
（1）使用电化学工作站的三电极体系进行测试，Ag/AgCl电极作为参比电极，铂丝电极为对电极，所制备的胃癌抗原电致化学发光传感器为工作电极，将电化学工作站和化学发光检测仪连接在一起将光电倍增管的高压设置为800 V，扫描电压设置为-1.7~0 V，扫描速率0.1 V/s；(1) The three-electrode system of the electrochemical workstation was used for testing, the Ag/AgCl electrode was used as the reference electrode, the platinum wire electrode was used as the counter electrode, and the prepared gastric cancer antigen electrochemiluminescence sensor was used as the working electrode. The luminescence detector is connected together, the high voltage of the photomultiplier tube is set to 800 V, the scanning voltage is set to -1.7~0 V, and the scanning rate is 0.1 V/s;
（2）在10 mL、pH 6.4~8.4的含50~60 mmol/L过硫酸钾的PBS缓冲溶液中，通过电致化学发光系统检测对0.01 pg/mL~50 ng/mL一系列不同浓度的胃癌抗原CA72-4标准溶液，产生的电致化学发光信号强度，绘制工作曲线；(2) In 10 mL of PBS buffer solution containing 50-60 mmol/L potassium persulfate at pH 6.4-8.4, a series of different concentrations of 0.01 pg/mL-50 ng/mL were detected by electrochemiluminescence system Stomach cancer antigen CA72-4 standard solution, the intensity of the electrochemiluminescence signal generated, and the working curve is drawn;
（3）将待测样品溶液代替胃癌抗原CA72-4标准溶液进行检测。(3) The test sample solution was used instead of the gastric cancer antigen CA72-4 standard solution for detection.
本发明的有益成果Beneficial results of the present invention
（1）电致化学发光免疫传感器制备方法,以Ag2SeCdSe纳米大头针为发光材料，利用其良好的光学性能，构建的传感器具有更高的灵敏度；(1) Electrochemiluminescence immunosensor preparation method, using Ag2 SeCdSe nano-pins as the luminescent material, taking advantage of its good optical properties, the sensor constructed has higher sensitivity;
（2）利用PPy-NH2GO大的比表面积将Ag2SeCdSe固载在PPy-NH2GO表面制得抗体捕获基底，PPy-NH2GO优良的导电性可以有效加强传感器的发光信号；(2) Using the large specific surface area of PPy-NH2 GO to immobilize Ag2 SeCdSe on the surface of PPy-NH2 GO to prepare the antibody capture substrate, the excellent conductivity of PPy-NH2 GO can effectively enhance the luminescent signal of the sensor;
（3））本发明制备的电致化学发光免疫传感器用于CA72-4的检测，操作简单，无放射性污染，信号响应范围宽，线性范围为0.1 pg/mL~20 ng/mL，检测限为21 fg/mL，可以实现简单、快速、高灵敏检测。(3) The electrochemiluminescence immunosensor prepared by the present invention is used for the detection of CA72-4, with simple operation, no radioactive contamination, wide signal response range, a linear range of 0.1 pg/mL~20 ng/mL, and a detection limit of 21 fg/mL, which can realize simple, rapid and highly sensitive detection.
实施例1 一种基于PPy-NH2GO-Ag2SeCdSe的胃癌抗原电致化学发光传感器的制备方法Example 1 Preparation method of gastric cancer antigen electrochemiluminescence sensor based on PPy-NH2 GO-Ag2 SeCdSe
（1）将直径4 mm的玻碳电极依次用1.0 μm、0.3 μm、0.05 μm氧化铝抛光粉依次做抛光处理，用乙醇超声清洗，再用超纯水冲洗干净；(1) Polish the glassy carbon electrode with a diameter of 4 mm with 1.0 μm, 0.3 μm, and 0.05 μm alumina polishing powder in sequence, clean it ultrasonically with ethanol, and rinse it with ultrapure water;
（2）滴涂4 μL、0.5 mg mL-1的PPy-NH2GO-Ag2SeCdSe-Ab抗体孵化溶液于电极表面，4℃保存至干燥；(2) Drop-coat 4 μL, 0.5 mg mL-1 PPy-NH2 GO-Ag2 SeCdSe-Ab antibody incubation solution on the surface of the electrode, and store at 4°C until dry;
（3）滴加4 μL、质量分数为1 %的牛血清白蛋白溶液，以封闭电极表面的非特异性活性位点，用pH 6.4的PBS缓冲溶液冲洗电极表面，4℃晾干；(3) Add 4 μL bovine serum albumin solution with a mass fraction of 1% dropwise to seal the non-specific active sites on the electrode surface, rinse the electrode surface with PBS buffer solution with pH 6.4, and dry at 4°C;
（4）滴加4 μL的胃癌抗原CA72-4溶液，用pH 6.4的PBS缓冲溶液冲洗电极表面，放置在4 ℃的冰箱中孵化3 h晾干，制得一种基于PPy-NH2GO-Ag2SeCdSe电致化学发光免疫传感器。(4) 4 μL of gastric cancer antigen CA72-4 solution was added dropwise, the surface of the electrode was washed with PBS buffer solution of pH 6.4, and placed in a refrigerator at 4 °C for 3 h to dry to prepare a PPy-NH2 GO-based Ag2 SeCdSe electrochemiluminescent immunosensor.
实施例2 一种基于PPy-NH2GO-Ag2SeCdSe的胃癌抗原电致化学发光传感器的制备方法Example 2 A preparation method of a gastric cancer antigen electrochemiluminescence sensor based on PPy-NH2 GO-Ag2 SeCdSe
（1）将直径4 mm的玻碳电极依次用1.0 μm、0.3 μm、0.05 μm氧化铝抛光粉依次做抛光处理，用乙醇超声清洗，再用超纯水冲洗干净；(1) Polish the glassy carbon electrode with a diameter of 4 mm with 1.0 μm, 0.3 μm, and 0.05 μm alumina polishing powder in sequence, clean it ultrasonically with ethanol, and rinse it with ultrapure water;
（2）滴涂4 μL、1 mg mL-1的PPy-NH2GO-Ag2SeCdSe-Ab抗体孵化溶液于电极表面，4℃保存至干燥；(2) Drop-coat 4 μL, 1 mg mL-1 PPy-NH2 GO-Ag2 SeCdSe-Ab antibody incubation solution on the surface of the electrode, and store at 4°C until dry;
（3）滴加4 μL、质量分数为2 %的牛血清白蛋白溶液，以封闭电极表面的非特异性活性位点，用pH 7.4的PBS缓冲溶液冲洗电极表面，4℃晾干；(3) Add 4 μL bovine serum albumin solution with a mass fraction of 2% dropwise to seal the non-specific active sites on the electrode surface, rinse the electrode surface with PBS buffer solution with pH 7.4, and dry at 4°C;
（4）滴加4 μL的胃癌抗原CA72-4溶液，用pH 7.4的PBS缓冲溶液冲洗电极表面，放置在4 ℃的冰箱中孵化7 h晾干，制得一种基于PPy-NH2GO-Ag2SeCdSe电致化学发光免疫传感器。(4) 4 μL of gastric cancer antigen CA72-4 solution was added dropwise, the surface of the electrode was rinsed with PBS buffer solution of pH 7.4, and placed in a refrigerator at 4 °C for 7 h to dry to prepare a PPy-NH2 GO-based Ag2 SeCdSe electrochemiluminescent immunosensor.
实施例3 一种基于PPy-NH2GO-Ag2SeCdSe的胃癌抗原电致化学发光传感器的制备方法Example 3 A preparation method of a gastric cancer antigen electrochemiluminescence sensor based on PPy-NH2 GO-Ag2 SeCdSe
（1）将直径4 mm的玻碳电极依次用1.0 μm、0.3 μm、0.05 μm氧化铝抛光粉依次做抛光处理，用乙醇超声清洗，再用超纯水冲洗干净；(1) Polish the glassy carbon electrode with a diameter of 4 mm with 1.0 μm, 0.3 μm, and 0.05 μm alumina polishing powder in sequence, clean it ultrasonically with ethanol, and rinse it with ultrapure water;
（2）滴涂4 μL、2.0 mg mL-1的PPy-NH2GO-Ag2SeCdSe-Ab抗体孵化溶液于电极表面，4℃保存至干燥；(2) Drop-coat 4 μL, 2.0 mg mL-1 PPy-NH2 GO-Ag2 SeCdSe-Ab antibody incubation solution on the surface of the electrode, and store at 4°C until dry;
（3）滴加4 μL、质量分数为3%的牛血清白蛋白溶液，以封闭电极表面的非特异性活性位点，用pH 8.4的PBS缓冲溶液冲洗电极表面，4℃晾干；(3) Add 4 μL bovine serum albumin solution with a mass fraction of 3% dropwise to seal the non-specific active sites on the electrode surface, rinse the electrode surface with PBS buffer solution with pH 8.4, and dry at 4°C;
（4）滴加4 μL的胃癌抗原CA72-4溶液，用pH 8.4的PBS缓冲溶液冲洗电极表面，放置在4 ℃的冰箱中孵化12 h晾干，制得一种基于PPy-NH2GO-Ag2SeCdSe电致化学发光免疫传感器。(4) 4 μL of gastric cancer antigen CA72-4 solution was added dropwise, the surface of the electrode was washed with PBS buffer solution of pH 8.4, and placed in a refrigerator at 4 °C for 12 h to dry to prepare a PPy-NH2 GO-based Ag2 SeCdSe electrochemiluminescent immunosensor.
实施例4 PPy-NH2GO-Ag2SeCdSe-Ab抗体孵化溶液的制备Example 4 Preparation of PPy-NH2 GO-Ag2 SeCdSe-Ab antibody incubation solution
0.05 mL、1 μg/mL CA72-4抗体Ab加入到PPy-NH2GO-Ag2SeCdSe纳米复合物溶液中，在4 ℃下振荡孵化12 h，离心除去过量的CA72-4抗体Ab，将产物分散到1 mL、pH 7.4的PBS中，制得PPy-NH2GO-Ag2SeCdSe-Ab抗体孵化溶液，储存在4 ℃下备用。Add 0.05 mL, 1 μg/mL CA72-4 antibody Ab to the PPy-NH2 GO-Ag2 SeCdSe nanocomposite solution, shake and incubate at 4 °C for 12 h, centrifuge to remove excess CA72-4 antibody Ab, and the product Disperse into 1 mL of PBS with pH 7.4 to prepare PPy-NH2 GO-Ag2 SeCdSe-Ab antibody incubation solution, and store it at 4 °C for future use.
实施例5 PPy-NH2GO-Ag2SeCdSe-Ab抗体孵化溶液的制备Example 5 Preparation of PPy-NH2 GO-Ag2 SeCdSe-Ab antibody incubation solution
0.1 mL、1 μg/mL CA72-4抗体Ab加入到PPy-NH2GO-Ag2SeCdSe纳米复合物溶液中，在4 ℃下振荡孵化18 h，离心除去过量的CA72-4抗体Ab，将产物分散到1 mL、pH 7.4的PBS中，制得PPy-NH2GO-Ag2SeCdSe-Ab抗体孵化溶液，储存在4 ℃下备用。Add 0.1 mL, 1 μg/mL CA72-4 antibody Ab to the PPy-NH2 GO-Ag2 SeCdSe nanocomposite solution, shake and incubate at 4 °C for 18 h, centrifuge to remove excess CA72-4 antibody Ab, and the product Disperse into 1 mL of PBS with pH 7.4 to prepare PPy-NH2 GO-Ag2 SeCdSe-Ab antibody incubation solution, and store it at 4°C for future use.
实施例6 PPy-NH2GO-Ag2SeCdSe-Ab抗体孵化溶液的制备Example 6 Preparation of PPy-NH2 GO-Ag2 SeCdSe-Ab antibody incubation solution
0.15 mL、1 μg/mL CA72-4抗体Ab加入到PPy-NH2GO-Ag2SeCdSe纳米复合物溶液中，在4 ℃下振荡孵化24 h，离心除去过量的CA72-4抗体Ab，将产物分散到1 mL、pH 7.4的PBS中，制得PPy-NH2GO-Ag2SeCdSe-Ab抗体孵化溶液，储存在4 ℃下备用。Add 0.15 mL, 1 μg/mL CA72-4 antibody Ab to the PPy-NH2 GO-Ag2 SeCdSe nanocomposite solution, shake and incubate at 4 °C for 24 h, centrifuge to remove excess CA72-4 antibody Ab, and the product Disperse into 1 mL of PBS with pH 7.4 to prepare PPy-NH2 GO-Ag2 SeCdSe-Ab antibody incubation solution, and store it at 4°C for future use.
实施例7 PPy-NH2GO-Ag2SeCdSe纳米复合物溶液的制备Example 7 Preparation of PPy-NH2 GO-Ag2 SeCdSe Nanocomposite Solution
（1）氨基化石墨烯NH2GO的制备(1) Preparation of aminated graphene NH2 GO
将90 mg 氧化石墨烯分散到30 mL乙二醇中，边超声边滴加0.8 mL 氨水，将得到的深褐色溶液转入聚四氟乙烯内衬高压反应釜中，170 ℃下反应8h，过滤并用超纯水洗涤，50 ℃干燥12 h；Disperse 90 mg of graphene oxide into 30 mL of ethylene glycol, add 0.8 mL of ammonia water dropwise while ultrasonicating, transfer the resulting dark brown solution into a polytetrafluoroethylene-lined autoclave, react at 170 °C for 8 hours, and filter and washed with ultrapure water, and dried at 50 °C for 12 h;
（2）聚吡咯插层氨基化石墨烯PPy-NH2GO的制备(2) Preparation of polypyrrole-intercalated aminated graphene PPy-NH2 GO
将氨基化石墨烯NH2GO分散到20 mL超纯水中，超声25 min，边超声边加入90 mL、0.13 g/mL的十二烷基苯磺酸钠，超声0.5 h后，将温度降至0 ℃，滴加200 μL吡咯，磁力搅拌20 min，边搅拌边加入20 mL、2 mol/L的三氯化铁溶液，搅拌反应2 h，过滤洗涤至中性，40 ℃干燥36 h；Disperse the aminated graphene NH2 GO in 20 mL of ultrapure water, sonicate for 25 min, add 90 mL of 0.13 g/mL sodium dodecylbenzenesulfonate while sonicating, after 0.5 h of sonication, lower the temperature to To 0 °C, add 200 μL of pyrrole dropwise, stir magnetically for 20 min, add 20 mL, 2 mol/L ferric chloride solution while stirring, stir for 2 h, filter and wash until neutral, and dry at 40 °C for 36 h;
（3）Ag2SeCdSe纳米大头针溶液的制备(3) Preparation of Ag2 SeCdSe nano-pin solution
将20 mg二氯化镉，10 mg二氧化硒和0.1 g聚乙烯吡咯烷酮k-30溶入8 mL的二甲基甲酰胺中，边搅拌边加入4 mL油酸，0.1 mL、4 mmol/L的硝酸银水溶液，0.06 g的六偏磷酸钠和50 μL的巯基乙酸，将得到的混合溶液转入聚四氟乙烯内衬高压反应釜中，90 ℃下反应15 h, 离心分离并用异丙醇洗涤，将产物分散到异丙醇中，制得Ag2SeCdSe纳米大头针溶液；Dissolve 20 mg of cadmium dichloride, 10 mg of selenium dioxide and 0.1 g of polyvinylpyrrolidone k-30 into 8 mL of dimethylformamide, add 4 mL of oleic acid while stirring, 0.1 mL, 4 mmol/L Silver nitrate aqueous solution, 0.06 g of sodium hexametaphosphate and 50 μL of mercaptoacetic acid were transferred to a polytetrafluoroethylene-lined autoclave, reacted at 90 °C for 15 h, centrifuged and washed with isopropanol Washing, dispersing the product in isopropanol to prepare Ag2 SeCdSe nano pin solution;
（4）PPy-NH2GO-Ag2SeCdSe纳米复合物溶液的制备(4) Preparation of PPy-NH2 GO-Ag2 SeCdSe nanocomposite solution
在Ag2SeCdSe纳米大头针溶液中加入1 mL、2 mg/mL的聚吡咯插层氨基化石墨烯PPy-NH2GO溶液，振荡孵化12 h，制得PPy-NH2GO-Ag2SeCdSe纳米复合物溶液。PPy-NH2 GO-Ag2 SeCdSe nanocomposites were prepared by adding 1 mL, 2 mg/mL polypyrrole-intercalated aminated graphene PPy-NH2 GO solution to the Ag2 SeCdSe nano-pin solution, shaking and incubating for 12 h substance solution.
实施例8上述所述的PPy-NH2GO-Ag2SeCdSe纳米复合物溶液的制备Example 8 Preparation of PPy-NH2 GO-Ag2 SeCdSe nanocomposite solution described above
（1）氨基化石墨烯NH2GO的制备(1) Preparation of aminated graphene NH2 GO
将100 mg 氧化石墨烯分散到40 mL乙二醇中，边超声边滴加1.0 mL 氨水，将得到的深褐色溶液转入聚四氟乙烯内衬高压反应釜中，180 ℃下反应10 h，过滤并用超纯水洗涤，60 ℃干燥24 h；Disperse 100 mg of graphene oxide into 40 mL of ethylene glycol, add 1.0 mL of ammonia water dropwise while ultrasonicating, transfer the obtained dark brown solution into a polytetrafluoroethylene-lined autoclave, and react at 180 °C for 10 h. Filter, wash with ultrapure water, and dry at 60 °C for 24 h;
（2）聚吡咯插层氨基化石墨烯PPy-NH2GO的制备(2) Preparation of polypyrrole-intercalated aminated graphene PPy-NH2 GO
将氨基化石墨烯NH2GO分散到25 mL超纯水中，超声30 min，边超声边加入100 mL、0.14 g/mL的十二烷基苯磺酸钠，超声1.0 h后，将温度降至0 ℃，滴加200 μL吡咯，磁力搅拌30 min，边搅拌边加入30 mL、2.5 mol/L的三氯化铁溶液，搅拌反应2.5 h，过滤洗涤至中性，50 ℃干燥42 h；Disperse the aminated graphene NH2 GO in 25 mL of ultrapure water, sonicate for 30 min, add 100 mL of 0.14 g/mL sodium dodecylbenzenesulfonate while sonicating, after sonicating for 1.0 h, lower the temperature to To 0 °C, add 200 μL of pyrrole dropwise, stir magnetically for 30 min, add 30 mL, 2.5 mol/L ferric chloride solution while stirring, stir for 2.5 h, filter and wash until neutral, and dry at 50 °C for 42 h;
（3）Ag2SeCdSe纳米大头针溶液的制备(3) Preparation of Ag2 SeCdSe nano-pin solution
将23 mg二氯化镉，11 mg二氧化硒和0.2 g聚乙烯吡咯烷酮k-30溶入10 mL的二甲基甲酰胺中，边搅拌边加入5 mL油酸，0.2 mL、4 mmol/L的硝酸银水溶液，0.07 g的六偏磷酸钠和50 μL的巯基乙酸，将得到的混合溶液转入聚四氟乙烯内衬高压反应釜中，100 ℃下反应15 h, 离心分离并用异丙醇洗涤，将产物分散到异丙醇中，制得Ag2SeCdSe纳米大头针溶液；Dissolve 23 mg of cadmium dichloride, 11 mg of selenium dioxide and 0.2 g of polyvinylpyrrolidone k-30 into 10 mL of dimethylformamide, add 5 mL of oleic acid while stirring, 0.2 mL, 4 mmol/L Silver nitrate aqueous solution, 0.07 g of sodium hexametaphosphate and 50 μL of mercaptoacetic acid were transferred to a polytetrafluoroethylene-lined autoclave, reacted at 100 °C for 15 h, centrifuged and washed with isopropanol Washing, dispersing the product in isopropanol to prepare Ag2 SeCdSe nano pin solution;
（4）PPy-NH2GO-Ag2SeCdSe纳米复合物溶液的制备(4) Preparation of PPy-NH2 GO-Ag2 SeCdSe nanocomposite solution
在Ag2SeCdSe纳米大头针溶液中加入1 mL、2 mg/mL的聚吡咯插层氨基化石墨烯PPy-NH2GO溶液，振荡孵化18 h，制得PPy-NH2GO-Ag2SeCdSe纳米复合物溶液。PPy-NH2 GO-Ag2 SeCdSe nanocomposites were prepared by adding 1 mL, 2 mg/mL polypyrrole-intercalated aminated graphene PPy-NH2 GO solution into the Ag2 SeCdSe nano-pin solution, shaking and incubating for 18 h substance solution.
实施例9上述所述的PPy-NH2GO-Ag2SeCdSe纳米复合物溶液的制备Example 9 Preparation of PPy-NH2 GO-Ag2 SeCdSe nanocomposite solution described above
（1）氨基化石墨烯NH2GO的制备(1) Preparation of aminated graphene NH2 GO
将110 mg 氧化石墨烯分散到50 mL乙二醇中，边超声边滴加1.2 mL 氨水，将得到的深褐色溶液转入聚四氟乙烯内衬高压反应釜中，190 ℃下反应12 h，过滤并用超纯水洗涤，70 ℃干燥36 h；Disperse 110 mg of graphene oxide into 50 mL of ethylene glycol, add 1.2 mL of ammonia water dropwise while ultrasonicating, transfer the obtained dark brown solution into a polytetrafluoroethylene-lined autoclave, and react at 190 °C for 12 h. Filter, wash with ultrapure water, and dry at 70 °C for 36 h;
（2）聚吡咯插层氨基化石墨烯PPy-NH2GO的制备(2) Preparation of polypyrrole-intercalated aminated graphene PPy-NH2 GO
将氨基化石墨烯NH2GO分散到30 mL超纯水中，超声35 min，边超声边加入110 mL、0.15 g/mL的十二烷基苯磺酸钠，超声1.5 h后，将温度降至0 ℃，滴加200 μL吡咯，磁力搅拌40 min，边搅拌边加入40 mL、3 mol/L的三氯化铁溶液，搅拌反应3 h，过滤洗涤至中性，60 ℃干燥64 h；Disperse the aminated graphene NH2 GO in 30 mL of ultrapure water, sonicate for 35 min, add 110 mL, 0.15 g/mL sodium dodecylbenzenesulfonate while sonicating, after 1.5 h of sonication, lower the temperature to To 0 °C, add 200 μL of pyrrole dropwise, stir magnetically for 40 min, add 40 mL, 3 mol/L ferric chloride solution while stirring, stir for 3 h, filter and wash until neutral, and dry at 60 °C for 64 h;
（3）Ag2SeCdSe纳米大头针溶液的制备(3) Preparation of Ag2 SeCdSe nano-pin solution
将26 mg二氯化镉，12 mg二氧化硒和0.3 g聚乙烯吡咯烷酮k-30溶入12 mL的二甲基甲酰胺中，边搅拌边加入6 mL油酸，0.3 mL、4 mmol/L的硝酸银水溶液，0.09 g的六偏磷酸钠和50 μL的巯基乙酸，将得到的混合溶液转入聚四氟乙烯内衬高压反应釜中，110 ℃下反应15 h, 离心分离并用异丙醇洗涤，将产物分散到异丙醇中，制得Ag2SeCdSe纳米大头针溶液；Dissolve 26 mg of cadmium dichloride, 12 mg of selenium dioxide and 0.3 g of polyvinylpyrrolidone k-30 into 12 mL of dimethylformamide, add 6 mL of oleic acid while stirring, 0.3 mL, 4 mmol/L silver nitrate aqueous solution, 0.09 g of sodium hexametaphosphate and 50 μL of mercaptoacetic acid, transfer the resulting mixed solution into a polytetrafluoroethylene-lined autoclave, react at 110 °C for 15 h, centrifuge and wash with isopropanol Washing, dispersing the product in isopropanol to prepare Ag2 SeCdSe nano pin solution;
（4）PPy-NH2GO-Ag2SeCdSe纳米复合物溶液的制备(4) Preparation of PPy-NH2 GO-Ag2 SeCdSe nanocomposite solution
在Ag2SeCdSe纳米大头针溶液中加入1 mL、2 mg/mL的聚吡咯插层氨基化石墨烯PPy-NH2GO溶液，振荡孵化24 h，制得PPy-NH2GO-Ag2SeCdSe纳米复合物溶液。PPy-NH2 GO-Ag2 SeCdSe nanocomposites were prepared by adding 1 mL, 2 mg/mL polypyrrole-intercalated aminated graphene PPy-NH2 GO solution into the Ag2 SeCdSe nano-pin solution, shaking and incubating for 24 h substance solution.
实施例10 CA72-4的检测Example 10 Detection of CA72-4
（1）使用电化学工作站的三电极体系进行测试，Ag/AgCl电极作为参比电极，铂丝电极为对电极，所制备的胃癌抗原电致化学发光传感器为工作电极，将电化学工作站和化学发光检测仪连接在一起将光电倍增管的高压设置为800 V，扫描电压设置为-1.7~0 V，扫描速率0.1 V/s；(1) The three-electrode system of the electrochemical workstation was used for testing, the Ag/AgCl electrode was used as the reference electrode, the platinum wire electrode was used as the counter electrode, and the prepared gastric cancer antigen electrochemiluminescence sensor was used as the working electrode. The luminescence detector is connected together, the high voltage of the photomultiplier tube is set to 800 V, the scanning voltage is set to -1.7~0 V, and the scanning rate is 0.1 V/s;
（2）在10 mL、pH 6.4~8.4的含50~60 mmol/L过硫酸钾的PBS缓冲溶液中，通过电致化学发光系统检测对0.01 pg/mL~50 ng/mL一系列不同浓度的CA72-4标准溶液，产生的电致化学发光信号强度，绘制工作曲线；(2) In 10 mL of PBS buffer solution containing 50-60 mmol/L potassium persulfate at pH 6.4-8.4, a series of different concentrations of 0.01 pg/mL-50 ng/mL were detected by electrochemiluminescence system CA72-4 standard solution, the intensity of the electrochemiluminescence signal produced, draw the working curve;
（3）将待测样品溶液代替胃癌抗原CA72-4标准溶液进行检测，测得线性范围为0.1 pg/mL~20 ng/mL，检测限为21 fg/mL。(3) The sample solution to be tested was used instead of the gastric cancer antigen CA72-4 standard solution for detection. The linear range was 0.1 pg/mL~20 ng/mL, and the detection limit was 21 fg/mL.
| Application Number | Priority Date | Filing Date | Title |
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| CN201410623368.3ACN104391117B (en) | 2014-11-08 | 2014-11-08 | Preparation method and application of gastric cancer antigen electrogenerated chemiluminescence sensor based on PP<y>-NH2GO-Ag2Se@CdSe |
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|---|---|---|---|
| CN201410623368.3ACN104391117B (en) | 2014-11-08 | 2014-11-08 | Preparation method and application of gastric cancer antigen electrogenerated chemiluminescence sensor based on PP<y>-NH2GO-Ag2Se@CdSe |
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| CN201410623368.3AExpired - Fee RelatedCN104391117B (en) | 2014-11-08 | 2014-11-08 | Preparation method and application of gastric cancer antigen electrogenerated chemiluminescence sensor based on PP<y>-NH2GO-Ag2Se@CdSe |
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