技术领域technical field
本发明涉及一种电化学发光免疫传感器，尤其是涉及一种基于多功能化氮化碳材料的肿瘤标志物电化学发光免疫传感器的制备方法及其应用。The invention relates to an electrochemiluminescence immunosensor, in particular to a preparation method and application of a tumor marker electrochemiluminescence immunosensor based on a multifunctional carbon nitride material.
背景技术Background technique
据WHO统计与预测：2008年全世界有760万人死于肿瘤疾病，2030年预计将有1100万人死于肿瘤疾病，肿瘤疾病已经成为公共卫生的头号公敌。肿瘤专家认为，实现对肿瘤的早期发现、早期诊断、早期治疗，可以有效降低肿瘤发病率、减少病死人数、控制医治费用，其中早期诊断是关键。然而，肿瘤疾病早期症状不明显，实现稳定、准确、可靠的早期诊断并不容易，肿瘤标志物的发现使癌症的早期诊断成为可能。肿瘤标志物是指在肿瘤的发生和增殖过程中产生的、反映肿瘤存在和生长的一类物质，包括蛋白质、激素、酶、基因等。一般而言，肿瘤细胞越多，恶性程度越高，越晚期，肿瘤标志物的浓度越高。因此，患者血液或组织中肿瘤标志物的检测，对肿瘤的辅助诊断、鉴别诊断、疗效观察、病情监测以及预后的评价具有很高价值。According to WHO statistics and predictions: in 2008, 7.6 million people died of tumor diseases in the world, and it is estimated that 11 million people will die of tumor diseases in 2030. Tumor diseases have become the number one public enemy of public health. Oncologists believe that early detection, early diagnosis, and early treatment of tumors can effectively reduce the incidence of tumors, reduce the number of deaths, and control medical expenses, among which early diagnosis is the key. However, the early symptoms of tumor diseases are not obvious, and it is not easy to achieve a stable, accurate, and reliable early diagnosis. The discovery of tumor markers makes early diagnosis of cancer possible. Tumor markers refer to a class of substances produced during the occurrence and proliferation of tumors that reflect the existence and growth of tumors, including proteins, hormones, enzymes, genes, etc. Generally speaking, the more tumor cells there are, the higher the degree of malignancy, and the higher the advanced stage, the higher the concentration of tumor markers. Therefore, the detection of tumor markers in the patient's blood or tissue is of great value for the auxiliary diagnosis, differential diagnosis, observation of curative effect, disease monitoring and prognosis evaluation of tumors.
目前检测肿瘤标志物的方法主要有：放射免疫分析法（RIA）、酶联免疫吸附分析法（ELISA）、化学发光免疫分析法（CLIA）、电化学免疫分析法（ECIA）、电化学发光免疫（ECLIA）和免疫电镜法等，这些方法都有一定的灵敏度和准确度，但也各有一定的不足之处：有的仪器昂贵、操作复杂、技术要求高，有的步骤繁多、易出现假阳性和假阴性，有的使用放射性试剂，有的特异性不强、灵敏度不高，有的响应时间长、无法重复测量。因此，开发灵敏、准确、快速、简便的肿瘤标志物检测方法是迫切需求。At present, the methods for detecting tumor markers mainly include: radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), chemiluminescence immunoassay (CLIA), electrochemical immunoassay (ECIA), and electrochemiluminescence immunoassay. (ECLIA) and immunoelectron microscopy, etc. These methods have certain sensitivity and accuracy, but they also have certain shortcomings: some instruments are expensive, the operation is complicated, and the technical requirements are high, and some have many steps and are prone to false positives. Positive and false negative, some use radioactive reagents, some have low specificity and sensitivity, and some have long response time and cannot be repeated. Therefore, it is an urgent need to develop sensitive, accurate, fast and simple tumor marker detection methods.
免疫传感器是利用抗体与抗原之间的特异性识别与结合而研制成的一类生物传感器，电化学发光（ECL）免疫传感器是电化学发光和免疫传感器相结合的产物，其中无标记型电化学发光免疫传感器以具备快速、稳定、选择性强、重现性好、易于操作、步骤简单等优点被广泛运用，具有良好的应用前景。Immunosensors are a type of biosensor developed by using the specific recognition and binding between antibodies and antigens. Electrochemiluminescence (ECL) immunosensors are the product of a combination of electrochemiluminescence and immunosensors. The label-free electrochemical Luminescent immunosensors are widely used due to their advantages of rapidity, stability, strong selectivity, good reproducibility, easy operation, and simple steps, and have good application prospects.
氮化碳材料（g-C3N4）是一种类似石墨的纳米薄膜，具有比表面积大、稳定性强、硬度大、导电性好、生物相容性良好等特点，并且氮化碳材料本身具有极强的电化学发光的能力，非常适合用于开发电化学发光免疫传感器。目前未见基于多功能化氮化碳材料的肿瘤标志物电化学发光免疫传感器的相关研究报道。Carbon nitride material (gC3 N4 ) is a nano-film similar to graphite, which has the characteristics of large specific surface area, strong stability, high hardness, good electrical conductivity, and good biocompatibility, and the carbon nitride material itself has The extremely strong electrochemiluminescence ability is very suitable for the development of electrochemiluminescence immunosensors. At present, there are no relevant research reports on tumor marker electrochemiluminescence immunosensors based on multifunctional carbon nitride materials.
发明内容Contents of the invention
本发明所要解决的技术问题是提供一种特异性好、灵敏度高、结果准确可靠、成本低、快速，且制备过程极其简单的基于多功能化氮化碳材料的肿瘤标志物电化学发光免疫传感器的制备方法及其应用。The technical problem to be solved by the present invention is to provide an electrochemiluminescent immunosensor for tumor markers based on multifunctional carbon nitride materials with good specificity, high sensitivity, accurate and reliable results, low cost, fast, and extremely simple preparation process The preparation method and its application.
本发明解决上述技术问题所采用的技术方案为：一种基于多功能化氮化碳材料的肿瘤标志物电化学发光免疫传感器的制备方法，具体步骤如下：The technical scheme adopted by the present invention to solve the above-mentioned technical problems is: a preparation method of a tumor marker electrochemiluminescence immunosensor based on a multifunctional carbon nitride material, and the specific steps are as follows:
（1）多功能化氮化碳材料的制备(1) Preparation of multifunctional carbon nitride materials
a. 将三聚氰胺在500～600℃下加热3～5 h，真空干燥后得到氮化碳（g-C3N4）粉末；a. Heat melamine at 500-600°C for 3-5 hours, and dry it in vacuum to obtain carbon nitride (gC3 N4 ) powder;
b. 取0.8～1.5 g 氮化碳粉末加入到80～120 mL 4～6 mol/L HNO3中，于120～150℃下回流24～48小时后，自然冷却至室温，水洗涤至pH=7，离心，将沉淀在35～40℃下真空干燥12～20小时，得到羧基化的氮化碳（g-C3N4）；b. Add 0.8-1.5 g of carbon nitride powder into 80-120 mL of 4-6 mol/L HNO3 , reflux at 120-150°C for 24-48 hours, cool naturally to room temperature, and wash with water to pH =7, centrifuge, and vacuum-dry the precipitate at 35-40°C for 12-20 hours to obtain carboxylated carbon nitride (gC3 N4 );
c. 取0.2～0.5 g FeCl2·4H2O和0.6～1.0 g FeCl3·6H2O溶于50～100 mL除氧的二次水中，然后在氮气氛保护下逐滴加入到三口烧瓶中，磁力搅拌使其充分混合均匀，然后将25wt%～28wt%的氨水逐滴缓慢加入到三口烧瓶中直至溶液的pH为8.0～10.0，将三口烧瓶内的溶液加热到70～100℃后，于70～100℃搅拌回流1～3 h，然后停止加热在氮气氛保护下搅拌、回流冷却至室温，得到黑色沉淀物纳米四氧化三铁；将纳米四氧化三铁用水清洗至中性，定容至50 mL，超声30 min～1 h后，加入0.1～0.2 mL 3-氨丙基三乙氧基硅烷（APTES），室温下搅拌 6～8 h，得到氨基化的纳米四氧化三铁悬浊液；c. Dissolve 0.2-0.5 g FeCl2 4H2 O and 0.6-1.0 g FeCl3 6H2 O in 50-100 mL of deaerated secondary water, and add them dropwise into a three-necked flask under nitrogen atmosphere protection , magnetic stirring to make it fully mixed evenly, then slowly add 25wt% to 28wt% ammonia water dropwise into the three-necked flask until the pH of the solution is 8.0-10.0, heat the solution in the three-necked flask to 70-100°C, and then Stir and reflux at 70-100°C for 1-3 hours, then stop heating, stir under the protection of nitrogen atmosphere, reflux and cool to room temperature to obtain a black precipitate of nano-ferric oxide; wash the nano-ferric oxide with water to neutral, and constant volume to 50 mL, after ultrasonication for 30 min to 1 h, add 0.1 to 0.2 mL of 3-aminopropyltriethoxysilane (APTES), and stir at room temperature for 6 to 8 h to obtain aminated nano-ferric oxide suspension liquid;
d. 取150～250 µL 0.5～1.5 mg/mL羧基化的氮化碳加入150～250 µL偶联试剂后，用0.8～1.2 mol/L盐酸调pH为4.0～6.0，振摇 1～2 h，离心取沉淀加水洗涤后，再离心取沉淀加水洗涤，再次离心取沉淀加水洗涤后，定容至200～300 µL，用0.1～0.2 mol/L NaOH溶液调pH为8.0～10.0，然后加入50～100 µL 0.1～1 µg/mL肿瘤标志物抗体溶液，摇床孵育1～2 h后再加入150～250 µL氨基化的纳米四氧化三铁悬浊液，继续孵育3～5 h，再加入50～100 µL 2%牛血清白蛋白溶液以封闭非特异性吸附位点，继续孵育1～2 h，用磁铁吸引分离，水清洗至中性后，定容至100～200 µL，即得到多功能化氮化碳材料溶液；d. Take 150-250 µL of 0.5-1.5 mg/mL carboxylated carbon nitride and add 150-250 µL of coupling reagent, adjust the pH to 4.0-6.0 with 0.8-1.2 mol/L hydrochloric acid, and shake for 1-2 h , centrifuged to take the precipitate and washed it with water, then centrifuged again to take the precipitate and added water to wash it, centrifuged again to take the precipitate and added water to wash it, then set the volume to 200-300 µL, adjusted the pH to 8.0-10.0 with 0.1-0.2 mol/L NaOH solution, then added 50 ～100 μL 0.1～1 μg/mL tumor marker antibody solution, incubate on a shaker for 1～2 h, then add 150～250 μL aminated nano-ferric oxide suspension, continue to incubate for 3～5 h, then add 50-100 µL 2% bovine serum albumin solution to block the non-specific adsorption sites, continue to incubate for 1-2 h, attract and separate with a magnet, wash with water until neutral, and dilute to 100-200 µL to obtain multifunctional Nitride carbon material solution;
（2）电化学发光免疫传感器的组装(2) Assembly of electrochemiluminescence immunosensor
a. 将直径为3～5 mm的磁性玻碳电极依次用1.0、0.3和0.05 μm的Al2O3抛光粉在麂皮上抛光至镜面，超声清洗2 min，然后依次用乙醇水溶液、硝酸水溶液和蒸馏水超声清洗；a. The magnetic glassy carbon electrode with a diameter of 3-5 mm was polished to a mirror surface on the suede with 1.0, 0.3 and 0.05 μm Al2 O3 polishing powder in sequence, ultrasonically cleaned for 2 min, and then washed with ethanol aqueous solution and nitric acid aqueous solution and distilled water ultrasonic cleaning;
b. 取5～10 µL步骤（1）所得的多功能化氮化碳材料溶液，滴在磁性玻碳电极表面，多功能化氮化碳材料即被均匀、牢固地吸附于电极表面，即得到用于检测肿瘤标志物的电化学发光免疫传感器。b. Take 5-10 µL of the multifunctional carbon nitride material solution obtained in step (1), and drop it on the surface of the magnetic glassy carbon electrode. The multifunctional carbon nitride material is evenly and firmly adsorbed on the electrode surface, and the obtained Electrochemiluminescent immunosensors for the detection of tumor markers.
步骤（1）中所述的偶联试剂为1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐与N-羟基琥珀酰亚胺溶于水中得到，所述的偶联试剂中1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐的摩尔浓度为10～100 mmol/L，所述的N-羟基琥珀酰亚胺摩尔浓度为1～10 mmol/L。The coupling reagent described in step (1) is obtained by dissolving 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide in water. The molar concentration of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride in the coupling reagent is 10～100 mmol/L, and the described N-hydroxysuccinimide molar The concentration is 1-10 mmol/L.
步骤（1）中所述的肿瘤标志物为：甲胎蛋白（AFP）、癌胚抗原（CEA)、糖类抗原15-3（CA15-3）、糖类抗原125（CA125）、糖类抗原19-9（CA19-9）或前列腺特异性抗原（PSA）。The tumor markers described in step (1) are: alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), carbohydrate antigen 15-3 (CA15-3), carbohydrate antigen 125 (CA125), carbohydrate antigen 19-9 (CA19-9) or prostate-specific antigen (PSA).
步骤（2）中所述的乙醇水溶液由乙醇和水按体积比1:1混合而成；所述的硝酸水溶液由硝酸和水按体积比1:1混合而成。The ethanol aqueous solution described in step (2) is formed by mixing ethanol and water in a volume ratio of 1:1; the described nitric acid aqueous solution is formed by mixing nitric acid and water in a volume ratio of 1:1.
上述基于多功能化氮化碳材料的肿瘤标志物电化学发光免疫传感器用于检测肿瘤标志物的方法，具体步骤如下：将用于检测肿瘤标志物的电化学发光免疫传感器浸泡在不同浓度的肿瘤标志物溶液中，于37℃下温育1 h后取出，水清洗后，作为工作电极；采用铂电极作为对电极，Ag/AgCl电极或者饱和甘汞电极作为参比电极，构成三电极体系；将三电极体系放入缓冲溶液，启动电化学反应，测量电化学发光强度；获得一系列不同浓度的肿瘤标志物溶液对应的电化学发光强度值，建立电化学发光强度值与肿瘤标志物溶液浓度之间的定量关系；根据这个定量关系检测待测样品中肿瘤标志物的浓度。The method for using the above-mentioned multifunctional carbon nitride material-based tumor marker electrochemiluminescence immunosensor for detecting tumor markers, the specific steps are as follows: soak the electrochemiluminescence immunosensor for detecting tumor markers in different concentrations of tumor In the marker solution, incubate at 37°C for 1 h, take it out, wash with water, and use it as a working electrode; use a platinum electrode as a counter electrode, and an Ag/AgCl electrode or a saturated calomel electrode as a reference electrode to form a three-electrode system; Put the three-electrode system into the buffer solution, start the electrochemical reaction, and measure the intensity of electrochemiluminescence; obtain the values of the intensity of electrochemiluminescence corresponding to a series of tumor marker solutions with different concentrations, and establish the value of the intensity of electrochemiluminescence and the concentration of the tumor marker solution Quantitative relationship among them; according to this quantitative relationship, the concentration of the tumor marker in the sample to be tested is detected.
所述的缓冲溶液为：pH = 7.5～8.5的磷酸盐缓冲溶液，含有10～30 mmol/L K2S2O8和80～100 mmol/L KCl。The buffer solution is: a phosphate buffer solution with pH = 7.5-8.5, containing 10-30 mmol/L K2 S2 O8 and 80-100 mmol/L KCl.
所述的电化学反应的条件如下：电位阶跃计时电流法，脉冲宽度：0.25秒；脉冲间隔：30秒；初始电压：0V；脉冲电压:−1.1V。The conditions of the electrochemical reaction are as follows: potential step chronoamperometry, pulse width: 0.25 seconds; pulse interval: 30 seconds; initial voltage: 0V; pulse voltage: −1.1V.
所述的肿瘤标志物为：甲胎蛋白（AFP）、癌胚抗原（CEA)、糖类抗原15-3（CA15-3）、糖类抗原125（CA125）、糖类抗原19-9（CA19-9）或前列腺特异性抗原（PSA）。The tumor markers are: alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), carbohydrate antigen 15-3 (CA15-3), carbohydrate antigen 125 (CA125), carbohydrate antigen 19-9 (CA19 -9) or prostate-specific antigen (PSA).
发明原理：多功能化氮化碳材料，在氮化碳上同时负载有纳米四氧化三铁、肿瘤标志物抗体，具有多种功能：（1）氮化碳材料表面积大、羧基官能团多，可以负载大量的纳米四氧化三铁、肿瘤标志物抗体；（2）氮化碳材料自身具有极强的电化学发光，不需要另外标记电化学发光体，大大简化了制备步骤；（3）纳米四氧化三铁，可以使得平面结构的多功能化氮化碳材料均匀、牢固地吸附于电极表面，实现电化学发光免疫传感器的一步制备，进一步简化了传感器制备方法，且纳米四氧化三铁可以大大提高多功能化氮化碳材料的导电性，提高电化学发光性能；（4）肿瘤标志物抗体，可以特异性识别肿瘤标志物；（5）多功能化氮化碳材料，具有平面结构和良好的导电性能，在磁力吸引下完全处于电极表面的外赫姆霍兹面之内，大大增强电化学发光信号。Invention principle: Multifunctional carbon nitride material, loaded with nano-ferric oxide and tumor marker antibody on the carbon nitride, has multiple functions: (1) The carbon nitride material has a large surface area and many carboxyl functional groups, which can Load a large amount of nano-ferric oxide and tumor marker antibody; (2) Carbon nitride material itself has extremely strong electrochemiluminescence, and does not require additional labeling of electrochemiluminescence, which greatly simplifies the preparation steps; (3) Nano-4 Ferric oxide can make the multifunctional carbon nitride material with planar structure evenly and firmly adsorbed on the surface of the electrode, realize the one-step preparation of electrochemiluminescence immunosensor, further simplify the sensor preparation method, and the nanometer ferric oxide can be greatly improved. Improve the conductivity of multifunctional carbon nitride materials and improve the performance of electrochemiluminescence; (4) tumor marker antibodies can specifically recognize tumor markers; (5) multifunctional carbon nitride materials have a planar structure and good Under the magnetic attraction, it is completely inside the outer Helmholtz surface of the electrode surface, which greatly enhances the electrochemiluminescent signal.
本发明将多功能化氮化碳材料滴涂于磁性玻碳电极表面之后，即被均匀、牢固地吸附于电极表面，构建成电化学发光免疫传感器，电化学反应激发下，多功能化氮化碳材料即会产生稳定的电化学发光；如果样品中含有肿瘤标志物，肿瘤标志物就会被电化学发光免疫传感器中的肿瘤标志物抗体俘获；肿瘤标志物是大分子蛋白，被结合到电极表面形成免疫复合物后，阻碍了电极表面的物质传递、电子传递和电化学发光，使得电化学发光强度降低；肿瘤标志物浓度越大，电化学发光强度越低，电化学发光强度与肿瘤标志物浓度的对数之间呈线性关系。据此可以实现样品中肿瘤标志物的未知浓度的检测。In the present invention, after the multifunctional carbon nitride material is drip-coated on the surface of the magnetic glassy carbon electrode, it is evenly and firmly adsorbed on the electrode surface, and an electrochemiluminescent immune sensor is constructed. Under the excitation of the electrochemical reaction, the multifunctional carbon nitride The carbon material will produce stable electrochemiluminescence; if the sample contains tumor markers, the tumor markers will be captured by the tumor marker antibodies in the electrochemiluminescence immunosensor; tumor markers are macromolecular proteins that are bound to the electrodes After the immune complex is formed on the surface, it hinders the material transfer, electron transfer and electrochemiluminescence on the electrode surface, which reduces the intensity of electrochemiluminescence; the greater the concentration of tumor markers, the lower the intensity of electrochemiluminescence, and the intensity of electrochemiluminescence is related to that of tumor markers. There is a linear relationship between the logarithms of the concentrations. Accordingly, the detection of the unknown concentration of the tumor marker in the sample can be realized.
与现有技术相比，本发明的优点在于：Compared with the prior art, the present invention has the advantages of:
（1）高灵敏度，本发明的检测灵敏度大约是现有方法的10倍以上，原因在于：首先，传统电化学发光免疫分析方法，是将电化学发光物质标记在肿瘤标志物抗体或者纳米材料上，可以标记的数量很有限，本发明使用的氮化碳材料，自身具有极强的电化学发光；其次，纳米四氧化三铁负载在氮化碳材料上，不仅使得传感器制备更简单，更重要的是，增强了功能化氮化碳材料的导电性；再次，纳米四氧化三铁使得整个多功能化氮化碳材料完全处于电极表面的外赫姆霍兹面之内，大大提高多功能化氮化碳材料的整体电化学发光性能。(1) High sensitivity. The detection sensitivity of the present invention is about 10 times higher than that of existing methods. The reasons are: First, the traditional electrochemiluminescence immunoassay method is to mark electrochemiluminescent substances on tumor marker antibodies or nanomaterials , the number of labels that can be marked is very limited. The carbon nitride material used in the present invention itself has extremely strong electrochemiluminescence; secondly, the nano-ferric oxide is loaded on the carbon nitride material, which not only makes the sensor preparation simpler, but also more important The most important thing is that the conductivity of the functionalized carbon nitride material is enhanced; again, the nano-ferric oxide makes the entire multifunctional carbon nitride material completely within the outer Helmholtz surface of the electrode surface, greatly improving the multifunctionality. Bulk electrochemiluminescent properties of carbon nitride materials.
（2）高特异性，常见其他肿瘤标志物对本检测体系均无干扰。原因在于：本发明是基于肿瘤标志物抗体与肿瘤标志物之间的特异性识别与结合而构建的电化学发光免疫传感器，干扰物质不是抗体的目标物，因此待测液中的干扰物质并不能与抗体结合，故对本检测体系无干扰。(2) High specificity, other common tumor markers do not interfere with this detection system. The reason is that the present invention is an electrochemiluminescent immunosensor based on the specific recognition and binding between tumor marker antibodies and tumor markers, and the interfering substances are not the target of the antibodies, so the interfering substances in the test solution cannot Combined with the antibody, so there is no interference to the detection system.
（3）结果准确，回收率均在90%～110%之间。(3) The results are accurate, and the recoveries are all between 90% and 110%.
（4）制备与检测方法简单、快速。将多功能化氮化碳材料滴涂于磁性玻碳电极表面之后，即可一步构建成电化学发光免疫传感器，制备方法极其简单；孵育完成后，即可检测得到即时的电化学发光信号，实现定量检测。(4) The preparation and detection methods are simple and rapid. After the multifunctional carbon nitride material is drip-coated on the surface of the magnetic glassy carbon electrode, an electrochemiluminescence immunosensor can be constructed in one step, and the preparation method is extremely simple; after the incubation is completed, an instant electrochemiluminescence signal can be detected, realizing Quantitative detection.
综上所述，本发明一步制备一种基于多功能化氮化碳材料的检测肿瘤标志物的电化学发光免疫传感器，兼具免疫分析的高选择性、电化学发光分析与功能化氮化碳结合的高灵敏度，具有高灵敏度、高特异性、简单、快速、易于操作等优点，可以实现对超低浓度肿瘤标志物的检测，具有良好的应用前景。In summary, the present invention prepares an electrochemiluminescent immunosensor for detecting tumor markers based on multifunctional carbon nitride materials in one step, which has the high selectivity of immunoassay, electrochemiluminescence analysis and functionalized carbon nitride Combined with high sensitivity, it has the advantages of high sensitivity, high specificity, simplicity, speed, and easy operation, etc. It can realize the detection of ultra-low concentration tumor markers, and has a good application prospect.
附图说明Description of drawings
图1为本发明的多功能化氮化碳材料的制备流程图；Fig. 1 is the preparation flowchart of multifunctional carbon nitride material of the present invention;
图2为不同浓度糖类抗原125的电化学发光强度(y)—浓度(x)对数线性图；Fig. 2 is the logarithmic linear diagram of the electrochemiluminescent intensity (y)-concentration (x) of different concentrations of carbohydrate antigen 125;
图3为不同浓度糖类抗原15-3的电化学发光强度 (y)—浓度(x)对数线性图；Fig. 3 is the logarithmic linear diagram of the electrochemiluminescent intensity (y)-concentration (x) of different concentrations of carbohydrate antigen 15-3;
图4为不同浓度糖类抗原19-9的电化学发光强度 (y)—浓度(x)对数线性图；Fig. 4 is the logarithmic linear diagram of the electrochemiluminescence intensity (y)-concentration (x) of different concentrations of carbohydrate antigen 19-9;
图5为不同浓度甲胎蛋白的电化学发光强度 (y)—浓度(x)对数线性图；Fig. 5 is the logarithmic linear diagram of the electrochemiluminescence intensity (y)-concentration (x) of different concentrations of alpha-fetoprotein;
图6为不同浓度癌胚抗原的电化学发光强度 (y)—浓度(x)对数线性图；Fig. 6 is the electrochemiluminescence intensity (y)-concentration (x) logarithmic linear graph of different concentrations of carcinoembryonic antigen;
图7为不同浓度前列腺特异性抗原的电化学发光强度 (y)—浓度(x)对数线性图。Fig. 7 is a logarithmic linear graph of the electrochemiluminescent intensity (y)-concentration (x) of different concentrations of prostate specific antigen.
具体实施方式detailed description
以下结合附图实施例对本发明作进一步详细描述。The present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments.
具体实施例一Specific embodiment one
一种基于多功能化氮化碳材料的肿瘤标志物电化学发光免疫传感器的制备方法，具体步骤如下：A method for preparing a tumor marker electrochemiluminescence immunosensor based on a multifunctional carbon nitride material, the specific steps are as follows:
（1）多功能化氮化碳材料的制备，工艺流程如图1所示(1) Preparation of multifunctional carbon nitride material, the process flow is shown in Figure 1
a. 将三聚氰胺在500～600℃下加热3～5 h，真空干燥后得到氮化碳（g-C3N4）粉末；a. Heat melamine at 500-600°C for 3-5 hours, and dry it in vacuum to obtain carbon nitride (gC3 N4 ) powder;
b. 取0.8～1.5 g 氮化碳粉末加入到80～120 mL 4～6 mol/L HNO3中，于120～150℃下回流24～48小时后，自然冷却至室温，水洗涤至pH=7，离心，将沉淀在35～40℃下真空干燥12～20小时，得到羧基化的氮化碳（g-C3N4）；b. Add 0.8-1.5 g of carbon nitride powder into 80-120 mL of 4-6 mol/L HNO3 , reflux at 120-150°C for 24-48 hours, cool naturally to room temperature, and wash with water to pH =7, centrifuge, and vacuum-dry the precipitate at 35-40°C for 12-20 hours to obtain carboxylated carbon nitride (gC3 N4 );
c. 取0.2～0.5 g FeCl2·4H2O和0.6～1.0 g FeCl3·6H2O溶于50～100 mL除氧的二次水中，然后在氮气氛保护下逐滴加入到三口烧瓶中，磁力搅拌使其充分混合均匀，然后将25wt%～28wt%的氨水逐滴缓慢加入到三口烧瓶中直至溶液的pH为8.0～10.0，将三口烧瓶内的溶液加热到70～100℃后，于70～100℃搅拌回流1～3 h，然后停止加热在氮气氛保护下搅拌、回流冷却至室温，得到的黑色沉淀物纳米四氧化三铁；将纳米四氧化三铁用水清洗至中性，定容至50 mL，超声30 min～1 h后，加入0.1～0.2 mL 3-氨丙基三乙氧基硅烷（APTES），室温下搅拌 6～8 h，得到氨基化的纳米四氧化三铁悬浊液；c. Dissolve 0.2-0.5 g FeCl2 4H2 O and 0.6-1.0 g FeCl3 6H2 O in 50-100 mL of deaerated secondary water, and add them dropwise into a three-necked flask under nitrogen atmosphere protection , magnetic stirring to make it fully mixed evenly, then slowly add 25wt% to 28wt% ammonia water dropwise into the three-necked flask until the pH of the solution is 8.0-10.0, heat the solution in the three-necked flask to 70-100°C, and then Stir and reflux at 70-100°C for 1-3 h, then stop heating, stir under the protection of a nitrogen atmosphere, reflux and cool to room temperature, and obtain a black precipitate of nano-ferric oxide; wash the nano-ferric oxide with water until neutral, and set to 50 mL, after ultrasonication for 30 min to 1 h, add 0.1 to 0.2 mL of 3-aminopropyltriethoxysilane (APTES), and stir at room temperature for 6 to 8 h to obtain aminated nanometer ferric oxide suspension turbid liquid;
d. 取150～250 µL 0.5～1.5 mg/mL羧基化的氮化碳加入150～250 µL偶联试剂后，用0.8～1.2mol/L盐酸调pH为4.0～6.0，振摇 1～2 h，离心取沉淀加水洗涤后，再离心取沉淀加水洗涤，再次离心取沉淀加水洗涤后，定容至200～300 µL，用0.1～0.2mol/L NaOH溶液调pH为8.0～10.0，然后加入50～100 µL 0.1～1 µg/mL肿瘤标志物抗体溶液（抗体冻干粉溶于水配制而成），摇床孵育1～2 h后再加入150～250 µL氨基化的纳米四氧化三铁悬浊液，继续孵育3～5 h，再加入50～100 µL 2%牛血清白蛋白溶液以封闭非特异性吸附位点，继续孵育1～2 h，用磁铁吸引分离，水清洗至中性后，定容至100～200µL，即得到多功能化氮化碳材料溶液；其中偶联试剂为1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐与N-羟基琥珀酰亚胺溶于水中得到，偶联试剂中1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐的摩尔浓度为10～100 mmol/L，N-羟基琥珀酰亚胺摩尔浓度为1～10 mmol/L；d. Take 150-250 µL of 0.5-1.5 mg/mL carboxylated carbon nitride and add 150-250 µL of coupling reagent, adjust the pH to 4.0-6.0 with 0.8-1.2 mol/L hydrochloric acid, and shake for 1-2 h , centrifuged to take the precipitate and washed it with water, then centrifuged again to take the precipitate and added water to wash it, centrifuged again to take the precipitate and added water to wash it, set the volume to 200-300 µL, adjusted the pH to 8.0-10.0 with 0.1-0.2mol/L NaOH solution, then added 50 ～100 μL 0.1～1 μg/mL tumor marker antibody solution (prepared by dissolving antibody freeze-dried powder in water), incubate on a shaker for 1～2 h, and then add 150～250 μL aminated nanometer ferric oxide suspension Then add 50-100 μL 2% bovine serum albumin solution to block the non-specific adsorption sites, continue to incubate for 1-2 h, separate with magnet, wash with water until neutral, Set the volume to 100-200 µL to obtain a multifunctional carbon nitride material solution; the coupling reagent is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxy Succinimide is obtained by dissolving in water, the molar concentration of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride in the coupling reagent is 10-100 mmol/L, N-hydroxy The molar concentration of succinimide is 1-10 mmol/L;
（2）电化学发光免疫传感器的组装(2) Assembly of electrochemiluminescence immunosensor
a. 将直径为3～5 mm的磁性玻碳电极依次用1.0、0.3和0.05 μm的Al2O3抛光粉在麂皮上抛光至镜面，超声清洗2 min，然后依次用乙醇水溶液（乙醇与水体积比为1:1）、硝酸水溶液（硝酸与水体积比为1:1）和蒸馏水超声清洗；a. The magnetic glassy carbon electrode with a diameter of 3-5 mm was polished to a mirror surface on the suede with 1.0, 0.3 and 0.05 μm Al2 O3 polishing powder in sequence, ultrasonically cleaned for 2 min, and then sequentially washed with ethanol aqueous solution (ethanol and Water volume ratio is 1:1), nitric acid aqueous solution (nitric acid and water volume ratio is 1:1) and distilled water ultrasonic cleaning;
b. 取5～10 µL步骤（1）所得的多功能化氮化碳材料溶液，滴在磁性玻碳电极表面，多功能化氮化碳材料即被均匀、牢固地吸附于电极表面，即得到用于检测肿瘤标志物的电化学发光免疫传感器。b. Take 5-10 µL of the multifunctional carbon nitride material solution obtained in step (1), and drop it on the surface of the magnetic glassy carbon electrode. The multifunctional carbon nitride material is evenly and firmly adsorbed on the electrode surface, and the obtained Electrochemiluminescent immunosensors for the detection of tumor markers.
上述肿瘤标志物为：甲胎蛋白（AFP）、癌胚抗原（CEA)、糖类抗原15-3（CA15-3）、糖类抗原125（CA125）、糖类抗原19-9（CA19-9）或前列腺特异性抗原（PSA）。The above tumor markers are: alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), carbohydrate antigen 15-3 (CA15-3), carbohydrate antigen 125 (CA125), carbohydrate antigen 19-9 (CA19-9 ) or prostate-specific antigen (PSA).
具体实施例二 Specific embodiment two
上述具体实施例一制备得到的基于多功能化氮化碳材料的肿瘤标志物电化学发光免疫传感器用于检测肿瘤标志物的方法，具体步骤如下：将用于检测肿瘤标志物的电化学发光免疫传感器浸泡在不同浓度的肿瘤标志物溶液中，于37℃下温育1 h后取出，水清洗后，作为工作电极；采用铂电极作为对电极，Ag/AgCl电极或者饱和甘汞电极作为参比电极，构成三电极体系；将三电极体系放入缓冲溶液，启动电化学反应，测量电化学发光强度；获得一系列不同浓度的肿瘤标志物溶液对应的电化学发光强度值，建立电化学发光强度值与肿瘤标志物溶液浓度之间的定量关系；根据这个定量关系检测待测样品中肿瘤标志物的浓度。The method for using the tumor marker electrochemiluminescence immunosensor based on the multifunctional carbon nitride material prepared in the above specific example 1 to detect tumor markers, the specific steps are as follows: the electrochemiluminescence immunosensor for detecting tumor markers The sensor was soaked in different concentrations of tumor marker solutions, incubated at 37°C for 1 h, taken out, washed with water, and used as a working electrode; a platinum electrode was used as a counter electrode, and an Ag/AgCl electrode or a saturated calomel electrode was used as a reference The electrodes constitute a three-electrode system; put the three-electrode system into the buffer solution, start the electrochemical reaction, and measure the electrochemiluminescence intensity; obtain a series of electrochemiluminescence intensity values corresponding to tumor marker solutions with different concentrations, and establish the electrochemiluminescence intensity The quantitative relationship between the value and the concentration of the tumor marker solution; according to this quantitative relationship, the concentration of the tumor marker in the sample to be tested is detected.
上述缓冲溶液为：pH = 7.5～8.5的磷酸盐缓冲溶液，含有10～30 mmol/L K2S2O8和80～100 mmol/L KCl。The above buffer solution is: a phosphate buffer solution with pH = 7.5-8.5, containing 10-30 mmol/L K2 S2 O8 and 80-100 mmol/L KCl.
上述电化学反应的条件如下：电位阶跃计时电流法，脉冲宽度：0.25秒；脉冲间隔：30秒；初始电压：0V；脉冲电压：-1.1V。The conditions of the above electrochemical reaction are as follows: potential step chronoamperometry, pulse width: 0.25 seconds; pulse interval: 30 seconds; initial voltage: 0V; pulse voltage: -1.1V.
上述肿瘤标志物为：甲胎蛋白（AFP）、癌胚抗原（CEA)、糖类抗原15-3（CA15-3）、糖类抗原125（CA125）、糖类抗原19-9（CA19-9）或前列腺特异性抗原（PSA）。The above tumor markers are: alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), carbohydrate antigen 15-3 (CA15-3), carbohydrate antigen 125 (CA125), carbohydrate antigen 19-9 (CA19-9 ) or prostate-specific antigen (PSA).
具体实施例三 Specific embodiment three
一种基于多功能化氮化碳材料的糖类抗原125（CA125）电化学发光免疫传感器的制备方法，具体步骤如下：A method for preparing a carbohydrate antigen 125 (CA125) electrochemiluminescence immunosensor based on a multifunctional carbon nitride material, the specific steps are as follows:
（1）多功能化氮化碳材料的制备(1) Preparation of multifunctional carbon nitride materials
a. 将三聚氰胺在550℃下加热4h，真空干燥后得到氮化碳（g-C3N4）粉末；a. Heat melamine at 550°C for 4 hours and dry it in vacuum to obtain carbon nitride (gC3 N4 ) powder;
b. 取1.2 g 氮化碳粉末加入到100 mL 5 mol/L HNO3中，于135℃下回流36小时后，自然冷却至室温，水洗涤至pH=7，离心，将沉淀在38℃下真空干燥16小时，得到羧基化的氮化碳（g-C3N4）；b. Add 1.2 g of carbon nitride powder into 100 mL of 5 mol/L HNO3 , reflux at 135°C for 36 hours, cool to room temperature naturally, wash with water until pH=7, centrifuge, and store the precipitate at 38°C Dry under vacuum for 16 hours to obtain carboxylated carbon nitride (gC3 N4 );
c. 取0.35 g FeCl2·4H2O和0.8 g FeCl3·6H2O溶于75 mL除氧的二次水中，然后在氮气氛保护下逐滴加入到三口烧瓶中，磁力搅拌使其充分混合均匀，然后将26.5wt%的氨水逐滴缓慢加入到三口烧瓶中直至溶液的pH为9.0，将三口烧瓶内的溶液加热到85℃后，于85℃搅拌回流2 h，然后停止加热在氮气氛保护下搅拌回流冷却至室温，得到的黑色沉淀物纳米四氧化三铁；将纳米四氧化三铁用水清洗至中性，定容至50 mL，超声45 min后，加入0.15 mL 3-氨丙基三乙氧基硅烷（APTES），室温下搅拌 7 h，得到氨基化的纳米四氧化三铁悬浊液；c. Dissolve 0.35 g FeCl2 4H2 O and 0.8 g FeCl3 6H2 O in 75 mL of deoxygenated secondary water, then add them dropwise into a three-necked flask under the protection of a nitrogen atmosphere, and stir magnetically to make them fully Mix evenly, then slowly add 26.5wt% ammonia water dropwise into the three-necked flask until the pH of the solution is 9.0, heat the solution in the three-necked flask to 85°C, stir and reflux at 85°C for 2 h, then stop heating under nitrogen Stir and reflux under the protection of the atmosphere and cool to room temperature to obtain black precipitate nano-ferric oxide; wash the nano-ferric oxide with water until neutral, set the volume to 50 mL, and after ultrasonication for 45 min, add 0.15 mL of 3-aminopropanol triethoxysilane (APTES), stirred at room temperature for 7 h to obtain aminated nano-ferric oxide suspension;
d. 取200 µL 1.0 mg/mL羧基化的氮化碳加入200 µL偶联试剂后，用1.0 mol/L盐酸调pH为5.0，振摇 1.5 h，离心取沉淀加水洗涤后，再离心取沉淀加水洗涤，再次离心取沉淀加水洗涤后，定容至250 µL，用0.15mol/L NaOH溶液调pH为9.0，然后加入75 µL 0.5µg/mL糖类抗原125抗体溶液，摇床孵育1.5 h后再加入200 µL氨基化的纳米四氧化三铁悬浊液，继续孵育4 h，再加入75 µL 2%牛血清白蛋白溶液以封闭非特异性吸附位点，继续孵育1.5h，用磁铁吸引分离，水清洗至中性后，定容至150µL，即得到多功能化氮化碳材料溶液；其中偶联试剂为1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐与N-羟基琥珀酰亚胺溶于水中得到，偶联试剂中1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐的摩尔浓度为50 mmol/L，所述的N-羟基琥珀酰亚胺摩尔浓度为5 mmol/L；d. Take 200 µL of 1.0 mg/mL carboxylated carbon nitride and add 200 µL of coupling reagent, adjust the pH to 5.0 with 1.0 mol/L hydrochloric acid, shake for 1.5 h, centrifuge to get the precipitate, wash with water, and then centrifuge to get the precipitate Add water to wash, centrifuge again to get the precipitate, add water and wash, dilute to 250 µL, adjust pH to 9.0 with 0.15mol/L NaOH solution, then add 75 µL 0.5 µg/mL carbohydrate antigen 125 antibody solution, and incubate on a shaking table for 1.5 h Then add 200 µL of aminated nano-iron ferric oxide suspension, continue to incubate for 4 h, then add 75 µL of 2% bovine serum albumin solution to block the non-specific adsorption sites, continue to incubate for 1.5 h, and separate with a magnet. After washing with water to neutrality, set the volume to 150 µL to obtain a multifunctional carbon nitride material solution; wherein the coupling reagent is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride Salt and N-hydroxysuccinimide are dissolved in water, and the molar concentration of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride in the coupling reagent is 50 mmol/L, The molar concentration of N-hydroxysuccinimide is 5 mmol/L;
（2）电化学发光免疫传感器的组装(2) Assembly of electrochemiluminescence immunosensor
a. 将直径为3～5 mm的磁性玻碳电极依次用1.0、0.3和0.05 μm的Al2O3抛光粉在麂皮上抛光至镜面，超声清洗2 min，然后依次用乙醇水溶液（乙醇与水体积比为1:1）、硝酸水溶液（硝酸与水体积比为1:1）和蒸馏水超声清洗；a. The magnetic glassy carbon electrode with a diameter of 3-5 mm was polished to a mirror surface on the suede with 1.0, 0.3 and 0.05 μm Al2 O3 polishing powder in sequence, ultrasonically cleaned for 2 min, and then sequentially washed with ethanol aqueous solution (ethanol and Water volume ratio is 1:1), nitric acid aqueous solution (nitric acid and water volume ratio is 1:1) and distilled water ultrasonic cleaning;
b. 取8 µL步骤（1）所得的多功能化氮化碳材料溶液，滴在磁性玻碳电极表面，多功能化氮化碳材料即被均匀、牢固地吸附于电极表面，即得到用于检测糖类抗原125的电化学发光免疫传感器。b. Take 8 µL of the multifunctional carbon nitride material solution obtained in step (1), and drop it on the surface of the magnetic glassy carbon electrode. The multifunctional carbon nitride material is evenly and firmly adsorbed on the electrode surface, and the An electrochemiluminescent immunosensor for the detection of carbohydrate antigen 125.
将用于检测糖类抗原125的电化学发光免疫传感器浸泡在不同浓度的糖类抗原125溶液中，于37℃下温育1 h后取出，水清洗后，作为工作电极；采用铂电极作为对电极，Ag/AgCl电极或者饱和甘汞电极作为参比电极，构成三电极体系；将三电极体系放入缓冲溶液，启动电化学反应，测量电化学发光强度；获得一系列不同浓度的糖类抗原125溶液对应的电化学发光强度值，建立电化学发光强度值与糖类抗原125溶液浓度之间的定量关系；根据这个定量关系检测待测样品中糖类抗原125的浓度。图3 不同浓度CA125的ECL信号(y)—浓度(x)对数线性关系如图2所示。线性线性方程为： y=-1635.66*logx+1839.02，相关系数R2=0.9932；线性范围为0.001～10 U/mL，检测限为0.3 mU/mL，线性良好，可用于检测待测样品中糖类抗原125的浓度。Soak the electrochemiluminescent immunosensor for detecting carbohydrate antigen 125 in different concentrations of carbohydrate antigen 125 solutions, take it out after incubating at 37°C for 1 h, wash it with water, and use it as a working electrode; a platinum electrode is used as a counter electrode. Electrode, Ag/AgCl electrode or saturated calomel electrode as a reference electrode to form a three-electrode system; put the three-electrode system into the buffer solution, start the electrochemical reaction, and measure the intensity of electrochemiluminescence; obtain a series of carbohydrate antigens with different concentrations 125 solution corresponding to the electrochemiluminescence intensity value, and establish a quantitative relationship between the electrochemiluminescence intensity value and the concentration of the carbohydrate antigen 125 solution; according to this quantitative relationship, detect the concentration of the carbohydrate antigen 125 in the sample to be tested. Figure 3 The logarithmic linear relationship between the ECL signal (y) and the concentration (x) of different concentrations of CA125 is shown in Figure 2. Linear The linear equation is: y=-1635.66*logx+1839.02, the correlation coefficientR2 =0.9932; the linear range is 0.001～10 U/mL, the detection limit is 0.3 mU/mL, the linearity is good, and it can be used to detect the sugar in the sample to be tested The concentration of antigen-like 125.
具体实施例四 Specific embodiment four
同上述具体实施例三，其区别在于：With the above-mentioned specific embodiment three, its difference is:
步骤（1）多功能化氮化碳材料的制备中：Step (1) In the preparation of multifunctional carbon nitride material:
a. 将三聚氰胺在500℃下加热5 h，真空干燥后得到氮化碳粉末；a. Heat melamine at 500°C for 5 h, and dry it in vacuum to obtain carbon nitride powder;
b. 取0.8 g 氮化碳粉末加入到80 mL 6 mol/L HNO3中，于120℃下回流48小时后，自然冷却至室温，水洗涤至pH=7，离心，将沉淀在35℃下真空干燥20小时，得到羧基化的氮化碳；b. Add 0.8 g of carbon nitride powder into 80 mL of 6 mol/L HNO3 , reflux at 120°C for 48 hours, cool to room temperature naturally, wash with water until pH = 7, centrifuge, and place the precipitate at 35°C Under vacuum drying for 20 hours, obtain carboxylated carbon nitride;
c. 取0.2 g FeCl2·4H2O和0.6 g FeCl3·6H2O溶于50 mL除氧的二次水中磁力搅拌使其充分混合均匀，然后将25wt%的氨水逐滴缓慢加入到三口烧瓶中直至溶液的pH为8.0，将三口烧瓶内的溶液加热到70℃后，于70℃搅拌回流3 h；将得到纳米四氧化三铁用水清洗至中性，定容至50 mL，超声30 min后，加入0.1 mL 3-氨丙基三乙氧基硅烷，室温下搅拌 6 h，得到氨基化的纳米四氧化三铁悬浊液；c. Dissolve 0.2 g FeCl2 4H2 O and 0.6 g FeCl3 6H2 O in 50 mL of deaerated secondary water with magnetic stirring to mix well, then slowly add 25wt% ammonia water dropwise to the three ports In the flask until the pH of the solution is 8.0, heat the solution in the three-neck flask to 70°C, then stir and reflux at 70°C for 3 h; wash the obtained nano-ferric oxide with water until neutral, set the volume to 50 mL, and sonicate for 30 After 1 min, add 0.1 mL of 3-aminopropyltriethoxysilane and stir at room temperature for 6 h to obtain aminated nano-ferric oxide suspension;
d. 取150 µL 1.5 mg/mL羧基化的氮化碳加入150 µL偶联试剂后，用0.8mol/L盐酸调pH为6.0，振摇 1h；离心洗涤后定容至200 µL，用0.1mol/L NaOH溶液调pH为8.0，然后加入50 µL 1 µg/mL CA125抗体溶液，摇床孵育1h后再加入150 µL氨基化的纳米四氧化三铁悬浊液，继续孵育3 h，再加入50 µL牛血清白蛋白溶液继续孵育1 h，用磁铁吸引分离，水清洗至中性后，定容至100µL，即得到多功能化氮化碳材料溶液；其中偶联试剂中1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐的摩尔浓度为10 mmol/L，所述的N-羟基琥珀酰亚胺摩尔浓度为10 mmol/L；d. Take 150 µL of 1.5 mg/mL carboxylated carbon nitride and add 150 µL of coupling reagent, adjust the pH to 6.0 with 0.8mol/L hydrochloric acid, shake for 1 hour; /L NaOH solution to adjust the pH to 8.0, then add 50 µL 1 µg/mL CA125 antibody solution, incubate on a shaker for 1 h, then add 150 µL aminated nano-ferric oxide suspension, continue to incubate for 3 h, then add 50 µL bovine serum albumin solution continued to incubate for 1 h, separated by magnet attraction, washed with water until neutral, and then adjusted to 100 µL to obtain a multifunctional carbon nitride material solution; the coupling reagent contained 1-(3-di The molar concentration of methylaminopropyl)-3-ethylcarbodiimide hydrochloride is 10 mmol/L, and the molar concentration of described N-hydroxysuccinimide is 10 mmol/L;
步骤（2）电化学发光免疫传感器的组装中取5 µL多功能化氮化碳材料溶液，滴在磁性玻碳电极表面。Step (2) In the assembly of the electrochemiluminescent immunosensor, take 5 µL of the multifunctional carbon nitride material solution and drop it on the surface of the magnetic glassy carbon electrode.
具体实施例五 Specific embodiment five
同上述具体实施例三，其区别在于：With the above-mentioned specific embodiment three, its difference is:
步骤（1）多功能化氮化碳材料的制备中：Step (1) In the preparation of multifunctional carbon nitride material:
a. 将三聚氰胺在600℃下加热3 h；a. Heat melamine at 600°C for 3 h;
b. 取1.5 g 氮化碳粉末加入到120 mL 4 mol/L HNO3中，于150℃下回流24小时；将沉淀在40℃下真空干燥12小时；b. Add 1.5 g of carbon nitride powder into 120 mL of 4 mol/L HNO3 , reflux at 150°C for 24 hours; vacuum-dry the precipitate at 40°C for 12 hours;
c. 取0.5 g FeCl2·4H2O和1.0 g FeCl3·6H2O溶于100 mL除氧的二次水中；将28wt%的氨水逐滴缓慢加入到三口烧瓶中直至溶液的pH为10.0，将三口烧瓶内的溶液加热到100℃后，于100℃搅拌回流1 h；将得到纳米四氧化三铁用水清洗至中性定容，超声1 h后，加入0.2 mL 3-氨丙基三乙氧基硅烷，室温下搅拌 8 h；c. Dissolve 0.5 g FeCl2 4H2 O and 1.0 g FeCl3 6H2 O in 100 mL of deaerated secondary water; slowly add 28 wt% ammonia water dropwise into the three-necked flask until the pH of the solution is 10.0 , heated the solution in the three-necked flask to 100°C, stirred and refluxed at 100°C for 1 h; washed the obtained nanometer iron ferric oxide with water to a neutral volume, ultrasonicated for 1 h, and added 0.2 mL of 3-aminopropyl tris Ethoxysilane, stirred at room temperature for 8 h;
d. 取250 µL 0.5 mg/mL羧基化的氮化碳加入250 µL偶联试剂后，用1.2mol/L盐酸调pH为4.0，振摇 2 h；离心洗涤定容至300 µL，用0.2mol/L NaOH溶液调pH为10.0，然后加入100 µL 0.1µg/mL CA125抗体溶液，摇床孵育2 h后再加入250 µL氨基化的纳米四氧化三铁悬浊液，继续孵育5 h，再加入100 µL牛血清白蛋白溶液继续孵育2 h，用磁铁吸引分离水清洗至中性后，定容至200µL，即得到多功能化氮化碳材料溶液；其中偶联试剂中1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐的摩尔浓度为100 mmol/L，所述的N-羟基琥珀酰亚胺摩尔浓度为1 mmol/L；d. Take 250 µL of 0.5 mg/mL carboxylated carbon nitride and add 250 µL of coupling reagent, adjust the pH to 4.0 with 1.2mol/L hydrochloric acid, shake for 2 h; /L NaOH solution to adjust the pH to 10.0, then add 100 µL 0.1 µg/mL CA125 antibody solution, incubate on a shaker for 2 h, then add 250 µL aminated nano-iron ferric oxide suspension, continue to incubate for 5 h, then add Continue to incubate 100 µL of bovine serum albumin solution for 2 h, use a magnet to attract separation water and wash to neutral, and then set the volume to 200 µL to obtain a multifunctional carbon nitride material solution; in which the coupling reagent contains 1-(3-di The molar concentration of methylaminopropyl)-3-ethylcarbodiimide hydrochloride is 100 mmol/L, and the described N-hydroxyl succinimide molar concentration is 1 mmol/L;
步骤（2）电化学发光免疫传感器的组装中取10 µL多功能化氮化碳材料溶液，滴在磁性玻碳电极表面。Step (2) In the assembly of the electrochemiluminescence immunosensor, take 10 µL of the multifunctional carbon nitride material solution and drop it on the surface of the magnetic glassy carbon electrode.
具体实施例六 Specific embodiment six
基于多功能化氮化碳材料的糖类抗原15-3（CA15-3）电化学发光免疫传感器，其具体制备方法同上述具体实施例三，其区别在于：肿瘤标志物为糖类抗原15-3，不同浓度CA15-3的ECL信号(y)—浓度(x)对数线性关系如图3所示，线性方程为：y=-1603.51*logx+1903.56，相关系数R2=0.9989，线性范围为0.001～10 U/mL，检测限为0.3 mU/mL。线性良好，可用于检测待测样品中糖类抗原15-3的浓度。Carbohydrate antigen 15-3 (CA15-3) electrochemiluminescent immunosensor based on multifunctional carbon nitride material, its specific preparation method is the same as the above specific example 3, the difference is that the tumor marker is carbohydrate antigen 15- 3. The ECL signal (y)-concentration (x) logarithmic linear relationship of different concentrations of CA15-3 is shown in Figure 3. The linear equation is: y=-1603.51*logx+1903.56, the correlation coefficientR2 =0.9989, and the linear range It is 0.001～10 U/mL, and the detection limit is 0.3 mU/mL. The linearity is good, and it can be used to detect the concentration of carbohydrate antigen 15-3 in the test sample.
具体实施例七 Specific embodiment seven
基于多功能化氮化碳材料的糖类抗原19-9（CA19-9）电化学发光免疫传感器，其具体制备方法同上述具体实施例三，其区别在于：肿瘤标志物为糖类抗原19-9（CA19-9），不同浓度糖类抗原19-9的ECL信号(y)—浓度(x)对数线性关系如图4所示，线性方程为：y=-1739.39*logx+1860.24，相关系数R2=0.9984，线性范围为0.001～10 U/mL，检测限为0.3 mU/mL。线性良好，可用于检测待测样品中糖类抗原19-9的浓度。Carbohydrate antigen 19-9 (CA19-9) electrochemiluminescence immunosensor based on multifunctional carbon nitride material, its specific preparation method is the same as the above specific example 3, the difference is that the tumor marker is carbohydrate antigen 19- 9 (CA19-9), the ECL signal (y)-concentration (x) logarithmic linear relationship of different concentrations of carbohydrate antigen 19-9 is shown in Figure 4, the linear equation is: y=-1739.39*logx+1860.24, correlation The coefficientR2 =0.9984, the linear range is 0.001～10 U/mL, and the detection limit is 0.3 mU/mL. The linearity is good, and it can be used to detect the concentration of carbohydrate antigen 19-9 in the test sample.
具体实施例八 Embodiment 8
基于多功能化氮化碳材料的甲胎蛋白（AFP）电化学发光免疫传感器，其具体制备方法同上述具体实施例三，其区别在于：肿瘤标志物为甲胎蛋白，不同浓度甲胎蛋白的ECL信号(y)—浓度(x)对数线性关系如图5所示，线性方程为：y=-1994.52*logx+4782.94，相关系数R2=0.9905，线性范围为0.01～10 ng/mL，检测限为3 pg/mL。线性良好，可用于检测待测样品中甲胎蛋白（AFP）的浓度。Alpha-fetoprotein (AFP) electrochemiluminescence immunosensor based on multifunctional carbon nitride materials, its specific preparation method is the same as the above-mentioned specific example 3, the difference is that: the tumor marker is alpha-fetoprotein, different concentrations of alpha-fetoprotein The logarithmic linear relationship between ECL signal (y)-concentration (x) is shown in Figure 5. The linear equation is: y=-1994.52*logx+4782.94, the correlation coefficientR2 =0.9905, and the linear range is 0.01-10 ng/mL. The detection limit was 3 pg/mL. It has good linearity and can be used to detect the concentration of alpha-fetoprotein (AFP) in the sample to be tested.
具体实施例九 Specific embodiment nine
基于多功能化氮化碳材料的癌胚抗原（CEA)电化学发光免疫传感器，其具体制备方法同上述具体实施例三，其区别在于：肿瘤标志物为癌胚抗原（CEA)，不同浓度CEA的ECL信号(y)—浓度(x)对数线性关系如图6所示，线性方程为：y=-1995.52*logx+2049.94,相关系数R2=0.9805，线性范围为0.01～10 ng/mL，检测限为3 pg/mL。线性良好，可用于检测待测样品中癌胚抗原的浓度。Carcinoembryonic antigen (CEA) electrochemiluminescent immunosensor based on multifunctional carbon nitride material, its specific preparation method is the same as the above-mentioned specific embodiment three, the difference is that: the tumor marker is carcinoembryonic antigen (CEA), different concentrations of CEA The ECL signal (y)-concentration (x) logarithmic linear relationship is shown in Figure 6, the linear equation is: y=-1995.52*logx+2049.94, the correlation coefficientR2 =0.9805, and the linear range is 0.01～10 ng/mL , with a detection limit of 3 pg/mL. It has good linearity and can be used to detect the concentration of carcinoembryonic antigen in the sample to be tested.
具体实施例十 Specific embodiment ten
基于多功能化氮化碳材料的前列腺特异性抗原（PSA）电化学发光免疫传感器，其具体制备方法同上述具体实施例三，其区别在于：肿瘤标志物为前列腺特异性抗原（PSA），不同浓度PSA的ECL信号(y)—浓度(x)对数线性关系如图7所示，线性方程为：y=-3338.80*logx+4993.87，相关系数R2=0.9945，线性范围为0.01～10 ng/mL，检测限为3 pg/mL。线性良好，可用于检测待测样品中前列腺特异性抗原的浓度。Prostate-specific antigen (PSA) electrochemiluminescence immunosensor based on multifunctional carbon nitride material, its specific preparation method is the same as the above-mentioned specific example 3, the difference is that the tumor marker is prostate-specific antigen (PSA), different The logarithmic linear relationship between ECL signal (y) and concentration (x) of PSA concentration is shown in Figure 7. The linear equation is: y=-3338.80*logx+4993.87, the correlation coefficientR2 =0.9945, and the linear range is 0.01 to 10 ng /mL, the detection limit was 3 pg/mL. Good linearity, can be used to detect the concentration of prostate specific antigen in the sample to be tested.
具体实施例十一 Specific Embodiment Eleven
将具体实施例三制备的基于多功能化氮化碳材料的糖类抗原125（CA125）电化学发光免疫传感器，分别测定10ng/mL的AFP、CEA、PSA和10 U/mL CA15-3、CA19-9，对应的电化学发光强度在7000以上，与空白值大致相当；而测定10-2 U/mL的CA125，对应的电化学发光强度明显降低，在3000左右，结果表明：选择性良好，常见其它肿瘤标志物无显著性干扰。The carbohydrate antigen 125 (CA125) electrochemiluminescence immunosensor based on the multifunctional carbon nitride material prepared in specific example 3 was used to detect 10 ng/mL of AFP, CEA, PSA and 10 U/mL of CA15-3 and CA19 respectively. -9, the corresponding electrochemiluminescence intensity is above 7000, which is roughly equivalent to the blank value; and the corresponding electrochemiluminescence intensity is significantly lower when measuring 10-2 U/mL of CA125, which is about 3000, the results show that the selectivity is good, Common other tumor markers had no significant interference.
同理选取甲胎蛋白（AFP）、癌胚抗原（CEA)、糖类抗原15-3（CA15-3）、糖类抗原125（CA125）、糖类抗原19-9（CA19-9）或前列腺特异性抗原（PSA）七种肿瘤标志物来验证上述具体实施例六-十的电化学发光免疫传感器均选择性良好，常见其它肿瘤标志物无显著性干扰。Similarly, alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), carbohydrate antigen 15-3 (CA15-3), carbohydrate antigen 125 (CA125), carbohydrate antigen 19-9 (CA19-9) or prostate Specific antigen (PSA) seven tumor markers were used to verify that the electrochemiluminescent immunosensors in the above specific examples 6-10 had good selectivity, and there was no significant interference with common other tumor markers.
具体实施例十二Specific embodiment twelve
取空白血清样品，采用标准加入法分别配制了不同浓度的肿瘤标志物溶液，按照发明具体实施例三、六、十中的具体实验步骤构建电化学发光传感器并对加标样品按具体实施例二方法进行了检测。检测结果如表1所示，Take a blank serum sample, prepare tumor marker solutions with different concentrations by using the standard addition method, construct an electrochemiluminescence sensor according to the specific experimental steps in the third, sixth, and tenth specific examples of the invention, and standardize the sample according to the second example method was tested. The test results are shown in Table 1,
表1人血清中多种肿瘤标志物的检测结果Table 1 The detection results of various tumor markers in human serum
由表1检测结果可知，结果的相对标准偏差（RSD）小于7.5%，回收率为96.6～102%，表明本发明对于血清中多种肿瘤标志物的检测精密度高，结果准确可靠。 It can be seen from the test results in Table 1 that the relative standard deviation (RSD) of the results is less than 7.5%, and the recovery rate is 96.6-102%, indicating that the present invention has high detection precision for various tumor markers in serum, and the results are accurate and reliable.
以上结果说明，本发明构建的检测肿瘤标志物的电化学发光免疫传感器，灵敏度高、检测限低、选择性高、操作简单、结果准确可靠。只需改变本电化学发光免疫传感器中的抗体，即可实现对不同目标肿瘤标志物的高灵敏度、特异性检测。The above results show that the electrochemiluminescent immunosensor for detecting tumor markers constructed by the present invention has high sensitivity, low detection limit, high selectivity, simple operation, and accurate and reliable results. Highly sensitive and specific detection of different target tumor markers can be achieved only by changing the antibodies in the electrochemiluminescence immunosensor.
当然，上述说明并非对本发明的限制，本发明也并不限于上述举例。本技术领域的普通技术人员在本发明的实质范围内，作出的变化、改型、添加或替换，也应属于本发明的保护范围。Of course, the above descriptions are not intended to limit the present invention, and the present invention is not limited to the above examples. Changes, modifications, additions or substitutions made by those skilled in the art within the essential scope of the present invention shall also belong to the protection scope of the present invention.
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510035191.XACN104655855B (en) | 2015-01-23 | 2015-01-23 | Preparation method and application of tumor marker electrochemiluminescence immunosensor based on multifunctional carbon nitride material |
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510035191.XACN104655855B (en) | 2015-01-23 | 2015-01-23 | Preparation method and application of tumor marker electrochemiluminescence immunosensor based on multifunctional carbon nitride material |
| Publication Number | Publication Date |
|---|---|
| CN104655855A CN104655855A (en) | 2015-05-27 |
| CN104655855Btrue CN104655855B (en) | 2016-06-08 |
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510035191.XAActiveCN104655855B (en) | 2015-01-23 | 2015-01-23 | Preparation method and application of tumor marker electrochemiluminescence immunosensor based on multifunctional carbon nitride material |
| Country | Link |
|---|---|
| CN (1) | CN104655855B (en) |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105115961B (en)* | 2015-08-07 | 2018-02-02 | 上海师范大学 | A kind of preparation method of the electrochemical luminescence sensor of nano composite material |
| CN105301241B (en)* | 2015-10-22 | 2017-02-01 | 宁波大学 | Preparing method and application of sandwich type electrochemical luminescence immunosensor for detecting tumor marker |
| CN105891473B (en)* | 2016-04-06 | 2017-12-26 | 宁波大学 | The preparation method and applications of food-borne pathogens immunosensor based on gold label silver stain signal amplification technique |
| CN106645737A (en)* | 2016-06-30 | 2017-05-10 | 深圳市亚辉龙生物科技股份有限公司 | S-100 chemiluminescence immunoassay kit and preparation method thereof |
| CN109633151B (en)* | 2018-12-26 | 2022-03-11 | 西北农林科技大学 | A kind of Salmonella Enteritidis detection method, test strip and application |
| CN109946355A (en)* | 2019-04-11 | 2019-06-28 | 青岛农业大学 | An electrochemiluminescence method for detecting tumor markers |
| CN110501502A (en)* | 2019-09-02 | 2019-11-26 | 嘉兴学院 | Preparation and application method of graphitized carbon-chitosan CA125 immunosensor |
| CN113433320B (en)* | 2021-07-19 | 2023-03-14 | 长春理工大学 | Method for detecting tumor marker CA19-9 by using magnetic separation fluorescence enhancement type aptamer sensor |
| CN113736305A (en)* | 2021-09-14 | 2021-12-03 | 沈阳先进涂层材料产业技术研究院有限公司 | Tripolyphosphate/carbon nitride nano composite material, preparation method thereof and application thereof in water-based anticorrosive paint |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102707049B (en)* | 2012-05-14 | 2014-12-10 | 宁波大学 | Preparation method and application of magnetic sandwich nano immunosensor |
| CN103116023B (en)* | 2013-01-25 | 2015-04-15 | 宁波大学 | ECL (electrochemiluminescence) immunosensor for detecting tumor markers and preparation method and applications thereof |
| CN104132934B (en)* | 2014-07-31 | 2016-10-12 | 济南大学 | The preparation of the molecular engram electroluminescent paper chip that the detection agriculture of a kind of Multi-example is residual |
| CN104297479B (en)* | 2014-09-24 | 2016-06-22 | 济南大学 | The preparation of detection tumor markers electrochemiluminescimmunosensor immunosensor and application |
| CN104297323B (en)* | 2014-11-01 | 2016-01-20 | 济南大学 | The carboxylated C of a kind of ZnO CdTe- 3n 4the preparation of photoelectricity DNA sensor and application thereof |
| Publication number | Publication date |
|---|---|
| CN104655855A (en) | 2015-05-27 |
| Publication | Publication Date | Title |
|---|---|---|
| CN104655855B (en) | Preparation method and application of tumor marker electrochemiluminescence immunosensor based on multifunctional carbon nitride material | |
| CN105301241B (en) | Preparing method and application of sandwich type electrochemical luminescence immunosensor for detecting tumor marker | |
| CN103116023B (en) | ECL (electrochemiluminescence) immunosensor for detecting tumor markers and preparation method and applications thereof | |
| CN102636649B (en) | Kit for detecting carcinoembryonic antigen based on antibody functionalized magnetic nanometer material and up-conversion fluorescence nanometer material | |
| CN104122309B (en) | A kind of preparation of cyclodextrin-Cu@Ag electrochemical immunosensor | |
| CN103472121B (en) | Sandwich type electrochemical immunosensor for simultaneously detecting markers of cervical cancer | |
| CN102967706A (en) | Preparation method and application of flow injection chemiluminiscence immuno sensor for detecting tumor marker | |
| CN104655616A (en) | Preparation method and application of electrochemiluminescence aptamer sensor for detecting tumor marker MUC1 | |
| CN104297479B (en) | The preparation of detection tumor markers electrochemiluminescimmunosensor immunosensor and application | |
| CN104391117B (en) | Preparation method and application of gastric cancer antigen electrogenerated chemiluminescence sensor based on PP<y>-NH2GO-Ag2Se@CdSe | |
| CN102226807B (en) | Electrochemical Immunoassay Based on Au-PB-SiO2 Composite Nanoparticles | |
| CN114295694A (en) | An electrochemiluminescence aptamer sensor for breast cancer HER-2 detection and its detection method | |
| CN109613244B (en) | A kind of preparation method and application of Ag@Pt-CuS-labeled immunosensor | |
| CN109839501A (en) | A kind of electrochemiluminescimmunosensor immunosensor and the preparation method and application thereof measuring circulating tumor cell | |
| CN113267632B (en) | A label-free electrochemiluminescence immuno-array sensor for detection of myocardial infarction markers mediated by multifunctional polyethyleneimine | |
| CN107422008A (en) | A kind of electrochemical immunosensor for determining alpha-fetoprotein and preparation method and application | |
| CN106526183A (en) | Preparation method and application of electrochemical luminescence immune sensor for potentiometric resolution of double tumor marker | |
| Ge et al. | Ultra-sensitive magnetic immunoassay of HE4 based on surface enhanced Raman spectroscopy | |
| You et al. | Sensitive detection of SARS-CoV-2 spike protein based on electrochemical impedance spectroscopy of Fe3O4@ SiO2–Au/GCE biosensor | |
| CN106093396A (en) | A kind of preparation method and application of immunosensor based on Au GQD@PtPd | |
| CN106771199B (en) | A kind of colorimetric immunoassay analysis method of detection tumor markers for non-diagnostic purpose | |
| CN106124487B (en) | Electrochemiluminescence multi-component immunodetection method based on spectral resolution principle | |
| Fu et al. | A high sensitivity prostate-specific antigen SERS detection platform based on laser resonance nanoparticles | |
| CN104198563B (en) | Preparation method and the application of lead ion gold-supported magnetic multi-wall carbon nano-tube tube sensor | |
| CN102435736A (en) | Method for measuring antigen of ovarian cancer embryo by electrochemical luminescence (ECL) immunosensor |
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right | Effective date of registration:20200729 Address after:Room 1,020, Nanxun Science and Technology Pioneering Park, No. 666 Chaoyang Road, Nanxun District, Huzhou City, Zhejiang Province, 313000 Patentee after:Huzhou You Yan Intellectual Property Service Co.,Ltd. Address before:315211 Zhejiang Province, Ningbo Jiangbei District Fenghua Road No. 818 Patentee before:Ningbo University | |
| TR01 | Transfer of patent right | ||
| EE01 | Entry into force of recordation of patent licensing contract | Application publication date:20150527 Assignee:Wuxing Zhili Atai Garment Factory Assignor:Huzhou You Yan Intellectual Property Service Co.,Ltd. Contract record no.:X2024330000372 Denomination of invention:Preparation method and application of tumor biomarker electrochemiluminescence immunosensor based on multifunctional nitride carbon materials Granted publication date:20160608 License type:Common License Record date:20240808 Application publication date:20150527 Assignee:Huzhou Zhili Jinshengwa Garment Factory Assignor:Huzhou You Yan Intellectual Property Service Co.,Ltd. Contract record no.:X2024330000371 Denomination of invention:Preparation method and application of tumor biomarker electrochemiluminescence immunosensor based on multifunctional nitride carbon materials Granted publication date:20160608 License type:Common License Record date:20240808 | |
| EE01 | Entry into force of recordation of patent licensing contract |