技术领域technical field
本发明涉及电化学发光免疫传感器，尤其是涉及用于检测海洋致病菌的夹心式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 sandwich type electrochemiluminescence immunosensor for detecting marine pathogenic bacteria.
背景技术Background technique
海洋致病菌是一种原核单细胞生物，呈球状、杆状、螺旋状等。海洋致病菌会导致海洋水产动植物患病，例如：副溶血弧菌可引起凡纳滨对虾幼虾发生毁灭性死亡。一些海洋致病菌是人畜共患病的重要病原，人类通过进食受污染的海产品或破损的皮肤接触海水感染致病菌，可引发中耳炎、泌尿系统感染、败血症等疾病甚至死亡。因此快速准确地检测海洋致病菌是保护人类健康和减少经济损失的重要途径。Marine pathogenic bacteria are prokaryotic single-celled organisms that are spherical, rod-shaped, helical, etc. Marine pathogenic bacteria can cause diseases in marine aquatic animals and plants, for example: Vibrio parahaemolyticus can cause devastating death of juvenile Litopenaeus vannamei. Some marine pathogenic bacteria are important pathogens of zoonotic diseases. Humans are infected with pathogenic bacteria through eating contaminated seafood or contacting seawater with damaged skin, which can cause diseases such as otitis media, urinary system infection, sepsis and even death. Therefore, rapid and accurate detection of marine pathogens is an important way to protect human health and reduce economic losses.
目前对于海洋致病菌的测定，常规培养方法包括增菌培养、选择性培养等过程，耗时长、操作繁琐、检出率低。以免疫学为基础的检测方法利用抗原抗体识别的特异性，包括酶联免疫吸附(ELISA)法等，但是灵敏度需要进一步提高。基于核酸的检测方法如聚合酶链式反应(PCR)是一种灵敏的检测方法，但较易出现假阳性结果。因此，开发快速、灵敏、准确、操作简便的海洋致病菌检测技术非常重要。At present, for the determination of marine pathogenic bacteria, conventional culture methods include enrichment culture, selective culture and other processes, which are time-consuming, cumbersome to operate and low in detection rate. Immunology-based detection methods utilize the specificity of antigen-antibody recognition, including enzyme-linked immunosorbent (ELISA) methods, etc., but the sensitivity needs to be further improved. Nucleic acid-based detection methods such as polymerase chain reaction (PCR) are sensitive detection methods, but are prone to false positive results. Therefore, it is very important to develop rapid, sensitive, accurate, and easy-to-operate detection techniques for marine pathogens.
电化学发光(Electrochemiluminescence,ECL)是一种在电极表面由电化学引发的化学发光反应，是化学发光与电化学相结合的产物。电化学发光免疫传感器是集电化学发光与免疫传感器为一体的技术，具有灵敏度高、操作简便等优点。氧化石墨烯是一种性能优异的新型碳材料，具有较高的比表面积和丰富的官能团，基于氧化石墨烯制备的功能化氧化石墨烯，可有效延展电极表面的赫姆霍兹面，显著增强电化学发光，提高灵敏度，是构建电化学发光传感器标记物的理想材料。免疫磁珠是近年来发展起来的一项免疫学技术，它将固化试剂特有的优点与免疫学反应的高度特异性结合于一体，运用核-壳的合成方法合成超顺磁性功能化四氧化三铁小球，利用其表面基团与抗体进行偶联，可结合相应的抗原。目前以免疫磁珠捕获细菌，以多功能化氧化石墨烯为标记物，用于检测海洋致病菌的夹心式电化学发光免疫传感器未见报道。Electrochemiluminescence (ECL) is a chemiluminescence reaction induced by electrochemistry on the electrode surface, and is the product of the combination of chemiluminescence and electrochemistry. Electrochemiluminescence immunosensor is a technology integrating electrochemiluminescence and immunosensor, which has the advantages of high sensitivity and easy operation. Graphene oxide is a new type of carbon material with excellent performance. It has a high specific surface area and abundant functional groups. The functionalized graphene oxide prepared based on graphene oxide can effectively extend the Helmholtz surface of the electrode surface and significantly enhance the Electrochemiluminescence improves sensitivity and is an ideal material for constructing electrochemiluminescence sensor markers. Immunomagnetic beads are an immunological technology developed in recent years. It combines the unique advantages of solidified reagents with the high specificity of immunological reactions, and uses the core-shell synthesis method to synthesize superparamagnetic functionalized trioxide Iron spheres, which are coupled to antibodies using their surface groups, can bind to corresponding antigens. At present, there is no report on the sandwich-type electrochemiluminescence immunosensor for detecting marine pathogenic bacteria by capturing bacteria with immunomagnetic beads and using multifunctional graphene oxide as a marker.
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检测速度快、检测结果灵敏度和准确性高、特异性强的用于检测海洋致病菌的夹心式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 a preparation method and application of a sandwich-type electrochemiluminescence immunosensor for detecting marine pathogenic bacteria with fast detection speed, high sensitivity and accuracy of detection results, and strong specificity.
本发明解决上述技术问题所采用的技术方案为：一种用于检测海洋致病菌的夹心式电化学发光免疫传感器的制备方法，包括以下步骤：The technical solution adopted by the present invention to solve the above-mentioned technical problems is: a preparation method of a sandwich-type electrochemiluminescence immunosensor for detecting marine pathogenic bacteria, comprising the following steps:
(1)免疫磁珠的合成(1) Synthesis of immunomagnetic beads
在干净的烧瓶中加入5～10mg的氨基化磁珠，再加入2～5mL含有3wt％戊二醛的0.1mol/LpH7.4的磷酸盐缓冲溶液，37℃孵育3h后，外加磁铁磁性分离后用0.1mol/LpH7.4的磷酸盐缓冲溶液洗涤3～5次，去除上清液后，0.1mol/LpH7.4的磷酸盐缓冲溶液定容至2～5mL；加入50～150μL0.5mg/mL海洋致病菌一抗溶液，37℃孵育1h，外加磁铁磁性分离后用0.1mol/LpH7.4的磷酸盐缓冲溶液洗涤3～5次，去除上清液后，用0.1mol/LpH7.4的磷酸盐缓冲溶液定容至2～5mL；加入2wt％的牛血清白蛋白溶液50～150μL以封闭非特异性活性位点，37℃孵育1h，外加磁铁磁性分离后用0.1mol/LpH7.4的磷酸盐缓冲溶液洗涤3～5次，去除上清液后，用0.1mol/LpH7.4的磷酸盐缓冲溶液定容至2～5mL，即可得到表面覆盖有海洋致病菌抗体的免疫磁珠溶液，4℃下储存备用；Add 5-10 mg of aminated magnetic beads to a clean flask, then add 2-5 mL of 0.1 mol/L pH7.4 phosphate buffer solution containing 3 wt % glutaraldehyde, incubate at 37 ° C for 3 h, and then add a magnet for magnetic separation Wash with 0.1mol/L pH7.4 phosphate buffer solution for 3 to 5 times, remove the supernatant, and dilute to 2~5mL with 0.1mol/LpH7.4 phosphate buffer solution; add 50~150μL of 0.5mg/mL Marine pathogen primary antibody solution, incubate at 37°C for 1 hour, add a magnet for magnetic separation, wash with 0.1mol/L pH7.4 phosphate buffer solution for 3 to 5 times, remove the supernatant, and wash with 0.1mol/LpH7.4 phosphate buffer solution Dilute the phosphate buffer solution to 2-5mL; add 50-150μL of 2wt% bovine serum albumin solution to block the non-specific active sites, incubate at 37°C for 1h, add a magnet for magnetic separation, and use 0.1mol/L phosphoric acid at pH7.4 Wash with salt buffer solution for 3 to 5 times, remove the supernatant, and dilute to 2 to 5 mL with 0.1mol/L phosphate buffer solution at pH 7.4 to obtain an immunomagnetic bead solution covered with antibodies to marine pathogens , stored at 4°C for later use;
(2)多功能化氧化石墨烯的合成(2) Synthesis of multifunctional graphene oxide
在干净的试管中加入150～250μL的1mg/mL氧化石墨烯(GO)，超声1h，然后加入100～300μL偶联试剂，混合均匀，同时滴加0.1mol/L稀盐酸至溶液pH＝4～6，振荡孵育1h后，8000r离心15min，去除上清液后，0.1mol/LpH7.4的磷酸盐缓冲溶液定容至0.5～1mL；加入40～60μL10-5～0.001mol/L电化学发光体溶液和40～60μL0.01～0.1mg/mL海洋致病菌二抗溶液，混合均匀，同时滴加0.1mol/L氢氧化钠溶液至溶液pH＝8～10，常温振荡孵育4h，8000r离心15min，去除上清液后，用0.1mol/LpH7.4的磷酸盐缓冲溶液定容至0.5～1mL；再加入2wt％的牛血清白蛋白溶液80～120μL以封闭非特异性活性位点，常温振荡孵育1h，8000r离心15min，去除上清液后，用0.1mol/LpH7.4的磷酸盐缓冲溶液定容至0.5～1mL，即可得到海洋致病菌抗体和电化学发光体同时化学键合的多功能化氧化石墨烯溶液，4℃下储存备用；Add 150-250 μL of 1 mg/mL graphene oxide (GO) into a clean test tube, sonicate for 1 hour, then add 100-300 μL of coupling reagent, mix well, and at the same time add 0.1mol/L dilute hydrochloric acid until the solution pH=4～ 6. After shaking and incubating for 1 hour, centrifuge at 8000r for 15 minutes. After removing the supernatant, adjust the volume to 0.5-1 mL with 0.1 mol/L pH7.4 phosphate buffer solution; add 40-60 μL of 10-5 ～0.001 mol/L electrochemiluminescence solution and 40-60μL 0.01-0.1mg/mL marine pathogenic bacteria secondary antibody solution, mix well, and at the same time add dropwise 0.1mol/L sodium hydroxide solution to pH=8-10, incubate with shaking at room temperature for 4h, centrifuge at 8000r for 15min , after removing the supernatant, use 0.1mol/L pH7.4 phosphate buffer solution to dilute to 0.5-1mL; then add 80-120μL of 2wt% bovine serum albumin solution to block non-specific active sites, and incubate with shaking at room temperature 1h, centrifuge at 8000r for 15min, remove the supernatant, and dilute to 0.5-1mL with 0.1mol/L pH7.4 phosphate buffer solution to obtain a multifunctional chemically bonded marine pathogenic bacteria antibody and electrochemiluminescent body at the same time Graphene oxide solution, stored at 4°C for future use;
(3)电极修饰(3) Electrode modification
将直径为3～5mm的磁性玻碳电极依次用1.0μm、0.3μm、0.05μm的三氧化二铝浆液抛光处理，用乙醇、水依次超声2min，洗净，氮气吹干；取5～10μL步骤(1)得到的免疫磁珠溶液，滴涂于处理过的磁性玻碳电极中心，免疫磁珠即被均匀地牢固吸附在电极表面；用水洗净后即得固载有免疫磁珠的磁性玻碳电极；Polish the magnetic glassy carbon electrode with a diameter of 3-5mm with 1.0μm, 0.3μm, 0.05μm Al2O3 slurry in sequence, ultrasonically clean it with ethanol and water for 2min, and dry it with nitrogen gas; take 5-10μL step (1) The obtained immunomagnetic bead solution is drip-coated on the center of the processed magnetic glassy carbon electrode, and the immunomagnetic beads are evenly and firmly adsorbed on the electrode surface; carbon electrode;
(4)夹心式电化学发光免疫传感器的组装(4) Assembly of sandwich electrochemiluminescence immunosensor
将步骤(3)所得的固载有免疫磁珠的磁性玻碳电极浸泡在含有待测海洋致病菌的溶液中，37℃下孵育1h；清洗后，再滴涂5～10μL步骤(2)得到的多功能化氧化石墨烯溶液中，37℃下孵育1h；清洗后，即组装成用于检测海洋致病菌的夹心式电化学发光免疫传感器。Soak the magnetic glassy carbon electrode immobilized with immunomagnetic beads obtained in step (3) in the solution containing the marine pathogenic bacteria to be tested, and incubate at 37°C for 1 h; The obtained multifunctional graphene oxide solution was incubated at 37° C. for 1 h; after washing, it was assembled into a sandwich-type electrochemiluminescent immunosensor for detecting marine pathogenic bacteria.
所述的电化学发光体为含氨基官能团的三联吡啶钌衍生物，溶剂为0.1mol/LpH＝7.4的Tris-HCl缓冲液。The electrochemiluminescent substance is a ruthenium derivative of terpyridine containing an amino functional group, and the solvent is a Tris-HCl buffer solution with a pH of 7.4 at 0.1 mol/L.
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(EDC)与N-羟基琥珀酰亚胺(NHS)按摩尔比10：1混合后溶于水中得到，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(EDC)的摩尔浓度为10～100mmol/L，所述的N-羟基琥珀酰亚胺(NHS)摩尔浓度为1～10mmol/L。The coupling reagent is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) in a molar ratio of 10:1 Dissolved in water after mixing to obtain, the molar concentration of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) in the coupling reagent is 10-100mmol/L, The molar concentration of N-hydroxysuccinimide (NHS) is 1-10 mmol/L.
所述的磷酸盐溶液为Na2HPO4·12H2O-NaH2PO4·2H2O体系缓冲溶液。The phosphate solution is a Na2 HPO4 ·12H2 O-NaH2 PO4 ·2H2 O system buffer solution.
所述的海洋致病菌为副溶血弧菌(VP)、创伤弧菌(VV)、哈维氏弧菌(VH)、阴沟肠杆菌(EC)或黄海希瓦氏菌(SM)。The marine pathogenic bacteria are Vibrio parahaemolyticus (VP), Vibrio vulnificus (VV), Vibrio harveyi (VH), Enterobacter cloacae (EC) or Shewanella yellowsea (SM).
利用上述夹心式电化学发光免疫传感器检测海洋致病菌的方法，具体步骤如下：将上述夹心式电化学发光免疫传感器作为工作电极；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；将上述三电极体系放入含共反应试剂的缓冲溶液，启动电化学反应，测量电化学发光强度，获得待测海洋致病菌溶液对应的电化学发光强度值；根据电化学发光强度值与海洋致病菌溶液浓度对数之间的定量关系可计算得到待测样品溶液中海洋致病菌的准确浓度。The method for detecting marine pathogenic bacteria by using the above-mentioned sandwich type electrochemiluminescence immunosensor, the specific steps are as follows: using the above-mentioned sandwich type electrochemiluminescence immunosensor as a working electrode; using a platinum electrode as a counter electrode, an Ag/AgCl electrode or a saturated calomel electrode As a reference electrode, a three-electrode system is formed; put the above-mentioned three-electrode system into a buffer solution containing co-reaction reagents, start an electrochemical reaction, measure the intensity of electrochemiluminescence, and obtain the intensity of electrochemiluminescence corresponding to the solution of marine pathogenic bacteria to be tested value; according to the quantitative relationship between the electrochemiluminescent intensity value and the logarithm of the concentration of the marine pathogenic bacteria solution, the exact concentration of the marine pathogenic bacteria in the sample solution to be tested can be calculated.
所述的含共反应试剂的缓冲溶液为含20～40mmol/L的三丙胺的0.1mol/LpH7～8的Na2HPO4·12H2O-NaH2PO4·2H2O体系缓冲溶液。The buffer solution containing co-reaction reagent is a 0.1 mol/L pH7-8 Na2 HPO4 ·12H2 O-NaH2 PO4 ·2H2 O system buffer solution containing 20-40 mmol/L tripropylamine.
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：1V；脉冲电压：1.5V。The conditions of the electrochemical reaction are as follows: potential step chronoamperometry, pulse width: 0.25 seconds; pulse interval: 30 seconds; initial voltage: 1V; pulse voltage: 1.5V.
原理：本发明将海洋致病菌第一抗体标记于氨基化磁珠表面制备了免疫磁珠，用于捕获海洋致病菌；将电化学发光体与海洋致病菌第二抗体一起化学键合到氧化石墨烯表面制备了功能化氧化石墨烯；然后采用夹心式的方法构建了检测海洋致病菌的电化学免疫传感器。功能化氧化石墨烯具有巨大表面积及良好的导电性，可有效延展电极的赫姆霍兹面，使得所有负载的电化学发光体都处于电极的赫姆霍兹面之内，不影响电化学发光体与电极之间的能量、电子传递，也不会影响第二抗体活性，最终实现大幅度提高检测灵敏度的目的。本发明制备的检测海洋致病菌的夹心式电化学发光免疫传感器采用“signal-on”(信号开，就是指检测对象存在时，检测信号增强)模式，即抗体与海洋致病菌反应后，电化学发光强度随着海洋致病菌的浓度增大而增强，相比于电化学发光强度随着海洋致病菌的浓度增大而减弱的“signal-off”(信号关，就是指检测对象存在时，检测信号减弱)模式，具有稳定性好、背景电流小、检出限低、灵敏度高等优点。Principle: In the present invention, the first antibody of marine pathogenic bacteria is marked on the surface of aminated magnetic beads to prepare immunomagnetic beads for capturing marine pathogenic bacteria; the electrochemiluminescent body and the second antibody of marine pathogenic bacteria are chemically bonded to Functionalized graphene oxide was prepared on the surface of graphene oxide; then an electrochemical immunosensor for detecting marine pathogenic bacteria was constructed by a sandwich method. Functionalized graphene oxide has a huge surface area and good conductivity, which can effectively extend the Helmholtz surface of the electrode, so that all the loaded ECLs are within the Helmholtz surface of the electrode, without affecting the electrochemiluminescence The energy and electron transfer between the body and the electrode will not affect the activity of the second antibody, and finally achieve the purpose of greatly improving the detection sensitivity. The sandwich-type electrochemiluminescent immunosensor for detecting marine pathogenic bacteria prepared by the present invention adopts the "signal-on" (signal-on, meaning that when the detection object exists, the detection signal is enhanced) mode, that is, after the antibody reacts with the marine pathogenic bacteria, The intensity of electrochemiluminescence increases as the concentration of marine pathogens increases, compared to the "signal-off" (signal off, which refers to the detection object When it exists, the detection signal weakens) mode, which has the advantages of good stability, small background current, low detection limit, and high sensitivity.
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。本发明的检测限达到1CFU/mL，而目前传统的检测方法检测限为104～10CFU/mL。原因在于：采用“signal-on”模式的电化学发光免疫方法、使用多功能化氧化石墨烯。(1) High sensitivity. The detection limit of the present invention reaches 1 CFU/mL, while the detection limit of the current traditional detection method is 104 -10 CFU/mL. The reason is: the electrochemiluminescence immunoassay using the "signal-on" mode and the use of multifunctional graphene oxide.
(2)高选择性。本发明基于海洋致病菌抗体与海洋致病菌之间的特异性识别与结合构建的夹心式电化学发光免疫传感器，干扰菌种不被海洋致病菌抗体所捕获，故对本检测体系无干扰。(2) High selectivity. The present invention is based on the specific recognition and combination between the marine pathogenic bacteria antibody and the marine pathogenic bacteria. The sandwich type electrochemiluminescence immunosensor is constructed. The interfering bacteria are not captured by the marine pathogenic bacteria antibodies, so there is no interference to the detection system. .
(3)高准确度。本发明制备的夹心式电化学发光传感器采用“signal-on”模式，与“signal-off”模式相比较具有稳定性好、背景电流小、检出限低、灵敏度高的优点，回收率均>92％。(3) High accuracy. The sandwich-type electrochemiluminescence sensor prepared by the present invention adopts the "signal-on" mode, and compared with the "signal-off" mode, it has the advantages of good stability, small background current, low detection limit and high sensitivity, and the recovery rate is > 92%.
综上所述，本发明制备了以免疫磁珠捕获细菌，以多功能化氧化石墨烯为标记物，用于检测海洋致病菌的夹心式电化学发光免疫传感器。本传感器采用了“signal-on”夹心式电化学发光免疫方法，具有高灵敏度、高特异性、快速、稳定、重现性好等优点，可以实现对超低浓度海洋致病菌的检测，具有良好的应用前景。In summary, the present invention has prepared a sandwich-type electrochemiluminescent immunosensor for detecting marine pathogenic bacteria by capturing bacteria with immunomagnetic beads and using multifunctional graphene oxide as a marker. The sensor adopts the "signal-on" sandwich electrochemiluminescence immunoassay method, which has the advantages of high sensitivity, high specificity, rapidity, stability, and good reproducibility, and can detect ultra-low concentrations of marine pathogenic bacteria. Good application prospects.
附图说明Description of drawings
图1为夹心式电化学发光免疫传感器检测海洋致病菌的原理图；Figure 1 is a schematic diagram of the detection of marine pathogenic bacteria by a sandwich electrochemiluminescent immunosensor;
图2为不同浓度副溶血弧菌(VP)的ECL强度(y)—浓度(x)对数关系图；Fig. 2 is the ECL intensity (y)-concentration (x) logarithmic relationship diagram of different concentrations of Vibrio parahaemolyticus (VP);
图3为不同浓度创伤弧菌(VV)的ECL强度(y)—浓度(x)对数关系图；Fig. 3 is the ECL intensity (y)-concentration (x) logarithmic relationship diagram of different concentrations of Vibrio vulnificus (VV);
图4为不同浓度哈维氏弧菌(VH)的ECL强度(y)—浓度(x)对数关系图；Fig. 4 is the ECL intensity (y)-concentration (x) logarithmic relationship diagram of different concentrations Vibrio harveyi (VH);
图5为不同浓度阴沟肠杆菌(EC)的ECL强度(y)—浓度(x)对数关系图；Fig. 5 is the ECL intensity (y)-concentration (x) logarithmic relationship diagram of different concentrations of Enterobacter cloacae (EC);
图6为不同浓度黄海希瓦氏菌(SM)的ECL强度(y)—浓度(x)对数关系图。Fig. 6 is a logarithmic relationship diagram of ECL intensity (y)-concentration (x) of different concentrations of Shewanella yellowsea (SM).
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
一种用于检测海洋致病菌的夹心式电化学发光免疫传感器的制备方法，包括以下步骤：A method for preparing a sandwich type electrochemiluminescence immunosensor for detecting marine pathogenic bacteria, comprising the following steps:
(1)免疫磁珠的合成(1) Synthesis of immunomagnetic beads
在干净的烧瓶中加入5～10mg的氨基化磁珠，再加入2～5mL含有3wt％戊二醛的0.1mol/LpH7.4的磷酸盐缓冲溶液，37℃孵育3h后，外加磁铁磁性分离后用0.1mol/LpH7.4的磷酸盐缓冲溶液洗涤3～5次，去除上清液后，0.1mol/LpH7.4的磷酸盐缓冲溶液定容至2～5mL；加入50～150μL0.5mg/mL海洋致病菌一抗溶液，37℃孵育1h，外加磁铁磁性分离后用0.1mol/LpH7.4的磷酸盐缓冲溶液洗涤3～5次，去除上清液后，用0.1mol/LpH7.4的磷酸盐缓冲溶液定容至2～5mL；加入2wt％的牛血清白蛋白溶液50～150μL以封闭非特异性活性位点，37℃孵育1h，外加磁铁磁性分离后用0.1mol/LpH7.4的磷酸盐缓冲溶液洗涤3～5次，去除上清液后，用0.1mol/LpH7.4的磷酸盐缓冲溶液定容至2～5mL，即可得到表面覆盖有海洋致病菌抗体的免疫磁珠溶液，4℃下储存备用；Add 5-10 mg of aminated magnetic beads to a clean flask, then add 2-5 mL of 0.1 mol/L pH7.4 phosphate buffer solution containing 3 wt % glutaraldehyde, incubate at 37 ° C for 3 h, and then add a magnet for magnetic separation Wash with 0.1mol/L pH7.4 phosphate buffer solution for 3 to 5 times, remove the supernatant, and dilute to 2~5mL with 0.1mol/LpH7.4 phosphate buffer solution; add 50~150μL of 0.5mg/mL Marine pathogen primary antibody solution, incubate at 37°C for 1 hour, add a magnet for magnetic separation, wash with 0.1mol/L pH7.4 phosphate buffer solution for 3 to 5 times, remove the supernatant, and wash with 0.1mol/LpH7.4 phosphate buffer solution Dilute the phosphate buffer solution to 2-5mL; add 50-150μL of 2wt% bovine serum albumin solution to block the non-specific active sites, incubate at 37°C for 1h, add a magnet for magnetic separation, and use 0.1mol/L phosphoric acid at pH7.4 Wash with salt buffer solution for 3 to 5 times, remove the supernatant, and dilute to 2 to 5 mL with 0.1mol/L phosphate buffer solution at pH 7.4 to obtain an immunomagnetic bead solution covered with antibodies to marine pathogens , stored at 4°C for later use;
(2)多功能化氧化石墨烯的合成(2) Synthesis of multifunctional graphene oxide
在干净的试管中加入150～250μL的1mg/mL氧化石墨烯(GO)，超声1h，然后加入100～300μL偶联试剂，混合均匀，同时滴加0.1mol/L稀盐酸至溶液pH＝4～6，振荡孵育1h后，8000r离心15min，去除上清液后，0.1mol/LpH7.4的磷酸盐缓冲溶液定容至0.5～1mL；加入40～60μL10-5～0.001mol/L电化学发光体溶液和40～60μL0.01～0.1mg/mL海洋致病菌二抗溶液，混合均匀，同时滴加0.1mol/L氢氧化钠溶液至溶液pH＝8～10，常温振荡孵育4h，8000r离心15min，去除上清液后，用0.1mol/LpH7.4的磷酸盐缓冲溶液定容至0.5～1mL；再加入2wt％的牛血清白蛋白溶液80～120μL以封闭非特异性活性位点，常温振荡孵育1h，8000r离心15min，去除上清液后，用0.1mol/LpH7.4的磷酸盐缓冲溶液定容至0.5～1mL，即可得到海洋致病菌抗体和电化学发光体同时化学键合的多功能化氧化石墨烯溶液，4℃下储存备用；Add 150-250 μL of 1 mg/mL graphene oxide (GO) into a clean test tube, sonicate for 1 hour, then add 100-300 μL of coupling reagent, mix well, and at the same time add 0.1mol/L dilute hydrochloric acid until the solution pH=4～ 6. After shaking and incubating for 1 hour, centrifuge at 8000r for 15 minutes. After removing the supernatant, adjust the volume to 0.5-1 mL with 0.1 mol/L pH7.4 phosphate buffer solution; add 40-60 μL of 10-5 ～0.001 mol/L electrochemiluminescence solution and 40-60μL 0.01-0.1mg/mL marine pathogenic bacteria secondary antibody solution, mix well, and at the same time add dropwise 0.1mol/L sodium hydroxide solution to pH=8-10, incubate with shaking at room temperature for 4h, centrifuge at 8000r for 15min , after removing the supernatant, use 0.1mol/L pH7.4 phosphate buffer solution to dilute to 0.5-1mL; then add 80-120μL of 2wt% bovine serum albumin solution to block non-specific active sites, and incubate with shaking at room temperature 1h, centrifuge at 8000r for 15min, remove the supernatant, and dilute to 0.5-1mL with 0.1mol/L pH7.4 phosphate buffer solution to obtain a multifunctional chemically bonded marine pathogenic bacteria antibody and electrochemiluminescent body at the same time Graphene oxide solution, stored at 4°C for future use;
(3)电极修饰(3) Electrode modification
将直径为3～5mm的磁性玻碳电极依次用1.0μm、0.3μm、0.05μm的三氧化二铝浆液抛光处理，用乙醇、水依次超声2min，洗净，氮气吹干；取5～10μL步骤(1)得到的免疫磁珠溶液，滴涂于处理过的磁性玻碳电极中心，免疫磁珠即被均匀地牢固吸附在电极表面；用水洗净后即得固载有免疫磁珠的磁性玻碳电极；Polish the magnetic glassy carbon electrode with a diameter of 3-5mm with 1.0μm, 0.3μm, 0.05μm Al2O3 slurry in sequence, ultrasonically clean it with ethanol and water for 2min, and dry it with nitrogen gas; take 5-10μL step (1) The obtained immunomagnetic bead solution is drip-coated on the center of the processed magnetic glassy carbon electrode, and the immunomagnetic beads are evenly and firmly adsorbed on the electrode surface; carbon electrode;
(4)夹心式电化学发光免疫传感器的组装(4) Assembly of sandwich electrochemiluminescence immunosensor
将步骤(3)所得的固载有免疫磁珠的磁性玻碳电极浸泡在含有待测海洋致病菌的溶液中，37℃下孵育1h；清洗后，再滴涂5～10μL步骤(2)得到的多功能化氧化石墨烯溶液中，37℃下孵育1h；清洗后，即组装成用于检测海洋致病菌的夹心式电化学发光免疫传感器。Soak the magnetic glassy carbon electrode immobilized with immunomagnetic beads obtained in step (3) in the solution containing the marine pathogenic bacteria to be tested, and incubate at 37°C for 1 h; The obtained multifunctional graphene oxide solution was incubated at 37° C. for 1 h; after washing, it was assembled into a sandwich-type electrochemiluminescent immunosensor for detecting marine pathogenic bacteria.
其中电化学发光体为含氨基官能团的三联吡啶钌衍生物，溶剂为0.1mol/LpH＝7.4的Tris-HCl缓冲液；偶联试剂为1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC)与N-羟基琥珀酰亚胺(NHS)按摩尔比10：1混合后溶于水中得到，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(EDC)的摩尔浓度为10～100mmol/L，所述的N-羟基琥珀酰亚胺(NHS)摩尔浓度为1～10mmol/L；磷酸盐溶液为Na2HPO4·12H2O-NaH2PO4·2H2O体系缓冲溶液。Wherein the electrochemiluminescent substance is a terpyridine ruthenium derivative containing an amino functional group, the solvent is a Tris-HCl buffer solution of 0.1mol/LpH=7.4; the coupling reagent is 1-(3-dimethylaminopropyl)-3-ethyl Carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) are mixed in a molar ratio of 10:1 and then dissolved in water to obtain, the coupling reagent in which 1-(3-dimethylamino The molar concentration of propyl)-3-ethylcarbodiimide hydrochloride (EDC) is 10～100mmol/L, and the molar concentration of described N-hydroxysuccinimide (NHS) is 1～10mmol/L; The phosphate solution is a Na2 HPO4 ·12H2 O-NaH2 PO4 ·2H2 O system buffer solution.
上述海洋致病菌为副溶血弧菌(VP)、创伤弧菌(VV)、哈维氏弧菌(VH)、阴沟肠杆菌(EC)或黄海希瓦氏菌(SM)。The above-mentioned marine pathogenic bacteria are Vibrio parahaemolyticus (VP), Vibrio vulnificus (VV), Vibrio harveyi (VH), Enterobacter cloacae (EC) or Shewanella yellowsea (SM).
具体实施例二Specific embodiment two
用于检测海洋致病菌的夹心式电化学发光免疫传感器检测海洋致病菌的方法，具体步骤如下：The sandwich-type electrochemiluminescent immunosensor for detecting marine pathogenic bacteria is used to detect marine pathogenic bacteria. The specific steps are as follows:
将上述具体实施例一步骤(3)制备得到的表面固载有免疫磁珠的磁性玻碳电极分别浸泡在含有不同浓度的海洋致病菌溶液中，于37℃下温育1h，二次水清洗后，浸泡在上述具体实施例一步骤(2)制备得到的多功能化氧化石墨烯溶液，37℃下孵育1h后取出，二次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；将上述三电极体系放入含共反应试剂的缓冲溶液，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海洋致病菌溶液对应的电化学发光强度值，建立电化学发光强度值与海洋致病菌溶液浓度对数之间的定量关系；根据定量关系可计算得到待测样品溶液中海洋致病菌的准确浓度。Soak the magnetic glassy carbon electrodes with immunomagnetic beads immobilized on the surface prepared in step (3) of the above-mentioned specific example 1 respectively in solutions containing different concentrations of marine pathogenic bacteria, and incubate at 37° C. for 1 hour, and dilute with secondary water After cleaning, soak in the multifunctional graphene oxide solution prepared in the first step (2) of the above specific example, take it out after incubating at 37° C. for 1 h, and use it as a working electrode after washing with secondary water; use a platinum electrode as a counter electrode, Ag /AgCl electrode or saturated calomel electrode as a reference electrode to form a three-electrode system; put the above three-electrode system into the buffer solution containing co-reaction reagents, start the electrochemical reaction, measure the intensity of electrochemiluminescence, and obtain a series of different concentrations of The corresponding electrochemiluminescence intensity value of the marine pathogenic bacteria solution, establishes the quantitative relationship between the electrochemiluminescence intensity value and the logarithm of the concentration of the marine pathogenic bacteria solution; according to the quantitative relationship, the concentration of the marine pathogenic bacteria in the sample solution to be tested can be calculated exact concentration.
上述缓冲溶液为含20～40mmol/L的三丙胺的0.1mol/LpH7～8的Na2HPO4·12H2O-NaH2PO4·2H2O体系缓冲溶液。The above buffer solution is a 0.1 mol/L pH 7-8 Na2HPO4 ·12H2 O-NaH2 PO4 ·2H2 O system buffer solution containing 20-40 mmol/L tripropylamine.
上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：1V；脉冲电压：1.5V。The conditions of the above electrochemical reaction are as follows: potential step chronoamperometry, pulse width: 0.25 seconds; pulse interval: 30 seconds; initial voltage: 1V; pulse voltage: 1.5V.
上述海洋致病菌为副溶血弧菌(VP)、创伤弧菌(VV)、哈维氏弧菌(VH)、阴沟肠杆菌(EC)或黄海希瓦氏菌(SM)。The above-mentioned marine pathogenic bacteria are Vibrio parahaemolyticus (VP), Vibrio vulnificus (VV), Vibrio harveyi (VH), Enterobacter cloacae (EC) or Shewanella yellowsea (SM).
具体实施例三Specific embodiment three
一种用于检测副溶血弧菌(VP)的夹心式电化学发光免疫传感器的制备方法，包括以下步骤：A preparation method for detecting a sandwich type electrochemiluminescence immunosensor for Vibrio parahaemolyticus (VP), comprising the following steps:
(1)免疫磁珠的合成(1) Synthesis of immunomagnetic beads
在干净的烧瓶中加入8mg的氨基化磁珠，再加入3.5mL含有3wt％戊二醛的0.1mol/LpH7.4的磷酸盐缓冲溶液，37℃孵育3h后，外加磁铁磁性分离后用0.1mol/LpH7.4的磷酸盐缓冲溶液洗涤4次，去除上清液后，0.1mol/LpH7.4的磷酸盐缓冲溶液定容至2～5mL；加入100μL0.5mg/mL副溶血弧菌一抗溶液，37℃孵育1h，外加磁铁磁性分离后用0.1mol/LpH7.4的磷酸盐缓冲溶液洗涤4次，去除上清液后，用0.1mol/LpH7.4的磷酸盐缓冲溶液定容至3mL；加入2wt％的牛血清白蛋白溶液100μL以封闭非特异性活性位点，37℃孵育1h，外加磁铁磁性分离后用0.1mol/LpH7.4的磷酸盐缓冲溶液洗涤4次，去除上清液后，用0.1mol/LpH7.4的磷酸盐缓冲溶液定容至3mL，即可得到表面覆盖有副溶血弧菌一抗的免疫磁珠溶液，4℃下储存备用；Add 8mg of aminated magnetic beads to a clean flask, then add 3.5mL of 0.1mol/L pH7.4 phosphate buffer solution containing 3wt% glutaraldehyde, incubate at 37°C for 3h, add a magnet for magnetic separation and use 0.1mol /LpH7.4 phosphate buffer solution washed 4 times, remove the supernatant, 0.1mol/LpH7.4 phosphate buffer solution to 2 ~ 5mL; add 100μL 0.5mg/mL Vibrio parahaemolyticus primary antibody solution , incubate at 37°C for 1 hour, add magnetic separation, wash with 0.1mol/L pH7.4 phosphate buffer solution for 4 times, remove the supernatant, and dilute to 3mL with 0.1mol/L pH7.4 phosphate buffer solution; Add 100 μL of 2 wt% bovine serum albumin solution to block non-specific active sites, incubate at 37°C for 1 h, apply magnetic separation with a magnet, wash with 0.1 mol/L pH7.4 phosphate buffer solution 4 times, remove the supernatant, Dilute to 3mL with 0.1mol/L pH7.4 phosphate buffer solution to obtain the immunomagnetic bead solution covered with the primary antibody of Vibrio parahaemolyticus, store at 4°C for later use;
(2)多功能化氧化石墨烯的合成(2) Synthesis of multifunctional graphene oxide
在干净的试管中加入200μL的1mg/mL氧化石墨烯(GO)，超声1h，然后加入200μL偶联试剂，混合均匀，同时滴加0.1mol/L稀盐酸至溶液pH＝5，振荡孵育1h后，8000r离心15min，去除上清液后，0.1mol/LpH7.4的磷酸盐缓冲溶液定容至0.8mL；加入50μL10-4mol/L电化学发光体溶液和50μL0.05mg/mL副溶血弧菌二抗溶液，混合均匀，同时滴加0.1mol/L氢氧化钠溶液至溶液pH＝9，常温振荡孵育4h，8000r离心15min，去除上清液后，用0.1mol/LpH7.4的磷酸盐缓冲溶液定容至0.8mL；再加入2wt％的牛血清白蛋白溶液100μL以封闭非特异性活性位点，常温振荡孵育1h，8000r离心15min，去除上清液后，用0.1mol/LpH7.4的磷酸盐缓冲溶液定容至0.8mL，即可得到副溶血弧菌二抗和电化学发光体同时化学键合的多功能化氧化石墨烯溶液，4℃下储存备用；其中电化学发光体为含氨基官能团的三联吡啶钌衍生物，溶剂为0.1mol/LpH＝7.4的Tris-HCl缓冲液；偶联试剂为1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC)与N-羟基琥珀酰亚胺(NHS)按摩尔比10：1混合后溶于水中得到，偶联试剂中1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC)的摩尔浓度为50mmol/L，所述的N-羟基琥珀酰亚胺(NHS)摩尔浓度为5mmol/L；磷酸盐溶液为Na2HPO4·12H2O-NaH2PO4·2H2O体系缓冲溶液；Add 200 μL of 1 mg/mL graphene oxide (GO) to a clean test tube, sonicate for 1 h, then add 200 μL of coupling reagent, mix well, and at the same time add 0.1 mol/L dilute hydrochloric acid to the solution pH = 5, shake and incubate for 1 h , centrifuge at 8000r for 15min, remove the supernatant, dilute to 0.8mL with 0.1mol/L pH7.4 phosphate buffer solution; add 50μL 10-4 mol/L electrochemiluminescent solution and 50μL 0.05mg/mL Vibrio parahaemolyticus Secondary antibody solution, mix evenly, add 0.1mol/L sodium hydroxide solution dropwise until the solution pH=9, incubate with shaking at room temperature for 4h, centrifuge at 8000r for 15min, remove the supernatant, buffer with 0.1mol/LpH7.4 phosphate Dilute the solution to 0.8mL; then add 100μL of 2wt% bovine serum albumin solution to block non-specific active sites, incubate with shaking at room temperature for 1h, centrifuge at 8000r for 15min, remove the supernatant, and use 0.1mol/L pH7.4 phosphoric acid Set the volume of the salt buffer solution to 0.8mL to obtain a multifunctional graphene oxide solution in which the secondary antibody of Vibrio parahaemolyticus and the electrochemiluminescent substance are chemically bonded at the same time, and store it at 4°C for later use; the electrochemiluminescent substance is an amino functional group The terpyridine ruthenium derivatives, the solvent is the Tris-HCl buffer solution of 0.1mol/LpH=7.4; the coupling reagent is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride ( EDC) and N-hydroxysuccinimide (NHS) are mixed at a molar ratio of 10:1 and dissolved in water to obtain 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide in the coupling reagent The molar concentration of hydrochloride (EDC) is 50mmol/L, and the molar concentration of N-hydroxysuccinimide (NHS) is 5mmol/L; the phosphate solution is Na2 HPO4 ·12H2 O-NaH2 PO4. 2H2 O system buffer solution;
(3)电极修饰(3) Electrode modification
将直径为4mm的磁性玻碳电极依次用1.0μm、0.3μm、0.05μm的三氧化二铝浆液抛光处理，用乙醇、水依次超声2min，洗净，氮气吹干；取8μL步骤(1)得到的免疫磁珠溶液，滴涂于处理过的磁性玻碳电极中心，免疫磁珠即被均匀地牢固吸附在电极表面；用水洗净后即得固载有免疫磁珠的磁性玻碳电极；Polish a magnetic glassy carbon electrode with a diameter of 4 mm with 1.0 μm, 0.3 μm, and 0.05 μm aluminum oxide slurries in sequence, then ultrasonically clean it with ethanol and water for 2 minutes, and dry it with nitrogen; take 8 μL of step (1) to obtain The immune magnetic bead solution is dripped on the center of the treated magnetic glassy carbon electrode, and the immune magnetic beads are evenly and firmly adsorbed on the surface of the electrode; after washing with water, the magnetic glassy carbon electrode loaded with immune magnetic beads is obtained;
(4)夹心式电化学发光免疫传感器的组装(4) Assembly of sandwich electrochemiluminescence immunosensor
将步骤(3)所得的固载有免疫磁珠的磁性玻碳电极浸泡在含有待测副溶血弧菌的溶液中，37℃下孵育1h；清洗后，再滴涂8μL步骤(2)得到的多功能化氧化石墨烯溶液中，37℃下孵育1h；清洗后，即组装成用于检测副溶血弧菌的夹心式电化学发光免疫传感器。Soak the magnetic glassy carbon electrode immobilized with immunomagnetic beads obtained in step (3) in the solution containing Vibrio parahaemolyticus to be tested, and incubate at 37°C for 1 h; The multifunctional graphene oxide solution was incubated at 37°C for 1 hour; after washing, it was assembled into a sandwich-type electrochemiluminescence immunosensor for detecting Vibrio parahaemolyticus.
将上述用于检测副溶血弧菌的夹心式电化学发光免疫传感器作为工作电极；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，测定电化学发光强度；获得一系列不同浓度的副溶血弧菌溶液对应的电化学发光强度值，建立电化学发光强度值与副溶血弧菌溶液浓度之间的定量关系；不同浓度副溶血弧菌VP的ECL强度(y)—浓度(x)对数线性关系如图2所示。线性方程为：y＝960.7*logx+17.3，相关系数R2＝0.9823，线性范围为4–(1×108)CFU/mL，检测限为1CFU/mL。线性良好，可用于检测样品中副溶血弧菌的未知浓度。The sandwich-type electrochemiluminescence immunosensor for detecting Vibrio parahaemolyticus is used as a working electrode; a platinum electrode is used as a counter electrode, and an Ag/AgCl electrode or a saturated calomel electrode is used as a reference electrode to form a three-electrode system; the three-electrode Put the system into the buffer solution, start the electrochemical reaction, and measure the intensity of electrochemiluminescence; obtain the corresponding electrochemiluminescence intensity values of a series of Vibrio parahaemolyticus solutions with different concentrations, and establish the relationship between the electrochemiluminescence intensity value and the concentration of Vibrio parahaemolyticus solution. The quantitative relationship between the ECL intensity (y)-concentration (x) logarithmic linear relationship of different concentrations of Vibrio parahaemolyticus VP is shown in Figure 2. The linear equation is: y=960.7*logx+17.3, the correlation coefficient R2 =0.9823, the linear range is 4–(1×108 ) CFU/mL, and the detection limit is 1 CFU/mL. Good linearity and can be used to detect unknown concentrations of Vibrio parahaemolyticus in samples.
具体实施例四Specific embodiment four
一种用于检测创伤弧菌(VV)的夹心式电化学发光免疫传感器的制备方法，包括以下步骤：A preparation method for detecting a sandwich type electrochemiluminescent immunosensor for Vibrio vulnificus (VV), comprising the following steps:
(1)免疫磁珠的合成(1) Synthesis of immunomagnetic beads
在干净的烧瓶中加入5mg的氨基化磁珠，再加入2mL含有3wt％戊二醛的0.1mol/LpH7.4的磷酸盐缓冲溶液，37℃孵育3h后，外加磁铁磁性分离后用0.1mol/LpH7.4的磷酸盐缓冲溶液洗涤3次，去除上清液后，0.1mol/LpH7.4的磷酸盐缓冲溶液定容至2mL；加入50μL0.5mg/mL创伤弧菌一抗溶液，37℃孵育1h，外加磁铁磁性分离后用0.1mol/LpH7.4的磷酸盐缓冲溶液洗涤3次，去除上清液后，用0.1mol/LpH7.4的磷酸盐缓冲溶液定容至2mL；加入2wt％的牛血清白蛋白溶液50μL以封闭非特异性活性位点，37℃孵育1h，外加磁铁磁性分离后用0.1mol/LpH7.4的磷酸盐缓冲溶液洗涤3次，去除上清液后，用0.1mol/LpH7.4的磷酸盐缓冲溶液定容至2mL，即可得到表面覆盖有创伤弧菌一抗的免疫磁珠溶液，4℃下储存备用；Add 5mg of aminated magnetic beads to a clean flask, then add 2mL of 0.1mol/L pH7.4 phosphate buffer solution containing 3wt% glutaraldehyde, incubate at 37°C for 3h, add a magnet for magnetic separation and use 0.1mol/L Wash 3 times with LpH7.4 phosphate buffer solution, remove the supernatant, dilute to 2mL with 0.1mol/LpH7.4 phosphate buffer solution; add 50μL 0.5mg/mL Vibrio vulnificus primary antibody solution, and incubate at 37℃ 1h, after magnetic separation with an external magnet, wash 3 times with 0.1mol/L pH7.4 phosphate buffer solution, remove the supernatant, and dilute to 2mL with 0.1mol/LpH7.4 phosphate buffer solution; add 2wt% 50 μL of bovine serum albumin solution to block non-specific active sites, incubate at 37 °C for 1 h, add magnetic separation, wash with 0.1 mol/L pH7.4 phosphate buffer solution for 3 times, remove the supernatant, wash with 0.1 mol/L Dilute the phosphate buffer solution of LpH7.4 to 2mL to obtain the immunomagnetic bead solution covered with the primary antibody of Vibrio vulnificus, and store it at 4°C for later use;
(2)多功能化氧化石墨烯的合成(2) Synthesis of multifunctional graphene oxide
在干净的试管中加入150μL的1mg/mL氧化石墨烯(GO)，超声1h，然后加入100μL偶联试剂，混合均匀，同时滴加0.1mol/L稀盐酸至溶液pH＝4，振荡孵育1h后，8000r离心15min，去除上清液后，0.1mol/LpH7.4的磷酸盐缓冲溶液定容至0.5mL；加入40μL0.001mol/L电化学发光体溶液和40μL0.1mg/mL海洋致病菌二抗溶液，混合均匀，同时滴加0.1mol/L氢氧化钠溶液至溶液pH＝8，常温振荡孵育4h，8000r离心15min，去除上清液后，用0.1mol/LpH7.4的磷酸盐缓冲溶液定容至0.5mL；再加入2wt％的牛血清白蛋白溶液80μL以封闭非特异性活性位点，常温振荡孵育1h，8000r离心15min，去除上清液后，用0.1mol/LpH7.4的磷酸盐缓冲溶液定容至0.5mL，即可得到创伤弧菌二抗和电化学发光体同时化学键合的多功能化氧化石墨烯溶液，4℃下储存备用；其中电化学发光体为含氨基官能团的三联吡啶钌衍生物，溶剂为0.1mol/LpH＝7.4的Tris-HCl缓冲液；偶联试剂为1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC)与N-羟基琥珀酰亚胺(NHS)按摩尔比10：1混合后溶于水中得到，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(EDC)的摩尔浓度为10mmol/L，所述的N-羟基琥珀酰亚胺(NHS)摩尔浓度为10mmol/L；磷酸盐溶液为Na2HPO4·12H2O-NaH2PO4·2H2O体系缓冲溶液；Add 150 μL of 1 mg/mL graphene oxide (GO) to a clean test tube, sonicate for 1 h, then add 100 μL of coupling reagent, mix well, and at the same time add 0.1 mol/L dilute hydrochloric acid to the solution pH = 4, shake and incubate for 1 h , centrifuge at 8000r for 15min, remove the supernatant, dilute to 0.5mL with 0.1mol/L pH7.4 phosphate buffer solution; Antibiotic solution, mix well, add dropwise 0.1mol/L sodium hydroxide solution to the solution pH = 8, incubate with shaking at room temperature for 4 hours, centrifuge at 8000r for 15 minutes, remove the supernatant, and use 0.1mol/L pH7.4 phosphate buffer solution Dilute to 0.5mL; add 80μL of 2wt% bovine serum albumin solution to block the non-specific active sites, incubate with shaking at room temperature for 1h, centrifuge at 8000r for 15min, remove the supernatant, and use 0.1mol/L pH7.4 phosphate The buffer solution was adjusted to 0.5mL to obtain a multifunctional graphene oxide solution in which the Vibrio vulnificus secondary antibody and the electrochemiluminescent substance were simultaneously chemically bonded, and stored at 4°C for later use; Pyridine ruthenium derivatives, the solvent is 0.1mol/L Tris-HCl buffer solution with pH=7.4; the coupling reagent is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) It is obtained by mixing with N-hydroxysuccinimide (NHS) at a molar ratio of 10:1 and then dissolving in water. In the coupling reagent, 1-(3-dimethylaminopropyl)-3-ethylcarbodiethylene The molar concentration of amine hydrochloride (EDC) is 10mmol/L, and the molar concentration of N-hydroxysuccinimide (NHS) is 10mmol/L; the phosphate solution is Na2HPO4 ·12H2 O-NaH2 PO4 2H2 O system buffer solution;
(3)电极修饰(3) Electrode modification
将直径为3mm的磁性玻碳电极依次用1.0μm、0.3μm、0.05μm的三氧化二铝浆液抛光处理，用乙醇、水依次超声2min，洗净，氮气吹干；取5μL步骤(1)得到的免疫磁珠溶液，滴涂于处理过的磁性玻碳电极中心，免疫磁珠即被均匀地牢固吸附在电极表面；用水洗净后即得固载有免疫磁珠的磁性玻碳电极；Polish a magnetic glassy carbon electrode with a diameter of 3 mm with 1.0 μm, 0.3 μm, and 0.05 μm aluminum oxide slurries in sequence, then ultrasonically clean it with ethanol and water for 2 minutes, and dry it with nitrogen; take 5 μL of step (1) to obtain The immune magnetic bead solution is dripped on the center of the treated magnetic glassy carbon electrode, and the immune magnetic beads are evenly and firmly adsorbed on the surface of the electrode; after washing with water, the magnetic glassy carbon electrode loaded with immune magnetic beads is obtained;
(4)夹心式电化学发光免疫传感器的组装(4) Assembly of sandwich electrochemiluminescence immunosensor
将步骤(3)所得的固载有免疫磁珠的磁性玻碳电极浸泡在含有待测创伤弧菌的溶液中，37℃下孵育1h；清洗后，再滴涂5μL步骤(2)得到的多功能化氧化石墨烯溶液中，37℃下孵育1h；清洗后，即组装成用于检测创伤弧菌的夹心式电化学发光免疫传感器。Soak the magnetic glassy carbon electrode immobilized with immunomagnetic beads obtained in step (3) in the solution containing Vibrio vulnificus to be tested, and incubate at 37°C for 1 h; The functionalized graphene oxide solution was incubated at 37° C. for 1 h; after washing, it was assembled into a sandwich-type electrochemiluminescent immunosensor for detecting Vibrio vulnificus.
将上述用于检测创伤弧菌的夹心式电化学发光免疫传感器作为工作电极；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，测定电化学发光强度；获得一系列不同浓度的创伤弧菌溶液对应的电化学发光强度值，建立电化学发光强度值与创伤弧菌溶液浓度之间的定量关系；不同浓度创伤弧菌VV的ECL强度(y)—浓度(x)对数线性关系如图3所示。y＝1141.8*logx+335.4，相关系数R2＝0.9873，线性范围为4–(1×108)CFU/mL，检测限为1CFU/mL。线性良好，可用于检测样品中创伤弧菌的未知浓度。The above-mentioned sandwich type electrochemiluminescence immunosensor for detecting Vibrio vulnificus is used as a working electrode; a platinum electrode is used as a counter electrode, and an Ag/AgCl electrode or a saturated calomel electrode is used as a reference electrode to form a three-electrode system; the three-electrode system Put in the buffer solution, start the electrochemical reaction, and measure the intensity of electrochemiluminescence; obtain the corresponding electrochemiluminescence intensity values of a series of different concentrations of V. Relation; The ECL intensity (y)-concentration (x) logarithmic linear relationship of different concentrations of Vibrio vulnificus VV is shown in Figure 3. y=1141.8*logx+335.4, the correlation coefficient R2 =0.9873, the linear range is 4–(1×108 ) CFU/mL, and the detection limit is 1 CFU/mL. Good linearity and can be used to detect unknown concentrations of Vibrio vulnificus in samples.
具体实施例五Specific embodiment five
一种用于检测哈维氏弧菌(VH)的夹心式电化学发光免疫传感器的制备方法，包括以下步骤：A preparation method for detecting a sandwich type electrochemiluminescence immunosensor for Vibrio harveyi (VH), comprising the following steps:
(1)免疫磁珠的合成(1) Synthesis of immunomagnetic beads
在干净的烧瓶中加入10mg的氨基化磁珠，再加入5mL含有3wt％戊二醛的0.1mol/LpH7.4的磷酸盐缓冲溶液，37℃孵育3h后，外加磁铁磁性分离后用0.1mol/LpH7.4的磷酸盐缓冲溶液洗涤5次，去除上清液后，0.1mol/LpH7.4的磷酸盐缓冲溶液定容至5mL；加入150μL0.5mg/mL哈维氏弧菌一抗溶液，37℃孵育1h，外加磁铁磁性分离后用0.1mol/LpH7.4的磷酸盐缓冲溶液洗涤5次，去除上清液后，用0.1mol/LpH7.4的磷酸盐缓冲溶液定容至5mL；加入2wt％的牛血清白蛋白溶液150μL以封闭非特异性活性位点，37℃孵育1h，外加磁铁磁性分离后用0.1mol/LpH7.4的磷酸盐缓冲溶液洗涤5次，去除上清液后，用0.1mol/LpH7.4的磷酸盐缓冲溶液定容至5mL，即可得到表面覆盖有哈维氏弧菌一抗的免疫磁珠溶液，4℃下储存备用；Add 10 mg of aminated magnetic beads to a clean flask, then add 5 mL of 0.1 mol/L pH7.4 phosphate buffer solution containing 3 wt % glutaraldehyde, incubate at 37 ° C for 3 h, add a magnet for magnetic separation and use 0.1 mol/L Wash with LpH7.4 phosphate buffer solution for 5 times, remove the supernatant, dilute to 5mL with 0.1mol/LpH7.4 phosphate buffer solution; add 150μL 0.5mg/mL Vibrio harveyi primary antibody solution, 37 Incubate at ℃ for 1 hour, add a magnet for magnetic separation, wash 5 times with 0.1mol/L pH7.4 phosphate buffer solution, remove the supernatant, and dilute to 5mL with 0.1mol/L pH7.4 phosphate buffer solution; add 2wt % bovine serum albumin solution 150 μL to block the non-specific active sites, incubate at 37°C for 1 h, add a magnet for magnetic separation, wash 5 times with 0.1 mol/L pH7.4 phosphate buffer solution, remove the supernatant, wash with 0.1 Dilute the mol/L pH7.4 phosphate buffer solution to 5mL to obtain the immunomagnetic bead solution covered with primary antibody against Vibrio harveyi, and store it at 4°C for later use;
(2)多功能化氧化石墨烯的合成(2) Synthesis of multifunctional graphene oxide
在干净的试管中加入250μL的1mg/mL氧化石墨烯(GO)，超声1h，然后加入300μL偶联试剂，混合均匀，同时滴加0.1mol/L稀盐酸至溶液pH＝6，振荡孵育1h后，8000r离心15min，去除上清液后，0.1mol/LpH7.4的磷酸盐缓冲溶液定容至1mL；加入60μL10-5mol/L电化学发光体溶液和60μL0.01mg/mL哈维氏弧菌二抗溶液，混合均匀，同时滴加0.1mol/L氢氧化钠溶液至溶液pH＝8，常温振荡孵育4h，8000r离心15min，去除上清液后，用0.1mol/LpH7.4的磷酸盐缓冲溶液定容至0.5mL；再加入2wt％的牛血清白蛋白溶液80～120μL以封闭非特异性活性位点，常温振荡孵育1h，8000r离心15min，去除上清液后，用0.1mol/LpH7.4的磷酸盐缓冲溶液定容至1mL，即可得到哈维氏弧菌二抗和电化学发光体同时化学键合的多功能化氧化石墨烯溶液，4℃下储存备用；其中电化学发光体为含氨基官能团的三联吡啶钌衍生物，溶剂为0.1mol/LpH＝7.4的Tris-HCl缓冲液；偶联试剂为1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC)与N-羟基琥珀酰亚胺(NHS)按摩尔比10：1混合后溶于水中得到，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(EDC)的摩尔浓度为100mmol/L，N-羟基琥珀酰亚胺(NHS)摩尔浓度为1mmol/L；磷酸盐溶液为Na2HPO4·12H2O-NaH2PO4·2H2O体系缓冲溶液；Add 250 μL of 1 mg/mL graphene oxide (GO) to a clean test tube, sonicate for 1 h, then add 300 μL of coupling reagent, mix well, and at the same time add 0.1 mol/L dilute hydrochloric acid to the solution pH = 6, shake and incubate for 1 h , centrifuge at 8000r for 15min, remove the supernatant, dilute to 1mL with0.1mol /L phosphate buffer solution at pH7.4; Secondary antibody solution, mix evenly, add 0.1mol/L sodium hydroxide solution dropwise until the solution pH=8, incubate with shaking at room temperature for 4h, centrifuge at 8000r for 15min, remove the supernatant, buffer with 0.1mol/LpH7.4 phosphate Dilute the solution to 0.5mL; add 80-120μL of 2wt% bovine serum albumin solution to block non-specific active sites, incubate with shaking at room temperature for 1h, centrifuge at 8000r for 15min, remove the supernatant, and use 0.1mol/LpH7.4 The phosphate buffer solution was fixed to 1mL, and the multifunctional graphene oxide solution in which the Vibrio harveyi secondary antibody and the electrochemiluminescent substance were chemically bonded at the same time could be obtained, which was stored at 4°C for later use; the electrochemiluminescence substance contained The terpyridine ruthenium derivative of the amino functional group, the solvent is the Tris-HCl buffer solution of 0.1mol/LpH=7.4; the coupling reagent is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride Salt (EDC) and N-hydroxysuccinimide (NHS) are mixed at a molar ratio of 10:1 and then dissolved in water. The coupling reagent is 1-(3-dimethylaminopropyl)-3-ethane The molar concentration of carbodiimide hydrochloride (EDC) is 100mmol/L, and the molar concentration of N-hydroxysuccinimide (NHS) is 1mmol/L; the phosphate solution is Na2 HPO4 ·12H2 O-NaH2 PO4 ·2H2 O system buffer solution;
(3)电极修饰(3) Electrode modification
将直径为5mm的磁性玻碳电极依次用1.0μm、0.3μm、0.05μm的三氧化二铝浆液抛光处理，用乙醇、水依次超声2min，洗净，氮气吹干；取10μL步骤(1)得到的免疫磁珠溶液，滴涂于处理过的磁性玻碳电极中心，免疫磁珠即被均匀地牢固吸附在电极表面；用水洗净后即得固载有免疫磁珠的磁性玻碳电极；Polish a magnetic glassy carbon electrode with a diameter of 5 mm with 1.0 μm, 0.3 μm, and 0.05 μm aluminum oxide slurries in sequence, then ultrasonically clean it with ethanol and water for 2 minutes, and dry it with nitrogen; take 10 μL of step (1) to obtain The immune magnetic bead solution is dripped on the center of the treated magnetic glassy carbon electrode, and the immune magnetic beads are evenly and firmly adsorbed on the surface of the electrode; after washing with water, the magnetic glassy carbon electrode loaded with immune magnetic beads is obtained;
(4)夹心式电化学发光免疫传感器的组装(4) Assembly of sandwich electrochemiluminescence immunosensor
将步骤(3)所得的固载有免疫磁珠的磁性玻碳电极浸泡在含有待测哈维氏弧菌的溶液中，37℃下孵育1h；清洗后，再滴涂10μL步骤(2)得到的多功能化氧化石墨烯溶液中，37℃下孵育1h；清洗后，即组装成用于检测哈维氏弧菌的夹心式电化学发光免疫传感器。Soak the magnetic glassy carbon electrode immobilized with immunomagnetic beads obtained in step (3) in the solution containing Vibrio harveyi to be tested, and incubate at 37°C for 1 h; The multifunctional graphene oxide solution was incubated at 37°C for 1 h; after washing, it was assembled into a sandwich electrochemiluminescent immunosensor for detecting Vibrio harveyi.
将上述用于检测哈维氏弧菌的夹心式电化学发光免疫传感器作为工作电极；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，测定电化学发光强度；获得一系列不同浓度的哈维氏弧菌溶液对应的电化学发光强度值，建立电化学发光强度值与哈维氏弧菌溶液浓度之间的定量关系；不同浓度哈维氏弧菌VH的ECL强度(y)—浓度(x)对数线性关系如图4所示。y＝1091.4*logx+1265.1，相关系数R2＝0.9858，线性范围为4–(1×108)CFU/mL，检测限为1CFU/mL。线性良好，可用于检测样品中哈维氏弧菌的未知浓度。The above-mentioned sandwich electrochemiluminescence immunosensor for detecting Vibrio harveyi was 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 electrode to form a three-electrode system; Put the electrode system into the buffer solution, start the electrochemical reaction, and measure the intensity of electrochemiluminescence; obtain the corresponding electrochemiluminescence intensity values of a series of solutions of Vibrio harveyi with different concentrations, and establish the value of the intensity of electrochemiluminescence and that of Vibrio harveyi The quantitative relationship between the solution concentrations; the ECL intensity (y)-concentration (x) logarithmic linear relationship of different concentrations of Vibrio harveyi VH is shown in Figure 4. y=1091.4*logx+1265.1, the correlation coefficient R2 =0.9858, the linear range is 4–(1×108 ) CFU/mL, and the detection limit is 1 CFU/mL. Good linearity and can be used to detect unknown concentrations of Vibrio harveyi in samples.
具体实施例六Specific embodiment six
一种用于检测阴沟肠杆菌(EC)的夹心式电化学发光免疫传感器的制备方法同上述具体实施例三，其区别在于制备过程中采用的海洋致病菌为阴沟肠杆菌。A preparation method of a sandwich-type electrochemiluminescence immunosensor for detecting Enterobacter cloacae (EC) is the same as the above-mentioned specific embodiment 3, the difference is that the marine pathogenic bacteria used in the preparation process is Enterobacter cloacae.
将上述用于检测阴沟肠杆菌的夹心式电化学发光免疫传感器作为工作电极；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，测定电化学发光强度；获得一系列不同浓度的阴沟肠杆菌溶液对应的电化学发光强度值，建立电化学发光强度值与阴沟肠杆菌溶液浓度之间的定量关系；不同浓度阴沟肠杆菌EC的ECL强度(y)—浓度(x)对数线性关系如图5所示。y＝1047.5*logx+522.4，相关系数R2＝0.9805，线性范围为4–1×108CFU/mL，检测限为1CFU/mL。线性良好，可用于检测样品中阴沟肠杆菌的未知浓度。The above-mentioned sandwich type electrochemiluminescence immunosensor for detecting Enterobacter cloacae is used as a working electrode; a platinum electrode is used as a counter electrode, and an Ag/AgCl electrode or a saturated calomel electrode is used as a reference electrode to form a three-electrode system; the three-electrode system Put in 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 solutions of Enterobacter cloacae with different concentrations, and establish the quantification between the value of the intensity of electrochemiluminescence and the concentration of Enterobacter cloacae solution Relation; the ECL intensity (y)-concentration (x) logarithmic linear relationship of different concentrations of Enterobacter cloacae EC is shown in Figure 5. y=1047.5*logx+522.4, the correlation coefficient R2 =0.9805, the linear range is 4-1×108 CFU/mL, and the detection limit is 1 CFU/mL. Good linearity and can be used to detect unknown concentrations of Enterobacter cloacae in samples.
具体实施例七Specific embodiment seven
一种用于检测黄海希瓦氏菌(SM)的夹心式电化学发光免疫传感器的制备方法同上述具体实施例三，其区别在于制备过程中采用的海洋致病菌为黄海希瓦氏菌。The preparation method of a sandwich-type electrochemiluminescence immunosensor for detecting Shewanella yellowsea (SM) is the same as that of the third embodiment above, except that the marine pathogen used in the preparation process is Shewanella yellowsea.
将上述用于检测黄海希瓦氏菌的夹心式电化学发光免疫传感器作为工作电极；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，测定电化学发光强度；获得一系列不同浓度的黄海希瓦氏菌溶液对应的电化学发光强度值，建立电化学发光强度值与黄海希瓦氏菌溶液浓度之间的定量关系；不同浓度黄海希瓦氏菌SM的ECL强度(y)—浓度(x)对数线性关系如图6所示。y＝1237*logx+618.1，相关系数R2＝0.9807，线性范围为4–1×108CFU/mL，检测限为1CFU/mL。线性良好，可用于检测样品中黄海希瓦氏菌的未知浓度。The above-mentioned sandwich electrochemiluminescence immunosensor for detecting Shewanella yellow sea was 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 electrode to form a three-electrode system; Put the electrode system into the buffer solution, start the electrochemical reaction, and measure the intensity of electrochemiluminescence; obtain the corresponding electrochemiluminescence intensity values of a series of solutions of Shewanella yellowsea with different concentrations, and establish the value of electrochemiluminescence intensity and the value of Shewanella yellowsea. Quantitative relationship between solution concentrations; the ECL intensity (y)-concentration (x) logarithmic linear relationship of different concentrations of Shewanella flavumii SM is shown in Figure 6. y=1237*logx+618.1, the correlation coefficient R2 =0.9807, the linear range is 4-1×108 CFU/mL, and the detection limit is 1 CFU/mL. The linearity is good, and it can be used to detect the unknown concentration of Shewanella yellowsea in the sample.
具体实施例八Embodiment 8
特异性试验specificity test
选取创伤弧菌(VV)、哈维氏弧菌(VH)、阴沟肠杆菌(EC)、黄海希瓦氏菌(SM)、副溶血弧菌(VP)5种菌来证明按上述具体实施例三的方法制备的用于检测副溶血弧菌(VP)的夹心式电化学发光免疫传感器的选择性。将制备的副溶血弧菌电化学发光免疫传感器分别测定108CFU/mL的VV、VH、EC、SM及空白对照组，对应的电化学发光强度都在300左右，而测定4000CFU/mL的VP，对应的电化学发光强度明显增强，在4500左右；同时将副溶血弧菌电化学发光免疫传感器测定含108CFU/mL的VV、108CFU/mL的VH、108CFU/mL的EC、108CFU/mL的SM和4000CFU/mL的VP的混合溶液，也在4500左右。以上结果显示：其他菌种浓度很高，信号却和空白大致相当，表示其他菌种对该传感器检测无干扰。结果表明：副溶血弧菌电化学发光免疫传感器选择性良好，常见干扰菌种无显著性干扰。Select Vibrio vulnificus (VV), Vibrio harveii (VH), Enterobacter cloacae (EC), Shewanella yellow sea (SM), and Vibrio parahaemolyticus (VP) to prove that according to the above specific examples Selectivity of Sandwich Electrochemiluminescence Immunosensor for Detection of Vibrio parahaemolyticus (VP) Prepared by Three Methods. The prepared Vibrio parahaemolyticus electrochemiluminescence immunosensor was used to measure 108 CFU/mL of VV, VH, EC, SM and the blank control group respectively. , the corresponding electrochemiluminescence intensity was significantly enhanced, around 4500; at the same time, the Vibrio parahaemolyticus electrochemiluminescence immunosensor was used to measure the VV containing 108 CFU/mL, the VH of 108 CFU/mL, and the EC of 108 CFU/mL. , The mixed solution of 108 CFU/mL SM and 4000 CFU/mL VP is also around 4500. The above results show that the concentration of other bacteria is very high, but the signal is roughly equivalent to that of the blank, indicating that other bacteria have no interference with the detection of the sensor. The results showed that the electrochemiluminescence immunosensor for Vibrio parahaemolyticus had good selectivity, and common interfering bacteria had no significant interference.
同理，上述具体实施例四的方法制备的用于检测创伤弧菌(VV)的电化学发光免疫传感器、具体实施例五的方法制备的用于检测哈维氏弧菌(VH)的电化学发光免疫传感器、具体实施例六的方法制备的用于检测阴沟肠杆菌(EC)的电化学发光免疫传感器以及具体实施例七的方法制备的用于检测黄海希瓦氏菌(SM)的电化学发光免疫传感器均选择性良好，常见干扰菌种无显著性干扰。In the same way, the electrochemiluminescence immunosensor for detecting Vibrio vulnificus (VV) prepared by the method of the above-mentioned specific embodiment four, and the electrochemical sensor for detecting Vibrio harveyi (VH) prepared by the method of specific embodiment five Luminescence immunosensor, the electrochemiluminescence immunosensor for detecting Enterobacter cloacae (EC) prepared by the method of specific example six, and the electrochemiluminescent immunosensor for detection of Shewanella yellowsea (SM) prepared by the method of specific example seven All the luminescent immunosensors had good selectivity, and common interfering bacteria had no significant interference.
具体实施例九Specific embodiment nine
海水中海洋致病菌的检测Detection of marine pathogens in seawater
准确移取海水空白样品，进行加标回收检测，按照上述具体实施例三-七中的具体实验步骤构建传感器并按具体实施例二方法进行检测，检测结果见表1。Accurately pipette a seawater blank sample, carry out standard addition recovery detection, construct a sensor according to the specific experimental steps in the above-mentioned specific examples 3-7, and perform detection according to the method of specific embodiment 2. The test results are shown in Table 1.
表1多种海洋致病菌的检测结果Table 1 Detection results of various marine pathogenic bacteria
由表1检测结果可知，结果的相对标准偏差(RSD)小于11％，回收率为91.3～110.3％，表明本发明对于海水中海洋致病菌的检测精密度高，结果准确可靠。As can be seen from the test results in Table 1, the relative standard deviation (RSD) of the results is less than 11%, and the recovery rate is 91.3-110.3%, showing that the present invention has high detection precision for marine pathogenic bacteria in seawater, and the results are accurate and reliable.
以上结果说明，本发明构建的检测海洋致病菌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海洋致病菌的高灵敏度、特异性、简单、快速检测。The above results show that the electrochemiluminescent immunosensor for detecting marine pathogenic bacteria constructed by the present invention has high sensitivity, low detection limit, high selectivity, simple operation, and accurate and reliable results. Only by changing the antibody in the electrochemiluminescence immunosensor, high sensitivity, specificity, simple and rapid detection of different target marine pathogenic bacteria can be realized.
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.
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| CN201510690790.5ACN105300963B (en) | 2015-10-22 | 2015-10-22 | For the preparation method and applications for the sandwich electrochemical luminescence immunosensor for detecting Marine Pathogenic Bacteria |
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