CN115753936B - Electrochemical luminescence biosensor for detecting organophosphorus pesticide and preparation method and application thereof - Google Patents
Electrochemical luminescence biosensor for detecting organophosphorus pesticide and preparation method and application thereof Download PDFInfo
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Abstract
Description
技术领域Technical Field
本发明涉及新型功能纳米材料与电化学生物传感器分析农药残留领域，尤其涉及一种检测有机磷农药电化学发光生物传感器及制备方法与应用。The present invention relates to the field of novel functional nanomaterials and electrochemical biosensors for analyzing pesticide residues, and in particular to an electrochemical luminescence biosensor for detecting organophosphorus pesticides, and a preparation method and application thereof.
背景技术Background Art
有机磷农药（Organophosphorus pesticides, Ops）作为农药主要的类型之一，其广谱、高效、价格低廉，广泛应用在果蔬农产品种植中。然而，在实际生产中，不科学的使用方法，或没有严格控制使用量，常将多种药剂混合使用，随意混配、增加用药量、增加使用频次等不按农药标签使用的情况频有发生，药物残留更为严重。长期摄入有机磷农药残留超标的水果和蔬菜，会对人体健康造成严重影响，比如，人体的白细胞吞噬功能减弱、肝脏能力变差以及中枢神经紊乱等，甚至导致细胞的癌化和变异等问题。因此，加强有机磷农药残留的监管，对保证食品安全、保障人类健康有着重要而深远的意义。Organophosphorus pesticides (Ops) are one of the main types of pesticides. They are broad-spectrum, highly effective, and inexpensive, and are widely used in the cultivation of fruit and vegetable agricultural products. However, in actual production, unscientific methods of use or lack of strict control of usage often lead to the mixing of multiple agents, random mixing, increased dosage, increased frequency of use, and other situations that do not follow the pesticide labels, and drug residues are more serious. Long-term intake of fruits and vegetables with excessive organophosphorus pesticide residues can have serious effects on human health, such as weakened phagocytic function of human white blood cells, poor liver function, and central nervous system disorders, and can even lead to cell canceration and mutation. Therefore, strengthening the supervision of organophosphorus pesticide residues is of great and far-reaching significance for ensuring food safety and protecting human health.
目前用于检测果蔬农产品中的有机磷农药的方法主要有高效液相色谱质谱法（HPLC）、液相色谱-质谱法（HPLC-MS）和液相色谱-串联质谱（LC-MS/MS）等。这些方法虽然具有很高的灵敏度和准确度、且可以一次测定多种成分等优点，但需要大型昂贵的仪器设备，分析程序复杂，检测周期长，检测成本高，需要专业的技术人员来完成，难以应用于农残的快速检测和现场分析。At present, the main methods used to detect organophosphorus pesticides in fruit and vegetable agricultural products include high performance liquid chromatography mass spectrometry (HPLC), liquid chromatography-mass spectrometry (HPLC-MS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Although these methods have the advantages of high sensitivity and accuracy and can measure multiple components at one time, they require large and expensive instruments and equipment, complex analysis procedures, long detection cycles, high detection costs, and require professional technicians to complete, making them difficult to apply to rapid detection and on-site analysis of pesticide residues.
电化学和化学发光结合的电致化学发光（Electrochemiluminescence，ECL）分析方法有许多独特的优点，如快速、灵敏度高、线性范围宽、背景信号低、不需借助任何外部光源，以及简化的光学装置等优点，是一种发展迅速的技术。因此，构建一种快速、灵敏的电化学发光生物传感器用于有机磷农药残留检测具有重要的研究意义和市场价值。The electrochemiluminescence (ECL) analysis method, which combines electrochemistry and chemiluminescence, has many unique advantages, such as rapidity, high sensitivity, wide linear range, low background signal, no need for any external light source, and simplified optical devices. It is a rapidly developing technology. Therefore, constructing a fast and sensitive electrochemiluminescence biosensor for the detection of organophosphorus pesticide residues has important research significance and market value.
发明内容Summary of the invention
有鉴于此，本申请提供一种检测有机磷农药电化学发光生物传感器及制备方法与应用，可快速、灵敏的检测有机磷化合物。In view of this, the present application provides an electrochemiluminescent biosensor for detecting organophosphorus pesticides, and a preparation method and application thereof, which can rapidly and sensitively detect organophosphorus compounds.
为达到上述技术目的，本申请采用以下技术方案：In order to achieve the above technical objectives, this application adopts the following technical solutions:
第一方面，本申请提供一种检测有机磷农药电化学发光生物传感器，包括玻碳电极，以及从内到外依次修饰于玻碳电极表面的镉化物量子点@介孔聚多巴胺纳米材料、nafion膜层、分子探针、互补DNA；分子探针包括5'端修饰氨基和3'端修饰二硫基的核酸适配体，与核酸适配体的3'端偶联的贵金属纳米粒子，核酸适配体的二级结构为发夹结构；互补DNA与分子探针通过碱基互补配对。In the first aspect, the present application provides an electrochemiluminescent biosensor for detecting organophosphorus pesticides, comprising a glassy carbon electrode, and cadmium quantum dots@mesoporous polydopamine nanomaterials, nafion membrane layers, molecular probes, and complementary DNA modified on the surface of the glassy carbon electrode from the inside to the outside; the molecular probe comprises a nucleic acid aptamer modified with an amino group at the 5' end and a disulfide group at the 3' end, and a noble metal nanoparticle coupled to the 3' end of the nucleic acid aptamer, wherein the secondary structure of the nucleic acid aptamer is a hairpin structure; the complementary DNA and the molecular probe are paired through base complementarity.
优选地，镉化物量子点@介孔聚多巴胺纳米材料由水性镉化物量子点生长在介孔聚多巴胺纳米材料的多孔中而形成。Preferably, the cadmium compound quantum dots@mesoporous polydopamine nanomaterial is formed by growing aqueous cadmium compound quantum dots in the pores of the mesoporous polydopamine nanomaterial.
优选地，镉化物量子点包括碲化镉量子点、硒化镉量子点、硫化镉量子点中的一种。Preferably, the cadmium compound quantum dots include one of cadmium telluride quantum dots, cadmium selenide quantum dots, and cadmium sulfide quantum dots.
优选地，贵金属纳米粒子包括金纳米粒子、铂纳米粒子、银纳米粒子、金铂双金属纳米粒子、金银双金属纳米粒子中的一种。Preferably, the noble metal nanoparticles include one of gold nanoparticles, platinum nanoparticles, silver nanoparticles, gold-platinum bimetallic nanoparticles, and gold-silver bimetallic nanoparticles.
优选地，镉化物量子点的粒径为5-10nm。Preferably, the particle size of the cadmium compound quantum dots is 5-10 nm.
优选地，镉化物量子点@介孔聚多巴胺纳米材料的制备方法，包括如下步骤：Preferably, the preparation method of cadmium quantum dots@mesoporous polydopamine nanomaterials comprises the following steps:
S1.将嵌段式聚醚F-127与水性镉化物量子点混合分散于乙醇水溶液中，得到均一溶液；S1. The block polyether F-127 and the aqueous cadmium quantum dots were mixed and dispersed in an ethanol aqueous solution to obtain a uniform solution;
S2.在均一溶液中依次加入盐酸多巴胺及1, 3, 5-三甲苯，而后加入氨水，搅拌反应，得到中间产物；S2. dopamine hydrochloride and 1, 3, 5-trimethylbenzene are sequentially added to the homogeneous solution, and then aqueous ammonia is added, and the reaction is stirred to obtain an intermediate product;
S3.将中间产物离心并洗涤，再分散于乙醇水溶液中超声处理，而后于密封条件下，100℃加热过夜，即得镉化物量子点@介孔聚多巴胺纳米材料。S3. The intermediate product is centrifuged and washed, dispersed in an ethanol aqueous solution and ultrasonically treated, and then heated at 100° C. overnight under sealed conditions to obtain cadmium quantum dots@mesoporous polydopamine nanomaterials.
第二方面，本申请提供一种检测有机磷农药电化学发光生物传感器的制备方法，包括以下步骤：In a second aspect, the present application provides a method for preparing an electrochemiluminescent biosensor for detecting organophosphorus pesticides, comprising the following steps:
K1.抛光清洗玻碳电极；K1. Polishing and cleaning of glassy carbon electrodes;
K2.将镉化物量子点@介孔聚多巴胺纳米材料溶液滴加于抛光清洗后的玻碳电极表面，进行电极改性，室温晾干后，得到镉化物量子点@介孔聚多巴胺/玻碳电极；K2. Add the cadmium quantum dot @ mesoporous polydopamine nanomaterial solution dropwise onto the surface of the polished and cleaned glassy carbon electrode to modify the electrode, and then dry it at room temperature to obtain a cadmium quantum dot @ mesoporous polydopamine/glassy carbon electrode;
K3.在镉化物量子点@介孔聚多巴胺/玻碳电极表面滴涂nafion溶液，室温晾干后，用水冲洗，得到nafion/镉化物量子点@介孔聚多巴胺/玻碳电极；K3. Drop-coat the nafion solution on the surface of the cadmium quantum dots @ mesoporous polydopamine / glassy carbon electrode, dry it at room temperature, and then rinse it with water to obtain a nafion / cadmium quantum dots @ mesoporous polydopamine / glassy carbon electrode;
K4.将分子探针滴涂于nafion/镉化物量子点@介孔聚多巴胺/玻碳电极表面，孵育过夜，得到分子探针/nafion/镉化物量子点@介孔聚多巴胺/玻碳电极；K4. drop-coat the molecular probe on the surface of nafion/cadmium quantum dots@mesoporous polydopamine/glassy carbon electrode and incubate overnight to obtain molecular probe/nafion/cadmium quantum dots@mesoporous polydopamine/glassy carbon electrode;
K5.将封闭剂滴涂于分子探针/nafion/镉化物量子点@介孔聚多巴胺/玻碳电极表面，经孵育过夜、冲洗后，再滴涂互补DNA，再进行孵育、冲洗，得互补DNA/分子探针/nafion/镉化物量子点@介孔聚多巴胺/玻碳电极即检测有机磷农药电化学发光生物传感器。K5. Apply the sealing agent dropwise on the surface of the molecular probe/nafion/cadmium quantum dots@mesoporous polydopamine/glassy carbon electrode, incubate overnight, rinse, then apply complementary DNA, incubate and rinse again, and obtain complementary DNA/molecular probe/nafion/cadmium quantum dots@mesoporous polydopamine/glassy carbon electrode, which is an electrochemiluminescent biosensor for detecting organophosphorus pesticides.
优选地，封闭剂为6-巯基-1-己醇溶液。Preferably, the blocking agent is a 6-mercapto-1-hexanol solution.
第三方面，本申请提供一种检测有机磷农药电化学发光生物传感器在检测有机磷农药中的应用，检测的参比电极为银/氯化银电极，对电极为铂片电极或铂丝电极，工作电极为互补DNA/分子探针/nafion/镉化物量子点@介孔聚多巴胺/玻碳电极。In the third aspect, the present application provides an application of an electrochemiluminescent biosensor for detecting organophosphorus pesticides in detecting organophosphorus pesticides, wherein the reference electrode for detection is a silver/silver chloride electrode, the counter electrode is a platinum sheet electrode or a platinum wire electrode, and the working electrode is a complementary DNA/molecular probe/nafion/cadmium quantum dots@mesoporous polydopamine/glassy carbon electrode.
优选地，检测的激发电压为2.0~0V，扫描速率为50-100mV/s。Preferably, the excitation voltage of the detection is 2.0-0V, and the scanning rate is 50-100mV/s.
本申请的有益效果如下：The beneficial effects of this application are as follows:
本申请将镉化物量子点@介孔聚多巴胺复合材料应用于电化学发光生物传感器制备中，显著提高了传感器的分析性能，构建了一种新型的电化学发光生物传感器检测有机磷农药的方法，该方法可以在绝大多数实验室中轻易实现而不需要复杂繁琐的大型设备支持；本发明制备的传感器，成本低廉，制备步骤简单，检测速度快，可应用于便携式检测；The present application applies the cadmium quantum dot@mesoporous polydopamine composite material to the preparation of an electrochemiluminescent biosensor, significantly improving the analytical performance of the sensor, and constructing a new electrochemiluminescent biosensor method for detecting organophosphorus pesticides. The method can be easily implemented in most laboratories without the need for complex and cumbersome large-scale equipment support; the sensor prepared by the present invention has low cost, simple preparation steps, fast detection speed, and can be applied to portable detection;
本申请利用介孔聚多巴胺的多孔结构特性以及大的比表面积，使碲化镉量子点在介孔聚多巴胺的孔隙中原位包裹负载，有利于提高碲化镉量子点的负载，制备出碲化镉量子点@介孔聚多巴胺复合材料，作为基底材料修饰于电极表面，并使用nafion溶液固定复合材料，发光信号稳定，应用于电化学发光生物传感器构建；The present application utilizes the porous structure characteristics and large specific surface area of mesoporous polydopamine to load cadmium telluride quantum dots in situ in the pores of mesoporous polydopamine, which is beneficial to increase the loading of cadmium telluride quantum dots, prepares cadmium telluride quantum dots@mesoporous polydopamine composite materials, and uses nafion solution to fix the composite materials. The luminescent signal is stable and is applied to the construction of electrochemiluminescent biosensors.
本申请的分子探针为核酸适配体，对有机磷农药具有高特异性和亲和力，其二级结构为发夹结构，其与互补DNA碱基互补配对，以提高传感器的检测线性范围和灵敏度。The molecular probe of the present application is a nucleic acid aptamer, which has high specificity and affinity for organophosphorus pesticides, and its secondary structure is a hairpin structure, which complementarily pairs with complementary DNA bases to improve the detection linear range and sensitivity of the sensor.
附图说明BRIEF DESCRIPTION OF THE DRAWINGS
图1为电化学发光生物传感器的制备过程；FIG1 is a preparation process of an electrochemiluminescent biosensor;
图2为电化学发光生物传感器的检测机制示意图；FIG2 is a schematic diagram of the detection mechanism of an electrochemiluminescent biosensor;
图3为碲化镉量子点、介孔聚多巴胺和碲化镉量子点@介孔聚多巴胺的X射线衍射图谱；FIG3 is an X-ray diffraction pattern of cadmium telluride quantum dots, mesoporous polydopamine and cadmium telluride quantum dots@mesoporous polydopamine;
图4为碲化镉量子点的透射电镜图；FIG4 is a transmission electron microscope image of cadmium telluride quantum dots;
图5为介孔聚多巴胺的透射电镜图；FIG5 is a transmission electron micrograph of mesoporous polydopamine;
图6为碲化镉量子点@介孔聚多巴胺的EDS元素分析图；FIG6 is an EDS elemental analysis diagram of cadmium telluride quantum dots@mesoporous polydopamine;
图7为金纳米粒子的紫外可见分光光谱图；FIG7 is a UV-visible spectroscopic diagram of gold nanoparticles;
图8为nafion/碲化镉量子点@介孔聚多巴胺/玻碳电极在0.1mol/L的过硫酸钾溶液中的循环伏安和电化学发光（ECL）循环稳定性图；FIG8 is a graph showing the cyclic voltammetry and electrochemiluminescence (ECL) cycle stability of nafion/cadmium telluride quantum dots@mesoporous polydopamine/glassy carbon electrode in 0.1 mol/L potassium persulfate solution;
图9为电化学发光生物传感器制备过程的循环伏安表征图；FIG9 is a cyclic voltammetry characterization diagram of the electrochemiluminescent biosensor preparation process;
图10为制备的电化学发光生物传感器用于有机磷农药检测的可行性验证图。FIG10 is a diagram showing the feasibility of using the prepared electrochemiluminescent biosensor for organophosphorus pesticide detection.
具体实施方式DETAILED DESCRIPTION
为了使本发明的目的、技术方案及优点更加清楚明白，以下结合实施例，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明。In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.
术语简写：Terminology Abbreviations:
玻碳电极：GCE；Glassy carbon electrode: GCE;
6-巯基-1-己醇溶液：MCH；6-Mercapto-1-hexanol solution: MCH;
羧基碲化镉量子点：CdTe-COOH QDs；Carboxyl cadmium telluride quantum dots: CdTe-COOH QDs;
介孔聚多巴胺：MPDA；Mesoporous polydopamine: MPDA;
亚碲酸钠：Na2TeO3；Sodium tellurite: Na 2 TeO 3 ;
水合肼：N2H4•H2O；Hydrazine hydrate: N 2 H 4 •H 2 O;
巯基乙酸：TGA；Thioglycolic acid: TGA;
1, 3, 5-三甲苯：TMB：1,3,5-Trimethylbenzene: TMB:
互补DNA：ctDNA。Complementary DNA: ctDNA.
如图1所示，本申请提供一种检测有机磷农药电化学发光生物传感器，包括玻碳电极，以及从内到外依次修饰于玻碳电极表面的镉化物量子点@介孔聚多巴胺纳米材料、nafion膜层、分子探针、互补DNA。As shown in Figure 1, the present application provides an electrochemiluminescent biosensor for detecting organophosphorus pesticides, including a glassy carbon electrode, and cadmium quantum dots@mesoporous polydopamine nanomaterials, nafion membrane layers, molecular probes, and complementary DNA modified on the surface of the glassy carbon electrode from the inside to the outside.
在本方案中，镉化物量子点@介孔聚多巴胺纳米材料由水性镉化物量子点生长在介孔聚多巴胺纳米材料的多孔中而形成；镉化物量子点包括碲化镉量子点、硒化镉量子点、硫化镉量子点中的一种，镉化物量子点的粒径为5-10nm，优选地，镉化物量子点为水溶性碲化镉。In this embodiment, the cadmium compound quantum dots @mesoporous polydopamine nanomaterial is formed by growing aqueous cadmium compound quantum dots in the pores of the mesoporous polydopamine nanomaterial; the cadmium compound quantum dots include one of cadmium telluride quantum dots, cadmium selenide quantum dots, and cadmium sulfide quantum dots, and the particle size of the cadmium compound quantum dots is 5-10 nm. Preferably, the cadmium compound quantum dots are water-soluble cadmium telluride.
镉化物量子点@介孔聚多巴胺纳米材料的制备方法，包括如下步骤：The preparation method of cadmium quantum dot@mesoporous polydopamine nanomaterial comprises the following steps:
S1.将嵌段式聚醚F-127与水性镉化物量子点混合分散于乙醇水溶液中，得到均一溶液；S1. The block polyether F-127 and the aqueous cadmium quantum dots were mixed and dispersed in an ethanol aqueous solution to obtain a uniform solution;
S2.在均一溶液中依次加入盐酸多巴胺及1, 3, 5-三甲苯，而后加入氨水，搅拌反应，得到中间产物；S2. dopamine hydrochloride and 1, 3, 5-trimethylbenzene are sequentially added to the homogeneous solution, and then aqueous ammonia is added, and the reaction is stirred to obtain an intermediate product;
S3.将中间产物离心并洗涤，再分散于乙醇水溶液中超声处理，而后于密封条件下，100℃加热过夜，即得镉化物量子点@介孔聚多巴胺纳米材料。S3. The intermediate product is centrifuged and washed, dispersed in an ethanol aqueous solution and ultrasonically treated, and then heated at 100° C. overnight under sealed conditions to obtain cadmium quantum dots@mesoporous polydopamine nanomaterials.
以碲化镉量子点@介孔聚多巴胺为镉化物量子点@介孔聚多巴胺纳米材料为例，其制备方法如下：Taking cadmium telluride quantum dots @ mesoporous polydopamine as cadmium telluride quantum dots @ mesoporous polydopamine nanomaterials as an example, the preparation method is as follows:
将1.0g嵌段式聚醚F-127和0.36~0.144g的水溶性碲化镉量子点加入50~100mL乙醇水混合溶液中，剧烈搅拌，获得CdTe量子点分散在嵌段式聚醚F-127中的均一溶液。然后将1.0g盐酸多巴胺溶解在上述溶液中，再加入2.0mL的1, 3, 5-三甲苯到混合溶液中。随后，向反应的混合溶液中加入1.0~2.5mL氨水，搅拌反应4~12h。最后，使用乙醇水混合溶液离心和洗涤几次得到介孔聚多巴胺颗粒的产物，然后重新分散在一定量的水和乙醇混合溶液中超声处理一段时间，转移到一个密封的特氟隆内衬高压釜中，100℃加热24h，获得碲化镉量子点@介孔聚多巴胺复合材料。1.0g of block polyether F-127 and 0.36~0.144g of water-soluble cadmium telluride quantum dots were added to 50~100mL of ethanol-water mixed solution and stirred vigorously to obtain a uniform solution in which CdTe quantum dots were dispersed in block polyether F-127. Then 1.0g of dopamine hydrochloride was dissolved in the above solution, and 2.0mL of 1, 3, 5-trimethylbenzene was added to the mixed solution. Subsequently, 1.0~2.5mL of ammonia water was added to the reacting mixed solution, and the reaction was stirred for 4~12h. Finally, the product of mesoporous polydopamine particles was obtained by centrifugation and washing several times with the ethanol-water mixed solution, and then redispersed in a certain amount of water and ethanol mixed solution for ultrasonic treatment for a period of time, transferred to a sealed Teflon-lined autoclave, and heated at 100℃ for 24h to obtain cadmium telluride quantum dots@mesoporous polydopamine composite materials.
其中，水溶性碲化镉量子点的制备方法为：将氯化镉半（五水合物）（45.672mg，0.2mmol）和巯基乙酸（46.06mg，0.5mmol）依次分散到50mL蒸馏水中，然后滴加NaOH溶液调节Cd-TGA复合物溶液pH至7.0~11.0。通过将高纯度的N2鼓泡30min，使溶液脱氧。在剧烈搅拌下，依次加入亚碲酸钠溶液（0.01~0.04mmol）和50~85%水合肼溶液（0.1~0.5mL），然后，油浴锅中回流搅拌加热（80~100°C，1~4h），获得巯基乙酸封端的碲化镉量子点。最后，用过量的乙醇离心（8000~10000rpm，5~10min）纯化粗的水溶性羧基碲化镉量子点（CdTe-COOHQDs）。将洗涤后的水溶性羧基碲化镉量子点（CdTe-COOH QDs）分散在去离子水中，4℃黑暗条件下储存。The preparation method of water-soluble cadmium telluride quantum dots is as follows: cadmium chloride hemi (pentahydrate) (45.672 mg, 0.2 mmol) and thioglycolic acid (46.06 mg, 0.5 mmol) are dispersed in 50 mL of distilled water in sequence, and then NaOH solution is added dropwise to adjust the pH of the Cd-TGA complex solution to 7.0-11.0. The solution is deoxygenated by bubbling high-purity N2 for 30 min. Under vigorous stirring, sodium tellurite solution (0.01-0.04 mmol) and 50-85% hydrazine hydrate solution (0.1-0.5 mL) are added in sequence, and then, the mixture is refluxed and stirred in an oil bath (80-100°C, 1-4 h) to obtain thioglycolic acid-terminated cadmium telluride quantum dots. Finally, the crude water-soluble carboxyl cadmium telluride quantum dots (CdTe-COOH QDs) were purified by centrifugation with excess ethanol (8000-10000 rpm, 5-10 min). The washed water-soluble carboxyl cadmium telluride quantum dots (CdTe-COOH QDs) were dispersed in deionized water and stored at 4°C in the dark.
镉化物量子点@介孔聚多巴胺/玻碳电极的最外层修饰有nafion，以防止碲化镉量子点@介孔聚多巴胺复合材料从玻碳电极表面脱落；nafion溶液是由乙醇水混合溶液配制，去离子水和乙醇的体积比为1/9~9/1。The outermost layer of the cadmium telluride quantum dot@mesoporous polydopamine/glassy carbon electrode is modified with nafion to prevent the cadmium telluride quantum dot@mesoporous polydopamine composite material from falling off the surface of the glassy carbon electrode; the nafion solution is prepared from an ethanol-water mixed solution, and the volume ratio of deionized water to ethanol is 1/9~9/1.
核酸适配体（Aptmer，Apt）是通过指数富集配体的***进化技术筛选而得到的寡核苷酸序列（RNA或DNA），与相应的配体有严格的识别能力和高度的亲和力，且具有靶分子范围广、易于人工合成、稳定性好、便于修饰等优点，本方案的分子探针包括5'端修饰氨基和3'端修饰二硫基的核酸适配体，对有机磷农药具有高特异性，5'端的氨基可以通过与nafion的磺酸基形成共价键，与核酸适配体的3'端偶联的贵金属纳米粒子，核酸适配体的二级结构为发夹结构；优选的，分子探针为hairpin-DNA-AuNPs，核酸适配体为一段DNA，其5'端修饰有氨基（-NH2），3'端修饰有巯基（-SH），其5'端的氨基通过与nafion的磺酸基形成共价键固定在电极表面，金纳米粒子偶联核酸适配体通过与核酸适配体的3'端的巯基形成Au-S共价键；贵金属纳米粒子包括金纳米粒子、铂纳米粒子、银纳米粒子、金铂双金属纳米粒子、金银双金属纳米粒子中的一种，优选地，贵金属纳米粒子为金纳米粒子；以氯金酸作为金源制备AuNPs，量取200mL超纯水，添加3mL 1%的氯金酸溶液，冰箱中冷却至4℃。在4℃环境下，添加0.5mL的碳酸钾溶液（0.2mol/L）后持续搅拌，剧烈搅拌，最后快速加入9mL硼氢化钠（0.5mg/mL），剧烈搅拌5min。将所得的酒红色溶液保存在4℃冰箱中备用。图7为金纳米粒子的紫外可见分光光谱图，在520nm左右具有金纳米粒子的紫外特征吸收峰，金纳米粒子的制备是成功的。Nucleic acid aptamers (Aptmer, Apt) are oligonucleotide sequences (RNA or DNA) obtained by systematic evolution technology of exponential enrichment ligands. They have strict recognition ability and high affinity with the corresponding ligands, and have the advantages of wide target molecule range, easy artificial synthesis, good stability, and convenient modification. The molecular probe of this scheme includes nucleic acid aptamers with amino groups modified at the 5' end and disulfide groups modified at the 3' end, which have high specificity for organophosphorus pesticides. The amino group at the 5' end can form a covalent bond with the sulfonic acid group of nafion, and the noble metal nanoparticles coupled to the 3' end of the nucleic acid aptamer have a hairpin structure as the secondary structure. Preferably, the molecular probe is hairpin-DNA-AuNPs, and the nucleic acid aptamer is a piece of DNA with an amino group (-NH 2 ), the 3' end is modified with a thiol (-SH), and the amino group at its 5' end is fixed on the electrode surface by forming a covalent bond with the sulfonic acid group of nafion, and the gold nanoparticles coupled to the nucleic acid aptamer form an Au-S covalent bond with the thiol group at the 3' end of the nucleic acid aptamer; the noble metal nanoparticles include one of gold nanoparticles, platinum nanoparticles, silver nanoparticles, gold-platinum bimetallic nanoparticles, and gold-silver bimetallic nanoparticles. Preferably, the noble metal nanoparticles are gold nanoparticles; AuNPs are prepared using chloroauric acid as a gold source, 200 mL of ultrapure water is measured, 3 mL of 1% chloroauric acid solution is added, and the solution is cooled to 4°C in a refrigerator. At 4°C, 0.5 mL of potassium carbonate solution (0.2 mol/L) is added and stirred continuously and vigorously, and finally 9 mL of sodium borohydride (0.5 mg/mL) is quickly added and stirred vigorously for 5 minutes. The resulting wine red solution is stored in a 4°C refrigerator for later use. FIG7 is a UV-visible spectroscopic spectrum of gold nanoparticles, which has a characteristic UV absorption peak of gold nanoparticles at around 520 nm, indicating that the preparation of gold nanoparticles is successful.
互补DNA与分子探针通过碱基互补配对形成双链DNA，具有刚性结构；互补DNA可与分子探针核酸适配体通过碱基互补配对修饰在电极表面，双链DNA具有刚性结构，互补DNA的存在，有助于降低传感器的空白背景信号，传感器的线性范围更宽，以及提高传感器的检测灵度，最后，完成电化学发光生物传感器的制备，用于有机磷农药的检测。Complementary DNA and molecular probes form double-stranded DNA through base complementary pairing, which has a rigid structure; complementary DNA can be modified on the electrode surface with molecular probe nucleic acid aptamers through base complementary pairing. Double-stranded DNA has a rigid structure. The presence of complementary DNA helps to reduce the blank background signal of the sensor, the linear range of the sensor is wider, and the detection sensitivity of the sensor is improved. Finally, the preparation of the electrochemiluminescence biosensor is completed for the detection of organophosphorus pesticides.
本方案得到的检测有机磷农药电化学发光生物传感器基于共振能量转移机理对有机磷农药实现检测，具体为，当目标物存在，核酸适配体空间结构发生改变，与目标物有机磷农药形成复合物，互补DNA从电极表面脱落，3'端的金纳米粒子被拉近到电极表面，发生能量转移（荧光猝灭），发光体碲化镉量子点的发光强度降低。The electrochemiluminescent biosensor for detecting organophosphorus pesticides obtained in this scheme detects organophosphorus pesticides based on the resonance energy transfer mechanism. Specifically, when the target is present, the spatial structure of the nucleic acid aptamer changes, forming a complex with the target organophosphorus pesticide, the complementary DNA falls off the electrode surface, and the gold nanoparticles at the 3' end are pulled closer to the electrode surface, energy transfer occurs (fluorescence quenching), and the luminescence intensity of the luminescent cadmium telluride quantum dots decreases.
如图1的制备过程所示，本申请提供一种检测有机磷农药电化学发光生物传感器的制备方法，包括以下步骤：As shown in the preparation process of FIG1 , the present application provides a method for preparing an electrochemiluminescent biosensor for detecting organophosphorus pesticides, comprising the following steps:
K1.抛光清洗玻碳电极，具体步骤为：使用Al2O3浆料在麂皮上抛光玻碳电极至镜面，1：1的硝酸、1：1的乙醇和蒸馏水分别超声清洗5~10min，彻底洗涤后，玻碳电极在0.5~1.0mol/L H2SO4溶液中使用循环伏安法活化，反复扫描直至达到稳定的循环伏安图为止，超纯水清洗电极表面，氮气干燥；K1. Polishing and cleaning of glassy carbon electrodes, specifically, the following steps: using Al2O3 slurry to polish the glassy carbon electrode on suede to a mirror surface, ultrasonically cleaning with 1:1 nitric acid, 1:1 ethanol and distilled water for 5-10 min respectively, after thorough cleaning, the glassy carbon electrode is activated by cyclic voltammetry in 0.5-1.0 mol/L H2SO4 solution, repeatedly scanning until a stable cyclic voltammogram is achieved, cleaning the electrode surface with ultrapure water, and drying with nitrogen;
K2.移取5~10μL（0.05~0.5mg/mL）碲化镉量子点@介孔聚多巴胺复合材料垂直滴涂在工作电极表面，进行电极改性，室温晾干后，得到镉化物量子点@介孔聚多巴胺/玻碳电极；K2. Pipette 5~10μL (0.05~0.5mg/mL) of cadmium telluride quantum dots@mesoporous polydopamine composite material and vertically drop-coat it on the surface of the working electrode to modify the electrode. After drying at room temperature, a cadmium telluride quantum dots@mesoporous polydopamine/glassy carbon electrode is obtained.
K3.在镉化物量子点@介孔聚多巴胺/玻碳电极表面滴涂5~10μL（0.5wt.%）的nafion溶液固定复合材料，室温晾干后，用水冲洗，去除电极表面多余的nafion溶液，得到nafion/镉化物量子点@介孔聚多巴胺/玻碳电极；K3. 5~10 μL (0.5 wt.%) of nafion solution was drop-coated on the surface of the cadmium quantum dot @ mesoporous polydopamine/glassy carbon electrode to fix the composite material, and after drying at room temperature, the composite material was rinsed with water to remove the excess nafion solution on the electrode surface to obtain a nafion/cadmium quantum dot @ mesoporous polydopamine/glassy carbon electrode;
K4.移取5~10μL（0.1~1.0μmol/L）滴涂于nafion/镉化物量子点@介孔聚多巴胺/玻碳电极表面，孵育过夜，通过磺酸基和氨基形成共价键固定分子探针到nafion/碲化镉量子点@介孔聚多巴胺/玻碳电极的传感界面，得到分子探针/nafion/镉化物量子点@介孔聚多巴胺/玻碳电极；K4. Pipette 5~10μL (0.1~1.0μmol/L) and drop-coat on the surface of nafion/cadmium quantum dots@mesoporous polydopamine/glassy carbon electrode, incubate overnight, and fix the molecular probe to the sensing interface of nafion/cadmium telluride quantum dots@mesoporous polydopamine/glassy carbon electrode through covalent bonds formed by sulfonic acid groups and amino groups to obtain molecular probe/nafion/cadmium quantum dots@mesoporous polydopamine/glassy carbon electrode;
K5.将5~10μL（0.1mol/L）封闭剂滴涂于分子探针/nafion/镉化物量子点@介孔聚多巴胺/玻碳电极表面，封闭非特异性结合位点，孵育反应时间为0.5~1.0h，PBS缓冲溶液（pH7.4）轻微冲洗电极表面，去除电极表面多余的6-巯基-1-己醇溶液，滴涂5~10μL（0.1~1.0μmol/L）的互补DNA，孵育1~3h，PBS缓冲溶液（pH7.4）轻微冲洗，得互补DNA/分子探针/nafion/镉化物量子点@介孔聚多巴胺/玻碳电极即检测有机磷农药电化学发光生物传感器。K5. Apply 5~10μL (0.1mol/L) of blocking agent to the surface of molecular probe/nafion/cadmium quantum dots@mesoporous polydopamine/glassy carbon electrode to block nonspecific binding sites. Incubate the reaction for 0.5~1.0h. Gently rinse the electrode surface with PBS buffer solution (pH7.4) to remove excess 6-mercapto-1-hexanol solution on the electrode surface. Apply 5~10μL (0.1~1.0μmol/L) of complementary DNA. Incubate for 1~3h. Gently rinse with PBS buffer solution (pH7.4) to obtain complementary DNA/molecular probe/nafion/cadmium quantum dots@mesoporous polydopamine/glassy carbon electrode, which is an electrochemiluminescent biosensor for detecting organophosphorus pesticides.
优选地，封闭剂为6-巯基-1-己醇溶液，封闭剂6-巯基-1-己醇溶液（MCH）封闭非特异性结合位点条件：在修饰电极hairpin-DNA-AuNPs在nafion/碲化镉量子点@介孔聚多巴胺/玻碳电极表面滴涂MCH溶液（0.1mol/L），室温孵育0.5~1.0h，使用PBS缓冲溶液（pH7.4）轻微冲洗，除去电极表面多余的MCH溶液。Preferably, the blocking agent is a 6-mercapto-1-hexanol solution. The blocking agent 6-mercapto-1-hexanol solution (MCH) blocks nonspecific binding sites under the following conditions: MCH solution (0.1 mol/L) is drop-coated on the surface of the modified electrode hairpin-DNA-AuNPs nafion/cadmium telluride quantum dots@mesoporous polydopamine/glassy carbon electrode, incubated at room temperature for 0.5-1.0 h, and gently rinsed with PBS buffer solution (pH 7.4) to remove excess MCH solution on the electrode surface.
如图2的检测过程所示，本申请提供一种检测有机磷农药电化学发光生物传感器在检测有机磷农药中的应用，检测方法为电化学发光法，检测的参比电极为银/氯化银电极，对电极为铂片电极或铂丝电极，工作电极为互补DNA/分子探针/nafion/镉化物量子点@介孔聚多巴胺/玻碳电极，测试步骤为孵育不同浓度的有机磷农药在制备的电化学发光生物传感器，37℃孵育反应1.0~3.0h，PBS缓冲溶液轻微冲洗，进行电化学发光法检测，记录发光强度，获得发光强度与浓度之间的工作曲线，优选地，检测的激发电压为2.0~0V，扫描速率为50-100mV/s。As shown in the detection process of Figure 2, the present application provides an application of an electrochemiluminescent biosensor for detecting organophosphorus pesticides in detecting organophosphorus pesticides. The detection method is electrochemiluminescence, the reference electrode for detection is a silver/silver chloride electrode, the counter electrode is a platinum sheet electrode or a platinum wire electrode, and the working electrode is a complementary DNA/molecular probe/nafion/cadmium quantum dots@mesoporous polydopamine/glassy carbon electrode. The test steps are to incubate different concentrations of organophosphorus pesticides in the prepared electrochemiluminescent biosensor, incubate the reaction at 37°C for 1.0~3.0h, rinse lightly with PBS buffer solution, perform electrochemiluminescence detection, record the luminescence intensity, and obtain a working curve between the luminescence intensity and the concentration. Preferably, the excitation voltage for detection is 2.0~0V, and the scanning rate is 50-100mV/s.
以下通过具体实施例对本申请进行进一步说明。The present application is further described below through specific examples.
实施例1Example 1
原材料准备：Raw material preparation:
水溶性羧基碲化镉量子点（CdTe-COOH QDs）的制备，具体合成步骤如下：Preparation of water-soluble carboxyl cadmium telluride quantum dots (CdTe-COOH QDs), the specific synthesis steps are as follows:
将氯化镉半（五水合物）（45.672mg，0.2mmol）和巯基乙酸（46.06mg，0.5mmol）依次分散到50mL蒸馏水中，然后滴加NaOH溶液调节Cd-TGA复合物溶液pH至7.0~11.0，通过将高纯度的N2鼓泡30min，使溶液脱氧。在剧烈搅拌下，依次加入亚碲酸钠溶液（4.4316mg，0.02mmol）和85%水合肼（0.377mL），然后，油浴锅中回流搅拌加热（100°C，4h），获得TGA封端的CdTe-COOH QD，最后，用过量的乙醇离心（8000rpm，10min）纯化粗的CdTe-COOH QDs，将洗涤后的CdTe-COOH QD分散在去离子水中，4℃黑暗条件下储存，如图4为碲化镉量子点的透射电镜图，粒径在5~10nm；Cadmium chloride hemi(pentahydrate) (45.672 mg, 0.2 mmol) and thioglycolic acid (46.06 mg, 0.5 mmol) were dispersed in 50 mL of distilled water in sequence, and then NaOH solution was added dropwise to adjust the pH of the Cd-TGA complex solution to 7.0~11.0, and the solution was deoxygenated by bubbling high-purity N2 for 30 min. Under vigorous stirring, sodium tellurite solution (4.4316 mg, 0.02 mmol) and 85% hydrazine hydrate (0.377 mL) were added in sequence, and then, the mixture was refluxed and stirred in an oil bath (100°C, 4 h) to obtain TGA-terminated CdTe-COOH QDs. Finally, the crude CdTe-COOH QDs were purified by centrifugation with excess ethanol (8000 rpm, 10 min), and the washed CdTe-COOH QDs were dispersed in deionized water and stored in the dark at 4°C. Figure 4 shows a transmission electron micrograph of cadmium telluride quantum dots with a particle size of 5-10 nm.
碲化镉量子点@介孔聚多巴胺复合材料（CdTe@MPDA）的制备，具体合成步骤如下：Preparation of cadmium telluride quantum dots @ mesoporous polydopamine composite material (CdTe@MPDA), the specific synthesis steps are as follows:
将1g嵌段式聚醚F-127和0.144g的水溶性羧基碲化镉量子点加入50mL乙醇水混合溶液中，剧烈搅拌，获得水溶性CdTe量子点分散在嵌段式聚醚F-127中的均一溶液。然后将1.0g盐酸多巴胺溶解在上述溶液中，再加入2.0mL的1, 3, 5-三甲苯到混合溶液中。随后，向反应的混合溶液中加入2.5mL氨水，搅拌反应4h。最后，使用乙醇水混合溶液离心和洗涤几次得到介孔聚多巴胺颗粒的产物，然后重新分散在一定量的水和乙醇混合溶液中超声处理一段时间，转移到一个密封的特氟隆内衬高压釜中，100℃加热24h，获得碲化镉量子点@介孔聚多巴胺复合材料。图3为碲化镉量子点、介孔聚多巴胺和碲化镉量子点@介孔聚多巴胺的X射线衍射图谱，碲化镉和介孔聚多巴胺复合后，相比于单独的碲化镉和介孔聚多巴胺，特征峰发生向右偏移且减弱。图5为不存在碲化镉量子点时制备的介孔聚多巴胺的透射电镜图，类球形多孔结构，粒径均一；图6为碲化镉量子点@介孔聚多巴胺的EDS元素分析图，复合材料由C、N、O、Cd和Te五种元素组成；图8为nafion/碲化镉量子点@介孔聚多巴胺/玻碳电极在0.1mol/L的过硫酸钾溶液中的循环伏安和电化学发光（ECL）循环稳定性图，在激发电压：2.0~0V和扫描速率：100mV/s条件下，碲化镉量子点与过硫酸钾发生反应，产生的电化学发光信号强且稳定等优点。以上实验表征结果，符合预期，碲化镉量子点@介孔聚多巴胺成功制备。1g of block polyether F-127 and 0.144g of water-soluble carboxyl cadmium telluride quantum dots were added to 50mL of ethanol-water mixed solution and stirred vigorously to obtain a uniform solution in which water-soluble CdTe quantum dots were dispersed in block polyether F-127. Then 1.0g of dopamine hydrochloride was dissolved in the above solution, and 2.0mL of 1, 3, 5-trimethylbenzene was added to the mixed solution. Subsequently, 2.5mL of ammonia water was added to the reacting mixed solution and stirred for 4h. Finally, the product of mesoporous polydopamine particles was obtained by centrifugation and washing several times with the ethanol-water mixed solution, and then redispersed in a certain amount of water and ethanol mixed solution for ultrasonic treatment for a period of time, transferred to a sealed Teflon-lined autoclave, and heated at 100℃ for 24h to obtain cadmium telluride quantum dots@mesoporous polydopamine composite materials. Figure 3 shows the X-ray diffraction patterns of cadmium telluride quantum dots, mesoporous polydopamine and cadmium telluride quantum dots@mesoporous polydopamine. After cadmium telluride and mesoporous polydopamine are composited, the characteristic peak shifts to the right and weakens compared to the individual cadmium telluride and mesoporous polydopamine. Figure 5 is a transmission electron microscope image of mesoporous polydopamine prepared in the absence of cadmium telluride quantum dots, with a spherical porous structure and uniform particle size; Figure 6 is an EDS elemental analysis of cadmium telluride quantum dots @ mesoporous polydopamine, and the composite material is composed of five elements: C, N, O, Cd and Te; Figure 8 is a cyclic voltammetry and electrochemiluminescence (ECL) cycle stability diagram of nafion/cadmium telluride quantum dots @ mesoporous polydopamine/glassy carbon electrode in 0.1 mol/L potassium persulfate solution. Under the conditions of excitation voltage: 2.0~0V and scan rate: 100mV/s, cadmium telluride quantum dots react with potassium persulfate, and the generated electrochemiluminescence signal is strong and stable. The above experimental characterization results are in line with expectations, and cadmium telluride quantum dots @ mesoporous polydopamine is successfully prepared.
使用以上原料，本实施例提供一种检测有机磷农药电化学发光生物传感器，包括玻碳电极，以及从内到外依次修饰于玻碳电极表面的碲化镉量子点@介孔聚多巴胺纳米材料、nafion膜层、hairpin-DNA-AuNPs分子探针、互补DNA；hairpin-DNA-AuNPs分子探针包括5'端修饰氨基和3'端修饰二硫基的核酸适配体，与核酸适配体的3'端偶联的金纳米粒子，核酸适配体的二级结构为发夹结构；互补DNA与分子探针通过碱基互补配对。Using the above raw materials, this embodiment provides an electrochemiluminescent biosensor for detecting organophosphorus pesticides, including a glassy carbon electrode, and cadmium telluride quantum dots@mesoporous polydopamine nanomaterials, nafion membrane layers, hairpin-DNA-AuNPs molecular probes, and complementary DNA, which are sequentially modified on the surface of the glassy carbon electrode from the inside to the outside; the hairpin-DNA-AuNPs molecular probe includes a nucleic acid aptamer modified with an amino group at the 5' end and a disulfide group at the 3' end, and gold nanoparticles coupled to the 3' end of the nucleic acid aptamer, and the secondary structure of the nucleic acid aptamer is a hairpin structure; the complementary DNA and the molecular probe are paired through base complementarity.
检测有机磷农药电化学发光生物传感器的制备方法，包括以下步骤：The preparation method of the electrochemiluminescent biosensor for detecting organophosphorus pesticides comprises the following steps:
K1.玻碳电极预处理，使用Al2O3浆料在麂皮上抛光玻碳电极至镜面，1：1的硝酸、1：1的乙醇和蒸馏水分别超声清洗5min，彻底洗涤后，玻碳电极在0.5mol/L H2SO4溶液中使用循环伏安法活化，反复扫描直至达到稳定的循环伏安图为止；K1. Pretreatment of glassy carbon electrode: polish the glassy carbon electrode to a mirror surface using Al2O3 slurry on suede, and ultrasonically clean it for 5 min using 1:1 nitric acid, 1:1 ethanol and distilled water respectively. After thorough washing, the glassy carbon electrode is activated using cyclic voltammetry in 0.5 mol/L H2SO4 solution, and scanned repeatedly until a stable cyclic voltammogram is achieved.
K2.移取10μL（0.5mg/mL）碲化镉量子点@介孔聚多巴胺复合材料垂直滴涂到预处理后的电极表面，进行电极改性，室温自然晾干；K2. Pipette 10 μL (0.5 mg/mL) of cadmium telluride quantum dots@mesoporous polydopamine composite material and vertically drop-coat it onto the pretreated electrode surface to modify the electrode, and then dry it naturally at room temperature;
K3.滴涂5μL（0.5wt.%）的nafion溶液固定复合材料，蒸馏水轻微冲洗，室温自然晾干后，蒸馏水轻微冲洗，去除电极表面多余的nafion溶液；K3. Apply 5 μL (0.5 wt.%) of nafion solution to fix the composite material, rinse lightly with distilled water, dry naturally at room temperature, and then rinse lightly with distilled water to remove excess nafion solution on the electrode surface;
K4.固定分子探针hairpin-DNA-AuNPs在nafion/碲化镉量子点@介孔聚多巴胺/玻碳电极，移取10μL（1.0μmol/L）hairpin-DNA-AuNPs溶液，滴涂在修饰电极表面，通过磺酸基和氨基形成共价键固定分子探针到nafion/碲化镉量子点@介孔聚多巴胺/玻碳电极的传感界面，过夜孵育；K4. Immobilize the molecular probe hairpin-DNA-AuNPs on the nafion/cadmium telluride quantum dots@mesoporous polydopamine/glassy carbon electrode. Pipette 10 μL (1.0 μmol/L) hairpin-DNA-AuNPs solution and drop it on the surface of the modified electrode. Immobilize the molecular probe to the sensing interface of the nafion/cadmium telluride quantum dots@mesoporous polydopamine/glassy carbon electrode through the formation of covalent bonds between the sulfonic acid group and the amino group, and incubate overnight.
K5.滴涂5μL（0.1mol/L）6-巯基-1-己醇溶液在修饰电极表面，封闭非特异性结合位点，孵育反应时间为1.0h，PBS缓冲溶液（pH7.4）轻微冲洗电极表面，去除电极表面多余的6-巯基-1-己醇溶液；滴涂10μL（1.0μmol/L）的互补DNA在分子探针/nafion/碲化镉量子点@介孔聚多巴胺/玻碳电极修饰电极表面，孵育3h，传感器制备完成，用于有机磷农药的检测，不用时放在冰箱4℃保存备用。使用循环伏安法对电化学发光生物传感器制备过程进行表征，通过对传感器每一步制备过程进行表征（图9），传感器的制备是成功的。K5. 5 μL (0.1 mol/L) 6-mercapto-1-hexanol solution was applied to the modified electrode surface to block nonspecific binding sites. The incubation reaction time was 1.0 h. The electrode surface was lightly rinsed with PBS buffer solution (pH 7.4) to remove excess 6-mercapto-1-hexanol solution on the electrode surface. 10 μL (1.0 μmol/L) of complementary DNA was applied to the surface of the molecular probe/nafion/cadmium telluride quantum dots@mesoporous polydopamine/glassy carbon electrode modified electrode. The sensor was incubated for 3 h. The sensor was prepared and used for the detection of organophosphorus pesticides. When not in use, it was stored in a refrigerator at 4 °C for standby use. The preparation process of the electrochemiluminescence biosensor was characterized by cyclic voltammetry. By characterizing each step of the sensor preparation process (Figure 9), the preparation of the sensor was successful.
应用例Application Examples
利用实施例1制备的电化学发光生物传感器对有机磷农药检测的可行性分析：Feasibility analysis of organophosphorus pesticide detection using the electrochemiluminescent biosensor prepared in Example 1:
电化学发光生物传感器测试：使用经典的三电极***，Ag/AgCl电极(饱和甘汞电极)为参比电极，铂片电极（5×5×0.2mm）为对电极，6-巯基-1-己醇/nafion/碲化镉量子点@介孔聚多巴胺/玻碳电极修饰电极为工作电极，之后，将5μL不同浓度的有机磷农药，室温孵育反应1h，使用PBS缓冲溶液（pH7.4）轻微冲洗。在过硫酸钾溶液（0.1mol/L）中进行电化学发光测试，使用循环伏安法施加一定的电压，激发电压：2.0~0V，扫描速率：100mV/s，记录测试不同浓度有机磷农药获得的发光强度。结果如图10所示，由于有机磷农药的存在，核酸适配体构象发生改变，互补DNA从电极表面脱落，修饰在分子探针核酸适配体的3'端的金纳米粒子拉近到电极表面，猝灭效果增强，有机磷农药的浓度越大，发光强度越小。Electrochemiluminescence biosensor test: using the classic three-electrode system, Ag/AgCl electrode (saturated calomel electrode) as the reference electrode, platinum electrode (5×5×0.2mm) as the counter electrode, 6-mercapto-1-hexanol/nafion/cadmium telluride quantum dots@mesoporous polydopamine/glassy carbon electrode modified electrode as the working electrode, then 5μL of organophosphorus pesticides of different concentrations were incubated at room temperature for 1h and lightly rinsed with PBS buffer solution (pH7.4). Electrochemiluminescence test was carried out in potassium persulfate solution (0.1mol/L), and a certain voltage was applied using cyclic voltammetry, excitation voltage: 2.0~0V, scan rate: 100mV/s, and the luminescence intensity obtained by testing organophosphorus pesticides of different concentrations was recorded. The results are shown in Figure 10. Due to the presence of organophosphorus pesticides, the conformation of the aptamer changes, the complementary DNA falls off the electrode surface, and the gold nanoparticles modified at the 3' end of the molecular probe aptamer are pulled closer to the electrode surface, the quenching effect is enhanced, and the higher the concentration of the organophosphorus pesticide, the lower the luminescence intensity.
以上，仅为本发明较佳的具体实施方式，但本发明的保护范围并不局限于此，任何熟悉本技术领域的技术人员在本发明揭露的技术范围内，可轻易想到的变化或替换，都应涵盖在本发明的保护范围之内。The above are only preferred specific implementations of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by any technician familiar with the technical field within the technical scope disclosed by the present invention should be covered within the protection scope of the present invention.
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