CN103543180A - Immune impedance biosensor detection method and device for quick pesticide residue detection - Google Patents
Immune impedance biosensor detection method and device for quick pesticide residue detectionDownload PDFInfo
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Abstract
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Description
Translated fromChinese技术领域technical field
本发明涉及一种对农药残留速测的免疫阻抗生物传感器检测方法和检测装置。The invention relates to an immunoimpedance biosensor detection method and a detection device for rapid detection of pesticide residues.
背景技术Background technique
各国政府不断加强农药残留量的监测工作，制定的标准越来越严格。农药残留检测和定量分析的常规方法是色谱、色谱-质谱联用等。这些方法的测定结果准确可靠，是当前农残分析的主流技术。但这些方法也存在仪器较昂贵，前处理时间长、过程烦琐等不足，只适合于在专业实验室中使用。目前，国内市场上的农药速测方法以酶抑制法应用较多，它是利用农药对某些特定酶，特别是胆碱酯酶的抑制作用，通过对比检测水解后的底物或水解产物，以比色或分光光度分析的方法判定酶是否被抑制，从而测定农药残留。这种方法快速、简单、方便，但也存在如下不足：Governments of various countries continue to strengthen the monitoring of pesticide residues, and the standards established are becoming more and more stringent. Conventional methods for the detection and quantitative analysis of pesticide residues are chromatography, chromatography-mass spectrometry, etc. The determination results of these methods are accurate and reliable, and they are the mainstream techniques for the analysis of pesticide residues. However, these methods also have disadvantages such as relatively expensive instruments, long pretreatment time, and cumbersome processes, and are only suitable for use in professional laboratories. At present, the rapid detection method of pesticides in the domestic market is mostly used in the enzyme inhibition method, which uses the inhibitory effect of pesticides on certain specific enzymes, especially cholinesterase, and detects the hydrolyzed substrate or hydrolyzate by comparison. Determine whether the enzyme is inhibited by colorimetric or spectrophotometric analysis, thereby determining pesticide residues. This method is fast, simple and convenient, but it also has the following disadvantages:
1、只能检测农药的总残留量，不能够区分具体农药的种类；1. It can only detect the total residues of pesticides, but cannot distinguish the specific types of pesticides;
2、显色不稳定，容易受周围环境、温度等影响，给判断带来误差；2. The color rendering is unstable, and it is easily affected by the surrounding environment and temperature, which will bring errors to the judgment;
3、检测精密度不高，易出现假阳性、假阴性结果；3. The detection precision is not high, and false positive and false negative results are prone to occur;
4、酶反应条件苛刻，对操作要求高；4. Enzyme reaction conditions are harsh and require high operation;
中国专利ZL 01253887.6 公开了探测农药残留用的量热式生物传感器，它是通过测量流经固定化酶柱的待测液体的温度变化来检测待测液体中的农药含量。其不足之处在于：结构复杂，所用的固定化酶柱要求高。国际上研究快速检测农药的生物传感器论文在也有发表，主要还是基于酶生物传感器，未见有用于检测农药的免疫阻抗生物传感器。Chinese patent ZL 01253887.6 discloses a calorimetric biosensor for detecting pesticide residues, which detects the pesticide content in the liquid to be tested by measuring the temperature change of the liquid to be tested flowing through the immobilized enzyme column. Its disadvantages are that the structure is complex and the requirements for the immobilized enzyme column are high. International papers on biosensors for rapid detection of pesticides have also been published in China, mainly based on enzyme biosensors, and no immunoimpedance biosensors for pesticide detection have been seen.
发明内容Contents of the invention
本发明的目的在于提供一种用于液体中农药残留检测的免疫阻抗生物传感器的检测方法,它可以快速测定目标农药在样品中的残留量，准确度好，灵敏度高。The object of the present invention is to provide a detection method of an immunoimpedance biosensor for detecting pesticide residues in liquids, which can quickly measure the residues of target pesticides in samples with good accuracy and high sensitivity.
本发明的用于农药残留速测的免疫阻抗生物传感器检测方法，使用免疫阻抗生物传感器检测装置，免疫阻抗生物传感器检测装置包括与阻抗分析仪电连接的工作电极、对电极和参比电极伸入到电解槽内；阻抗分析仪与计算机相连；该检测方法包括下述步骤：The immunoimpedance biosensor detection method for rapid detection of pesticide residues of the present invention uses an immunoimpedance biosensor detection device, which includes a working electrode electrically connected to an impedance analyzer, a counter electrode and a reference electrode extending into the In the electrolyzer; Impedance analyzer is connected with computer; This detection method comprises the following steps:
a. 电极清洗a. Electrode cleaning
以清洗物质对工作电极、对电极和参比电极进行清洗；Clean the working electrode, counter electrode and reference electrode with cleaning substances;
b. 电极预处理b. Electrode pretreatment
以硅烷溶液对电极处理；再以甲醇和水清洗电极去除非特异性吸附；Treat the electrode with silane solution; then wash the electrode with methanol and water to remove non-specific adsorption;
c. 抗体固定c. Antibody immobilization
把农药抗体溶液注入电解槽内，使抗体通过交联固定在工作电极上；以磷酸盐缓冲液和去离子水，对电极进行清洗；Inject the pesticide antibody solution into the electrolytic cell to fix the antibody on the working electrode through cross-linking; wash the electrode with phosphate buffer and deionized water;
d. 阻抗测试d. Impedance test
把待测液体样本注入电解槽，使得待测液体样本与电极上的固定抗体发生特异性反应；然后以磷酸盐缓冲液通清洗电极，再把含有[Fe(CN)6] 3-或者[Fe(CN)6] 4-的电解液注入电解槽内，然后以阻抗分析仪和计算机检测并计算出电解液的法拉第阻抗；The liquid sample to be tested is injected into the electrolytic cell, so that the liquid sample to be tested reacts specifically with the immobilized antibody on the electrode; then the electrode is washed with phosphate buffer solution, and then the electrode containing [Fe(CN)6 ]3- or [Fe (CN)6 ] The electrolyte of4- is injected into the electrolytic cell, and then the Faraday impedance of the electrolyte is detected and calculated by an impedance analyzer and a computer;
e. 数据分析e. Data analysis
对不同浓度的农药标准溶液，分别按照上述a-d步骤，得到与各浓度的农药相应的法拉第阻抗，制定农药浓度与拉第阻抗的标准曲线；将待测液体样本按照上述a-d步骤得到的法拉第阻抗，根据标准曲线判断待测液体样本中的农药浓度。For the pesticide standard solutions of different concentrations, follow the steps a-d above to obtain the Faraday impedance corresponding to the pesticides of each concentration, and formulate the standard curve of pesticide concentration and Faraday impedance; the Faraday impedance obtained by the liquid sample to be tested according to the steps a-d above, Determine the pesticide concentration in the liquid sample to be tested according to the standard curve.
上述的检测方法，步骤a中，对电极清洗时，分别以丙酮、乙醇和去离子水进行清洗，再以1:1:5 (v/v) H2O2/NH4OH/H2O溶液清洗，10分钟后，再以去离子水冲洗，后通入氮气干燥；In the above detection method, in step a, when cleaning the electrodes, wash them with acetone, ethanol and deionized water respectively, and then wash them with 1:1:5 (v/v) H2 O2 /NH4 OH/H2 O Solution cleaning, after 10 minutes, rinse with deionized water, and then pass through nitrogen to dry;
步骤b中，硅烷溶液为3-氨丙基三乙氧基硅烷；In step b, the silane solution is 3-aminopropyltriethoxysilane;
步骤c中，含牛血清白蛋白的磷酸盐缓冲液中含有体积比为1%的牛血清白蛋白，磷酸盐缓冲液的PH值为 7.4；In step c, the phosphate buffer containing bovine serum albumin contains bovine serum albumin with a volume ratio of 1%, and the pH of the phosphate buffer is 7.4;
步骤d中，电解液中，[Fe(CN)6] 3-与[Fe(CN)6] 4-的浓度比1:1；法拉第阻抗测试频率1Hz -100kHz，振幅为±5mV。In step d, the concentration ratio of [Fe(CN)6 ]3- to [Fe(CN)6 ]4- in the electrolyte is 1:1; the frequency of the Faraday impedance test is 1 Hz-100 kHz, and the amplitude is ±5 mV.
本发明的有益效果：本发明的检测方法是以流动注射的方式，将农药抗体固定在工作电极上，作为生物传感器的敏感元件。固定抗体与溶液中的农药抗原发生特异性结合后，在电极表面形成“抗原—抗体”的复合物，改变双电层的电荷转移和表面层的电阻。通过测定固定抗体与不同浓度标准农药作用后系统的阻抗值的变化，制定标准曲线，再测量待测农药与固定抗体作用后的阻抗值，并与标准曲线比较，从而得出待测农药中的目标农药的含量。本发明的检测方法快速、简单、方便，同时准确度高，灵敏度高，重复性好。Beneficial effects of the present invention: the detection method of the present invention is to immobilize the pesticide antibody on the working electrode as the sensitive element of the biosensor in the way of flow injection. After the immobilized antibody specifically binds to the pesticide antigen in the solution, an "antigen-antibody" complex is formed on the electrode surface, which changes the charge transfer of the electric double layer and the resistance of the surface layer. By measuring the change of the impedance value of the system after the action of the immobilized antibody and different concentrations of standard pesticides, a standard curve is established, and then the impedance value after the action of the pesticide to be tested and the immobilized antibody is measured, and compared with the standard curve, so as to obtain the concentration of the pesticide to be tested. The content of the target pesticide. The detection method of the invention is fast, simple and convenient, and meanwhile has high accuracy, high sensitivity and good repeatability.
本发明同时提供一种为在上述检测方法中使用的免疫阻抗生物传感器检测装置，它结构简单、制作方便、成本低廉，可以反复使用，重复性好，能够保证检测的一致性。The present invention also provides an immunoimpedance biosensor detection device used in the above detection method, which has simple structure, convenient manufacture, low cost, can be used repeatedly, has good repeatability, and can ensure the consistency of detection.
本发明的在上述检测方法中使用的免疫阻抗生物传感器检测装置，包括注射泵、转盘、电解槽、排出泵；与阻抗分析仪电连接的工作电极、对电极和参比电极伸入到电解槽内；阻抗分析仪与计算机相连；转盘内具有沿着径向延伸的6条通道：电解液通道、磷酸盐缓冲液通道、待测样本通道、电极清洗通道、电极预处理通道、抗体注入通道；6条通道的入口和出口分别位于转盘的同一圆周上；当转盘绕中心转动时，一通道的入口和出口同时分别与注射泵的出口、电解槽的入口相通；电解槽的出口与排出泵的入口相通。The immunoimpedance biosensor detection device used in the above detection method of the present invention includes a syringe pump, a turntable, an electrolytic cell, and a discharge pump; the working electrode, the counter electrode, and the reference electrode that are electrically connected to the impedance analyzer extend into the electrolytic cell Inside; the impedance analyzer is connected to the computer; there are 6 channels extending radially in the turntable: electrolyte channel, phosphate buffer channel, sample channel to be tested, electrode cleaning channel, electrode pretreatment channel, antibody injection channel; The inlets and outlets of the six channels are respectively located on the same circumference of the turntable; when the turntable rotates around the center, the inlet and outlet of one channel are connected to the outlet of the injection pump and the inlet of the electrolytic cell respectively; the outlet of the electrolytic cell is connected to the discharge pump. The entrances are connected.
所述的免疫阻抗生物传感器检测装置，还包括与排出泵的出口相通的废液池。The immunoimpedance biosensor detection device also includes a waste liquid pool communicated with the outlet of the discharge pump.
所述的免疫阻抗生物传感器检测装置，工作电极为盘阵微电极或叉指式带列微电极。In the immunoimpedance biosensor detection device, the working electrode is a disk array microelectrode or an interdigitated strip microelectrode.
本免疫阻抗生物传感器检测装置的有益效果：使用时，均使用注射泵泵入各种物质；而且通过转盘的转动，能够使得不同的通道分别与注射泵和电解槽相通，以通过不同的通道注入不同的物质，保证检测步骤的一致性和重复性，使用检测的结果准确性高。The beneficial effect of the immunoimpedance biosensor detection device: when in use, various substances are pumped in by a syringe pump; and through the rotation of the turntable, different channels can be communicated with the syringe pump and the electrolytic cell respectively, so that the substances can be injected through different channels Different substances ensure the consistency and repeatability of the detection steps, and the accuracy of the detection results is high.
附图说明Description of drawings
图1是免疫阻抗生物传感器检测装置的示意图；1 is a schematic diagram of an immunoimpedance biosensor detection device;
图2是免疫阻抗生物传感器检测装置的原理框图。Fig. 2 is a functional block diagram of an immunoimpedance biosensor detection device.
图3是电解槽的等效电路图。Fig. 3 is an equivalent circuit diagram of an electrolytic cell.
图4是对硫磷农药浓度—阻抗标准曲线。Figure 4 is a parathion pesticide concentration-impedance standard curve.
具体实施方式Detailed ways
参见图1、2所示的用于农药残留速测的免疫阻抗生物传感器检测装置，由驱动系统、进样系统、反应系统、检测系统、控制系统组成。驱动系统由注射泵1，排出泵10组成，进样系统由转盘2、导管3组成，电解槽4的反应系统，检测系统由工作电极5，对电极6，参比电极7组成，控制系统由阻抗分析仪8，计算机9组成，从电解槽的出口流出的废液经排出泵10排入废液池11。工作电极可以为叉指式带列微电极或者盘阵微电极（圆盘电极），对电极为铂电极，参比电极为 Ag/AgCl 电极。Referring to Figures 1 and 2, the immunoimpedance biosensor detection device for rapid detection of pesticide residues is composed of a drive system, a sample injection system, a reaction system, a detection system, and a control system. The driving system is composed of a
与阻抗分析仪电连接的工作电极5、对电极6和参比电极7伸入到电解槽内；阻抗分析仪与计算机相连。转盘2内具有沿着径向延伸的6条通道：电解液通道A、磷酸盐缓冲液通道B、待测样本通道C、电极清洗通道D、电极预处理通道E、抗体注入通道F；6条通道的入口位于靠近转盘中心部的同一内圆的圆周上；注射泵的出口与所述6条通道入口所在内圆的圆周密封接触。6条通道的出口位于转盘外周的同一圆周上。导管的进口与所述6条通道出口所在外圆的圆周密封接触。导管的出口与电解槽的入口相通；电解槽的出口通过排液泵与废液池相通。口当转盘绕中心转动时，6条通道中任一通道的入口和出口可以同时分别与注射泵的出口、导管的入口相通。The working electrode 5, the counter electrode 6 and the reference electrode 7 electrically connected with the impedance analyzer extend into the electrolytic cell; the impedance analyzer is connected with the computer. There are 6 channels extending radially in the turntable 2: electrolyte channel A, phosphate buffer channel B, sample channel C to be tested, electrode cleaning channel D, electrode pretreatment channel E, antibody injection channel F; 6 channels The inlets of the channels are located on the same inner circle near the center of the turntable; the outlet of the syringe pump is in sealing contact with the inner circles where the inlets of the six channels are located. The outlets of the six passages are located on the same circle of the outer periphery of the turntable. The inlet of the conduit is in sealing contact with the circumference of the outer circle where the outlets of the six passages are located. The outlet of the conduit communicates with the inlet of the electrolyzer; the outlet of the electrolyzer communicates with the waste liquid pool through the drain pump. When the turntable rotates around the center, the inlet and outlet of any one of the six channels can communicate with the outlet of the syringe pump and the inlet of the catheter respectively at the same time.
对农药残留速测的免疫阻抗生物传感器检测方法，是对电极经过预处理后，固定农药抗体，加样检测得到交流阻抗谱，进行等效电路拟合，通过比较不同浓度农药作用的电荷转移电阻Ret值，制定标准曲线；测得待测农药作用下的电荷转移电阻Ret值，并与标准曲线比较，判定待测物中的目标农药残留浓度。其具体方法如下：The immunoimpedance biosensor detection method for rapid detection of pesticide residues is to fix the pesticide antibody after the electrode is pretreated, add samples to detect the AC impedance spectrum, perform equivalent circuit fitting, and compare the charge transfer resistance of different concentrations of pesticides. Ret value, formulate a standard curve; measure the charge transfer resistance Ret value under the action of the pesticide to be tested, and compare it with the standard curve to determine the target pesticide residue concentration in the test substance. The specific method is as follows:
1、建立对硫磷农药的浓度—阻抗标准曲线。1. Establish the concentration-impedance standard curve of parathion pesticide.
a. 电极清洗a. Electrode cleaning
以注射泵在2mL/min的速度下分别把4 mL丙酮、乙醇和去离子水通过电极清洗通道D注入电解槽内并通过排出泵排出，再以注射泵把4mL的1:1:5 (v/v) H2O2 /NH4OH/H2O混合溶液通过电极清洗通道D注入电解槽内并排出，10分钟后，再以注射泵注入去离子水彻底冲洗并排出，后通入氮气干燥；对工作电极、对电极和参比电极进行清洗并干燥。Use a syringe pump to inject 4 mL of acetone, ethanol and deionized water into the electrolytic cell through the electrode cleaning channel D at a speed of 2 mL/min and discharge it through the discharge pump, and then use a syringe pump to inject 4 mL of 1:1:5 (v /v) H2 O2 /NH4 OH/H2 O mixed solution is injected into the electrolytic cell through the electrode cleaning channel D and discharged. After 10 minutes, inject deionized water with a syringe pump to rinse thoroughly and discharge, and then pass nitrogen gas Drying; wash and dry the working, counter, and reference electrodes.
b. 电极预处理b. Electrode pretreatment
以注射泵把4mL 2% 3-氨丙基三乙氧基硅烷溶液通过电极预处理通道E注入电解槽内并排出，处理电极工作；再以注射泵分别把甲醇和水清洗通过电极清洗通道注入电解槽内并排出，清洗电极，去除非特异性吸附；Use a syringe pump to inject 4mL of 2% 3-aminopropyltriethoxysilane solution into the electrolytic cell through the electrode pretreatment channel E and discharge it to treat the electrode work; then use a syringe pump to clean methanol and water respectively and inject it through the electrode cleaning channel In the electrolytic cell and discharged, the electrode is cleaned to remove non-specific adsorption;
c. 抗体固定c. Antibody immobilization
以注射泵把1mg/mL浓度的对硫磷多克隆抗体溶液4mL，在0.5mL/min的速度下，通过抗体注入通道注入电解槽内并排出，使抗体通过交联固定在工作电极上；20分钟后以注射泵把含1%血清白蛋白的磷酸盐缓冲液（PH值为7.4）通过磷酸盐缓冲液通道注入电解槽内并排出，以注射泵把去离子水通过电极清洗通道注入电解槽内并排出，对电极进行清洗，去除未绑定的抗体。Use a syringe pump to inject 4 mL of parathion polyclonal antibody solution with a concentration of 1 mg/mL at a speed of 0.5 mL/min into the electrolytic cell through the antibody injection channel and discharge it, so that the antibody is fixed on the working electrode through cross-linking; 20 Minutes later, use a syringe pump to inject 1% serum albumin-containing phosphate buffer (pH 7.4) into the electrolytic cell through the phosphate buffer channel and discharge it, and use a syringe pump to inject deionized water into the electrolytic cell through the electrode cleaning channel Inside and out, the electrode is washed to remove unbound antibody.
d. 阻抗测试d. Impedance test
以注射泵把不同浓度的农药标准溶液通过待测样本通道注入电解槽并排出，使得农药标准溶液与电极上的固定抗体发生特异性反应；30分钟后以注射泵把磷酸盐缓冲液通过磷酸盐缓冲液通道注入电解槽内并排出，再以注射泵把0.01 mol/L 的[Fe (CN)6] 3-/4-（1:1）磷酸盐电解液通过电解液通道A泵入电解槽内，然后以阻抗分析仪和计算机检测电解液的交流阻抗谱，阻抗分析仪的采样频率范围1Hz-100 kHz，振幅为±5 mV。对交流阻抗谱采用图3所示的等效电路拟合，计算出与农药浓度相应的法拉第阻抗。图3中，Ret表示电荷转移电阻，Rt表示溶液电阻，Cdl表示双层电容，CPE表示常相位角元件, 代表氧化还原探针的属性。Use a syringe pump to inject pesticide standard solutions of different concentrations into the electrolytic cell through the sample channel to be tested and discharge, so that the pesticide standard solution reacts specifically with the immobilized antibody on the electrode; after 30 minutes, use a syringe pump to pass the phosphate buffer solution through the phosphate The buffer channel is injected into the electrolytic cell and discharged, and then the 0.01 mol/L [Fe (CN)6 ]3-/4- (1:1) phosphate electrolyte is pumped into the electrolytic cell through the electrolyte channel A by a syringe pump Then, the AC impedance spectrum of the electrolyte was detected by an impedance analyzer and a computer. The sampling frequency range of the impedance analyzer was 1 Hz-100 kHz, and the amplitude was ±5 mV. The equivalent circuit shown in Figure 3 is used to fit the AC impedance spectrum, and the Faraday impedance corresponding to the pesticide concentration is calculated. In Figure 3, Ret represents the charge transfer resistance, Rt represents the solution resistance, Cdl represents the double layer capacitance, and CPE represents the constant phase angle element, representing the properties of the redox probe.
e. 数据分析e. Data analysis
根据得到的与各浓度的农药相应的法拉第阻抗，制定农药浓度与拉第阻抗的标准曲线，如图4所示。图4中，纵坐标表示电荷转移电阻，单位欧姆；横坐标表示对硫磷农药的浓度，单位纳克/毫升。According to the obtained Faradaic impedance corresponding to each concentration of pesticides, a standard curve of pesticide concentration and Faradaic impedance was established, as shown in Figure 4. In Fig. 4, the ordinate represents the charge transfer resistance, the unit is ohm; the abscissa represents the concentration of parathion pesticide, the unit is nanogram/milliliter.
2、对硫磷农药的快速检测2. Rapid detection of parathion pesticide
步骤a、b、c、d与上述相同，只不过以待测对硫磷农药代替农药标准溶液，计数出与待测对硫磷农药的农药浓度相应的法拉第阻抗。然后以把待测对硫磷农药的法拉第阻抗与标准曲线比较，计算待测对硫磷农药中的农药浓度。Steps a, b, c, and d are the same as above, except that the pesticide standard solution is replaced by the parathion pesticide to be tested, and the Faraday impedance corresponding to the pesticide concentration of the parathion pesticide to be tested is counted. Then by comparing the Faraday impedance of the parathion pesticide to be tested with the standard curve, the pesticide concentration in the parathion pesticide to be tested is calculated.
为了达到传感器重复使用的目的，测试完后，在通过磷酸盐缓冲液通道泵入磷酸盐缓冲液冲洗，再通过电极清洗通道泵入蛋白质去垢剂，去除电极表面结合的抗原抗体复合物。In order to achieve the purpose of reusing the sensor, after the test, the phosphate buffer is pumped through the phosphate buffer channel to rinse, and then the protein detergent is pumped through the electrode cleaning channel to remove the antigen-antibody complex bound to the electrode surface.
本发明可以快速测定目标农药在样品中的残留量，准确度好，灵敏度高，可以重复使用。The invention can quickly measure the residual amount of the target pesticide in the sample, has good accuracy, high sensitivity and can be used repeatedly.
最后，还需要注意的是，以上列举的仅是本发明的具体实施例。显然，本发明不限于以上实施例，还可以有许多变形。Finally, it should also be noted that what is listed above are only specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments, and many variations are possible.
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| CN115968445A (en)* | 2020-04-08 | 2023-04-14 | 以色列农业和农村发展部农业研究组织(范卡尼中心) | System and method for determining the prevalence of SARS-CoV-2 in a population |
| CN113930816A (en)* | 2021-10-29 | 2022-01-14 | 无锡乐普金属科技有限公司 | Method for electroplating molybdenum-copper alloy surface |
| CN116148315A (en)* | 2021-11-19 | 2023-05-23 | 天津大学 | Nondestructive and in-situ detection method for predicting elongation change trend of 625 alloy after tensile fracture |
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