检测水中微囊藻毒素-LR电化学免疫传感器
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摘要
本研究成功开发了两种类型的基于丝网印刷碳电极的电化学免疫传感器。一是基于电催化还原与间接竞争免疫检测原理，通过自主制备锇氧化还原聚合物修饰丝网印刷碳电极，开发了电流型免疫传感器系统；二是研制了一种新型基于多壁碳纳米管修饰的阻抗型免疫传感器，上述两种传感器可对水中微量污染物实现快速定量检测。研究中在系统分析影响传感器性能的基础上，以微囊藻毒素-LR为目标物，建立了水中污染物电流型和阻抗型免疫传感器的修饰技术和检测方法，并用于实际水样检测。
主要研究成果是：（1）提出了锇氧化还原聚合物修饰丝网印刷碳电极的方法，即先将小分子污染物微囊藻毒素-LR与经戊二醛活化后的惰性鸡卵白蛋白结合制备成微囊藻毒素-LR的抗原复合物，再将该复合物与锇氧化还原聚合物、牛血清白蛋白通过混合交联固定的方式固定在丝网印刷碳电极表面上的传感器修饰方法。（2）建立了多壁碳纳米管修饰丝网印刷碳电极的方法，即采用聚乙烯亚胺作为多壁碳纳米管分散剂，先制备出沉降性质稳定的多壁碳纳米管悬浊液，再将其修饰在丝网印刷碳电极表面，活化的抗原复合物通过醛基与聚乙烯亚胺上的胺基发生共价缩合反应修饰在电极表面。（3）采用扫描电子显微镜、循环伏安以及阻抗谱扫描，综合表征并分析了两种传感器的表面形态和电化学性能。研究结果表明，修饰后的传感器对微囊藻毒素-LR的抗体表现出良好的特异性响应，而非特异性吸附很弱。（4）实际水样检测基质效应研究结果表明：受试的甲醇等基质对样品检测没有明显影响，样品检测适宜pH范围为6~8，腐殖酸和铜离子对检测影响较大；研究中通过在预温育反应混合物中添加1%螯合剂乙二胺四乙酸和10mg/mL牛血清白蛋白，能有效抑制样品中基质对免疫传感器检测的影响。（5）电流型免疫传感器检测微囊藻毒素-LR的检测限为0.17μg/L，定量检测区间为0.43~10.72μg/L，检测周期约为80min；阻抗型免疫传感器检测微囊藻毒素-LR的检测限为0.58μg/L，定量检测区间为0.76~7.40μg/L，检测周期约为55min。检测不同类型实际水样中的微囊藻毒素-LR的结果表明：研制的传感器的检测变异系数均在10%之内，回收率在83%~121%之间，与高效液相色谱的分析结果相比具有良好的线性相关性，且具有较好的精确度和稳定性，可满足环境检测的需要。
Two different electrochemical immunosensors based on screen-printedcarbon electrode (SPCE) have been successfully developed. The first one is anamperometric immunosensor system based on the integrated principle ofelectrocatalytic reduction and indirect competitive immunoassay, which hasbeen developed by using a self-made osmium redox polymer (OsPVP) modifiedon SPCE. The second one is a new impedimetric immunosensor based on themodification of multi-wall carbon nanotube (MWCNT). Both biosensors canachieve the rapid and accurate detection of trace pollutants in water samples.Based on the detailed evaluation of the factors affecting sensor performance,taken microcystin-(leucine-arginine)(MC-LR) for example, the modifyingscheme of amperometric and impedimetric immunosensor and the measurementmethod of the pollutants in water were established.
The results obtained from this study listed as follows:(1) OsPVP wascovalently co-immobilized on the surface of SPCE with bovine serum albumin(BSA) and hapten-carrier conjugates MC-LR-ovalbumin (MC-LR-OVA), whichwere synthesized by mixing MC-LR and OVA activated by glutaraldehyde.(2)MWCNT was modified on the surface of SPCE by preparing a stablepolyethyleneimine-MWCNT (MWCNT-PEI) suspension, in which the PEI wasapplied as the dispersant agent. After the mixture was immobilized, and then theMC-LR-OVA activated was covalently bound to electrode surface via thecondensation reaction between the aldehyde groups of conjugate and the aminegroups of MWCNT-PEI.(3) The morphology and electrochemical properties ofthe sensor modified was evaluated by scanning electron microscopy, cyclicvoltammetry, and electrochemical impedance spectroscopy. The results showthat good binding of monoclonal antibody against MC-LR (Anti-MC-LR) andlow non-specific adsorption was monitored.(4) The experimental results ofmatrix effect in real water sample indicated that the concentration of methanolunder the scope of the experimental conditions didn’t affect the monitoring ofsamples, the proposed pH was6~8, and the high concentration of Cu2+and humus on the immunoassay which could affect the performance ofimmunosensor could be effectively compromised when1%EDTA and10mg/mL BSA were added to the pre-incubated mixture.(5) The detection limit ofamperometric immunosensor for MC-LR was found to be0.17μg/L with thequantitative detection range of0.43~10.72μg/L, and the total analysis time wasapproximately80min. The detection limit of impedimetric immunosensor forMC-LR was found to be0.58μg/L with the quantitative detection range of0.76~7.40μg/L, and the total analysis time was approximately55min. Severalwater samples of different origins were measured with less than15%deviationof the detection and the recovery ratio of MC-LR was determined to be83%~121%. The determination results of the samples including MC-LR were ingood correlation by those obtained from high performance liquidchromatography. This indicates that the prepared immunosensor can beapplicable to meet requirements of environmental monitoring for the tracepollutants with good accuracy and stability.
主要研究成果是：（1）提出了锇氧化还原聚合物修饰丝网印刷碳电极的方法，即先将小分子污染物微囊藻毒素-LR与经戊二醛活化后的惰性鸡卵白蛋白结合制备成微囊藻毒素-LR的抗原复合物，再将该复合物与锇氧化还原聚合物、牛血清白蛋白通过混合交联固定的方式固定在丝网印刷碳电极表面上的传感器修饰方法。（2）建立了多壁碳纳米管修饰丝网印刷碳电极的方法，即采用聚乙烯亚胺作为多壁碳纳米管分散剂，先制备出沉降性质稳定的多壁碳纳米管悬浊液，再将其修饰在丝网印刷碳电极表面，活化的抗原复合物通过醛基与聚乙烯亚胺上的胺基发生共价缩合反应修饰在电极表面。（3）采用扫描电子显微镜、循环伏安以及阻抗谱扫描，综合表征并分析了两种传感器的表面形态和电化学性能。研究结果表明，修饰后的传感器对微囊藻毒素-LR的抗体表现出良好的特异性响应，而非特异性吸附很弱。（4）实际水样检测基质效应研究结果表明：受试的甲醇等基质对样品检测没有明显影响，样品检测适宜pH范围为6~8，腐殖酸和铜离子对检测影响较大；研究中通过在预温育反应混合物中添加1%螯合剂乙二胺四乙酸和10mg/mL牛血清白蛋白，能有效抑制样品中基质对免疫传感器检测的影响。（5）电流型免疫传感器检测微囊藻毒素-LR的检测限为0.17μg/L，定量检测区间为0.43~10.72μg/L，检测周期约为80min；阻抗型免疫传感器检测微囊藻毒素-LR的检测限为0.58μg/L，定量检测区间为0.76~7.40μg/L，检测周期约为55min。检测不同类型实际水样中的微囊藻毒素-LR的结果表明：研制的传感器的检测变异系数均在10%之内，回收率在83%~121%之间，与高效液相色谱的分析结果相比具有良好的线性相关性，且具有较好的精确度和稳定性，可满足环境检测的需要。
Two different electrochemical immunosensors based on screen-printedcarbon electrode (SPCE) have been successfully developed. The first one is anamperometric immunosensor system based on the integrated principle ofelectrocatalytic reduction and indirect competitive immunoassay, which hasbeen developed by using a self-made osmium redox polymer (OsPVP) modifiedon SPCE. The second one is a new impedimetric immunosensor based on themodification of multi-wall carbon nanotube (MWCNT). Both biosensors canachieve the rapid and accurate detection of trace pollutants in water samples.Based on the detailed evaluation of the factors affecting sensor performance,taken microcystin-(leucine-arginine)(MC-LR) for example, the modifyingscheme of amperometric and impedimetric immunosensor and the measurementmethod of the pollutants in water were established.
The results obtained from this study listed as follows:(1) OsPVP wascovalently co-immobilized on the surface of SPCE with bovine serum albumin(BSA) and hapten-carrier conjugates MC-LR-ovalbumin (MC-LR-OVA), whichwere synthesized by mixing MC-LR and OVA activated by glutaraldehyde.(2)MWCNT was modified on the surface of SPCE by preparing a stablepolyethyleneimine-MWCNT (MWCNT-PEI) suspension, in which the PEI wasapplied as the dispersant agent. After the mixture was immobilized, and then theMC-LR-OVA activated was covalently bound to electrode surface via thecondensation reaction between the aldehyde groups of conjugate and the aminegroups of MWCNT-PEI.(3) The morphology and electrochemical properties ofthe sensor modified was evaluated by scanning electron microscopy, cyclicvoltammetry, and electrochemical impedance spectroscopy. The results showthat good binding of monoclonal antibody against MC-LR (Anti-MC-LR) andlow non-specific adsorption was monitored.(4) The experimental results ofmatrix effect in real water sample indicated that the concentration of methanolunder the scope of the experimental conditions didn’t affect the monitoring ofsamples, the proposed pH was6~8, and the high concentration of Cu2+and humus on the immunoassay which could affect the performance ofimmunosensor could be effectively compromised when1%EDTA and10mg/mL BSA were added to the pre-incubated mixture.(5) The detection limit ofamperometric immunosensor for MC-LR was found to be0.17μg/L with thequantitative detection range of0.43~10.72μg/L, and the total analysis time wasapproximately80min. The detection limit of impedimetric immunosensor forMC-LR was found to be0.58μg/L with the quantitative detection range of0.76~7.40μg/L, and the total analysis time was approximately55min. Severalwater samples of different origins were measured with less than15%deviationof the detection and the recovery ratio of MC-LR was determined to be83%~121%. The determination results of the samples including MC-LR were ingood correlation by those obtained from high performance liquidchromatography. This indicates that the prepared immunosensor can beapplicable to meet requirements of environmental monitoring for the tracepollutants with good accuracy and stability.
引文
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