基于固定化酶-MWNT修饰铂电极检测黄曲霉毒素传感器的研制及其条件优化
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摘要
黄曲霉毒素(AFB_1)是一种强致癌致畸的真菌毒素。目前国内外已有多种AFB_1的检测方法的报道。其中基于黄曲霉毒素解毒酶(ADTZ)对黄曲霉毒素B_1和黄曲霉毒素B_1结构类似物杂色曲霉素(ST)的酶生物传感器检测，是本研究所新近报告的方法。
本研究的目的是针对组装的酶生物传感器检测AFB_1、ST尚未进行的稳定性、使用寿命连续检测特性，以及提高检测灵敏度等开展研究，探索可用于多次使用并能用于连续分析AFB_1的酶生物传感器的构建方法。
方法：制备高纯度和高比活的rADTZ，作为传感器的识别元件。组装和比较三种不同方式的传感器：①rADTZ／EDC&NHS／MWNT电极(共价交联固定rADTZ于MWNT修饰电极)②rADTZ&sol-gel电极(sol-gel溶胶凝胶包埋rADTZ直接固定于铂电极表面)③rADTZ&sol-gel／MWNT电极(sol-gel溶胶凝胶包埋rADTZ固定于MWNT修饰电极)用循环伏安法(CV)，微分脉冲伏安法(DPV)和计时电流分析法(TB)，表征修饰电极的检测性能及检测条件优化。
结果：
1．常压蛋白纯化系统两步法纯化出来的rADTZ酶活为95.8U／g，纯度达到76.10％，为制备酶修饰电极提供必要的实验材料。
2．rADTZ&sol-gel／MWNT修饰电极具有良好的抗甲醇干扰能力。良好的重复性和稳定性，4℃保存28天后，峰电流差值标准偏差为2.1％。
3．计时电流分析实验结果表明，此酶修饰电极具有较为快速的响应，平均响应时间为44s，当C_(AFB1)大于45μg／L以上时，电流响应时间小于30s。并且能连续稳定检测5小时以上。对AFB_1的检测线性范围为：5×10~(-6)g／L～2.5×10~(-5)g／L，3×10~(-5)g／L～7×10~(-5)g／L，表明rADTZ&sol-gel／MWNT修饰电极是一种理想的，且具有与流动注射FIA分析结合，进行AFB_1在线动态检测的手段和方式。
AFB_1 was a kind of mycotoxin having strong cancerigenic mutant abilities. Several methodsfor AFB_1 detecting had been reported in both domestic and foreign articles. Among the articles, thedetections of AFB_1 and its structural analogue ST with enzyme biosensor based on ADTZ was a newmethod firstly reported by our instititute.
The objectives of our study were developing researches on improving the constancy, servicelife, detecting continuity and sensitivity of the assembled enzyme biosensor for AFB_1 and STdetections, so as to find out a method for the construction of an enzyme biosensor which coulddetect AFB_1 continuously.
Methods: rADTZ with high purity and high special activity was used as a recognizing element toassemble and compare three biosensors constructed in three different ways①rADTZ/EDC&NHS/MWNT electrode (covalently cross linking rADTZ on MWNT modifiedelectrode)②rADTZ&sol-gel electrode(immobilizing the sol-gel which had embedded rADTZ inadvance directly on the surface of Pt electrode)③rADTZ&sol-gel/MWNT electrode(immobilizingsol-gel-embedded rADTZ on MWNT modified electrode). Cyclic voltammetry (CV), differentiatedpulse voltammetry (DPV) and single potential time base (TB) were employed to analyze electricalsignals detected, to characterize this modified electrode and to optimize detecting conditions of thebiosensor.
Results:
(1) rADTZ was purified with the special activity of 95.8U/g and the purity of 76.10% by atwo-step procedure using ordinary pressure purification system providing necessary materials for thepreparation of the enzyme-modified electrode.
(2) rADTZ/sol-gel/MWNT modified electrode could resist the interference of methanol fairlywell, having good reproducibility and constancy. After being placed at 4℃for 28 days, the electrodehad a stable response current with a standard deviation of 2.1%.
(3) The TB experiment showed that the enzyme-modified electrode had a rather sensitiveresponse with an average response time of 44s. Moreover when C_(AFB1) is more than 45μg·L~(-1), theresponse time will be less than 30s and the biosensor could last detecting stably for more than 5 hours. Two linear range of 5×10~(-6)g/L～2.5×10~(-5)g/L and 3×10~(-5)g/L～7×10~(-5)g/L were gotwhile AFB_1 was detected. The linear range was already within the detecting limit of AFB_1 allowedin feed and feed additives in China, which manifested that the rADTZ/sol-gel/MWNT modifiedelectrode was an ideal method and means to detect AFB1 online with the ability to combine withFIA.
本研究的目的是针对组装的酶生物传感器检测AFB_1、ST尚未进行的稳定性、使用寿命连续检测特性，以及提高检测灵敏度等开展研究，探索可用于多次使用并能用于连续分析AFB_1的酶生物传感器的构建方法。
方法：制备高纯度和高比活的rADTZ，作为传感器的识别元件。组装和比较三种不同方式的传感器：①rADTZ／EDC&NHS／MWNT电极(共价交联固定rADTZ于MWNT修饰电极)②rADTZ&sol-gel电极(sol-gel溶胶凝胶包埋rADTZ直接固定于铂电极表面)③rADTZ&sol-gel／MWNT电极(sol-gel溶胶凝胶包埋rADTZ固定于MWNT修饰电极)用循环伏安法(CV)，微分脉冲伏安法(DPV)和计时电流分析法(TB)，表征修饰电极的检测性能及检测条件优化。
结果：
1．常压蛋白纯化系统两步法纯化出来的rADTZ酶活为95.8U／g，纯度达到76.10％，为制备酶修饰电极提供必要的实验材料。
2．rADTZ&sol-gel／MWNT修饰电极具有良好的抗甲醇干扰能力。良好的重复性和稳定性，4℃保存28天后，峰电流差值标准偏差为2.1％。
3．计时电流分析实验结果表明，此酶修饰电极具有较为快速的响应，平均响应时间为44s，当C_(AFB1)大于45μg／L以上时，电流响应时间小于30s。并且能连续稳定检测5小时以上。对AFB_1的检测线性范围为：5×10~(-6)g／L～2.5×10~(-5)g／L，3×10~(-5)g／L～7×10~(-5)g／L，表明rADTZ&sol-gel／MWNT修饰电极是一种理想的，且具有与流动注射FIA分析结合，进行AFB_1在线动态检测的手段和方式。
AFB_1 was a kind of mycotoxin having strong cancerigenic mutant abilities. Several methodsfor AFB_1 detecting had been reported in both domestic and foreign articles. Among the articles, thedetections of AFB_1 and its structural analogue ST with enzyme biosensor based on ADTZ was a newmethod firstly reported by our instititute.
The objectives of our study were developing researches on improving the constancy, servicelife, detecting continuity and sensitivity of the assembled enzyme biosensor for AFB_1 and STdetections, so as to find out a method for the construction of an enzyme biosensor which coulddetect AFB_1 continuously.
Methods: rADTZ with high purity and high special activity was used as a recognizing element toassemble and compare three biosensors constructed in three different ways①rADTZ/EDC&NHS/MWNT electrode (covalently cross linking rADTZ on MWNT modifiedelectrode)②rADTZ&sol-gel electrode(immobilizing the sol-gel which had embedded rADTZ inadvance directly on the surface of Pt electrode)③rADTZ&sol-gel/MWNT electrode(immobilizingsol-gel-embedded rADTZ on MWNT modified electrode). Cyclic voltammetry (CV), differentiatedpulse voltammetry (DPV) and single potential time base (TB) were employed to analyze electricalsignals detected, to characterize this modified electrode and to optimize detecting conditions of thebiosensor.
Results:
(1) rADTZ was purified with the special activity of 95.8U/g and the purity of 76.10% by atwo-step procedure using ordinary pressure purification system providing necessary materials for thepreparation of the enzyme-modified electrode.
(2) rADTZ/sol-gel/MWNT modified electrode could resist the interference of methanol fairlywell, having good reproducibility and constancy. After being placed at 4℃for 28 days, the electrodehad a stable response current with a standard deviation of 2.1%.
(3) The TB experiment showed that the enzyme-modified electrode had a rather sensitiveresponse with an average response time of 44s. Moreover when C_(AFB1) is more than 45μg·L~(-1), theresponse time will be less than 30s and the biosensor could last detecting stably for more than 5 hours. Two linear range of 5×10~(-6)g/L～2.5×10~(-5)g/L and 3×10~(-5)g/L～7×10~(-5)g/L were gotwhile AFB_1 was detected. The linear range was already within the detecting limit of AFB_1 allowedin feed and feed additives in China, which manifested that the rADTZ/sol-gel/MWNT modifiedelectrode was an ideal method and means to detect AFB1 online with the ability to combine withFIA.
引文
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