基于免疫传感器测定2，4-二氯苯氧乙酸技术的研究
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摘要
本论文从纳米金材料的特性和抗原-抗体特异性着手,以获得高灵敏度、快速、简便、低成本的生物传感器为目的。利用自组装技术和静电吸附作用,基于电化学技术,设计建立了几种新型生物传感器技术。实现2,4-二氯苯氧乙酸(2,4-D)的准确、快速、简便的检测。主要结果如下：
(1)利用自组装技术和静电吸附作用,将2,4-二氯苯氧乙酸抗体(anti-2,4-D)固定在通过由自组装L-半胱氨酸和静电吸附多层辣根过氧化物酶(HRP)/纳米金修饰的金电极表面,获得了用于检测2,4-D的无试剂安培型免疫传感器。采用本方法得出如下结论：在2,4-D浓度为5ng/mL-5000 ng/mL范围内成线性关系,线性方程是y=0.0199x+21.811,相关系数R为0.9968,检测下限大约为0.1ng/ml。利用此种传感器测定表现出了良好的重复性和重现性,方法简单,灵敏度高。
(2)通过电沉积纳米金于玻碳电极表面所形成的多孔纳米金修饰层直接固定2,4-D抗体,研究出了一种直接测定2,4-D的阻抗型无试剂免疫传感器技术。采用本方法获得了2,4-D浓度在0.5 ng/mL-5000 ng/mL范围内与阻抗增长率成线性关系,线性方程是y=0.045x+62.55,相关系数R为0.9970,检测下限为0.1 ng/mL。本方法不同于其他的免疫传感器,本实验无须对抗原或抗体进行任何标记,可以直接通过阻抗变化值获得2,4-D浓度信息,无需其他电子媒介的引入及生物分子的标记,降低了外界对检测的干扰。
(3)通过由自组装1,6-己二硫醇和静电吸附纳米金修饰的金电极表面将2,4-D-BSA固定,在电极上进行高特异性的竞争免疫反应,用HRP标记的羊抗鼠IgG将信号进一步放大,提高检测灵敏度,得到了一个基于辣根过氧化物酶(HRP)的自由竞争免疫传感器。通过实验得出：在2,4-D浓度为0.5 ng/mL-2500 ng/mL范围内成线性关系,校正曲线是y=-18.45x+71.59,相关系数R为0.9960,检测下限大约为0.05ng/ml。经初步应用,证实此方法能较好的满足测定2,4-D的需要,提高了检测的灵敏度和准确性,方法简单,快速。
The purpose of the paper was to develop highly sensitive, fast, easy, affordable biosensor base on the specificity of antigen-antibody recognization. Several new bio-sensor technologies based on electrochemical technology were established by self-assembly and electrostatic adsorption. The dichlorophenoxyacefic acid (2,4-D) can be detected accurately, rapidly and conveniently by these technologies. The main results are shown as bellow:
(1) A novel amperometric reagentless immunosensor has been constructed for the detection of 2,4-D. In this reagentless immunosensor,2,4-D was fixed on gold electrode surface which is modified by L-cysteine and the multi-layer horseradish peroxidase (HRP)/nano-Au. The results showed good linearity with the content of 2,4-D in the range of 5ng/mL-5000ng/mL.The linear equation is y=0.0199x+21.811, and the correlation coefficient R is 0.9968, with a detection limit at about 0.1ng/mL. This reagentless immunosensor, was found to be highly reproducible.In addition, no other electronic media and bio-molecular markers were used in this sensor.The detection results by using the sensor was found to be consistant with HPLC results.
(2) The impedance reagentless immunosensor for the detection of 2,4-D was also studied. This method is realized mainly through porous nano-gold modified layer directly fixed 2,4-D antibody, which is formed by electro-deposition nano gold in the glassy carbon electrode surface. The results showed good linearity with the content of 2,4-D in the range of 0.5ng/mL-5000 ng/mL.The linear equation is y=0.045x+62.55, and the correlation coefficient R is 0.9970, with a detection limit at about 0.1ng/mL. This method is different from other immunosensors, because it can directly obtain the 2,4-D concentration information through the impedance changes. No antigen or antibody tags were needed in the sensor. Therefore, the interference was reduced.
(3) A novel free competition immunosensor for the detection of 2,4-D was developed.2,4-D-BSA fixed upon gold electrode surface which is modified by self-assembly 1,6-F 2 Mercaptans and electrostatic adsorption nano-Au, and highly-specific competition immune response on electrode surface. The detection sensitivity was improved by using HRP labeled goat anti-mouse IgG to enlarge the signal. The results showed good linearity with the content of 2,4-D in the range of 0.5ng/mL-2500ng/mL.The calibration curve is y=-18.45x+71.59,and the correlation coefficient R is 0.9960, with a detection limit at about 0.05ng/mL.
(1)利用自组装技术和静电吸附作用,将2,4-二氯苯氧乙酸抗体(anti-2,4-D)固定在通过由自组装L-半胱氨酸和静电吸附多层辣根过氧化物酶(HRP)/纳米金修饰的金电极表面,获得了用于检测2,4-D的无试剂安培型免疫传感器。采用本方法得出如下结论：在2,4-D浓度为5ng/mL-5000 ng/mL范围内成线性关系,线性方程是y=0.0199x+21.811,相关系数R为0.9968,检测下限大约为0.1ng/ml。利用此种传感器测定表现出了良好的重复性和重现性,方法简单,灵敏度高。
(2)通过电沉积纳米金于玻碳电极表面所形成的多孔纳米金修饰层直接固定2,4-D抗体,研究出了一种直接测定2,4-D的阻抗型无试剂免疫传感器技术。采用本方法获得了2,4-D浓度在0.5 ng/mL-5000 ng/mL范围内与阻抗增长率成线性关系,线性方程是y=0.045x+62.55,相关系数R为0.9970,检测下限为0.1 ng/mL。本方法不同于其他的免疫传感器,本实验无须对抗原或抗体进行任何标记,可以直接通过阻抗变化值获得2,4-D浓度信息,无需其他电子媒介的引入及生物分子的标记,降低了外界对检测的干扰。
(3)通过由自组装1,6-己二硫醇和静电吸附纳米金修饰的金电极表面将2,4-D-BSA固定,在电极上进行高特异性的竞争免疫反应,用HRP标记的羊抗鼠IgG将信号进一步放大,提高检测灵敏度,得到了一个基于辣根过氧化物酶(HRP)的自由竞争免疫传感器。通过实验得出：在2,4-D浓度为0.5 ng/mL-2500 ng/mL范围内成线性关系,校正曲线是y=-18.45x+71.59,相关系数R为0.9960,检测下限大约为0.05ng/ml。经初步应用,证实此方法能较好的满足测定2,4-D的需要,提高了检测的灵敏度和准确性,方法简单,快速。
The purpose of the paper was to develop highly sensitive, fast, easy, affordable biosensor base on the specificity of antigen-antibody recognization. Several new bio-sensor technologies based on electrochemical technology were established by self-assembly and electrostatic adsorption. The dichlorophenoxyacefic acid (2,4-D) can be detected accurately, rapidly and conveniently by these technologies. The main results are shown as bellow:
(1) A novel amperometric reagentless immunosensor has been constructed for the detection of 2,4-D. In this reagentless immunosensor,2,4-D was fixed on gold electrode surface which is modified by L-cysteine and the multi-layer horseradish peroxidase (HRP)/nano-Au. The results showed good linearity with the content of 2,4-D in the range of 5ng/mL-5000ng/mL.The linear equation is y=0.0199x+21.811, and the correlation coefficient R is 0.9968, with a detection limit at about 0.1ng/mL. This reagentless immunosensor, was found to be highly reproducible.In addition, no other electronic media and bio-molecular markers were used in this sensor.The detection results by using the sensor was found to be consistant with HPLC results.
(2) The impedance reagentless immunosensor for the detection of 2,4-D was also studied. This method is realized mainly through porous nano-gold modified layer directly fixed 2,4-D antibody, which is formed by electro-deposition nano gold in the glassy carbon electrode surface. The results showed good linearity with the content of 2,4-D in the range of 0.5ng/mL-5000 ng/mL.The linear equation is y=0.045x+62.55, and the correlation coefficient R is 0.9970, with a detection limit at about 0.1ng/mL. This method is different from other immunosensors, because it can directly obtain the 2,4-D concentration information through the impedance changes. No antigen or antibody tags were needed in the sensor. Therefore, the interference was reduced.
(3) A novel free competition immunosensor for the detection of 2,4-D was developed.2,4-D-BSA fixed upon gold electrode surface which is modified by self-assembly 1,6-F 2 Mercaptans and electrostatic adsorption nano-Au, and highly-specific competition immune response on electrode surface. The detection sensitivity was improved by using HRP labeled goat anti-mouse IgG to enlarge the signal. The results showed good linearity with the content of 2,4-D in the range of 0.5ng/mL-2500ng/mL.The calibration curve is y=-18.45x+71.59,and the correlation coefficient R is 0.9960, with a detection limit at about 0.05ng/mL.
引文
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