Department of Biochemistry, School of Life Sciences, University of Hyderabad, Hyderabad, Andhra Pradesh, 500046, India.
Biosens Bioelectron. 2010 May 15;25(9):2016-20. doi: 10.1016/j.bios.2010.01.029. Epub 2010 Feb 2.
In this report we have used gold nanoparticles (AuNPs) to covalently attach an antibody (Ab(1)) using a spacer arm. The AuNPs/Ab(1) modified gold electrode was used for a sandwich electrochemical immunoassay. The detection was done using cyclic voltammetry and impedance measurements using Horse Radish Peroxidase (HRP) as enzyme label on secondary antibody (Ab(2)) and 3,3', 5,5'-tertramethyl benzidine (TMB) as an electroactive dye. The cyclic voltammetric experiments showed three clear peaks at potentials 154 mV, -33 mV and -156 mV. There was an increase in the both anodic and cathodic current values for the peak at potential -33 mV, when H2O2 was added and the other peaks observed at potential 154 mV and -156 mV resulted due to the oxidation and reduction of TMB. The detection limit of this electrode was 2 ng/mL or 10 pg/5 microL of the analyte. The electrochemical impedance spectroscopy studies demonstrate that the formation of antigen-antibody complexes increases the series resistance and thus confirms the assembly on the electrode. This study showed that AuNPs was efficient in preserving the activity and orientation of the antibody and it can form a major platform in many clinical immunoassays.
在本报告中，我们使用金纳米粒子 (AuNPs) 通过间隔臂共价连接抗体 (Ab(1))。AuNPs/Ab(1)修饰的金电极用于夹心型电化学免疫分析。使用辣根过氧化物酶 (HRP) 作为二级抗体 (Ab(2)) 的酶标记物和 3,3',5,5'-四甲基联苯胺 (TMB) 作为电化学活性染料进行循环伏安法和阻抗测量进行检测。循环伏安实验在 154 mV、-33 mV 和-156 mV 的电势下显示出三个清晰的峰。当加入 H2O2 时，在电势-33 mV 处的峰的阳极和阴极电流值均增加，而在电势 154 mV 和-156 mV 处观察到的其他峰则归因于 TMB 的氧化和还原。该电极的检测限为 2ng/mL 或 10pg/5μL 的分析物。电化学阻抗谱研究表明，抗原-抗体复合物的形成增加了串联电阻，从而证实了在电极上的组装。这项研究表明，AuNPs 有效地保持了抗体的活性和定向性，并且可以成为许多临床免疫分析中的主要平台。
