Key Laboratory on Luminescence and Real-Time Analysis, Ministry of Education, School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, PR China.
Biosens Bioelectron. 2013 May 15;43:19-24. doi: 10.1016/j.bios.2012.11.035. Epub 2012 Dec 4.
In this work, we describe a new sensitive strategy for electrochemical detection of protein via small molecule/protein interactions. This assay is based on a terminal protection mechanism that small molecule-linked single-stranded DNA (ssDNA) is protected against hydrolysis by exonuclease I when the target protein is captured by the corresponding small molecule recognition element. Positively charged gold nanoparticles (AuNPs) are attached to the termini-protected and negatively charged ssDNA through electrostatic interactions. Subsequent AuNP-catalyzed silver enhancement followed by a highly characteristic and sensitive solid-state Ag/AgCl process is introduced to the sensing platform to amplify the signal output. By combining the amplification ability resulting from the silver deposition on the surface-captured AuNPs with the inherent high sensitivity of the electrochemical solid-state Ag/AgCl process, our method expands its range to the detection of macromolecules that bind to specific small molecules and enables low picomolar detection of protein. As a model of biotin/streptavidin interaction, a detection limit of 10 pM for streptavidin is readily achieved with desirable sensitivity and specificity, which indicates that the terminal protection assay coupled with the electrochemical solid-state Ag/AgCl process can offer a promising platform for the determination of various of types of proteins or small molecule-protein interactions.
在这项工作中，我们描述了一种通过小分子/蛋白质相互作用电化学检测蛋白质的新的灵敏策略。该测定法基于末端保护机制，当目标蛋白质被相应的小分子识别元件捕获时，小分子连接的单链 DNA（ssDNA）通过核酸外切酶 I 的水解被保护。带正电荷的金纳米粒子（AuNPs）通过静电相互作用附着在末端保护的带负电荷的 ssDNA 上。随后，通过 AuNP 催化的银增强以及引入到传感平台中的高特征性和灵敏的固态 Ag/AgCl 过程，对信号输出进行放大。通过将银在表面捕获的 AuNPs 上沉积所产生的放大能力与电化学固态 Ag/AgCl 过程固有的高灵敏度相结合，我们的方法将其检测范围扩展到了结合特定小分子的大分子，并实现了对蛋白质的低皮摩尔检测。作为生物素/链霉亲和素相互作用的模型，我们可以轻松地以理想的灵敏度和特异性实现对链霉亲和素的 10 pM 检测限，这表明末端保护测定法与电化学固态 Ag/AgCl 过程相结合可以为各种类型的蛋白质或小分子-蛋白质相互作用的测定提供有前景的平台。
