Fang Lanyun, Lü Zhaozi, Wei Hui, Wang Erkang
Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Graduate School of the Chinese Academy of Sciences, Changchun 130022, People's Republic of China.
Biosens Bioelectron. 2008 Jun 15;23(11):1645-51. doi: 10.1016/j.bios.2008.01.023. Epub 2008 Feb 7.
Quantitative electrochemiluminescence (ECL) detection of a model protein, bovine serum albumin (BSA) was achieved via biotin-avidin interaction using an avidin-based sensor and a well-developed ECL system of tris(2,2'-bipyridine) ruthenium(II) derivative as label and tri-n-propylamine (TPA) as coreactant. To detect the protein, avidin was linked to the glassy carbon electrode through passive adsorptions and covalent interaction with carboxylate-terminated carbon nanotubes that was used as binder to immobilize avidin onto the electrode. Then, biotinylated BSA tagged with tris(2,2'-bipyridine) ruthenium(II) label was attached to the prepared avidin surface. After binding of BSA labeled with tris(2,2'-bipyridine) ruthenium(II) derivative to the surface-immobilized avidin through biotin, ECL response was generated when the self-assembled modified electrode was immersed in a TPA-containing electrolyte solution. Such double protein labeling protocol with a biotin label for biorecognition and ruthenium label for ECL detection facilitated the detection of protein compared to the classical double antibody sandwich format. The ECL intensity was linearly proportional to the feed concentration of BSA over two orders of magnitude in the range of 15nM to 7.5microM. The detection limit was estimated to be 1.5nM. Further application to the lysozyme analysis was carried out to validate the present approach for an effective and favorable protocol for the quantitative detection of proteins. The dynamic range of lysozyme was from 0.001gL(-1) to 0.1gL(-1) and the detection limit was 0.1mgL(-1). Electrochemical impedance and cyclic voltammetric measurements along with some necessary control experiments were conducted to characterize the successful formation of self-assembled modified electrodes and to grant the whole detection process.
通过生物素-抗生物素蛋白相互作用，利用基于抗生物素蛋白的传感器和成熟的以三（2,2'-联吡啶）钌（II）衍生物为标记物、三正丙胺（TPA）为共反应剂的电化学发光（ECL）体系，实现了对模型蛋白牛血清白蛋白（BSA）的定量ECL检测。为检测该蛋白，抗生物素蛋白通过被动吸附以及与用作粘合剂将抗生物素蛋白固定在电极上的羧基末端碳纳米管的共价相互作用，连接到玻碳电极上。然后，用三（2,2'-联吡啶）钌（II）标记的生物素化BSA附着到制备好的抗生物素蛋白表面。在用三（2,2'-联吡啶）钌（II）衍生物标记的BSA通过生物素与表面固定的抗生物素蛋白结合后，将自组装修饰电极浸入含TPA的电解质溶液中时会产生ECL响应。与经典的双抗体夹心形式相比，这种用于生物识别的生物素标记和用于ECL检测的钌标记的双蛋白标记方案有助于蛋白质的检测。在15 nM至7.5 μM范围内，ECL强度与BSA的进料浓度在两个数量级上呈线性比例关系。检测限估计为1.5 nM。进一步将其应用于溶菌酶分析，以验证本方法是一种有效且良好的蛋白质定量检测方案。溶菌酶的动态范围为0.001 gL⁻¹至0.1 gL⁻¹，检测限为0.1 mgL⁻¹。进行了电化学阻抗和循环伏安测量以及一些必要的对照实验，以表征自组装修饰电极的成功形成并确保整个检测过程。
