Department of Chemistry and Chemistry Institute for Functional Materials, Pusan National University, Busan 609-735, Korea.
Anal Chem. 2011 May 15;83(10):3926-33. doi: 10.1021/ac200447b. Epub 2011 Apr 27.
The authors herein report optimized conditions for ultrasensitive phosphatase-based immunosensors (using redox cycling by a reducing agent) that can be simply prepared and readily applied to microfabricated electrodes. The optimized conditions were applied to the ultrasensitive detection of cardiac troponin I in human serum. The preparation of an immunosensing layer was based on passive adsorption of avidin (in carbonate buffer (pH 9.6)) onto indium-tin oxide (ITO) electrodes. The immunosensing layer allows very low levels of nonspecific binding of proteins. The optimum conditions for the enzymatic reaction were investigated in terms of the type of buffer solution, temperature, and concentration of MgCl(2), and the optimum conditions for antigen-antibody binding were determined in terms of incubation time, temperature, and concentration of phosphatase-conjugated IgG. Very importantly, the antigen-antibody binding at 4 °C is extremely important in obtaining reproducible results. Among the four phosphatase substrates (L-ascorbic acid 2-phosphate (AAP), 4-aminophenyl phosphate, 1-naphthyl phosphate, 4-amino-1-naphthyl phosphate) and four phosphatase products (L-ascorbic acid (AA), 4-aminophenol, 1-naphthol, 4-amino-1-naphthol), AAP and AA meet the requirements most for obtaining easy dissolution and high signal-to-background ratios. More importantly, fast AA electrooxidation at the ITO electrodes does not require modification with any electrocatalyst or electron mediator. Furthermore, tris(2-carboxyethyl)phosphine (TCEP) as a reducing agent allows fast redox cycling, along with very low anodic currents at the ITO electrodes. Under these optimized conditions, the detection limit of an immunosensor for troponin I obtained without redox cycling of AA by TCEP is ca. 100 fg/mL, and with redox cycling it is ca. 10 fg/mL. A detection limit of 10 fg/mL was also obtained even when an immunosensing layer was simply formed on a micropatterned ITO electrode. From a practical point of view, it is of great importance that ultralow detection limits can be obtained with simply prepared enzyme-based immunosensors.
本文作者报告了用于基于磷酸酶的超灵敏免疫传感器（使用还原剂的氧化还原循环）的优化条件，该条件可以简单制备并易于应用于微制造电极。优化条件应用于人血清中心肌钙蛋白 I 的超灵敏检测。免疫传感层的制备基于亲和素（在碳酸盐缓冲液（pH 9.6）中）被动吸附到氧化铟锡（ITO）电极上。免疫传感层允许蛋白质的非特异性结合非常低。从缓冲溶液的类型、温度和 MgCl2 的浓度方面研究了酶反应的最佳条件，并从孵育时间、温度和磷酸酶结合 IgG 的浓度方面研究了抗原-抗体结合的最佳条件。非常重要的是，在 4°C 下的抗原-抗体结合对于获得可重复的结果非常重要。在四种磷酸酶底物（L-抗坏血酸 2-磷酸（AAP）、4-氨基苯磷酸、1-萘基磷酸、4-氨基-1-萘基磷酸）和四种磷酸酶产物（L-抗坏血酸（AA）、4-氨基酚、1-萘酚、4-氨基-1-萘酚）中，AAP 和 AA 最符合获得易溶解和高信号-背景比的要求。更重要的是，AA 在 ITO 电极上的快速电氧化不需要任何电催化剂或电子介体进行修饰。此外，三（2-羧乙基）膦（TCEP）作为还原剂允许 AA 快速氧化还原循环，同时在 ITO 电极上的阳极电流非常低。在这些优化条件下，未经 TCEP 还原循环的 AA 的免疫传感器检测肌钙蛋白 I 的检测限约为 100 fg/mL，而经还原循环的检测限约为 10 fg/mL。即使在简单地在微图案化 ITO 电极上形成免疫传感层的情况下，也获得了 10 fg/mL 的检测限。从实际角度来看，用简单制备的基于酶的免疫传感器获得超低检测限非常重要。
