Department of Chemistry and Chemistry Institute of Functional Materials, Pusan National University, Busan, Republic of Korea.
Anal Chem. 2013 May 21;85(10):4863-8. doi: 10.1021/ac400573j. Epub 2013 May 10.
Catalytic reactions of enzyme labels in enzyme-linked immunosorbent assays require a long incubation period to obtain high signal amplification. We present herein a simple immunosensing scheme in which the incubation period is minimized without a large increase in the detection limit. This scheme is based on electrochemical-enzymatic (EN) redox cycling using glucose oxidase (GOx) as an enzyme label, Ru(NH3)6(3+) as a redox mediator, and glucose as an enzyme substrate. Fast electron mediation of Ru(NH3)6(3+) between the electrode and the GOx label attached to the electrode allows high signal amplification. The acquisition of chronocoulometric charges at a potential in the mass transfer-controlled region excludes the influence of the kinetics of Ru(NH3)6(2+) electrooxidation and also facilitates high signal-to-background ratios. The reaction between reduced GOx and Ru(NH3)6(3+) is rapid even in air-saturated Tris buffer, where the faster competitive reaction between reduced GOx and dissolved oxygen also occurs. The direct electrooxidation of glucose at the electrode and the direct electron transfer between glucose and Ru(NH3)6(3+) that undesirably increase background levels occur relatively slowly. The detection limit for the EN redox cycling-based detection of cancer antigen 125 (CA-125) in human serum is slightly higher than 0.1 U/mL for the incubation period of 0 min, and the detection limits for the incubation periods of 5 and 10 min are slightly lower than 0.1 U/mL, indicating that the detection limits are almost similar irrespective of the incubation period and that the immunosensor is highly sensitive.
酶标记物在酶联免疫吸附测定中的催化反应需要较长的孵育时间才能获得高信号放大。本文提出了一种简单的免疫传感方案，在不显著增加检测限的情况下，将孵育时间最小化。该方案基于电化学-酶（EN）氧化还原循环，使用葡萄糖氧化酶（GOx）作为酶标记物、Ru(NH3)6(3+)作为氧化还原介体、葡萄糖作为酶底物。Ru(NH3)6(3+)在电极和电极上附着的 GOx 标记物之间的快速电子介导允许高信号放大。在传质控制区的电位下采集计时库仑电荷排除了 Ru(NH3)6(2+)电化学氧化动力学的影响，也有利于高信号与背景的比值。即使在充满空气的 Tris 缓冲液中，还原的 GOx 和 Ru(NH3)6(3+)之间的反应也很快，在该缓冲液中，还原的 GOx 和溶解氧之间的竞争反应也更快。葡萄糖在电极上的直接电氧化和葡萄糖与 Ru(NH3)6(3+)之间的直接电子转移会导致背景水平升高，这两个过程相对较慢。在孵育期为 0 分钟时，基于 EN 氧化还原循环检测人血清中癌抗原 125（CA-125）的检测限略高于 0.1 U/mL，孵育期为 5 分钟和 10 分钟时的检测限略低于 0.1 U/mL，表明检测限几乎不受孵育期的影响，且该免疫传感器具有高灵敏度。
