†Department of Chemistry and Chemistry Institute for Functional Materials, Pusan National University, Busan 609-735, Korea.
‡Department of Laboratory Medicine, Yonsei University College of Medicine, Seoul 135-720, Korea.
Anal Chem. 2015 Apr 7;87(7):3574-8. doi: 10.1021/ac504485a. Epub 2015 Mar 9.
In washing-free electrochemical detection, various redox and reactive species cause significant interference. To minimize this interference, we report a washing-free electrochemical immunosensor using flavin adenine dinucleotide (FAD)-dependent glycerol-3-phosphate dehydrogenase (GPDH) and glycerol-3-phosphate (GP) as an enzyme label and its substrate, respectively, because the reaction of FAD-dependent dehydrogenases with dissolved O2 is slow and the level of GP preexisting in blood is low (<0.1 mM). A combination of a low electrocatalytic indium-tin oxide (ITO) electrode and fast electron-mediating Ru(NH3)6(3+) is employed to obtain a high signal-to-background ratio via proximity-dependent electron mediation of Ru(NH3)6(3+) between the ITO electrode and the GPDH label. Electrochemical oxidation of GPDH-generated Ru(NH3)6(2+) is performed at 0.05 V vs Ag/AgCl, at which point the electrochemical interference is very low. When a washing-free immunosensor is applied to cardiac troponin I detection in human serum, the calculated detection limit is approximately 10 pg/mL, indicating that the immunosensor is very sensitive in spite of the use of washing-free detection with a short detection period (10 min for incubation and 100 s for electrochemical measurement). The low-interference washing-free electrochemical immunosensor shows good promise for fast and simple point-of-care testing.
在免洗电化学检测中，各种氧化还原和反应性物质会引起显著的干扰。为了最小化这种干扰，我们报告了一种免洗电化学免疫传感器，该传感器使用黄素腺嘌呤二核苷酸（FAD）依赖性甘油-3-磷酸脱氢酶（GPDH）和甘油-3-磷酸（GP）分别作为酶标记物及其底物，因为 FAD 依赖性脱氢酶与溶解氧的反应速度较慢，而且血液中 GP 的预存水平较低（<0.1mM）。低电催化氧化铟锡（ITO）电极和快速电子中介 Ru(NH3)6(3+)的组合被用来通过 Ru(NH3)6(3+)在 ITO 电极和 GPDH 标记物之间的近程电子中介作用，获得高的信号与背景比。在 0.05 V 相对于 Ag/AgCl 对 GPDH 生成的 Ru(NH3)6(2+)进行电化学氧化，此时电化学干扰非常低。当将免洗免疫传感器应用于人血清中心肌肌钙蛋白 I 的检测时，计算出的检测限约为 10pg/mL，表明尽管采用了短检测周期（孵育 10 分钟，电化学测量 100 秒）的免洗检测，但该免疫传感器非常灵敏。这种低干扰的免洗电化学免疫传感器有望用于快速、简单的即时检测。
