Prayikaputri Putu Udiyani, Park Seonhwa, Kim Seonghye, Yoon Young Ho, Kim Suhkmann, Yang Haesik
Department of Chemistry and Chemistry Institute for Functional Materials, Pusan National University, Busan, 46241, Republic of Korea.
EONE Laboratories, Incheon, 22014, Rebulic of Korea.
Biosens Bioelectron. 2023 Mar 15;224:115058. doi: 10.1016/j.bios.2022.115058. Epub 2023 Jan 5.
Amide hydrolysis using enzyme labels, such as proteases, occurs at a slower rate than phosphoester and carboxyl ester hydrolysis. Thus, it is not very useful for obtaining high signal amplification in biosensors. However, amide hydrolysis is less sensitive to nonenzymatic spontaneous hydrolysis, allowing for lower background levels. Herein, we report that amide hydrolysis by DT-diaphorase (DT-D) occurs rapidly and that its combination with five redox-cycling reactions allows for the development of a highly sensitive electrochemical immunosensor. DT-D rapidly generates ortho-aminohydroxy-naphthalene (oAN) from its amide substrate via amide hydrolysis, which not even trypsin, a highly catalytic protease, can achieve. NADH, which is required for amide hydrolysis, advantageously acts as a reducing agent for rapid electrooxidation-based redox-cycling reactions. In the presence of oAN, DT-D, and NADH, two redox-cycling reactions rapidly occur. In the additional presence of an electron mediator, Ru(NH) [Ru(III)], three more redox-cycling reactions occur because Ru(III) reacts rapidly with oAN and DT-D. Although the O-related redox-cycling reactions and redox reaction decrease electrochemical signals, this signal-decreasing effect is not significant in air-saturated solutions. The slow electrooxidation of NADH at an indium tin oxide electrode and sluggish reaction between NADH and Ru(III) allow for low electrochemical backgrounds. When the developed signal amplification scheme is tested for the sandwich-type electrochemical detection of parathyroid hormone (PTH), a detection limit of ∼1 pg/mL is obtained. The detection method is highly sensitive and can accurately measure PTH in serum samples.
使用酶标记物（如蛋白酶）进行酰胺水解的速率比磷酸酯和羧酸酯水解的速率要慢。因此，它在生物传感器中获得高信号放大方面不是很有用。然而，酰胺水解对非酶促自发水解不太敏感，从而可实现较低的背景水平。在此，我们报告称，DT - 黄递酶（DT - D）介导的酰胺水解反应迅速，并且其与五个氧化还原循环反应相结合可用于开发高灵敏度的电化学免疫传感器。DT - D通过酰胺水解从其酰胺底物迅速生成邻氨基羟基萘（oAN），这甚至是高催化活性的蛋白酶胰蛋白酶都无法实现的。酰胺水解所需的NADH有利地充当用于基于快速电氧化的氧化还原循环反应的还原剂。在存在oAN、DT - D和NADH的情况下，两个氧化还原循环反应迅速发生。在额外存在电子媒介体Ru(NH) [Ru(III)]的情况下，又会发生另外三个氧化还原循环反应，因为Ru(III)与oAN和DT - D反应迅速。尽管与氧相关的氧化还原循环反应和氧化还原反应会降低电化学信号，但在空气饱和溶液中这种信号降低效应并不显著。NADH在氧化铟锡电极上的缓慢电氧化以及NADH与Ru(III)之间的缓慢反应使得电化学背景较低。当将所开发的信号放大方案用于甲状旁腺激素（PTH）的夹心型电化学检测时，获得了约1 pg/mL的检测限。该检测方法具有高灵敏度，能够准确测量血清样品中的PTH。
