Park Seonhwa, Yang Haesik
Department of Chemistry and Chemistry Institute of Functional Materials, Pusan National University, Busan 609-735, Korea.
Analyst. 2014 Aug 21;139(16):4051-5. doi: 10.1039/c4an00465e.
We report a simple, sensitive, and selective electrochemical method for trypsin detection that can cover a wide range of concentrations. The method is based on the proteolytic generation of an electroactive species (P) by trypsin followed by a signal-amplified electrochemical measurement of P using electrochemical-chemical (EC) or electrochemical-chemical-chemical (ECC) redox cycling. The detection is performed using bare indium-tin oxide (ITO) electrodes without washing steps. P is generated by the cleavage of an amide bond between P and oligopeptide (Gly-Pro-Arg) at the C-terminal of Gly-Pro-Arg-P. Four trypsin products including 4-amino-1-naphthol (AN) and their trypsin substrates are investigated to obtain a high signal-to-background ratio in ECC redox cycling. AN and its trypsin substrate produce the highest signal-to-background ratio. The detection limits obtained with ECC redox cycling involving AN (approximately 1 ng mL(-1) and 100 ng mL(-1) with an incubation period of 2 h and 30 min, respectively) in Tris buffer (pH 8.0) are lower than those obtained with EC redox cycling involving AN (approximately 5 ng mL(-1) and 200 ng mL(-1) with an incubation period of 2 h and 30 min, respectively). In trypsin detection using ECC redox cycling, the interference effects of electroactive species such as l-ascorbic acid and uric acid are not significant.
我们报道了一种简单、灵敏且具有选择性的用于检测胰蛋白酶的电化学方法，该方法可覆盖较宽的浓度范围。该方法基于胰蛋白酶对电活性物质（P）的蛋白水解生成，随后使用电化学 - 化学（EC）或电化学 - 化学 - 化学（ECC）氧化还原循环对P进行信号放大的电化学测量。检测使用裸氧化铟锡（ITO）电极进行，无需清洗步骤。P是通过在甘氨酰 - 脯氨酰 - 精氨酸 - P的C末端切断P与寡肽（甘氨酰 - 脯氨酰 - 精氨酸）之间的酰胺键而生成的。研究了包括4 - 氨基 - 1 - 萘酚（AN）在内的四种胰蛋白酶产物及其胰蛋白酶底物，以在ECC氧化还原循环中获得高信噪比。AN及其胰蛋白酶底物产生的信噪比最高。在Tris缓冲液（pH 8.0）中，涉及AN的ECC氧化还原循环（分别在2小时和30分钟的孵育期下，检测限约为1 ng mL⁻¹和100 ng mL⁻¹）所获得的检测限低于涉及AN的EC氧化还原循环（分别在2小时和30分钟的孵育期下，检测限约为5 ng mL⁻¹和200 ng mL⁻¹）所获得的检测限。在使用ECC氧化还原循环进行胰蛋白酶检测时，诸如l - 抗坏血酸和尿酸等电活性物质的干扰作用不显著。
