Abstract

Beta-galactosidase (β-Gal) is an important enzyme utilized to determine cell senescence and understand disease pathology, among being utilized for several biosensing applications. This key biomarker can be measured indirectly using electrochemical methods by detecting the electroactive p-aminophenol (PAP) produced from the enzymatic conversion of a substrate. Here, we present a quick electrochemical method based on cyclic voltammograms (CVs) to quantify the amount of β-Gal in aqueous solution in under 5 minutes. Using timed CVs, the enzyme kinetics of β-Gal versus 4-aminophenyl-β-D-galactopyranoside (PAPG) was estimated. The detection limit for β-Gal was 40 ng ml⁻¹ using the timed quick CV method. K_M was found to be 18.7 ± 0.7 µM, and V_max 10.1 ± 0.6 µmol min⁻¹ mg⁻¹, for β-Gal with PAPG as a substrate. By adding divalent salts Mg²⁺ and Ca²⁺, K_M was lowered to nearly half, and V_max increased by 20%. The electrochemical protocol utilized neutral pH and ambient room temperature benchtop conditions, making it amenable to scanning electrochemical microscopy and lab-on-chip detection platforms. In addition to cell senescence, this quick and simple electrochemical method can be used in broad biosensing applications, including the detection of water and foodborne pathogens and the identification of toxic contaminants.

摘要

β-半乳糖苷酶（β-Gal）是一种重要的酶，可用于确定细胞的衰老并了解疾病的病理状况，并被用于多种生物传感应用中。通过检测由底物的酶促转化产生的电活性对氨基苯酚（PAP），可以使用电化学方法间接测量该关键生物标志物。在这里，我们提出了一种基于循环伏安图（CV）的快速电化学方法，用于在5分钟内定量测定水溶液中β-Gal的量。使用定时CV，估计了β-Gal与4-氨基苯基-β-D-吡喃半乳糖苷（PAPG）的酶动力学。使用定时快速CV方法测得的β-Gal的检出限为40 ng ml ^-1。发现K _M为18.7±0.7 µM，V _max为10.1±0.6 µmol min^-1  mg ^-1，对于以PAPG为底物的β-Gal而言。通过添加二价盐Mg ²⁺和Ca 2 ⁺，K _M降低至近一半，并且V _max增加20％。电化学方案利用中性pH和室温室温台式条件，使其适合于扫描电化学显微镜和芯片实验室检测平台。除细胞衰老外，这种快速简便的电化学方法还可用于广泛的生物传感应用中，包括水和食源性病原体的检测以及有毒污染物的鉴定。