Herein, we report an electrochemical biosensor for the measurement of proteins and protease activity using carbon nanopowder paste electrode modified with tyrosinase. The measurement of proteins is based on the amperometric measurement of the enzymatic product, tyrosine, at low applied potential (i.e +0.05 V vs. Ag/AgCl), in a linear range from 1 μg/mL to 10 μg/mL for casein and albumin bovine serum. This biosensor is also used to assess the enzymatic activity of the enzymes namely trypsin and proteinase K, observing a detection limit of 0.02 mU/mL (S/N=3). The ability of this assay to monitor the trypsin activity is exploited to investigate its inhibition by leupeptin. The trypsin kinetic interactions reveal uncompetitive binding of leupeptin with 50 % inhibition equal to 3.75 μM, a dynamic range of 0.25–10 μM, and a detection limit of 0.25 μM. The assay developed can be considered as general activity assay of any protease with the advantages of good storage stability (few weeks), low detection limit and ability to analyse turbid and colored samples.

中文翻译：

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用于蛋白酶活性测定和抑制剂筛选的计时电流生物传感器

在此，我们报告了一种使用酪氨酸酶修饰的碳纳米粉糊电极测量蛋白质和蛋白酶活性的电化学生物传感器。蛋白质的测量基于酶产物酪氨酸的安培测量，在低施加电位（即 +0.05 V vs. Ag/AgCl）下，酪蛋白的线性范围为 1 μg/mL 至 10 μg/mL，白蛋白牛血清。该生物传感器还用于评估胰蛋白酶和蛋白酶 K 等酶的酶活性，检测限为 0.02 mU/mL (S/N=3)。该测定监测胰蛋白酶活性的能力被用来研究亮肽素对胰蛋白酶的抑制作用。胰蛋白酶动力学相互作用显示亮抑酶肽的非竞争性结合，50% 抑制等于 3.75 μM，动态范围为 0.25–10 μM，检测限为 0.25 μM。