Chronoamperometric Biosensor for Protease Activity Assay and Inhibitor Screening

Abstract

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.

Recent Advances in Electrochemical Biosensors Based on Enzyme Inhibition for Clinical and Pharmaceutical Applications

“…Many researchers have linked the presence of protease to diseases (e.g., AIDS, neurodegenerative diseases, cancer) [ 114 , 115 , 116 ]. Recently, our group proposed a new and a sensitive approach to assay protease activity and inhibition using a tyrosinase biosensor [ 22 ]. The biosensor was elaborated using carbon nanopowder electrode modified with tyrosinase.…”

“…These drug targets act as enzyme inhibitors; consequently, the inhibitor concentration affecting the rate of this enzymatic transformation can be measured. Electrochemical biosensors perform measurements using small sample volumes, low concentrations of biological components and sometimes miniaturized analytical devices [ 20 , 21 , 22 , 23 , 24 ].…”