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Chronoamperometric Biosensor for Protease Activity Assay and Inhibitor Screening
Author(s) -
El Harrad Loubna,
Amine Aziz
Publication year - 2017
Publication title -
electroanalysis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.574
H-Index - 128
eISSN - 1521-4109
pISSN - 1040-0397
DOI - 10.1002/elan.201700340
Subject(s) - leupeptin , detection limit , biosensor , chemistry , trypsin , bovine serum albumin , chromatography , amperometry , protease , casein , tyrosinase , enzyme , biochemistry , electrochemistry , electrode
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.