Rational design of bienzyme nanoparticles-based total cholesterol electrochemical sensors and the construction of cholesterol oxidase expression system

ABSTRACT In this study, we demonstrate the first report of bienzyme nanoparticles (BENP)–based cholesterol electrochemical sensor. The developed BENP consists of cholesterol oxidase (ChOx) and horseradish peroxidase (HRP), and these enzymes were crosslinked by glutaraldehyde treatment. An alternative expression system of ChOx is also firstly introduced through this work by constructing its production system in Pichia pastoris exploiting ChOx expressing gene taken from Streptomyces sp. The obtained ChOx is then applied to synthesize BENP with HRP at different ratios to attain the best condition that can yield in enhanced enzymatic activity. To utilize the material for electrochemical sensors, the BENP is furtherly modified with cysteamine to graft thiol (-SH) group that facilitates BENP immobilization on gold electrode through Au-S bond. The kinetic study of the BENP was conducted using cholesterol and H2O2, which are substrates of the BENP constituents, ChOx and HRP, respectively. Moreover, its characterizations, catalytic activity, specificity, and selectivity toward cholesterol also has been investigated. The developed BENP-based cholesterol electrochemical sensor exhibited a long dynamic range (0-675 mg/dL) with a low detection limit (0.18 mg/dL), high selectivity, and long-term stability (> 98% of its initial activity over 25 days).