Ionic liquid modified chloroperoxidase is immobilized on gold nanoparticles-reduced graphene oxide nanocomposites for efficient sensoring of nitrite by electroenzymatic catalysis

Abstract

Electroenzymatic biosensors have wide applications due to its fastness and sensitivity. Here we propose a bio-nanocomposite to be dropped on glassy carbon electrode (GCE) for nitrite sensoring by electroenzymatic catalysis. The composite is constructed by ionic liquids (1-butyl-3-methylimidazolium bromide, ILBMB) modified chloroperoxidase (CPO) immobilized on gold nanoparticles (Au NPs) modified reduced graphene oxide (rGO). The ILBMB can facilitate the direct electron transfer (DET) of CPO on GCE without a redox mediator. The molecular docking indicates ILBMB is located at the opening of substrate channel rather than occupy the active site of CPO, which is beneficial to its DET. Circular dichroic spectrum (CD) verifies the α-helix content of CPO is increased, and ultraviolet-visible spectrum (UV–vis) demonstrates the heme prosthetic group become more exposed by ILBMB. These changes result in the enhancement of CPO activity. The rGO-Au NPs plays two roles enhancing the detection signal and improving the loading of CPO on GCE. The sensor (CPO-ILBMB/rGO-Au NPs/GCE) provides two models for nitrite detection as cyclic voltammetry and amperometry. The detection limit is 0.25 μmol·L−1 and 0.049 μmol·L−1, respectively with corresponding detection range of 2.0−2500 μmol·L−1 and 0.5–300 μmol·L−1. The high sensitivity, stability, and selectivity of this electroenzymatic biosensor ensure its potential practical application.