Direct electrochemical detection of extracellular nitric oxide in Arabidopsis protoplast based on cytochrome P450 55B1 biosensor

Abstract

Cytochrome P450 55B1(CYP55B1) from Chlamydomonas reinhardtii reduces nitric oxide (NO) to dinitrogen oxide (N2O) with the electron supply from NAD(P)H in vivo. Here a novel nitric oxide biosensor was developed by immobilized CYP55B1 on the surface of pyrolytic graphite electrode (PGE) by cross-linking with glutaraldehyde (GA) and bovine serum albumin (BSA). The direct electrochemistry of CYP55B1 was realized with the redox peak potential of −0.355 V and −0.385 V and the catalytic reduction peak of NO by CYP55B1 is at −0.85 V at the scan rate of 0.5 V S−1 in pH 7.0 phosphate buffer. The apparent coverage (Γ = 1.43 × 10−11 mol cm−2), the electron transfer rate constant (ks = 17.39 s−1) and apparent affinity to NO (Kmapp = 11.64 nM) of CYP55B1 in GA/BSA film were obtained. The catalytic mechanism of CYP55B1 towards NO with NADH was examined by the biosensor. The linear range of NO detection was investigated by differential pulse voltammetry with the results of 5–50 nM and the detection limit of 0.5 nM (S/N = 3). The selectivity and stability of the electrochemical biosensor were investigated. Furthermore, the CYP55B1electrochemical biosensor was applied to monitor NO release from Arabidopsis protoplasts with the average content of 0.848 fmol per cell under anaerobic condition.