Amperometric L-arginine biosensor based on a novel recombinant arginine deiminase

Abstract

The authors describe an amperometric biosensor for the amino acid L-arginine (L-Arg). It is based on the use of a Nafion/Polyaniline (PANi) composite on a platinum screen-printed electrode (Pt-SPE) using a novel recombinant arginine deiminase isolated from Mycoplasma hominis. The protein was over-expressed, purified and employed as a biorecognition element of the sensor. Enzymatic hydrolysis of L-Arg leads to the formation of ammonium ions which diffuse into the Nafion/PANi layer and induce the electroreduction of PANi at a potential of −0.35 V (vs Ag/AgCl). L-Arg sensitivity is 684 ± 32 A·M−1·m−2, and the apparent Michaelis-Menten constant (KM app) is 0.31 ± 0.05 mM. The calibration plot is linear over the range 3–200 μM L-Arg, the limit of detection is 1 μM, and the response time (for 90% of the total signal change to occur) is 15 s. The sensor is selective and exhibits good storage stability (> 1 month without loss in signal). The biosensor was applied to the analysis of L-Arg in pharmaceutical samples and of ammonium and L-Arg in spiked human plasma obtained from blood of healthy volunteers and those with a hepatic disorder. Data generated were found to be in good agreement with a reference fluorometric enzymatic assay.