One-step fabrication of integrated disposable biosensor based on ADH/NAD+/meldola's blue/graphitized mesoporous carbons/chitosan nanobiocomposite for ethanol detection

Abstract

A novel strategy to simplify the dehydrogenase-based electrochemical biosensor fabrication through one-step drop-coating nanobiocomposite on a screen printed electrode (SPE) was developed. The nanobiocomposite was prepared by successively adding graphitized mesoporous carbons (GMCs), meldola's blue (MDB), alcohol dehydrogenase (ADH) and cofactor nicotinamide adenine dinucleotide (NAD+) in chitosan (CS) solution. MDB/GMCs/CS film was prepared. Cyclic voltammetry measurements demonstrated that MDB was strongly adsorbed on GMCs. After optimizing the concentration of MDB and the working potential, the MDB/GMCs/CS film presented a fast amperometric response (5s), excellent sensitivity (10.36nAμM−1), wide linear range (10–410μM) toward NADH and without any other interference signals (such as AA, UA, DA, H2O2 and metal ions). Furthermore, concentrations of ADH and NAD+ in nanobiocomposite and the detection conditions (temperature and pH) were also optimized. The constructed disposable ethanol biosensor showed an excellent linear response ranged from 0.5 to 15mM with high sensitivity (67.28nAmM−1) and a low limit of detection (80μM) and a remarkable long-term stability (40 days). The intra-batch and inter-batch variation coefficients were both less than 5% (n=5). The ethanol recovery test demonstrated that the proposed biosensor offered a remarkable and accurate method for ethanol detection in the real blood samples.