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

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), meldolaʹs 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 (5 s), excellent sensitivity (10.36 nA μM−1), wide linear range (10–410 μM) 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 15 mM with high sensitivity (67.28 nA mM−1) and a low limit of detection (80 μM) 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.