Low potential detection of NADH based on Fe3O4 nanoparticles/multiwalled carbon nanotubes composite: Fabrication of integrated dehydrogenase-based lactate biosensor

► GC electrode modified with multiwalled carbon nanotube and Fe3O4 nanoparticles. ► Fe3O4/MWCNTs nanocomposite shows well defined redox process at ca. −0.2V. ► Redox system shows excellent electrocatalytic activity toward NADH oxidation.► With entrapment of alcohol dehydrogenase enzyme a stable lactate biosensor fabricated.► The proposed biosensor was used for detection of lactate in human serum samples. Fe3O4 magnetic nanoparticles were in situ loaded on the surface of multiwalled carbon nanotubes (MWCNTs) by a simple coprecipitation procedure. The resulting Fe3O4/MWCNTs nanocomposite brings new capabilities for electrochemical sensing by combining the advantages of Fe3O4 magnetic nanoparticles and MWCNTs. It was found that Fe3O4 has redox properties similar to those of frequently used mediators used for electron transfer between NADH and electrode. The cyclic voltammetric results indicated the ability of Fe3O4/MWCNTs modified GC electrode to catalyze the oxidation of NADH at a very low potential (0.0mV vs. Ag/AgCl) and subsequently, a substantial decrease in the overpotential by about 650mV compared with the bare GC electrode. The catalytic oxidation current allows the stable and selective amperometric detection of NADH at an applied potential of 0.0mV (Ag/AgCl) with a detection limit of 0.3μM and linear response up to 300μM. This modified electrode can be used as an efficient transducer in the design of biosensors based on coupled dehydrogenase enzymes. Lactate dehydrogenase (LDH) and NAD+ were subsequently immobilized onto the Fe3O4/MWCNTs nanocomposite film by covalent bond formation between the amine groups of enzyme or NAD+ and the carboxylic acid groups of the Fe3O4/MWCNT film. Differential pulse voltammetric detection of lactate on Fe3O4/MWCNT/LDH/NAD+ modified GC electrode gives linear responses over the concentration range of 50–500μM with the detection limit of 5μM and sensitivity of 7.67μAmM−1. Furthermore, the applicability of the sensor for the analysis of lactate concentration in human serum samples has been successfully demonstrated.