| Summary: | Flavin adenine dinucleotide (FAD) is a coenzyme and acts as a redox cofactor in metabolic process. Owing to such problems as poor electron transfer properties, unfavorable adsorption, and lack of stability on rigid electrodes, the bio-electrochemical applications of FAD have been limited. Herein, a novel fabrication method was developed for the immobilization process using 2D MXene (Ti<sub>3</sub>C<sub>2</sub>T<sub>x</sub>), which enhanced the redox property of FAD and improved the electro-catalytic reduction of hydrogen peroxide (H<sub>2</sub>O<sub>2</sub>) in neutral medium. The FAD-immobilized Ti<sub>3</sub>C<sub>2</sub>T<sub>x</sub> electrode (FAD/Ti<sub>3</sub>C<sub>2</sub>T<sub>x</sub>) was studied by UV-Visible and Raman spectroscopies, which confirmed the successful adsorption of FAD on the Ti<sub>3</sub>C<sub>2</sub>T<sub>x</sub> surface. The surface morphology and the elemental composition of Ti<sub>3</sub>C<sub>2</sub>T<sub>x</sub> were investigated by high resolution transmission electron microscopy and the energy dispersive X-ray analysis. The redox property of the FAD/Ti<sub>3</sub>C<sub>2</sub>T<sub>x</sub> modified glassy carbon electrode (FAD/Ti<sub>3</sub>C<sub>2</sub>T<sub>x</sub>/GCE) was highly dependent on pH and exhibited a stable redox peak at −0.455 V in neutral medium. Higher amounts of FAD molecules were loaded onto the 2D MXene (Ti<sub>3</sub>C<sub>2</sub>T<sub>x</sub>)-modified electrode, which was two times higher than the values in the reported work, and the surface coverage (ᴦ<sub>FAD</sub>) was 0.8 × 10<sup>−10</sup> mol/cm<sup>2</sup>. The FAD/Ti<sub>3</sub>C<sub>2</sub>T<sub>x</sub> modified sensor showed the electrocatalytic reduction of H<sub>2</sub>O<sub>2</sub> at −0.47 V, which was 130 mV lower than the bare electrode. The FAD/Ti<sub>3</sub>C<sub>2</sub>T<sub>x</sub>/GCE sensor showed a linear detection of H<sub>2</sub>O<sub>2</sub> from 5 nM to 2 µM. The optimization of FAD deposition, amount of Ti<sub>3</sub>C<sub>2</sub>T<sub>x</sub> loading, effect of pH and the interference study with common biochemicals such as glucose, lactose, dopamine (DA), potassium chloride (KCl), ascorbic acid (AA), amino acids, uric acid (UA), oxalic acid (OA), sodium chloride (NaCl) and acetaminophen (PA) have been carried out. The FAD/Ti<sub>3</sub>C<sub>2</sub>T<sub>x</sub>/GCE showed high selectivity and reproducibility. Finally, the FAD/Ti<sub>3</sub>C<sub>2</sub>T<sub>x</sub> modified electrode was successfully applied to detect H<sub>2</sub>O<sub>2</sub> in ovarian cancer cell lines.
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