H5N1 flu is a highly virulent and variable subtype of influenza with significant epidemic and pandemic potential. In this study, we introduce a novel, maskless, and rapid manufacturing process for a microfluidic chip integrated with electrodes for the quantitative detection of H5N1-DNA sequences. This detection leverages a catalytic redox-recycling signal via a novel Fe₃O₄@TMU-8 nanocomposite, which facilitates the turnover of the oxidation state of [Ru(NH₃)₆]³⁺, thereby amplifying the electrochemical signal output. The positively charged [Ru(NH₃)₆]³⁺ molecule associates with the phosphate backbone of the nucleic acids in H5N1-DNA. Changes in the aptasensor’s redox-recycling signal, due to the hybridization of DNA sequences with [Ru(NH₃)₆]³⁺, were used as the elec trochemical sensing response. Under optimal conditions, the signal exhibited a linear relationship with H5N1- DNA concentration, ranging from 1 fM to 1 nM, with a detection limit of 0.16 fM. This report details the fabrication of the microfluidic device using Poly(methyl methacrylate) (PMMA) sheet substrates. A laser system was employed to generate microfluidic patterns directly on the PMMA sheet. This biosensing device demon strated long-term stability and good reproducibility, making it suitable for the quantitative assay of H5N1-DNA sequences. The results from food sample analyses further confirmed the applicability and effectiveness of the resulting biosensor
