Site-Selective Electrodeposition on a Microelectrode Array Chip for Parallel Detection of Staphylococcus aureus and Escherichia coli

Microelectrode arrays (MEAs) hold inherent potential for the parallel analysis of foodborne pathogens; however, their miniaturized scale introduces critical challenges for multitarget detection, including cross-talk between adjacent units during functionalization and a severely limited sensing area that restricts probe immobilization and ultimate sensitivity. To overcome these limitations, a MEA-based aptasensor is constructed by integrating site-selective electrodeposition with a directed molecular allocation strategy for highly sensitive and specific simultaneous detection of Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli). A graphene oxide-polypyrrole nanocomposite and an AuNPs/chitosan-gold composite were precisely deposited on different microelectrode units, enabling the spatially resolved immobilization of aptamers against S. aureus and E. coli, respectively. Fluorescence and Raman spectroscopy confirmed the selective allocation of the respective aptamers to their targeted electrode units, effectively preventing cross-talk. Using methylene blue as an electroactive probe, the binding of target pathogens to their respective aptamers was quantified by square wave voltammetry. With optimized conditions, the limit of detection for S. aureus and E. coli was as low as 25.4 CFU mL–1 and 8.8 CFU mL–1. Real-sample assays confirmed that the MEA-based aptasensor is a precise and viable tool for high-speed quantification of S. aureus and E. coli, exhibiting considerable potential for applications in environmental and food safety monitoring practices.