Silicon Micropillar-Enhanced CRISPR Biosensor for Rapid and Sensitive Detection of Drug-Resistant Bacteria

1 The growing threat of antibiotic-resistant pathogens, such as methicillin-resistant Staphylococcus aureus (MRSA), underscores the urgent need for rapid, sensitive, and field-deployable diagnostic technologies. Here, we present a silicon micropillar-enhanced CRISPR biosensor that integrates high-aspect-ratio microstructures with a one-pot RPA/CRISPR-Cas12a assay for ultrasensitive and specific detection of MRSA. Micropillar arrays with fixed diameters and varying heights (100 µm, 300 µm, and 500 µm) were fabricated via deep reactive ion etching and functionalized for surface probe immobilization. Suboptimal crRNA design was employed to modify Cas12a activation kinetics, enabling declined trans-cleavage and enhanced end-point signal accumulation. The 500 µm micropillar configuration demonstrated a tenfold improvement in sensitivity compared to the 100 µm array, with a limit of detection reaching 10 ³ CFU mL ^-1 . The platform also showed high specificity against non-target bacterial strains. These findings highlight the potential of combining microstructured chips with one-pot CRISPR diagnostics to advance next-generation point-of-care tools for infectious disease monitoring.