Screening of highly-specific aptamers and their applications in paper-based microfluidic chips for rapid diagnosis of multiple bacteria

•A novel “systematic evolution of ligands by exponential enrichment” (SELEX) protocol was developed to screen bacterial aptamers.•A new dual-aptamer, paper-based microfluidic chip was developed for fast diagnosis of three common nosocomial bacteria.•The system possesses many advantages such as faster detection times, smaller size and capability to detect multiple pathogens simultaneously. A bacterial “systematic evolution of ligands by exponential enrichment” protocol was developed herein to identify nucleic acid aptamers capable of binding molecules from three common nosocomial and antibiotic-resistant bacteria: Acinetobacter baumannii, Escherichia coli, and multidrug-resistant Staphylococcus aureus. This high-affinity and high-specificity process featured three selection stages for screening of bacteria-specific aptamers, and the aptamers identified were integrated into a microfluidic system. The biotin-labeled aptamers were first bound to nitrocellulose membranes housed within the chip and then incubated with bacteria; a tetramethyl benzidine (TMB)-streptavidin (blue) color reaction was next exploited upon binding of secondary aptamers to primary ones, thereby permitting bacterial detection. This new dual-aptamer microfluidic chip possesses many advantages over its traditional-scale counterparts, such as faster detection times (35 min), smaller size (7.0 cm × 5.0 cm × 1.2 cm), higher specificity, and the capability to detect multiple pathogens simultaneously; it may therefore be promising for point-of-care bacterial diagnostics.