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

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.