Rapid and sensitive multiplex molecular diagnosis of respiratory pathogens using plasmonic isothermal RPA array chip

Abstract

The current clinically available multiplex molecular diagnostic technologies are difficult to apply to onsite diagnostic platforms due to their large and sophisticated instrumentation, long amplification times and limited number of simultaneous detections. We developed a plasmonic isothermal recombinase polymerase amplification (RPA) array chip for rapid and sensitive multiplex molecular detection. The 3D plasmonic substrate composed of Au nanoparticles (NPs) on dense Au nanopillars (NPOP) showed highly enhanced plasmon-enhanced fluorescence (PEF) of RPA products with long DNA amplicons (~200 bp). The plasmonic 4-plex RPA array chip successfully detected bacterial DNA within 30 min and viral RNA within 40 min; the sensitivity of the plasmonic RPA array chip was comparable to or 10-fold higher than that of the 4-pelx liquid-phase RPA and 4-plex liquid-phase PCR techniques. Additionally, no cross-reactivity was observed. The 4-plex plasmonic RPA array chip was preliminary evaluated using clinical respiratory viral-positive nasopharyngeal swab samples. This approach enables rapid, sensitive and high-multiplex molecular detection and can be used in the realization of a simplified and miniaturized platform for onsite multiplex molecular diagnostics.

Keywords: Multiplex detection; Onsite diagnostics; Plasmon-enhanced fluorescence; RPA; Solid-phase amplification.