Digital-resolution and Highly Sensitive Detection of Multiple Exosomal Small RNAs by DNA Toehold Probe-based Photonic Resonator Absorption Microscopy

Small noncoding RNAs (snRNA) have been emerging as promising diagnostic biomarkers for detecting early stage cancer. Currently existing methods for snRNA detection, including northern blot, reverse transcription-polymerase chain reaction, microarrays and RNA-Seq, are limited to time-consuming, low sensitivity, expensive instrumentation or complex analysis of data. Herein, we present a rapid quantitative analysis of multiple liver cancer-associated exosomal snRNA by a nucleic acid toehold probe-based photonic resonator absorption microscopy (PRAM) assay, with digital resolution and high sensitivity. The assay relies on the use of three toehold probe-encoded gold nanoparticles (AuNPs) and addressable photonic crystal (PC) sensing chips. The presence of target snRNA will initiate toehold-mediated strand displacement reactions that trigger the capture of gold particles onto the PC surface, which is subsequently imaged by PRAM for digital counting of detected snRNA molecules. We achieved highly sensitive and selective detection of three snRNA targets in buffer with a 30 min assay protocol, with detection limits of 4.56 fM, 4.68 fM and 0.69 pM. Having confirmed our assay's performance for detection of snRNA targets spiked into exosomal RNA extracts, we demonstrated its capability for quantitative detection of the same targets from patient blood plasma samples. The approach offers a rapid, simple workflow that operates at room temperature with a single step without enzymatic amplification, while the detection instrument can be implemented as a low-cost portable system for point of care environments.