Rolling Circle Amplification-Coupled Glass Nanopore Counting of Mild Traumatic Brain Injury-Related Salivary miRNAs

Mild traumatic brain injury (mTBI) could be underdiagnosed and underreported due to the delayed onset of symptoms and the conventional subjective assessment. Recent studies have suggested that salivary microRNAs (miRNAs) could be reliable biomarkers for objective mTBI diagnosis. In this work, we demonstrated a rolling circle amplification (RCA)-coupled resistive pulse-counting platform for profiling mTBI-related miRNAs, using easy-to-fabricate large glass nanopores (200 nm diameter). The method relies on the linear and specific elongation of the miRNA to a much larger RCA product, which the large glass nanopore can digitally count with a high signal-to-noise ratio. We developed and validated the RCA assay against let-7a, miR-30e, and miR-21. We demonstrated the quantification capability of this large glass nanopore counting platform for purified miRNAs as well as miRNAs in salivary total RNA background. Finally, we quantitatively evaluated the performance of profiling each individual miRNAs in a mixed analyte. Our results showed that the RCA-coupled large glass nanopore counting provides a promising and accessible alternative toward the clinical diagnosis of mTBI using salivary miRNAs.