Amplification-Free Detection of Micrornas Related to Clear Cell Renal Cell Carcinoma Utilizing a Novel Nanopore-Based Sensor

Abstract

MicroRNAs (miRs) are small noncoding RNAs that play an important role in gene regulation. Recent studies have shown the correlation of the miRs expression level to carcinogenesis. Currently, qRT-PCR technology is considered as the ‘gold standard’ for miR detection due to its high sensitivity and specificity. However, this technique requires time-consuming and expensive amplification steps along with labeling and enzymatic reactions. In this work, a new nanopore-based detection scheme utilizing a borosilicate micropipette and an assay of complementary gamma-peptide nucleic acid (γ-PNA) probes conjugated to polystyrene beads have been reported for the detection of miR-204 and miR-210 related to the clear cell Renal Cell Carcinoma (ccRCC). Electroosmotic flow (EOF) was induced as the driving force to transport PNA-beads harboring target miRs to the tip of the pore (sensing zone) which resulted in pore blockades with unique and easily distinguishable serrated shape electrical signals. However, in the case of the control experiments ionic current blockades with right-angled shape were detected. The results showed 1 to 10 fM concentration detection limit and 97.6% detection accuracy in 87 experiments. This simple, PCR-free, and robust platform has a technological appeal to be evolved into a quantitative measurement tool for analysis of miR biomarkers in basic and clinical research by correlating the dwelling time of the particles translocation through the pore with the concentration of the RNA oligomers bound to their surface.