Recent advances in molecular technologies have provided various assay strategies for monitoring biomarkers, such as miRNAs for early detection of various diseases and cancers. However, there is still an urgent unmet need to develop practical and accurate miRNA analytical tools that could facilitate the incorporation of miRNA biomarkers into clinical practice and management. In this study, we demonstrate the feasibility of using a cascade amplification method, referred to as the "Cascade Amplification by Recycling Trigger Probe" (CARTP) strategy, to improve the detection sensitivity of the inverse Molecular Sentinel (iMS) nanobiosensor. The iMS nanobiosensor developed in our laboratory is a unique homogeneous multiplex bioassay technique based on surface-enhanced Raman scattering (SERS) detection, and was used to successfully detect miRNAs from clinical samples. The CARTP strategy based on the toehold-mediated strand displacement reaction is triggered by a linear DNA strand, called the "Recycling Trigger Probe" (RTP) strand, to amplify the iMS SERS signal. Herein, by using the CARTP strategy, we show a significantly improved detection sensitivity with the limit of detection (LOD) of 45 fM, which is 100-fold more sensitive than the non-amplified iMS assay used in our previous report. We envision that the further development and optimization of this strategy ultimately will allow multiplexed detection of miRNA biomarkers with ultra-high sensitivity for clinical translation and application.
Keywords: SERS; microRNA detection; molecular diagnostics; nanobiosensors; nanoprobes; nucleic acid detection; plasmonics; signal amplification; surface-enhanced Raman scattering.