Absolute quantification of microRNAs based on mass transport limitation under a laminar flow SPR system

As an important biomarker for diagnostics and therapeutics of various diseases, the low-cost, quantitative detection method of microRNAs (miRNAs) has recently caught broad attention. However, their small size and low abundance still derive challenges to quantification detection. In this study, we developed an ultrasensitive and multiplexed surface plasmon resonance (SPR) biosensor for quantifying miRNAs without standard. We introduced the mass transport limitation (MTL) strategy for the absolute quantification of miRNAs. We first explore the mechanism of DNA capture and the condition for triggering MTL on the SPR biosensor. We demonstrated that probes of 22-25 nt in length with fewer influences of the secondary structure provide better triggering of MTL. For proof of concept studies, let-7a, miR-155 and miR-21 were selected as candidate targets. Based on the structure and kinetics analysis, we demonstrate the best capture probe efficiency, and this biosensor's limit of detection (LOD) is 500 fM without any signal amplification. Furthermore, our biosensor achieves multiplex detection, which could detect three targets simultaneously. The quantitative results of miRNA indicated the great prospects of our biosensor in nucleic acid-related early diagnosis and biosensing.