Kinetically Regulated Isothermal Amplification Enables Enhanced microRNA Sensing via Solid-State Nanopores.

Abstract

Solid-state nanopore sensing has become a common method for biomarker detection, but it still faces challenges with short nucleic acid markers (<50 nt) such as microRNAs (miRNAs). A simple but effective signal amplification strategy, specifically optimized for nanopore detection, is therefore needed. Here, we have developed an efficient workflow based on hybridization chain reaction (HCR) to specifically extend the target miRNA and conduct single-molecule analysis within solid-state nanopores. The miRNA translocation signals in nanopores were amplified with an enhanced signal-to-noise ratio (SNR) through multi-step optimization and screening of amplification system design, enabling effective analysis across a wide range of nanopore sizes and voltages. We then introduced kinetically regulated hairpin engineering, validated by thermodynamic and molecular dynamics simulations, to address the challenges posed by the high heterogeneity of standard amplification products. This modification enables controlled amplification to improve product length uniformity, promote natural folding of DNA concatemers, and increase inter-molecule repulsive forces, thereby enhancing signal quality. With further optimization of nanopore size, we achieved accurate detection of target miRNA below 10 pM and verified quantitative performance in serum samples. Our work is expected to provide an easy and robust pathway for enhanced sensing of small nucleic acids using solid-state nanopores.