Nanopore-based immunoassay based on rolling circle amplification and DNA fragmentations.

Abstract

In recent years, small-size portable nanopore-based sequencers have become a robust tool with unique advantages for genomics applications. However, a few challenges have impeded applying nanopore sequencers as sensitive, quantitative diagnostic tools. One of the major bottlenecks is the inefficient capture of molecules into a pore at pM or lower concentrations which are typical for disease biomarkers. To bridge this gap we have developed a signal amplification strategy utilizing immunocapture, isothermal rolling circle amplification, and sequence-specific fragmentation of the product to release multiple DNA reporter molecules for nanopore detection. This group of DNA reporter molecules that comprise different lengths and structures could produce a distinct nanopore cluster spectrum in the two-parameter space of dwell times and fractional current blockades. By identifying the nanopore cluster spectrum, we could identify the biomarker analytes and achieve quantification. As a proof of concept, we demonstrate the quantification of human epididymis protein 4 (HE4) to low pM levels using wild-type alpha-hemolysin. Future improvement that employ microfluidics and nanopore arrays can further reduce the limit of detection, allow multiplexed biomarker detection, and further, reduce the footprint, cost, and portability of existing laboratory and point-of-care devices.