Ultra-low concentration detection of DNA without its amplification using a biological nanopore.

Abstract

Nanopore sensing is a powerful tool for single-molecule detection. A single molecule is recognized and detected from the information obtained when the target molecule passes through the nanoscale pore by electrophoresis. However, practically it is challenging to measure the target molecules at ultra-low concentrations because it takes a long time for the target molecule to enter the pore. Several elaborate techniques with a biological nanopore, α-hemolysin (αHL), have been proposed to detect the subpicomolar or femtomolar nucleic acids. Wang et al. used a salt gradient between solutions that sandwiched the αHL pore to enhance the electric field and increased the capture frequency of the targets, resulting in the detection of nucleic acid molecules at 0.1 pM. Zhang et al. showed the detection of target nucleic acids at 1 fM using the αHL pore with isothermal amplification of the targets. In contrast, we have recently reported the detection of 0.5 fM nucleic acids without their amplification reaction in the presence of a 500 nM probe complementary binding to the target. The excess amount of the probe relative to the target molecules was assumed to increase the capture frequency of the target/probe duplexes. Here, we attempt to reveal the mechanism of detecting DNA molecules at ultra-low concentrations with the αHL nanopore using probes with complementary sequences to the target molecules. We found that the complementary strand of the target in the probe sequence enhances the capture frequency of the target/probe duplexes by interacting with the target/probe molecules. In addition, higher concentrations of the probe relative to the target molecule (>107-fold) resulted in more frequent capture. This approach potentially advances nanopore technology toward its application in the direct detection of nucleic acid biomarkers at ultra-low levels in body fluids.