Two-tiered electrical detection, purification, and identification of nucleic acids in complex media

We demonstrate a two-tiered platform for electrically detecting, purifying, and distinguishing mismatched nucleic acid sequences (DNA and RNA) in complex biological liquid samples. The first tier uses a nanoporous gold (np-Au) for the electrochemical detection and subsequent purification of nucleic acids. The sieve-like structure of np-Au blocks the permeation of large biomolecules and allows for selective transport of nucleic acids to the sensing surface, thereby obviating the need for prior purification steps. The second tier utilizes single-molecule electrical conductance measurements to verify sequence information and identify the presence of mismatches between the DNA probe and the RNA target. This step utilizes the single-molecule break junction approach to measure samples eluted from the first-tier to reduce the possibility of false positives from similar target sequences. In this report, the two-tiered approach is employed to detect a 15-base pair (bp) E. coli. RNA sequence in blood, and is further expanded to differentiate between the perfectly-matched case and partial mismatches of the DNA:RNA hybrids. (C) 2019 Elsevier Ltd. All rights reserved.