Engineering Proceedings (May 2023)
Detection of Pathogens and Antimicrobial Resistance Genes at Low Concentration via Electrochemical Oligonucleotide Tags
Abstract
Pathogens can be detected electrochemically by measuring guanine oxidation signals generated from RNA or DNA hybridized to a biosensor working electrode. However, the associated limit of detection (LOD) is not sufficiently low for widespread clinical use. Working electrodes employing nanomaterials such as carbon nanotubes successfully reduce the LOD, but nanosensors experience high variability, poor fabrication yield, and high production cost. Our work presented here demonstrates a novel approach for electrochemically detecting low-concentration pathogens and antimicrobial resistance genes that transfers the guanine oxidation source from naturally occurring RNA to synthetic oligonucleotides. In our assay, signal amplification is accomplished by binding RNA from lysed microbial cells to microparticles conjugated with millions of guanine-rich oligonucleotide tags. We employed a sandwich hybridization assay to bind RNA between a screen-printed carbon working electrode conjugated with recognition probes, and a microparticle conjugated with electrochemical oligonucleotide tags. These tags contained a polyguanine detection sequence and an RNA capture sequence on the same oligonucleotide. Single-stranded polyguanine was prefabricated into a quadruplex to enable 8-oxoguanine signals at 0.47 V. This eliminated nonspecific guanine oxidation signals from the RNA, while further reducing the LOD over guanine oxidation. A 70-mer capture sequence was found to be more selective and hybridized faster at room temperature than conventional 20-mer capture sequences. Particle sizes were evaluated from 100 nm to 1.5 µm in diameter, and the larger diameter particles produced greater detection signals. A better performance was obtained by employing magnetic microparticles and magnetically separating magnetic microparticle–RNA complexes from nonspecific materials, such as lysed cell constituents and cell debris, that can interfere with sandwich formation and detection. The high-density magnetic microparticles rested on the electrode surface, causing a portion of the oligonucleotides to adsorb to the working electrode surface.
Keywords
