Detection of low level pathogens and antimicrobial resistance genes using electrochemical oligonucleotide tags

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. This work demonstrates a new approach for electrochemically detecting pathogens and antimicrobial resistance genes that shifts the guanine oxidation source from naturally occurring RNA to synthetic oligonucleotides. Signal amplification is accomplished by binding RNA from lysed cells to microparticles conjugated with millions of guanine-rich oligonucleotide tags. A sandwich hybridization assay binds RNA between a screen printed carbon working electrode conjugated with recognition probes and a microparticle conjugated with electrochemical oligonucleotide tags. The tags contain a polyguanine detection sequence and an RNA capture sequence on the same oligonucleotide. Single stranded polyguanine is prefabricated into a quadruplex to enable 8-oxoguanine signals at 0.47V, which eliminates nonspecific guanine oxidation signals from the RNA while further reducing LOD over guanine oxidation. A 70 mer capture sequence was found to be more selective and hybridize faster at room temperature than conventional 20 mer capture sequences. Particle sizes were evaluated from 100 nm to 3 µm in diameter, and the larger diameter particles produced a greater detection signal. Better performance was obtained with magnetic microparticles which allowed magnetic separation of target RNA from nonspecific materials. The high density magnetic microparticles rested on the electrode surface causing a portion of the oligonucleotides to adsorb to the working electrode surface.