In this paper, we report the ultra-sensitive indirect electrochemical detection of E. coli O157:H7 using antibody functionalized primary (magnetic) beads for capture and polyguanine (polyG) oligonucleotide functionalized secondary (polystyrene) beads as an electrochemical tag. Vacuum filtration in combination with E. coli O157:H7 specific antibody modified magnetic beads were used for extraction of E. coli O157:H7 from 100 mL samples. The magnetic bead conjugated E. coli O157:H7 cells were then attached to polyG functionalized secondary beads to form a sandwich complex (magnetic bead/E. coli secondary bead). While the use of magnetic beads for immuno-based capture is well characterized, the use of oligonucleotide functionalized secondary beads helps combine amplification and potential multiplexing into the system. The antibody functionalized secondary beads can be easily modified with a different antibody to detect other pathogens from the same sample and enable potential multiplexing. The polyGs on the secondary beads enable signal amplification up to 10\(^{8}\) guanine tags per secondary bead (\(7.5\times10^{6}\) biotin-FITC per secondary bead, 20 guanines per oligonucleotide) bound to the target (E. coli). A single-stranded DNA probe functionalized reduced graphene oxide modified glassy carbon electrode was used to bind the polyGs on the secondary beads. Fluorescent imaging was performed to confirm the hybridization of the complex to the electrode surface. Differential pulse voltammetry (DPV) was used to quantify the amount of polyG involved in the hybridization event with tris(2,2'-bipyridine)ruthenium(II) (Ru(bpy)\(_{3}^{2+}\)) as the mediator. The amount of polyG signal can be correlated to the amount of E. coli O157:H7 in the sample. The method was able to detect concentrations of E. coli O157:H7 down to 3 CFU/100 mL, which is 67 times lower than the most sensitive technique reported in literature. The signal to noise ratio for this work was 3. We also demonstrate the use of the protocol for detection of E. coli O157:H7 seeded in waste water effluent samples.

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.