Sensors, Vol. 15, Pages 12034-12052: Highly Sensitive Bacteria Quantification Using Immunomagnetic Separation and Electrochemical Detection of Guanine-Labeled Secondary Beads (Sensors)

In this paper, we report the ultra-sensitive indirect electrochemical detection of extit{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 extit{E. coli} O157:H7 specific antibody modified magnetic beads were used for extraction of extit{E. coli} O157:H7 from 100 mL samples. The magnetic bead conjugated extit{E. coli} O157:H7 cells were then attached to polyG functionalized secondary beads to form a sandwich complex (magnetic bead/ extit{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 imes10^{6}) biotin-FITC per secondary bead, 20 guanines per oligonucleotide) bound to the target ( extit{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 extit{E. coli} O157:H7 in the sample. The method was able to detect concentrations of extit{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 extit{E. coli} O157:H7 seeded in waste water effluent samples.