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Sensors 2015, 15(5), 12034-12052; doi:10.3390/s150512034

Highly Sensitive Bacteria Quantification Using Immunomagnetic Separation and Electrochemical Detection of Guanine-Labeled Secondary Beads

1
Department of Mechanical Engineering, University of Utah, Salt Lake City, UT 84112, USA
2
Espira Inc., 825 N 300 W Suite N-223, Salt Lake City, UT 84103, USA
3
Department of Bioengineering, University of Utah, Salt Lake City, UT 84112, USA
4
Guanine Inc., Salt Lake City, UT 84103, USA
*
Authors to whom correspondence should be addressed.
Academic Editor: Stephane Evoy
Received: 23 March 2015 / Accepted: 7 May 2015 / Published: 22 May 2015
(This article belongs to the Special Issue Biosensors for Pathogen Detection)
View Full-Text   |   Download PDF [9047 KB, uploaded 22 May 2015]   |  

Abstract

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. View Full-Text
Keywords: Escherichia coli O157:H7 detection; biosensors; pathogen detection; electrochemical detection; differential pulse voltammetry; immunomagnetic separation Escherichia coli O157:H7 detection; biosensors; pathogen detection; electrochemical detection; differential pulse voltammetry; immunomagnetic separation
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

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MDPI and ACS Style

Jayamohan, H.; Gale, B.K.; Minson, B.; Lambert, C.J.; Gordon, N.; Sant, H.J. Highly Sensitive Bacteria Quantification Using Immunomagnetic Separation and Electrochemical Detection of Guanine-Labeled Secondary Beads. Sensors 2015, 15, 12034-12052.

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