Highly Sensitive Bacteria Quantification Using Immunomagnetic Separation and Electrochemical Detection of Guanine-Labeled Secondary BeadsReportar como inadecuado




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1

Department of Mechanical Engineering, University of Utah, Salt Lake City, UT 84112, USA

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Espira Inc., 825 N 300 W Suite N-223, Salt Lake City, UT 84103, USA

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Department of Bioengineering, University of Utah, Salt Lake City, UT 84112, USA

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Guanine Inc., Salt Lake City, UT 84103, USA





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Authors to whom correspondence should be addressed.



Academic Editor: Stephane Evoy

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 tris2,2-bipyridinerutheniumII Rubpy\ {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





Autor: Harikrishnan Jayamohan 1,* , Bruce K. Gale 1,2, Bj Minson 2, Christopher J. Lambert 3, Neil Gordon 4 and Himanshu J. Sant 1,2,*

Fuente: http://mdpi.com/



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