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Highly sensitive bacteria quantification using immunomagnetic separation and electrochemical detection of guanine-labeled secondary beads.
Sensors (Basel). 2015 May 22; 15(5):12034-52.S

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 10⁸ guanine tags per secondary bead (7.5 x 10⁶ 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.

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Authors+Show Affiliations

Jayamohan H
Department of Mechanical Engineering, University of Utah, Salt Lake City, UT 84112, USA. hari.jayamohan@utah.edu.
Gale BK
Department of Mechanical Engineering, University of Utah, Salt Lake City, UT 84112, USA. bjminson@gmail.com. Espira Inc., 825 N 300 W Suite N-223, Salt Lake City, UT 84103, USA. bjminson@gmail.com.
Minson B
Espira Inc., 825 N 300 W Suite N-223, Salt Lake City, UT 84103, USA. bjminson@gmail.com.
Lambert CJ
Department of Bioengineering, University of Utah, Salt Lake City, UT 84112, USA. chris.lambert@utah.edu.
Gordon N
Guanine Inc., Salt Lake City, UT 84103, USA. neil.gordon@guanineinc.com.
Sant HJ
Department of Mechanical Engineering, University of Utah, Salt Lake City, UT 84112, USA. himanshu.sant@utah.edu. Espira Inc., 825 N 300 W Suite N-223, Salt Lake City, UT 84103, USA. himanshu.sant@utah.edu.

MeSH

Bacteriological TechniquesBiosensing TechniquesElectrochemical TechniquesEscherichia coli O157GuanineImmunomagnetic Separation

Pub Type(s)

Journal Article
Research Support, U.S. Gov't, Non-P.H.S.

Language

eng

PubMed ID

26007743

Citation

Jayamohan, Harikrishnan, et al. "Highly Sensitive Bacteria Quantification Using Immunomagnetic Separation and Electrochemical Detection of Guanine-labeled Secondary Beads." Sensors (Basel, Switzerland), vol. 15, no. 5, 2015, pp. 12034-52.
Jayamohan H, Gale BK, Minson B, et al. Highly sensitive bacteria quantification using immunomagnetic separation and electrochemical detection of guanine-labeled secondary beads. Sensors (Basel). 2015;15(5):12034-52.
Jayamohan, H., Gale, B. K., Minson, B., Lambert, C. J., Gordon, N., & Sant, H. J. (2015). Highly sensitive bacteria quantification using immunomagnetic separation and electrochemical detection of guanine-labeled secondary beads. Sensors (Basel, Switzerland), 15(5), 12034-52. https://doi.org/10.3390/s150512034
Jayamohan H, et al. Highly Sensitive Bacteria Quantification Using Immunomagnetic Separation and Electrochemical Detection of Guanine-labeled Secondary Beads. Sensors (Basel). 2015 May 22;15(5):12034-52. PubMed PMID: 26007743.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Highly sensitive bacteria quantification using immunomagnetic separation and electrochemical detection of guanine-labeled secondary beads. AU - Jayamohan,Harikrishnan, AU - Gale,Bruce K, AU - Minson,Bj, AU - Lambert,Christopher J, AU - Gordon,Neil, AU - Sant,Himanshu J, Y1 - 2015/05/22/ PY - 2015/03/23/received PY - 2015/05/07/accepted PY - 2015/5/27/entrez PY - 2015/5/27/pubmed PY - 2015/12/15/medline KW - Escherichia coli O157:H7 detection KW - biosensors KW - differential pulse voltammetry KW - electrochemical detection KW - immunomagnetic separation KW - pathogen detection SP - 12034 EP - 52 JF - Sensors (Basel, Switzerland) JO - Sensors (Basel) VL - 15 IS - 5 N2 - 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 10⁸ guanine tags per secondary bead (7.5 x 10⁶ 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. SN - 1424-8220 UR - https://www.unboundmedicine.com/medline/citation/26007743/Highly_sensitive_bacteria_quantification_using_immunomagnetic_separation_and_electrochemical_detection_of_guanine_labeled_secondary_beads_ DB - PRIME DP - Unbound Medicine ER -