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Highly sensitive bacteria quantification using immunomagnetic separation and electrochemical detection of guanine-labeled secondary beads.

Jayamohan H, Gale BK, Minson B, Lambert CJ, Gordon N, Sant HJ - Sensors (Basel) (2015)

Bottom Line: A single-stranded DNA probe functionalized reduced graphene oxide modified glassy carbon electrode was used to bind the polyGs on the secondary beads.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 Article: PubMed Central - PubMed

Affiliation: Department of Mechanical Engineering, University of Utah, Salt Lake City, UT 84112, USA. hari.jayamohan@utah.edu.

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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Electrochemical signal corresponding to E. coli O157:H7 in waste water effluent samples. Negative control is in the form of DI water without any E. coli O157:H7 in it. EC measurement condition: pulse size: 20 mV, scan rate: 5 mV/s, scan range 0.5 V to 1.2 V (vs. Ag/AgCl reference electrode). Supporting electrolyte: 0.2 M acetate buffer solution (pH 5) containing 5 μM .
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f9-sensors-15-12034: Electrochemical signal corresponding to E. coli O157:H7 in waste water effluent samples. Negative control is in the form of DI water without any E. coli O157:H7 in it. EC measurement condition: pulse size: 20 mV, scan rate: 5 mV/s, scan range 0.5 V to 1.2 V (vs. Ag/AgCl reference electrode). Supporting electrolyte: 0.2 M acetate buffer solution (pH 5) containing 5 μM .

Mentions: Our assay was able to detect E. coli O157:H7 in waste water plant effluent (Figure 9). The amount of native E. coli O157:H7 in waste water effluent samples was unknown. The initial test yielded a 65 nA signal. The waste water effluent was then seeded with 300 CFU E. coli O157:H7 and tested. The results in Figure 8, show the electrochemical signal at 225 nA post-seeding with 300 CFU E. coli O157:H7. The difference in signal corresponds to 225 − 65 = 180 nA which is 95% of signal corresponding to 300 CFUs tested in PBS buffer solution (Figure 8a). The negative control (DI water) gave a signal of 20 nA which corresponds to signal range for 0 CFUs in buffer. Post autoclaving the waste water sample gave a detection signal, indicating that dead bacteria were also detected. One possible solution to fix this would be to run an additional scan after a prescribed time (about 1 h) to gauge the amount of live bacteria.


Highly sensitive bacteria quantification using immunomagnetic separation and electrochemical detection of guanine-labeled secondary beads.

Jayamohan H, Gale BK, Minson B, Lambert CJ, Gordon N, Sant HJ - Sensors (Basel) (2015)

Electrochemical signal corresponding to E. coli O157:H7 in waste water effluent samples. Negative control is in the form of DI water without any E. coli O157:H7 in it. EC measurement condition: pulse size: 20 mV, scan rate: 5 mV/s, scan range 0.5 V to 1.2 V (vs. Ag/AgCl reference electrode). Supporting electrolyte: 0.2 M acetate buffer solution (pH 5) containing 5 μM .
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4481928&req=5

f9-sensors-15-12034: Electrochemical signal corresponding to E. coli O157:H7 in waste water effluent samples. Negative control is in the form of DI water without any E. coli O157:H7 in it. EC measurement condition: pulse size: 20 mV, scan rate: 5 mV/s, scan range 0.5 V to 1.2 V (vs. Ag/AgCl reference electrode). Supporting electrolyte: 0.2 M acetate buffer solution (pH 5) containing 5 μM .
Mentions: Our assay was able to detect E. coli O157:H7 in waste water plant effluent (Figure 9). The amount of native E. coli O157:H7 in waste water effluent samples was unknown. The initial test yielded a 65 nA signal. The waste water effluent was then seeded with 300 CFU E. coli O157:H7 and tested. The results in Figure 8, show the electrochemical signal at 225 nA post-seeding with 300 CFU E. coli O157:H7. The difference in signal corresponds to 225 − 65 = 180 nA which is 95% of signal corresponding to 300 CFUs tested in PBS buffer solution (Figure 8a). The negative control (DI water) gave a signal of 20 nA which corresponds to signal range for 0 CFUs in buffer. Post autoclaving the waste water sample gave a detection signal, indicating that dead bacteria were also detected. One possible solution to fix this would be to run an additional scan after a prescribed time (about 1 h) to gauge the amount of live bacteria.

Bottom Line: A single-stranded DNA probe functionalized reduced graphene oxide modified glassy carbon electrode was used to bind the polyGs on the secondary beads.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 Article: PubMed Central - PubMed

Affiliation: Department of Mechanical Engineering, University of Utah, Salt Lake City, UT 84112, USA. hari.jayamohan@utah.edu.

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.

Show MeSH
Related in: MedlinePlus