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Fiber-Optic Fluoroimmunoassay System with a Flow-Through Cell for Rapid On-Site Determination of Escherichia coli O157:H7 by Monitoring Fluorescence Dynamics.

Miyajima K, Koshida T, Arakawa T, Kudo H, Saito H, Yano K, Mitsubayashi K - Biosensors (Basel) (2013)

Bottom Line: The measurement for each sample was completed within 12 min.This minimized the time for measurement down to 6 min.The system is suitable for rapid and direct determination for microorganisms or bacteria in food, clinical, and environmental sources.

View Article: PubMed Central - PubMed

Affiliation: Department of Advanced Sciences and Technology for Biomedical Sensors, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549, Japan; E-Mail: miya.bdi@tmd.ac.jp ; Department of Biomedical Devices and Instrumentation, Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, 2-3-10 Kanda-Surugadai, Chiyoda-ku, Tokyo 101-0062, Japan; E-Mails: arakawa.bdi@tmd.ac.jp (T.A.); kudo.bdi@tmd.ac.jp (H.K.).

ABSTRACT
Dynamic fluoroimmunoassay with a flow-through system using optical fiber probes consisting of polystyrene was developed and applied to a quantitative detection of E. coli O157:H7. The system measures E. coli as fluorescence of sandwich-type immune complexes formed by capture antibodies immobilized on the surface of the probe, E. coli cells, and fluorescently labeled detection antibodies. Excitation was carried out using an evanescent wave from the probe. Resulting fluorescence recoupled into the probe was detected by a photodiode. The assay system was constructed with a flow cell which was available for sequential injection of experimental reagents. In vitro characterization was performed using the flow cell, and the calibration range of E. coli O157:H7 was from 10(3) to 10(7) cells/mL. The measurement for each sample was completed within 12 min. Furthermore, it was also possible to estimate the concentrations of E. coli O157:H7 by the increasing rate of fluorescence during binding reaction of detection antibodies to antigens. This minimized the time for measurement down to 6 min. The system is suitable for rapid and direct determination for microorganisms or bacteria in food, clinical, and environmental sources.

No MeSH data available.


Related in: MedlinePlus

(a) Design of flow-through fluoroimmunoassay system. Bacteria solution and fluorescently labeled antibody are injected with syringes to the flow cell according to each measurement step. (b) The measurement step of the fiber-optic fluoroimmunoassay system. Fluorescent intensities are measured every measurement step, and the concentrations of E. coli O157:H7 are quantified.
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biosensors-03-00120-f003: (a) Design of flow-through fluoroimmunoassay system. Bacteria solution and fluorescently labeled antibody are injected with syringes to the flow cell according to each measurement step. (b) The measurement step of the fiber-optic fluoroimmunoassay system. Fluorescent intensities are measured every measurement step, and the concentrations of E. coli O157:H7 are quantified.

Mentions: Figure 3(a) represents the composition of the flow-through measurement system. E. coli O157:H7 cells and Cy5-labeled antibodies are put in the syringe by 800 µL and the optical fiber probe adsorbed with the capture antibodies is set in the flow cell. By controlling the injecting or collecting reagents in syringes, the antigen–antibody reaction is done and immune complexes are formed on the probe surface. Preliminarily, capture antibodies were immobilized by dipping the probe in the 100 μL of capture antibody solution, which contained 1.0 mg/mL capture antibodies, at 4 °C for 15 h. Then the probe was rinsed with phosphate buffer (PB; pH 7.4) and incubated with 1% BSA-PB to block the nonspecific binding site.


Fiber-Optic Fluoroimmunoassay System with a Flow-Through Cell for Rapid On-Site Determination of Escherichia coli O157:H7 by Monitoring Fluorescence Dynamics.

Miyajima K, Koshida T, Arakawa T, Kudo H, Saito H, Yano K, Mitsubayashi K - Biosensors (Basel) (2013)

(a) Design of flow-through fluoroimmunoassay system. Bacteria solution and fluorescently labeled antibody are injected with syringes to the flow cell according to each measurement step. (b) The measurement step of the fiber-optic fluoroimmunoassay system. Fluorescent intensities are measured every measurement step, and the concentrations of E. coli O157:H7 are quantified.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4263595&req=5

biosensors-03-00120-f003: (a) Design of flow-through fluoroimmunoassay system. Bacteria solution and fluorescently labeled antibody are injected with syringes to the flow cell according to each measurement step. (b) The measurement step of the fiber-optic fluoroimmunoassay system. Fluorescent intensities are measured every measurement step, and the concentrations of E. coli O157:H7 are quantified.
Mentions: Figure 3(a) represents the composition of the flow-through measurement system. E. coli O157:H7 cells and Cy5-labeled antibodies are put in the syringe by 800 µL and the optical fiber probe adsorbed with the capture antibodies is set in the flow cell. By controlling the injecting or collecting reagents in syringes, the antigen–antibody reaction is done and immune complexes are formed on the probe surface. Preliminarily, capture antibodies were immobilized by dipping the probe in the 100 μL of capture antibody solution, which contained 1.0 mg/mL capture antibodies, at 4 °C for 15 h. Then the probe was rinsed with phosphate buffer (PB; pH 7.4) and incubated with 1% BSA-PB to block the nonspecific binding site.

Bottom Line: The measurement for each sample was completed within 12 min.This minimized the time for measurement down to 6 min.The system is suitable for rapid and direct determination for microorganisms or bacteria in food, clinical, and environmental sources.

View Article: PubMed Central - PubMed

Affiliation: Department of Advanced Sciences and Technology for Biomedical Sensors, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549, Japan; E-Mail: miya.bdi@tmd.ac.jp ; Department of Biomedical Devices and Instrumentation, Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, 2-3-10 Kanda-Surugadai, Chiyoda-ku, Tokyo 101-0062, Japan; E-Mails: arakawa.bdi@tmd.ac.jp (T.A.); kudo.bdi@tmd.ac.jp (H.K.).

ABSTRACT
Dynamic fluoroimmunoassay with a flow-through system using optical fiber probes consisting of polystyrene was developed and applied to a quantitative detection of E. coli O157:H7. The system measures E. coli as fluorescence of sandwich-type immune complexes formed by capture antibodies immobilized on the surface of the probe, E. coli cells, and fluorescently labeled detection antibodies. Excitation was carried out using an evanescent wave from the probe. Resulting fluorescence recoupled into the probe was detected by a photodiode. The assay system was constructed with a flow cell which was available for sequential injection of experimental reagents. In vitro characterization was performed using the flow cell, and the calibration range of E. coli O157:H7 was from 10(3) to 10(7) cells/mL. The measurement for each sample was completed within 12 min. Furthermore, it was also possible to estimate the concentrations of E. coli O157:H7 by the increasing rate of fluorescence during binding reaction of detection antibodies to antigens. This minimized the time for measurement down to 6 min. The system is suitable for rapid and direct determination for microorganisms or bacteria in food, clinical, and environmental sources.

No MeSH data available.


Related in: MedlinePlus