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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.


Monitoring of fluorescence during the entire time of measurement. The amount of fluorescent intensity of each concentration of E. coli O157:H7 increased during binding reaction of Cy5-labeled antibodies to cell-surface antigen.
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biosensors-03-00120-f006: Monitoring of fluorescence during the entire time of measurement. The amount of fluorescent intensity of each concentration of E. coli O157:H7 increased during binding reaction of Cy5-labeled antibodies to cell-surface antigen.

Mentions: The flow-through system was constructed by employing the flow cell linked to the optical detection unit. Supplying the reagents sequentially into the flow cell, fluorescence was able to be measured throughout the immunoreaction. Changes of fluorescence during binding reaction of Cy5-labeled antibodies to E. coli O157:H7 cells were monitored. Figure 6 represents the changes of the fluorescence for the entire time of measurement. During binding reaction of Cy5-labeled antibodies, the amount of fluorescent intensity of each concentration of E. coli O157:H7 increased as the reaction proceeded. In addition, the increasing rate of the fluorescence was associated with the concentration of E. coli O157:H7. Figure 7 shows the relation between antigen concentrations, and each plot in the figure represents the increased rate of fluorescence at 1 min after injection of Cy5-labeled antibodies. The calibration range was almost the same as the quantification method described above. The coefficient of determination (R) was 1.000 by the regression analysis as shown by the following equation:(2)


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)

Monitoring of fluorescence during the entire time of measurement. The amount of fluorescent intensity of each concentration of E. coli O157:H7 increased during binding reaction of Cy5-labeled antibodies to cell-surface antigen.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

biosensors-03-00120-f006: Monitoring of fluorescence during the entire time of measurement. The amount of fluorescent intensity of each concentration of E. coli O157:H7 increased during binding reaction of Cy5-labeled antibodies to cell-surface antigen.
Mentions: The flow-through system was constructed by employing the flow cell linked to the optical detection unit. Supplying the reagents sequentially into the flow cell, fluorescence was able to be measured throughout the immunoreaction. Changes of fluorescence during binding reaction of Cy5-labeled antibodies to E. coli O157:H7 cells were monitored. Figure 6 represents the changes of the fluorescence for the entire time of measurement. During binding reaction of Cy5-labeled antibodies, the amount of fluorescent intensity of each concentration of E. coli O157:H7 increased as the reaction proceeded. In addition, the increasing rate of the fluorescence was associated with the concentration of E. coli O157:H7. Figure 7 shows the relation between antigen concentrations, and each plot in the figure represents the increased rate of fluorescence at 1 min after injection of Cy5-labeled antibodies. The calibration range was almost the same as the quantification method described above. The coefficient of determination (R) was 1.000 by the regression analysis as shown by the following equation:(2)

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.