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Proceedings Paper

Fluorescence quenching competitive fluoroimmunoassay in microdroplets
Author(s): Jun Feng; Guomin Shan; Bruce D. Hammock; Ian M. Kennedy
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Paper Abstract

The use of micro droplets as a medium for sensitive detection in fluorescence-based immunoassays has been explored in two contexts. The competitive immuno-reaction of a pesticide hapten, esfenvalerate, with its antibody was performed in micro droplets generated by a vibrating orifice aerosol generator system with a 10-micrometers diameter orifice. Fluorescence from Rhodamine 6G was excited by the second harmonic of a Nd:YAG laser and detected by a 1/8 m imaging spectrograph with a 512 X 512 thermoelectrically cooled, charged-coupled device (CCD) camera. The conjugate of esfenvalerate with rhodamine exhibited similar fluorescence to that of pure rhodamine 6G. When anti-esfenvalerate antibodies were added to the droplets, the fluorescence decreased due to quenching. When a sample of esfenvalerate was added to the droplets, esfenvalerate and esfenvalerate- rhodamine conjugate competed for binding with the anti- esfenvalerate antibody. The release of the conjugated rhodamine from the antigen-antibody complex allowed the fluorescence signal to recover. The assay in a picoliter droplet sample was shown to enable detection down to approximately 0.1 nM. Micro droplets also exhibit strong cavity-dependent optical behavior that gives rise to lasing action. Lasing from Rhodamine fluorescence was quenched by the addition of a second dye, oxonol, that absorbs in the spectral region where the Rh 6G fluorescence peaked. A very strong impact on the droplet resonances was observed, leading to the possible use of quenching as an assay for oxonol-labeled haptens.

Paper Details

Date Published: 28 March 2002
PDF: 10 pages
Proc. SPIE 4634, Methods for Ultrasensitive Detection II, (28 March 2002); doi: 10.1117/12.463830
Show Author Affiliations
Jun Feng, Univ. of California/Davis (United States)
Guomin Shan, Univ. of California/Davis (United States)
Bruce D. Hammock, Univ. of California/Davis (United States)
Ian M. Kennedy, Univ. of California/Davis (United States)

Published in SPIE Proceedings Vol. 4634:
Methods for Ultrasensitive Detection II
Charles W. Wilkerson, Editor(s)

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