Fluorescence quenching competitive fluoroimmunoassay in microdroplets

Jun Feng; Guomin Shan; Bruce D. Hammock; Ian M. Kennedy

doi:10.1117/12.463830

28 March 2002 Fluorescence quenching competitive fluoroimmunoassay in microdroplets

Jun Feng, Guomin Shan, Bruce D. Hammock, Ian M. Kennedy

Proceedings Volume 4634, Methods for Ultrasensitive Detection II; (2002) https://doi.org/10.1117/12.463830
Event: High-Power Lasers and Applications, 2002, San Jose, California, United States

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.

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Jun Feng, Guomin Shan, Bruce D. Hammock, and Ian M. Kennedy "Fluorescence quenching competitive fluoroimmunoassay in microdroplets", Proc. SPIE 4634, Methods for Ultrasensitive Detection II, (28 March 2002); https://doi.org/10.1117/12.463830

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KEYWORDS

Luminescence

Rhodamine

Molecules

Spectroscopy

CCD cameras

Cameras

Laser scattering

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