28 March 2002 Fluorescence quenching competitive fluoroimmunoassay in microdroplets
Author Affiliations +
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.
© (2002) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE). Downloading of the abstract is permitted for personal use only.
Jun Feng, Jun Feng, Guomin Shan, Guomin Shan, Bruce D. Hammock, Bruce D. Hammock, Ian M. Kennedy, Ian M. Kennedy, } "Fluorescence quenching competitive fluoroimmunoassay in microdroplets", Proc. SPIE 4634, Methods for Ultrasensitive Detection II, (28 March 2002); doi: 10.1117/12.463830; https://doi.org/10.1117/12.463830
PROCEEDINGS
10 PAGES


SHARE
Back to Top