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Proceedings Paper

BHHST: An improved lanthanide chelate for time-resolved fluorescence applications
Author(s): Russell Connally; Dayong Jin; James Piper
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Paper Abstract

The detection of the waterborne pathogens Giardia lamblia and Cryptosporidium parvum in environmental water bodies requires concentration of large volumes of water due to the low dose required for infection. The highly concentrated (10,000-fold) water sample is often rich in strongly autofluorescent algae, organic debris and mineral particles that can obscure immunofluorescently labeled (oo)cysts during analysis. Time-resolved fluorescence techniques exploit the long fluorescence lifetimes of lanthanide chelates (ms) to differentiate target fluorescence from background autofluorescence (ns). Relatively simple instrumentation can be used to enhance the signal-to-noise ratio (S/N) of labelled target. Time-resolved fluorescence techniques exploit the large difference in lifetime by briefly exciting fluorescence from the sample using a pulsed excitation source. Capture of the resulting fluorescence emission is delayed until the more rapidly decaying autofluorescence has faded beyond detection, whereon the much stronger and slower fading emission from labelled target is collected. BHHCT is a tetradentate beta-diketone chelate that is activated to bind with protein (antibody) as the chlorosulfonate. The high activity of this residue makes conjugations difficult to control and can lead to the formation of unstable immunoconjugates. To overcome these limitations a 5-atom hydrophylic molecular tether was attached to BHHCT via the chlorosulfonate and the BHHCT derivative was then activated to bind to proteins as the succinimide. The new compound (BHHST) could be prepared in high purity and was far more stable than the chlorosulfonate on storage. A high activity immunocojugate was prepared against Cryptosporidium that yielded an 8-fold increase in SNR using a lab-built time-resolved fluorescence microscope.

Paper Details

Date Published: 4 April 2005
PDF: 12 pages
Proc. SPIE 5704, Genetically Engineered and Optical Probes for Biomedical Applications III, (4 April 2005); doi: 10.1117/12.584818
Show Author Affiliations
Russell Connally, Macquarie Univ. (Australia)
Dayong Jin, Macquarie Univ. (Australia)
James Piper, Macquarie Univ. (Australia)


Published in SPIE Proceedings Vol. 5704:
Genetically Engineered and Optical Probes for Biomedical Applications III
Darryl J. Bornhop; Samuel I. Achilefu; Ramesh Raghavachari; Alexander P. Savitsky, Editor(s)

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