Spectral properties and biological applications of ELF enzyme substrates that yield fluorescent precipitates at the enzymatic activity sites

Zhenjun Diwu; Dieter H. Klaubert; Richard P. Haugland

doi:10.1117/12.347530

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3 May 1999 Spectral properties and biological applications of ELF enzyme substrates that yield fluorescent precipitates at the enzymatic activity sites

Zhenjun Diwu, Dieter H. Klaubert, Richard P. Haugland

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Proceedings Volume 3602, Advances in Fluorescence Sensing Technology IV; (1999) https://doi.org/10.1117/12.347530
Event: BiOS '99 International Biomedical Optics Symposium, 1999, San Jose, CA, United States

Abstract

Although coumarin- and fluorescein-based fluorogenic substrates are widely used in a variety of enzyme assays, they are still ill-adapted for continuous assays in the cases of enzymes that have optimal activity below pH 7.0 because coumarins and fluoresceins generally have the maximum fluorescence above pH 10. Additionally the enzymatic products of coumarin- and fluorescein-based fluorogenic substrates are soluble and readily diffuse away from the site of enzyme activity, and are especially troublesome for in vivo applications. We have now developed a variety of 2-(2'- hydroxyphenyl)-4(3H)-quinazolinone (HPQ)-based enzyme substrates that have overcome these limitations. The water- soluble, colorless, and nonfluorescent substrates generate highly fluorescent and photostable precipitates upon enzymatic action without the addition of the secondary trapping agents. Such fluorogenic precipitating substrates ensure fluorescent histochemical imaging of enzymes with cytologically fine resolution and sharp contrast over cellular autofluorescence and therefore inherently enhance sensitivity and specificity of the targeted analysis.

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Zhenjun Diwu, Dieter H. Klaubert, and Richard P. Haugland "Spectral properties and biological applications of ELF enzyme substrates that yield fluorescent precipitates at the enzymatic activity sites", Proc. SPIE 3602, Advances in Fluorescence Sensing Technology IV, (3 May 1999); https://doi.org/10.1117/12.347530

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