Fluorescence-lifetime-based determination of anions using a site-directed mutant enzyme transducer

Richard B. Thompson; Hai-Jui Lin; Zhengfang Ge; Kelly Johnson; Carol A. Fierke

doi:10.1117/12.273524

7 May 1997 Fluorescence-lifetime-based determination of anions using a site-directed mutant enzyme transducer

Richard B. Thompson, Hai-Jui Lin, Zhengfang Ge, Kelly Johnson, Carol A. Fierke

Proceedings Volume 2980, Advances in Fluorescence Sensing Technology III; (1997) https://doi.org/10.1117/12.273524
Event: BiOS '97, Part of Photonics West, 1997, San Jose, CA, United States

Abstract

In comparison to metal ions in aqueous solutions, there are few methods for analysis of small anions such as cyanide, cyanate, carbonate, sulfide, and nitrate. Yet such analytes are important as environmental pollutants and as reagents and byproducts of industrial processes, paper manufacture, and mining. For some time we have been developing fluorescence-based fiber optic biosensors for metal ions such as zinc, cobalt, copper, mercury, nickel and cadmium, using the unparalleled selectivity and avidity of a metalloenzyme, human carbonic anhydrase. In the cases of Cu²⁺, CO²⁺, and Ni²⁺, we made use of the characteristic weak d-d absorbance bands of these metals when bound in the active site of the enzyme to serve as a fluorescence energy transfer acceptor for a suitably positioned fluorescent label attached to the enzyme. For this approach the intensity and lifetime of the fluorophore reflect the degree of energy transfer, and therefore the concentration of the metal. To measure certain anions such as cyanide and cyanate, we made use of the well-known perturbation of the d-d absorbance of Co²⁺ when an anion inhibitor becomes bound and inhibits the enzyme. These changes in absorbance modify the overlap integral with a suitable fluorescent label, and thereby the degree of energy transfer, resulting in a perturbation of the intensity and lifetime.

Citation Download Citation

Richard B. Thompson, Hai-Jui Lin, Zhengfang Ge, Kelly Johnson, and Carol A. Fierke "Fluorescence-lifetime-based determination of anions using a site-directed mutant enzyme transducer", Proc. SPIE 2980, Advances in Fluorescence Sensing Technology III, (7 May 1997); https://doi.org/10.1117/12.273524

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