Enzymes oxygen quenching

The dye is excited by light suppHed through the optical fiber (see Fiber optics), and its fluorescence monitored, also via the optical fiber. Because molecular oxygen, O2, quenches the fluorescence of the dyes employed, the iatensity of the fluorescence is related to the concentration of O2 at the surface of the optical fiber. Any glucose present ia the test solution reduces the local O2 concentration because of the immobilized enzyme resulting ia an iacrease ia fluorescence iatensity. This biosensor has a detection limit for glucose of approximately 100 ]lM , response times are on the order of a miaute. 

The phosphorescence lifetimes of various proteins at room temperature are given in Table 3.1. Some variability in the lifetimes reported from lab to lab is evident, possibly due to different enzyme preparation, removal of oxygen (see below), or other conditions. Nevertheless, when measured under the same conditions, it is apparent that the tryptophan lifetimes vary dramatically from protein to protein. Alkaline phosphatase exhibits the longest lifetime from a protein in solution with a lifetime of 1.5—1.7 s at 22°C, approaching the lifetime of 5.5 s at 77 K. The lifetime of free indole in solution is 15—30 /is at 22°C.(38 39) Therefore, in the absence of other quenching mechanisms, the lower limit for the phosphorescence lifetime of a fully exposed tryptophan moiety in a protein should be about 20 /is. 

The carrier used for this purpose consisted of a 0.1 M phosphate buffer of pH 7. The appearance of the sensing microzone is shown in Fig. 5.5.B. The oxygen optrode used was based on a 10-pm silicone rubber film containing dissolved decacyclene as indicator (S) that was fixed on a 110-pm thick polyester support (PS). A 9-pm black PTFE membrane (I) was used for optical insulation. The dye fluorescence was found to be markedly dependent on the concentration of oxygen, which exerted a quenching effect on it. The enzyme (glutamate oxidase) was immobilized on a 150-pm thick immunoaffmity membrane (E). The sensor was prepared similarly as reported by Trettnak et al. [7]. 

Recently, Veldink et al.10s) reported the observation of chemiluminescence during the oxygenation of linoleic acid by lipoxygenase. This phenomenon is observed only during the aerobic phase of the reaction and is quenched by superoxide dismutase. This suggests the involvement of superoxide in this process, which is not unreasonable considering the participation of ferrous iron and free radicals in the enzyme reaction. 

Similar optical biosensors have been prepared for many other analytes. For example, a cholesterol optical biosensor has been devised based on fluorescence quenching of an oxygen-sensitive dye that is coupled to consumption of oxygen resulting from the enzyme-catalyzed oxidation of cholesterol by the enzyme cholesterol oxidase. Serum bilirubin has been detected using bilirubin oxidase, coimmobilized with a ruthenium dye, on an optical fiber.The bilirubin sensor was reported to exhibit a lower detection limit of iO Xmol/L, a linear range up to 30mmol/L, and a typical reproducibihty of 3% (CV), certainly adequate for clinical application. 

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