Effect of ethanol variation on the internal environment of sol-gel bulk and thin films with aging.

Sol-gel derived bulk and thin films were prepared from different compositions at low pH ( approximately 2.0) containing varying concentrations of ethanol from 15 to 60% at constant water (H(2)O)/tetraethyl-orthosilicate (TEOS) ratio (R=4). The fluorescence microscopic and spectroscopic measurements on fluorescent probe, Hoechst 33258 (H258) entrapped in these compositions were carried out at different days of storage to monitor the effects of concentration of ethanol on the internal environment of sol-gel materials. Fluorescence microscopic observations on sol-gel thin films, prepared by dip coating technique depicted uniform and cracked surface at withdrawal speed 1cm/min (high speed) and 0.1cm/min (low speed) respectively, which did not change during aging. Fluorescence spectral measurements showed emission maximum of H258 at approximately 535 nm in fresh sols at all concentrations of ethanol which depicted slight blue shift to 512 nm during aging in bulk. No such spectral shift has been observed in sol-gel thin films coated at high speed whereas thin films coated at low speed clearly showed an additional band at approximately 404 nm at 45 and 60% concentration of ethanol after about one month of storage. Analysis of the fluorescence lifetime data indicated single exponential decay (1.6-1.8 ns) in fresh sol and from third day onwards, invariably double exponential decay with a short (tau(1)) and a long (tau(2)) component were observed in sol-gel bulk with a dominant tau(1) at approximately 1.2 ns at all concentrations of ethanol. A double exponential decay consisting of a short component (tau(1)) at approximately 0.2 ns and a long component (tau(2)) at approximately 3.5 ns were observed at all ethanol concentrations in both fresh and aged sol-gel thin films. Further, distribution analysis of lifetimes of H258 showed two mean lifetimes with increased width in aged bulk and thin films. These results are likely to have strong implications in designing the internal environment for applications in biosensors.

Fluorescence spectroscopic studies to characterize the internal environment of tetraethyl-orthosilicate derived sol-gel bulk and thin films with aging.

Characterization of the internal environment of a sol-gel matrix is an important area of investigation in optical biosensors. In the present study, different sol-gel compositions were prepared by varying the water (H2O) to tetraethyl-orthosilicate (TEOS) ratio (R) from 1 to 16 and the changes in the internal environment of the sol-gel both in bulk and thin films as a function of aging (storage) were investigated using fluorescence spectroscopy. We focussed on the fluorescence characteristics , viz. emission and excited state lifetime of Hoechst 33258 (H258), a bisbenzimidazole derivative, which was used as fluorescence probe entrapped in the TEOS derived sol-gel bulk and thin films. These sols were prepared at a low pH (approximately 2.0) and the thin films were coated by dip coating technique at withdrawal speeds of 1 cm/min and 0.1cm/min. Usually, uniform thin films were obtained at a high speed (1 cm/min) and partially cracked film at a low speed (0.1 cm/min) as observed by fluorescence microscope. These observations did not change during aging. On the contrary, three months long observations on steady-state fluorescence emission measurements on H258 depicted a blue shift from 535 nm to 508 nm at R = 1 in the sol-gel bulk, whereas at higher ratios this was not prominent. At all ratios, dual emission bands were observed in thin films. This may be due to faster sol-gel to xerogel transition during aging depending on the ratio (R). Analysis of the excited state decay profiles of H258 revealed a double exponential fitting having a short (tau1) and a long (tau2) component in both fresh and during aging, in the sol-gel bulk and thin films, indicating heterogeneity in the internal environment. The value of tau1 increased from 0.4 ns to 1.2 ns whereas tau2 attained a value from 3.0 ns to 3.6 ns at R = 1 upon aging in the sol-gel bulk. The corresponding values of tau1 and tau2 in thin films were 0.3 ns and 3.5 ns, respectively. The values of these decay components in thin films did not alter much due to storage, but their relative contributions showed more systematic changes in the thin films. The observed changes could be correlated to rigidification in the bulk depending on the ratio (R). This process was very slow at R > or = 4. The heterogeneity in the internal environment of bulk and thin films upon aging appeared to be different as revealed from analysis of excited-state lifetime. Thus, the bisbenzimidazole derivative H258 appears to be very useful probe for characterizing the internal environment of both the sol-gel bulk and thin films.

Activity and Conformation of Yeast Alcohol Dehydrogenase (YADH) Entrapped in Reverse Micelles.

Yeast alcohol dehydrogenase (YADH) solubilized in reverse micelles of aerosol OT (i.e., AOT or sodium bis (2-ethyl hexyl) sulfosuccinate) in isooctane has been shown to be catalytically more active than that in aqueous buffer under optimum conditions of pH, temperature, and water content in reverse micelles. Studies of the secondary structure conformational changes of the enzyme in reverse micelles have been made from circular dichroism spectroscopy. It has been seen that the conformation of YADH in reverse micelles is extremely sensitive to pH, temperature, and water content. A comparison has been made between the catalytic activity of the enzyme and the alpha-helix content in the conformation and it has been observed that the enzyme is most active at the maximum alpha-helix content. While the beta-sheet content in the conformation of the entrapped enzyme was found to be dependent on the enzyme-micelle interface interaction, the alpha-helix and random coil conformations are governed by the degree of entrapment and the extent of rigidity provided by the micelle core to the enzyme structure. Copyright 2000 Academic Press.

Differentiation of substrate-binding sites in pyruvate kinase by selective photoaffinity labeling.

The four substrate-binding sites in porcine liver pyruvate kinase have been labeled with the photoaffinity reagent 8-azido-2'-O-dansyl-[alpha-32P]ATP (AD-ATP) under different experimental conditions. In the dark, the native pyruvate kinase was reversibly and competitively inhibited by AD-ATP, with KI = 2.8 microM and KADP = 0.18 mM. Under UV-irradiation, the enzyme was covalently labeled in the presence of Mg2+ by AD-ATP and inactivated irreversibly. Measurement of this photoinactivation process in the presence of various concentrations of ADP gave KI = 4.0 microM and KADP = 0.2 mM. A linear plot of the relative specific activity of the partially photolabeled enzyme after gel-filtration vs. the number of label per pyruvate kinase molecule introduced in the presence of Mg2+ shows that each covalent label completely inactivates a tetrameric pyruvate kinase molecule. In the strict absence of Mg2+, up to three substrate-binding sites in each pyruvate kinase molecule can be labeled by AD-ATP without decreasing enzyme activity. Subsequent addition of Mg2+ enables AD-ATP to label the remaining site and inactivate the enzyme. These observations show that there are one catalytic and three non-catalytic substrate-binding sites in each pyruvate kinase molecule. A probable structural reason for possible functional differentiation of intrinsically identical substrate-binding sites in all tetrameric enzymes is suggested.

Skin friction drag measurements by LDV.

A laser Doppler velocimeter employing a microscope objective as the receiving lens has been developed for measuring fluid velocity inside the boundary layer flow field with a spatial resolution of 40 microm. The method was applied for direct measurement of aerodynamic skin friction drag from the measured velocity gradient at the wall. Experimental results obtained on skin friction and on velocity components in a turbulent boundary layer on a low speed wind tunnel showed good agreement with previously reported data using conventional instruments such as hot-wire anemometers and Preston tubes. The method thus provides a tool for measurement and control of skin friction on aerodynamic bodies without perturbing the flow field.