Systems Immobilized on Quartz Slides

With an aim to establish a basis for a fibro-optic biosensoring, a number of fluorescence molecular systems were immobilized on quartz surface and investigated. 

A series of substituted trans-stilbene derivatives have been prepared and immobilized on a quartz surface [19]. Several immobilization methods have been tried including the silanization technique, cross-linking with cyanuric chloride, surface activation with cyanogen bromide, and surface smoothing with coating proteins. 

Studies of solvent polar effects on the fluorescence spectrum of immobilized stilbenes indicated that the maximum wavelength of the fluorescence emission is not very sensitive to solvent polarity. The apparent local polarity of the medium in the vicinity of the stilbene label was estimated. The trans-cis photoisomerization kinetics of the stilbene derivatives in the immobilized and free state in a medium with different viscosity was monitored by fluorescence technique at constant illumination conditions. Investigation of the microviscosity effect on the photoisomerization of the immobilized and free stilbene label was carried out by changing the relative concentration of glycerin in a glycerin-water mixture used as a solvent. With an appropriate calibration, the microviscosity in the vicinity of the stilbene label was estimated. The apparent photoisomerization rate constant of the process was found to be 3-4 times less for the immobilized label than in a free state. 

A new photochrome-fluorescence-spin method for the simultaneous quantitative analysis of the redox status and viscosity of a medium has been developed [20], The method of the viscosity measurement is based on the use of double fluorescence-nitroxide molecules. 

In such hybrid compounds, the nitroxide moiety quenches the fluorescence of the fluorophore (stilbene moiety). The reduction of nitroxide segment by an antioxidant (ascorbic acid) caused a rise in fluorescence of the fluorophore. The rate constant of the stilbene fragment photoisomerization in such systems depended on the viscosity of the medium. The synthesized dual stilbene-nitroxide probe was covalently immobilized onto the surface of a quartz plate as an eventual sensor. The immobilization procedure included a cyanogen bromide surface activation followed by smoothing with a protein tether. The rate of fluorescence change was monitored in aqueous glycerol solution of different viscosities and content of ascorbic acid. The dependence of kapp on the reciprocal absolute viscosity 1 /T] of the bulk mixture glycerin-water and the dependence of the initial intensity of fluorescence (/o) on solution viscosity were also studied. 

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