Sol-gel technique has been applied to encapsulation of biomolecules, such as enzymes and coenzymes, because this method is a low temperature process and obtains a transparent porous matrix. In particular, the matrix prepared from organically substituted silanes may influence the typical chemical activity of organic molecules entrapped. To fabricate enzyme encapsulated sol-gel silica gel, thetramethyl orthosilicate (TMOS), 3-aminopropyltrimethoxysilane (APTMS), and methyltriethoxysilane (MTES) were used as precursors. Alcohol dehydrogenase (ADH) and β-nicotinamide adenine dinucleotide ($NAD^+$) were also used as an enzyme and a coenzyme encapsulated in gel matrix. The reduced form of coenzyme (NADH) absorbs strongly around 340nm and fluoresces with a 440~460nm fluorescent maximum so that the application to an optical biosensor is possible. Thus ADH can be employed as a sensor for alcohol that takes advantage of NADH fluorescence.
Using precursors with different functional group, transparent porous glasses in which NADH or $NAD^+$ /ADH were encapsulated were fabricated. Measurements of fluorescence and absorbance of enzyme encapsulated in gel were carried out with a spectrofluorimeter and a UV spectrophotometer, respectively. Thorough measurements, we confirmed that 450nm fluorescence intensity increased by enzyme reaction and enzyme activity was retained in gel. The pore size and microstructure of NADH encapsulated gel were measured using DSC, XRD, SEM, TEM. In summary, the enzyme activity depends on the microstructure of the gel influenced by the process conditions.
