A novel fiber-optic probe for detection of enzyme activity using conventional substrates.

摘要

The thesis describes the fabrication and the characterization of a new fiber-optic probe which can form an integrated fluorescence-detecting system with a miniaturized fiber-optic spectrometer to detect enzyme-substrate reactions as well as the activities of enzymes, including enzymes produced by E. coli and Total Coliform.; Polydimethylsiloxane (PDMS) was used to produce round beads which were subsequently modified by 3-aminopropyl triethoxysilane (APTES) and then cut into hemispheres to glue to fibers. Qualitative tests using 2,4-dinitrofluorobenzene (DNFB) and IR spectra confirmed that an APTES-rich coating was formed on the PDMS surface.; Fluorescence detection showed the new probes could adsorb 4-methylumbelliferone (MU), a product of some specific enzyme-substrate reactions, though the signals produced included fluorescence both from adsorbed MU and MU in the solution. Sequential MU addition tests showed that the signal from adsorbed MU was consistent with a Langmuir adsorption isotherm. Photodegradation was observed during the sequential addition tests, and methods were developed to avoid it. Probes prepared from different APTES concentration reagent solutions (1%, 5%, 10%, 20%) were used to determine that the 5% APTES solution gave the probe with the best performance. Optimizing the adsorbed signal provided a probe which could detect MU even in opaque solutions.; Enzyme reactions where MU was the product were studied under different substrate concentrations to demonstrate enzyme activity detection with the probe. Michaelis-Menten parameters were determined and were in agreement with literature values. Enzyme activities of two enzymes were measured using different enzyme concentrations, and as little as 1 g of enzyme could be detected. Finally, qualitative tests for the detection of E. coli and Total Coliform in real lake water samples showed positive results.