engleski

OPTICAL SENSOR FOR AMYLASE ACTIVITY MEASUREMENT

Alpha-amylase (EC 3.2.1.1), enzyme is widely used in industrial conversion of starch in to the sugars. It cleaves internal the -1, 4- glycosidic bonds in starch to produce glucose, maltose, or dextrins. The aim of this work was the development of a new optical sensing scheme for the determination of amylase activity (concentration). Luminescent beads were introduced as indicator for the enzyme activity measurements. They were immobilized in a partly cross linked starch layer. Amylase uses starch as a substrate and converts it into smaller polyglucose units. As a consequence of the enzymatic process the luminescent beads are liberated that makes the determination of amylase activity plausible by measuring the fluorescence change of the starch layer. Instead of measuring the fluorescence intensity, dual life-time referencing (DLR) method was chosen to follow the biocatalytic reaction, where rhodamine B (RB) was used as indicator and ruthenium-tris(diphenyl phenantroline) (Ru(dpp)3) as reference. The sensing layer(s) was prepared on glass support as follows. The reference layer was made using sol-gel technique. 0.18 mg Ru(dpp) was dissolved in a mixture of 0.2 ml of ethanol and 0.1 ml distilled water, and 0.247 ml tetraethoxysilane (TEOS) was added to the stirred solution. The sol gel process was then initiated by the addition of 0.01 ml of 0.1 M HCl. Condensation was allowed to proceed for 10 min and then the sol was spin casted on the glass support. It was dried at room temperature for 3 hours and then at 80°C for 2 hours in an annealing-furnace. The resulting reference layer was placed in a glass vessel and tempered at 200 °C overnight in argon atmosphere. The glassy layer prepared this way was impermeable for oxygen. The cocktail for the sensing layer consisted of 0.2 ml saturated water soluble starch solution, 0.2 ml of rhodamine B containing PVP nanobeads emulsion, and 0.1 mg of different UV cross linking agents. The sensing starch layer was spin casted on the reference layer and was irradiated by a UV lamp (366 nm), and left to dry at room temperature for 1 day. Before measurements the sensor was dipped in the deionized water and left to swell for 1 hour. The sensors were placed in a holder in front of a bifurcated optical fiber. The phase-shift measuring setup was based on an SRS830 lock-in amplifier and a photo multiplier tube (Hamamatsu), the sensors were illuminated by LED (470 nm) modulated by the lock-in. Two broadband interference filters, 500 and 600 nm CWL, were used to separate the excitation and emission light, respectively. Measurements were performed in blank and enzyme containing solutions, the enzyme activities obtained were compared with those obtained by standard method. It was found that the proper cross linking of the originally water soluble starch layer was essential for the amylase sensing. Insufficient cross linking resulted in the delamination of the sensing layer, while well cross linked starch was not available for the enzymatic reaction. Therefore different cross linking compounds and curing times were used and tested to find out the best layer by keeping the amylase activity constant.

optical sensor ; fluorescence ; amylase ; dual-lifetime

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