Study On Establishment Of Molecular Imprinting-flunrescence Quenching Method And Application Of Metronidazole Drug Analysis

Fluorescence quenching analysis provides an important tool for the analysis anddetermination of trace analytes in complex samples, due to its many features such ashigh sensitivity, selectivity, the less using of the sample, the simple instrument, rapiddetermination and so on. Integrated with fluorescence quenching method andmolecular imprinting technique provides even broader prospects for drug analysis. Inthis research, preperation of the fluorescence glass slide and combined withfluorescence quenching method and molecular imprinting technique were studied.MPB-immobilized glass slide, MPB+AAF-immobilized ratiometric glass slide,MIP/CTA-MPB fluorescence sensing membrane were prepared. And NBAA wasimmobilized on the polystyrene （PS） surface of96-well microtiter plate.Obtaining of the recearch findings as follows:1.We report the fabrication of a new sensing membrane for fluorescence detectionof metronidazole （MNZ）. Briefly, a pyrenebutyric acid derivative,2-（methacryloyloxy）ethyl-4-（1-pyrenyl） butanoate （MPB） with a double bond, was synthesized andcopolymerized with2-hydroxyethylmethacrylate （HEMA） on the activated glasssurface by thermal initiation in the presence of cross-linker. The sensor respondslinearly to metronidazole in the concentration range of1.23³5.48mg/L in aqueoussolution with a detection limit of0.36mg/L. The lifetime is enhanced by covalentlyimmobilizing the pyrenebutyric acid derivative on glass slide, which hinders leachingof the dye from the membrane. The sensor could be regenerated after use by washingin methanol （RSD=2.42%）, and it shows sufficient stability, and selectivity.Interference of other pharmaceuticals on membrane performance is discussed. Thedeveloped membrane has been successfully applied for the direct determination ofmetronidazole in human serum sample without pretreatment. And the recovery rangewas84.63%～104.62%, RSD was0.99%～1.83%.2.5-fluoresceinamine was chosen from9fluorescent reagent, and it was used as a ratio fluorescent reagent. MPB+AAF-immobilized ratiometric glass slide wasprepared. And it was used the sensing of the metronidazole. Briefly, the derivative of5-fluoresceinamine, acrylamidofluorescein （AAF） with a double bond, wassynthesized and copolymerized with MPB and HEMA on the activated glass surfaceby thermal initiation in the presence of cross-linker. The membrane has goodselectivity to metronidazole, the value of the （F₅25-F₃78）0/（F₅25-F₃78） was11.05,which was much higher than other common interference drugs. In addition, themembrane has good stability, reproducibility, and selectivity. The quantitativecorrelation was created as described in Equation: Ln[（F₅25－F₃78）0/（F₅25－F₃78）]=0.031[MNZ]+0.015，R²=0.987. To test the applicability of MPB+AAF-immobilizedglass slide, metronidazole measurement was performed in5%human serum. And therecovery range was92.2%～117.3%.3. MIP florescence sensing membrane （MIP/CTA-MPB） was prepared byPhotopolymerization method. And its composition were that MNZ-Es as template,MAA as functional monomer, EDMA as a cross-linking agent, CTA for the solidphase carrier, MPB as fluorescence reagent. The quenching constant Ksv ofMIP/CTA-MPB membrane and NIP/CTA-MPB membrane was0.033,0.004respectively, Imprinting factor K‵was8.25.4. The ester （NBAA） of the4-nitrophenol（4-NP） was synthesized and wasimmobilized on the polystyrene （PS） surface of a standard96-well microtiter plate byphoto-polymerization method. And it was used the detection of the a-chymotrypsin.The linear equation was Y=0.2374X+0.1302（Y is the absorbance value, X is theconcentration of a-chymotrypsin） and the correlation coefficient: R²=0.9880.