| Polycyclic aromatic hydrocarbons(PAHs) are a large group of organic compounds with two or more fused aromatic rings,and are one kind of environmental hormones.Due to the widespread distribution of PAHs in the environment and the carcinogenic and mutagenic nature of PAHs,the U.S.Environmental Protection Agency(EPA) has identified 16 unsubstituted PAHS as priority pollutants,which are monitored routinely for regulatory purposes,and so does our country.So there has been increased concern in PAHs' monitoring and analysis in recent years. Here we want to study a new method on determinations of BSA,also on Naphthalene(NA) and Phenanthrene(PH) used the fluorescent substance——Lumichrome(LC) as the fluorescent probe in the analysis of fluorescence immunity assay.Three aspect research have been carried out in this paper:Part 1:On the bases of the previous study,we find the factors of the synthesis of LC,then to optimize.So we used the H3BO3-NaOH as the hydrolyzed solution for 6,7-Dimethyl-9-formylmethylisoalloxazine(FMF) with pH 7.6,extracted the solution with chloroform,and then separated with the Column Chromatography to obtain LC.Part 2:It was found that BSA had the ability to quench the fluorescence of LC,under pH 7.8 Tris-HCL buffer,the quenched intensity of fluorescence was proportional to the concentration of BS.Based on this fact,LC had been used as the fluorescent probe for detection of BSA.Under optimal conditions,the calibration graphs are linear up from 0.05mg/L to 1.0 mg/L for BSA,and the detection limit is 0.008 mg/L,then a new method for detecting protein has been built.At the same time,the fluorescence quenching mechanism of BSA to LC was discussed,and it was proved that the quenching type was static quenching.The apparent binding constant and the binding sites were calculated,also the main type of force between BSA and LC was hydrophobic force by calculated the thermodynamic constants.Part 3:Using LC as fluorescent marker,we obtained the tagged antibody and hapten of NA and PH.Then the direct competitive immunofluorescence and antibody-coated immunofluorescence were built to detect the NA and PH.For the direct competitive immunofluorescence:the linear calibration range for the determination of NA was 1×10-7-1×10-2 mg/mL,the detection limit was 5.0×10-8 mg/mL, R2=0.9917,the recovery of NA in three kinds of soil samples(the living sludge,the plating sludge and the printing sludge) was between 97.53%and 107.57%;the linear calibration range for the determination of PH was 10-6-10-2 mg/mL,the detection limit was 1.4×10-7 mg/mL, R2=0.9939,the recovery of PH in three kinds of soil samples was between 95.43%and 104.26%.For the antibody-coated immunofluorescence:the linear calibration range for the determination of NA was 1×10-7-1×10-2 mg/mL,the detection limit was 3.04×10-8 mg/mL, R2=0.9924,the recovery of NA in three kinds of soil samples(the living sludge,the plating sludge and the printing sludge) was between 97.91%and 107.11%;the linear calibration range for the determination of PH was 10-7—10-3 mg/mL,the detection limit was 5.76×10-8 mg/mL, R2=0.9942,the recovery of PH in three kinds of soil samples was between 95.22%and 104.7t%.These two methods were high in sensitivity,good in choice,wide in linear range and fast and easy in operation,also the results were satisfactory. |
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