Research Of A Highly Sensitive Determination Of Protein By Fluorescence Probe

Protein is one of the most important components in creatures, material foundation of biology processes and basic materials of all organism in living body. It has the crucial functions in the life process. It is also closed related to nutrition, enzyme, virus, immunity, material transportation and heredity etc. Quantitative analysis and special recognition to protein is important to the exploration to bio-mechanism. The exploration to the concentration and structure of protein exhibits very important meaning in medicine, immunity, clinic diagnosis and drugs screening etc.Fluorescent assay is widely used in the determination of protein for its high sensitivity, good selectivity, wide responding range and similarity of determination conditions to the physiological environment of the living body. Fluorescent probe technique is a highly sensitive assay which is used to research protein molecular in the solution, and is based on photochemical and photophysical characteristics of the molecular. As the development of the technology in recent years, fluorescent probe technique which is a novel method for determination of protein receives much concern for its little interference, quick determination , high sensitivity, accuracy and practicality.In our present work, we intend to use a dye molecule -brilliant cresol blue(BCB) and acridine orange(AO) as the fluorescence probe, to explore to the detection of the Tau protein. By systematic investigation we found out the optimal control conditions for two detecting systems. The main results are summarized as follows:Brilliant cresol blue(BCB) and Acridine orange(AO) are used as the fluorescence probes ,HSA is used as the model of the protein and Tris-HCl is used as the buffer solution . By investigating the experimental conditions such as PH of solution, amount of the probe, consequence of the reagent addition,, time of the reaction and effect to fluorescence intensity by ion intensity of the solution ,we optimal the experimental conditions. By systematic investigation we found that the combination between BCB and HSA can enhance fluorescence intensity of the BCB solution. The extent of the enhancement indicated that HSA could be quantified with good linear range of 1×l0^-*5mg/ml-2×l0^-2mg/ml,R²=0.9932, , with a low detection limit of 9.93ng/ml, The relative standard deviation(n=6) is 3.09% at 1.0ng/ml HSA concentration level, the method is not only sensitive,but also simply.By systematic investigation to the AO-SDS-HSA system, we found that the disaggregation of AO polymer which is caused by the addition of protein can enhance the fluorescence intensity of the system. The extent of the enhancement indicated that protein could be quantified with good linear range of 2×l0^-6mg/ml-2×l0^-2mg/ml, R²=0.9984, with a low detection limit of 1.82ng/ml, The relative standard deviation(n=6) is 1.61% at 4.0×l0^-5mg/ml HSA concentration level, the method is more highly sensitive than the former one.The formal fluorescence probes are used to detecting the R3 pepitide, BCB does not have good performance in the determination of the R3, but AO fluorescence probe manifest more sensitively in the determination of the R3, R²=0.9951, , with a low detection limit of 1.33ng/ml.