Studies On Novel Fluorescence And Colorimetric Methods For The Detection Of Alkaline Phosphatase Activity

Alkaline phosphatase(ALP)is a kind of non-specific phosphate monolipase,which can catalyze the hydrolysis of phosphate monolipid to produce inorganic phosphate and corresponding alcohols,phenols and sugars.ALP is widely found in bacteria,fungi and animals,and widely distributed in human liver,bone,intestine,kidney and placenta.The abnormal level of alkaline phosphatase in serum is closely related to many diseases,such as bone disease,diabetes,breast cancer,prostate and hepatitis.The concentration of ALP in water environment is closely related to eutrophication,which can transform organophosphorus into reactive phosphorus which can be directly utilized by phytoplankton.In addition,the activity of ALP is also one of the important indexes to evaluate soil quality.UV-vis and fluorescence analysis technology,which is the most widely used in optical detection technology,has the advantages of short analysis time,low cost,simple operation and high sensitivity.it has been used for the analysis and determination of heavy metals,pesticides,antibiotics and other environmental pollutants,which has greatly promoted the application of analytical chemistry in the field of environmental science.In this paper,three novel ALP analysis methods based on fluorescence spectrum,fluorescence polarization and UV-vis detection techniques are established,which provide some technical support for the clinical diagnosis and environmental index analysis of cancer and other diseases.The specific research contents are as follows:Part one:Using copper-catalyzed DNA chemical ligation and fluorescence quenching mechanism,a new fluorescence analysis method was developed for the sensitive detection of alkaline phosphatase activity.When ALP is present,the copper-catalyzed click reaction can be promoted,and the fluorescence signal of the system changes through the chain displacement reaction.The results showed that there was a good linear relationship between the logarithm of concentration of ALP and the fluorescence polarization signal of the system in the range of 0～400 U/L under the optimized experimental conditions.This method has good selectivity and high sensitivity with a detection limit of 1 U/L.This method utilizes the signal amplification effect of copper catalysis and has the advantage of wide analysis and detection range,and successfully applied to the detection of alkaline phosphatase in river water samples.Part two:In order to further improve the detection sensitivity,taking graphene oxide nanosheets(GO)as the signal amplification element,a highly sensitive fluorescence polarization assay was developed for the determination of alkaline phosphatase activity by using ALP to hydrolyze phosphate groups and lambda exonuclease(λexo)to degrade phosphate-modified nucleic acid substrates.The fluorescence polarization value induced by different concentrations of ALP was investigated.The effects of the reaction time of ALP,the dosage and reaction time ofλexo,and the concentration and reaction time of GO were studied.The results showed that there was a good linear relationship between the logarithm of concentration of ALP and the fluorescence polarization signal of the system in the range of 0.05～60U/L under the optimized experimental conditions.This method has good selectivity and high sensitivity with a detection limit of 0.05 U/L.Compared with the previous work,the sensitivity of this work is increased by 20 times.At the same time,this method was successfully used for the determination of ALP in bovine serum samples and lake samples.Part three:Based on the advantages of colorimetry,such as simplicity,rapidity and visual visibility,a UV-visible analysis method using Ag(I)to catalyze the color reaction of tetramethylbenzidine(TMB)was constructed to detect alkaline phosphatase activity.Ag(I)has strong oxidase-like activity and can catalyze the color reaction of TMB,showing blue oxidized TMB.There is an obvious visible light absorption peak at the wavelength 652nm.ALP can hydrolyze the substrate ascorbic acid 2-phosphate(AAP)to ascorbic acid(AA),AA.It can reduce the Ag~+to Ag~0in the system,so that the oxidase activity of Ag~+is inhibited.The color reaction would not be carried out,and the visible light signal is weakened.Under the optimized conditions,the absorbance and the concentration of ALP show a good linear relationship in the range of 0.5～100 U/L,and the detection limit is 0.5 U/L.The method has the advantages of simple operation,rapid detection,low cost and good selectivity.This method has been applied to the determination of endogenous ALP in eutrophicated river water and bovine serum samples with good results.Therefore,the proposed method has a certain potential in environmental monitoring and clinical diagnosis.