Quantitative Detection Of Methylglyoxal In Biological Samples Using A Ratiometric Fluorescent Probe

Excessive methylglyoxal(MG)in organisms can cause a series of diseases.The relationship between diabetes and MG's high level is important but unclear.Therefore,quantitative detection of MG concentration in living organisms is of great significance for the research of diabetes.Chromatographic methods such as HPLC,and LC-MS,GC-MS are used to detect MG levels,however they typically require expensive instrumentation and operating costs as well as prolonged derivatization.Therefore,it is important to develop a simple and efficient detection method to detect MG's concentration.Fluorescent probes with high selectivity,sensitivity and imaging capability have been widely used for chemical detection in biological samples.The ratiometric fluorescence detection is a novel technique that uses the ratio of the fluorescence intensities at two different wavelengths as a response signal to determine the target concentration.The singal is not affected by the multiple factors in the environment,allowing better accuracy of the quantitative detection.In this thesis,we mainly finished three parts of work,in order to detect concentrations of MG in biological samples using the ratiometric fluorescent probe WHL45.(1)Construction of quantitative methods and application of WHL45 to the determination of MG concentration in animal serum.In this thesis,we used the advantages of ratiometric fluorescent probe WHL45 to specifically recognize MG and characteized kinetic parameters of reaction between WHL45 and MG in buffer solution.We developed a quantitative method to detect MG's concetration based on the standard addition method..The feasibility of the method was tested by fetal bovine serum(FBS).(2)Quantitative detection of MG in human urine and plasma.For urine samples,we diluted samples to reduce the fluorescence background and adjusted the salt ion concentration to increase the reactivity of WHL45 and MG.The quantitative result of MG concentration in urine was 4.73 ± 1.02 ?M For plasma samples,matrix interference was removed by deproteinization of trichloroacetic acid(TCA).The quantitative results in plasma were,the P value was less than 0.01,indicating a significant difference in MG concentrations in plasma of diabetic and non-diabetic individuals.(3)Application of WHL45 to the determination of MG concentration in blood cells.We detected the total MG's concentration and the MG's concentration in white blood cells,respectively.Then the MG's concentrations in each white blood cell and red blood cell were calculated based on these data.The results indicate that MG presents in blood cells and mainly in leukocytes.And the MG's concentrations in diabetic human leukocytes are significantly higher than non-diabetic human leukocytes(P<0.01).