Study On Methodologies And Determination Of Biochemical Samples Based On Microchip Capillary Electrophoresis

Microfluidic chip laboratory,as known as microfluidic chip or chip laboratory,is chemical or biological laboratories built in a small chip.It integrates the basic operating units such as sample preparation,reaction,separation and detection,forms a microchannel network,and uses controlled fluids through the system to achieve the functions of conventional chemical or biological laboratories.It begins to study mainly as the microchip capillary electrophoresis?MCE?and has many advantages,such as faster response,less sample consumption,higher throughput and easier to carry.MCE has been widely applied in many fields,such as environmental monitoring,food inspection,drug screening,disease treatment and so on.In recent years,analytical researchers have paid more and more attention to MCE.However,the size of the chip is too small,which results in small injection volumes and short detection optical path.The detection sensitivity cannot meet the requirements of trace detection in food,environment or other fields.In order to improve the detection sensitivity,in addition to improving the sensitivity of the detectors,various enrichment technologies can also be developed to enrich the sample and improve the detection sensitivity of the method.In this paper,MCE combined with laser induced fluorescence detector?LIF?and field-amplified stacking injection?FASI?were used to explore the applications in biochemical analysis.The main contents are as follows:Chapter 1 briefly introduced the history,basic characteristics,injection methods,detection technology,online enrichment technology and applications of microchip capillary electrophoresis.It also introduced the research contents and significances of the paper.Chapter 2,two kinds of?-casomorphins including?-CM-5 and?-CM-7 in cheese were detected by MCE-LIF using fluorescein isothiocyanate?FITC?as pre-column derivatization reagent based on FASI.?-CM-5 and?-CM-7 were successfully detected in two minutes.The results showed that the detection limits of?-CM-5 and?-CM-7 are 8.2 nM and 3.6 nM,respectively.The linear ranges were0.05-2?M and 0.02-1?M,respectively.The spiked recoveries were in the range of86.9%-107.5%.Chapter 3 described an ultrasensitive strategy developed for rapid detection of Escherichia coli?E.coli?by MCE-LIF combined with FASI.This strategy was based on the measurement of the activity of?-galactosidase??-gal?,an enzyme that was often used as a marker for coliforms.The enzyme activity was detected by the measurement of 4-aminophenol,which was produced upon hydrolysis of the substrate4-aminophenyl-?-D-galactopyranoside?PAPG?by?-gal.4-aminophenol was derivatized with fluorescence isothiocyanate?FITC?and detected by MCE-LIF.To obtain higher sensitivity of detection,FASI was employed in MCE.Under optimal conditions,the fluorescence responses of 4-aminophenol achieved were directly proportional to the concentrations of E.coli ranging from 20 to 500 CFU mL^-1 with a detection limit of 10 CFU mL^-1.The technique permitted detection of E.coli down to23 CFU mL^-1 in river water.The results confirmed that MCE in combination with LIF is a promising approach for detection of E.coli with high sensitivity.Chapter 4 established a method for rapid detection of the concentration of DNA methyltransferase by MCE-LIF.Dam MTase,a kind of DNA methyltransferase,can recognize the special site 5'-GATC-3'and methylated adenine in this site.After the methylation of the special site,the double-stranded DNA?dsDNA?was resistant to cleavage by Dpn II.Therefore,methylated ds DNA was whole but unmethylated dsDNA was cleavaged by Dpn II.Then,dsDNA was stained with SYBR Gold and detected by MCE-LIF,and its fluorescence intensity was found to be proportional to the concentration of Dam MTase in the range of 0.5-20 U/mL.And the detection limit of the method was as low as 0.12 U/m L.The method was simple and easy to do without excess sample pretreatments.Due to its simplicity and rapidity,the label-free method can be used for the primary screening of anticancer and antibacterial DNA methyltransferase inhibitors.