Study On Methodologies And Determination Based On Aptamer By Microchip Capillary Electrophoresis

Microfluidic chip analysis,which takes chip as operating platform and life science as the main application object at present,is the focus of development in the field of micro-total analysis system.Its goal is to integrate the functions of the whole laboratory on the microchip,including sampling,dilution,reagent addition,reaction,separation,detection and so on,which can be used many times.Microfluidic chips are widely used in biological detection,chemical analysis,clinical medicine,life sciences,pharmaceutical synthesis and so on.Based on the great advantages of MCE and a wide variety of research fields,microfluidic electrophoresis of macromolecules,ions and bacteria was carried out in this paper.The selectivity and sensitivity of the experiment to the target were greatly improved.The main contents of this paper include the following four parts:Chapter 1,the development background,main advantages,basic characteristics,detection methods and main application fields of microfluidic chip electrophoresis were introduced.Chapter 2,a method of microfluidic chip electrophoresis and polymerase chain reaction?PCR?was designed to test the methods of the study on the detection of three foodborne bacteria.In this study,multiplex PCR amplification combined with microchip electrophoresis?MCE?was studied to simultaneously and sensitively detect Staphylococcus aureus,Proteus mirabilis,and Enterobacter sakazakii.In order to simultaneously and accurately detect the aim bacteria,three pairs of primers were specially designed for the multiplex PCR amplification of the target genes of three bacteria,which were the specific genes corresponding to these bacteria respectively.The multiplex PCR products of the three food-borne pathogens were analyzed by MCE and the limits of detection of target DNA fragments were 1.2-2.2 ng?L^-1,?S/N=3?.The limits of detection of the aim bacteria were calculated as 53 CFU mL^-1 for Enterobacter sakazakii,32 CFU mL^-11 for Proteus mirabilis,28 CFU mL^-1 for Staphylococcus aureus,respectively.Satisfactory results were obtained when this method was applied to detect the three foodborne bacteria in milk samples.Chapter 3,a method for detecting cytochrome C in serum by microfluidic chip electrophoresis based on specific aptamer was introduced.In this study,selective and sensitive detection of Cyt C based on an aptamer strategy coupled with microchip electrophoresis?MCE?was developed.Following the binding of a specific aptamer to Cyt C,the aptamer exhibited an irregular state,reducing the binding affinity of a fluorescent probe,and thus preventing the aptamer-Cyt C complexes from detection within the MCE.Experimental conditions such as incubation time,pH,temperature,and ionic strength were optimized.A measurement of Cyt C concentration by MCE was achieved within 135 s,with a limit of detection as low as 0.4 nM.The proposed method has high selectivity and good stability for the detection of Cyt C.Chapter 4,a DNAzyme-based microfluidic chip electrophoresis method for the detection of lead ions in wastewater was designed.Heavy metal pollution has become one of the most serious environmental problems.In this chapter,a simple and rapid method for the detection of Pb???in sewage was established based on DNAzyme and MCE.In this paper,by using the binding double strand of DNAzyme and S-DNA,in the presence of Pb???,the standard curve of Pb???concentration corresponding to the fluorescence signal of DNAzyme in MCE detection was obtained.Pb???can be quantitatively detected by MCE within 135s,and the fluorescence signal of DNAzyme in MCE detection is corresponding to the concentration of Pb???.Detection limit as low as 2 nM.In practice,the concentration of DNAzyme can be quantified by the fluorescence signal of Pb???in MCE.