Application Of Microfluidic Chip In Biological And Pharmaceutical Analysis

Microfluidics is a rapidly developed interdisciplinary field, which cooperates with physics, chemistry, biomedicine and micro-system engineering, etc. In recent years, microchip technology, as a new type of micro analysis platform for chemistry, biology and medicine, has been revealing its great potential in life science and medical application. In order to develop the application of microfluidic chip, we carried out the following three works around this topic, which included gene transfection of cells in droplets, aptamer-based assay for thrombin and detection of sulfonamides. This paper includes the following several aspects:The first chapter reviewed the microfluidic platform in the application of analytical chemistry for life science, mainly divided into two parts, droplets on the microfluidic platform and aptamer-based detection on the microchip. In the first part, the formation and manipulation, as well as application of the droplets were described. The second part detailedly introduced all kinds of aptamer-based detection methods and its application on microfluidic platform.The second chapter described chemical transfection of cells in picoliter aqueous droplets on microfluidic chip. The manipulation of cells inside water-in-oil droplets is essential for high-throughput screening of cell-based assay using droplet microfluidics. Cell transfection inside droplets is a critical step involved in functional genomics studies that examine in situ functions of genes using the droplet platform. Conventional water-in-hydrocarbon oil droplets are not compatible with chemical transfection due to its damage to cell viability and extraction of organic transfection reagents from the aqueous phase. In this work, we studied chemical transfection of cells encapsulated in picoliter droplets in fluorocarbon oil. The use of fluorocarbon oil permitted high cell viability and little loss of the transfection reagent into the oil phase. We investigated the influence of incubation time inside droplets, DNA concentration and droplet size on transfection. After optimization, we were able to achieve similar transfection efficiency in droplets to that in the bulk solution. Interestingly, the transfection efficiency increased with smaller droplets, suggesting effects from either the microscale confinement or the surface-to-volume ratio.In the third chapter, we reported the development of an on-chip aptamer-based fluorescence assay for protein detection and quantification. Thrombin has two DNA aptamers which can recognize two different epitopes of the protein. Aptamer-functionalized magnetic beads were utilized to capture the target analyte in the microfluidic channel, while the second aptamer, functionalized with fluorescent dye Cy3, was employed for fluorescence detection. The approach enabled rapid thrombin detection with high specificity. Experimental conditions, such as DNA degeneration conditions, incubation time, etc., were optimized on the microchip platform. This method proved to be rapid and efficient, only requiring minimal reagent. This work demonstrated the successful application of on-chip aptamer-based assay for detection of thrombin. A linear response was obtained for thrombin in the 65-1000 ng/mL concentration range, with the detection limit of 30 ng/mL. We also fulfilled the aptamer-based detection of thrombin in serum on microfluidic platform.In the fourth chapter, a simple and sensitive microfluidic capillary electrophoresis method with laser-induced fluorescence detection was developed for the analysis of three sulfonamides using fluorescein isothiocyanate as derivatization reagent. The performance of the system was demonstrated through the separation and determination of sulfamethazine, sulfamerazine and sulfamethoxazole. The injection and separation voltages, the concentration of borax, pH of the buffer and the content of ethanol in a running buffer were optimized to get great influence on the separation with 1.6 minutes. This proposed method showed satisfactory sensitivity with the limits of detection (S/N=3) of 0.01 to 0.04 μmol/L for the sulfonamides. The method also exhibited very good reproducibility with the relative standard deviations of not more than 9.2% and 2.2% for fluorescence intensity and migration time, respectively.