Anti-Candida Albicans Drug Screening And The Study On Cell Metabolites Based On Microfluidic Chip

Microfluidic chip,also known as lab on a chip,is a technique which manipulate micro volume fluids to move in a microfluidic channel,by integrating several functional units such as sample preparation,reaction,separation,and detection module onto a single chip,it could minimize the traditional lab to a microfluidic chip.This technology focused on nanometer to micrometer-scale fluids,and has the unique advantages of high throughput,high sensitivity,miniaturization,high degree of functional integration,time and reagents saving.The droplet microfluidic chip is an important branch of the microfluidic chip.With droplets as the microreactor,a single experiment can be used to generate up to 10 million droplets,which is easy to achieve high-throughput screening.Since the size of droplets is similar to the size of cells.In recent years,the chip has been widely used in the research of microorganisms,including bacteria,yeast and cyanobacteria,which can be used for identification and detection of pathogens.At present,polydimethylsiloxane is the most commonly used chip material in the research of microfluidic chips in the laboratory.Due to its good biocompatibility,it is suitable for culturing cells,and then the cell metabolites are subjected to solid phase extraction and pretreatment to prepare for mass spectrometry analyze.Some progresses have been made in drug discovery and disease mechanism research which used microfluidic chip coupled with mass spectrometry.In this study,microfluidic chip technology was used for drug screening and to study the changes of cell metabolites by coupling with mass spectrometry.1.This study established a concentration gradient generation droplet microfluidic chip platform for drug screening of Candida albicans,and investigated the concentration gradient generation by the distribution of dye Alamar Blue and sodium fluorescein on the chip.The concentration gradient of the fluconazole in the chip was quantitatively analyzed by liquid chromatography.Finally,the ratio of the two-phase flow rate to 1:1 was determined for the follow-up drug screening studies.Using Alamar Blue as a cell viability indicator,the amphotericin B susceptibility test was performed through the platform.The MIC range of amphotericin B can be obtained in one experiment to be 0.5-1.6 ?g/ml,which means that this strain of Candida albicans is sensitive to amphotericin B and consistent with the CLSIrecommended standard indicating that the platform can quickly screen the range of MIC values of antibacterial drugs in one experiment.In addition,fluconazole and terbinafine were tested separately.The MIC values of fluconazole and other drugs were consistent with the standard and the strain of Candida albicans showed resistance to terbinafine and the results were consistent with the 96 wells plate method.This indicates that the method can also be used for rapid screening of resistant strains.2.The study of cell metabolites was carried out based on the microfluidic chip mass spectrometry platform.Using hBMEC as the research object,the cells were prepared by A?1-42 stimulation,and the normal group,AD model group and Danshen pre-protection group and the huperzine A pre-protection group were cultured in the chip channel,and the changes of metabolites among the groups were monitored by liquid chromatography-mass spectrometry.Among the 96 monitored metabolites,there were 12 significant differences between the normal group and the model group,mainly amino acids.After pathway analysis,the pathways involved in amino acid metabolism and biosynthesis were found.The most relevant ones were the pathways involved in the transport of RNA biosynthesis are consistent with those reported in the literatures.At the same time,it was found that 6 of the above 12 compounds showed a trend of callback under the effect of the total extract of Salvia miltiorrhiza Bge.,which partly reflected the protective effect of the drug on cells metabolism.Since the living environment of vascular cells in the microfluidic chip is closer to the blood flow state in the body,the online detection and analysis method of the chip mass spectrometry can better simulate the living state of the cells in the body,thereby this platform can play an important role in the research of the pathogenesis of AD and the mechanism of protection effect of Chinese medicine.