Research On Cells Metabolism And Interaction Based On Combination Technology Between Mass Spectrometry And Microfluidic Devices

Nowadays, life science is becoming a very important part of natural science. Cell isthe basic unit of organism, from which we start to crack the code of life entity.Microfluidic technology, which is being developed fast currently, is widely used in thecell research field, due to its micro-scale channels and flexible design. In this thesis, wefocused on the determination of molecular structure and contents of the cell secretionand signaling factor. Cells were manipulated and captured on the microfluidic devices,and mass spectrometry (MS) was employed for qualitative and quantitative detection.Our key issue is connecting the microchips with MS, realizing the cell culture and drugstimulation in microchannels, and purifying the cell secretion before being detected byMS.An integrated system combining microfluidic devices with electrosprayionization quadrupole time-of-flight mass spectrometer (ESI-Q-TOF-MS) wassuccessfully developed. A single C30bead was trapped in the microchannels to mimicthe cell for evaluating the analysis platform. A series of herbicides absorbed on thesingle C30bead was detected by MS within5min, and only2.5μL solvents wasconsumed. The herbicides in vegetable samples were determined. Molecular structureand chemical contents could be rapidly determined for several components with agreatly simplified pretreatment approach.In order to get the information from the cell secretions, the developedmicrofluidic-MS analysis platform was induced for monitoring cellular chemical release.PC12cells were cultured and stimulated in the poly-L-lysine coated microchannels. Amulti-channel miniaturized extraction chip was integrated to remove salts and proteininterference effects prior to MS detection. The glutamate releasing was monitored fromPC12cells by A42-induced neurotoxicity. Carnosine protection effect was evaluated inthis process. The limit of quantitation for glutamate was2.0μg/L. The time andchemical consumption was greatly reduced comparing to the conventional biologicalprotocol and chromatogram technology.Facing to the real biological samples, the complex substance and mixture of several types of cells should be separated before the next procedure. In order to sortcells by the size difference, a porous membrane has been fabricated to create a singlefilter that contains multiple pore sizes ranging from6.4to16.6μm inside a monolithicthree-dimensional microfluidic structure. Smaller pore size openings were able toachieve by overlapping two porous membranes. The white blood cells were separatedfrom the whole blood sample with the efficiency of99.7%. This filter operateswithout any detectable irreversible clogging, which is achieved using a cross-flowplaced in front of each filtration section.The interaction and signaling function between cells was studied based on theprevious work. PC12and GH3cells were co-cultured in microchannels which wereconnected with several much lower microchannels. The MS was induced to monitor thegrowth hormone secretion from GH3cells. The induction between different cell typeswas observed during the co-culture process. Different drugs were applied on PC12cellsto cause the difference of secreted neurotransmitters, while the contents of growthhormone was monitored to discuss the mechanism of signaling function between cells.