| After more than 20 years of development,the microfluidic chip,as a kind of revolutionary technology platform,has shown great market prospects.In order to step up the pace of going out of the professional labs and entering into the clinical applications and even ordinary families,the first problem to solve is the high cost of microchip fabrication.Plastic microchips appeared under this enormous demand.However,most of the plastic chips used currently suffer from the drawbacks such as hydrophobic surfaces and adsorption of molecules onto channel walls,which may greatly affect the accuracy and repeatability of the separation results.The surface modification is needed to control the electrosmotic flow(EOF)and suppress the interaction between analytes and channel surfaces.It is the most simple and practical method to add active materials into running buffer for surface modification.The buffer additives used in plastic electrophoretic chips are mainly surfactants and neutral polymers.Small molecular surfactants may cause some problems such as nonuniform coating,vulnerbility of channel blockage and bad stability.Neutral polymers lack the ability to control EOF.In order to improve the analysis performance of plastic chips and meet the needs of basic research and market developments,in this dissertation,we studied the effects of polymeric additives with different states of charge on EOF regulation and the improvement of electrophoretic performance in plastic microchips.Furthermore,during the separation process,gradients that have been widely used in chromatographic and electrophoretic technologies were applied to increase the resolutions and analysis speeds of different components in complex samples.The control of background electrolyte(BGE)flows through dynamic change of the high voltage output may construct the gradients of either concentration or pH,which may compensate the shortages of microchip electrophoresis caused by the short effective separation distance and enhance the separation capability of microchips.Therefore,it is also an important aspect of this work to construct separation modes with multiple gradients through a simple and practical ways and study their functions on electrophoretic separation on microchips.There are six chapters in this dissertation:Chapter 1: the basic conception of microfluidic chip was introduced in detail,including performances of the materials and technologies used for chip fabrication,the development status of microchip surface modification,sampling method,separation mode,detection system and application involved in microchip electrophoresis.Chapter 2: Using hydroxypropyl cellulose(HPC)as running buffer additives,xylose,glucose and lactose labeled with 8-aminonaphthalene-1,3,6-trisulfonate(ANTS)could be resolved in borate solutions within 30 s on cyclic olefin copolymer(COC)microchips with laser induce fluorescence(LIF)detection.Theoretical plate numbers of 1×106/m and limits of detection(LOD,S/N=3)of 0.15-0.23 ?mol/L were achieved.The linear range was in 0.5-1.0×103 ?mol/L for all analytes.The relative standard deviations(RSDs)of peak area and the migration time is not more than 3.1% and 1.9%.The microwave-assisted derivatization greatly improved the analysis speed of saccharides with microchip electrophoresis.The applicability of the proposed method was verified by the determination of glucose in human urine and serum,the standard addition recovery obtained was 92.7%-115.1%.Separation of oligosaccharides from konjac powder was also demonstrated and the results showed that this method can be regarded as a kind of effective means for qualitative and quantitative analysis of oligosaccharides.Chapter 3: Sodium polystyrene sulfonate(PSSNa)was used for electrophoretic separation of four fluorescein isothiocyanate(FITC)labeled biogenic amines.It can provide stable cathodic EOF and achieve the baseline separation of four biogenic amines under alkaline conditions.Because of the charges,strong surface adsorption,certain viscosity and surface active property of PSSNa with high molecular,it can play the roles of BGE,surface modifier,viscosity regulator and pseudo-stationary phases at the same time.Its addition in the COC microchip with the 75 ?m channel can realize fast separation of four kinds of biogenic amines and ensure the theoretical plate number reach more than 8×105/m.PSSNa solved the problems such as the poor stability of sodium dodecyl sulfate(SDS)and control surface charges abilities of HPC.Relying on LIF detection,the LODs of FITC-biogenic amines ranged from 1.3 to 3.6 nmol/L.The linear range is 0.01-20 ?mol/L.The RSDs not more than 3.7% and 2.7% were obtained for peak areas and migration times,respectively.Finally,the proposed method was applied to determine the content of four selected biogenic amines in fish and showed that PSSNa could prevent the interfering from FITC and sample matrices to analytes,standard addition recovery of 85.4%-112.0% was obtained.From these results,we can see the anionic polyelectrolyte has a great potential for practical application of MCE.Chapter 4: We used the cationic polymer polydiallyldimethylammonium chloride(PDDA)to produce anodic EOF during the COC microchip electrophoresis.In the presence of organic solvents,such as methanol,PDDA can effectively restrain the adsorption of hydrophobic analytes on COC surface,making rhodamine B,rhodamine 6G and rhodamine 123 migrate to positive electrode and be separated quickly and efficiently on COC microchip.The combination of 473 nm LIF detection and microchip electrophoresis can get the LOD range of 0.18-16 nmol/L,the RSDs of peak area and migration time were not more than 2.9% and 3.7%,respectively.The method was used in cosmetics and food rhodamine analysis,the standard addition recovery of 93.3%-107.3% was obtained finally.After the cationic polymer additive was replaced by anionic polymer polyacrylic acid(PAA)and neutral polymer HPC,we found that ionic polymers can effectively make up for the inadequacy of neutral polymers as additives to realize the flexible control of channel surface charges.Chapter 5: A double-cross channel configuration was processed on the low-cost COC microchips.A homemade two-channel high voltage power supply was used to realize the regulation and mixing of fluids.By investigating the performance of the instruments and the mixing results in microchannels,the basic condition of separation and detection under concentration gradient were verified.With isocratic mode,we first studied the effects of PDDA,acetonitrile concentration and acidity of BGE on migrating behaviors of proteins.The results indicated that the analysis speed was faster,but the resolution and signal intensity of the target analytes are hard to guarantee at the same time when the mixture of PDDA and acetonitrile was used as BGE for separation of isothiocyanate rhodamine B(RBITC)labeled thymopentin,ribonuclease A and lysozyme.The formation of voltage gradient and PDDA concentration gradient could provide certain improvement on the separation.However,the acetonitrile concentration gradient and pH gradient might be detrimental to the stability of the separation system.Chapter 6: Based on dynamic gradient as described in Chapter 5,we produced electric field,ionic strength,additive concentration and pH gradients for specific analytes by programmed voltage output.Glucosamine was chosen as a model analyte and the influences of ionic strength,HPC concentration and acidity of buffer on analytes migration behaviors were investigated under isocratic condition at first.Then different gradient modes were tested and the results indicated that resolution,selectivity and speed could be enhanced.Although FITC-labeled glucosamine,glycine,glutamic acid and leucine could be baseline separated within 40 s under isocratic voltage,the gradient mode could apparently improve the resolution of glucosamine and other impurities in the complex matrix. 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