Carrier Ampholyte-free Isoelectric Focusing On Paper-based Analytical Device And Fast Protyping Of Microfluidic Device With Dry Film Photoresist

Micro total analysis systems(μTAS)own lots of merits such as low sample consumption,high analytical sensitivity,short analysis time and easy integration.The application scope ofμTAS has covered a wide range of areas such as chemistry,biology,physics and electronic disciplines.Microfluidic paper-based analytical devices(μPADs)are introduced into μTAS in 2007,which obtain wide attentions due to its easy operation and low cost.Electrophoresis is one of the most widely used separation techniques.Traditionally,electrophoresis is implemented in closed fluidic channel like capillary and micro-channel,which is not beneficial for sample recovery and post analysis.μPADs with open fluidic channel are simple and easy to manipulate,possessing potential applications in point of care test.In the first chapter,principle of isoelectric focusing(IEF)and the types of pH gradient formation were simply introduced.Then,the materials of microchip,properties of substrates,and the fabrication methods of glass,silicon,polymers and paper microchip were briefly reviewed.In the second chapter,carrier ampholyte free IEF of proteins on μPADs was proposed.First of all,the materials of paper channel,electroosmotic flow and operation modes were optimized.The glass fiber was selected as paper channel,and 0.2%(m/v)hydroxyethyl cellulose was added into anolyte to suppress electroosmotic flow.The optimized operation procedure was that paper channel was pre-wetted by the anolyte,and that sample was added into cathodic reservoir.Secondly,the IEF process was visually demonstrated by colored protein,BHb,and IEF of the mixture of Phyco and BHb was demonstrated.The pH gradient along paper fluidic channel was characterized and tuned.Then,the recovered fractions were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE)and matrix-assisted laser desorption ionization-time-of-flight-mass spectrometry(MALDI-TOF-MS).Ultimately,IEF of BHb protein with Locke’s normal saline as sample medium was also successfully demonstrated.In the third chapter,microchip was fabricated using dry film photoresist by lithography and replication.Firstly,the substrate materials and lamination condition were optimized.PMMA slice was selected as the substrate,and office laminator was used for lamination of the dry film photoresist.The micro channel thus prepared was characterized by microscope imaging.Finally,the applicability of the prepared microchips was demonstrated by laser induced fluorescence detection of fluorescein sodium and contact conductivity detection of inorganic small ions.The established PAD-CAF-IEF method was fast,low comsupmtion and compatible with MS.Moreover,the fabrication of microfluidic chip,based on dry film photoresist,provided an alternative in non-clean room.