Ultralow Background Substrate for Protein Microarray Technology and On-chip Study of Macromolecular Crowding Effect on FRET

This thesis is about two techniques based on microfluidics: an ultralow background substrate for protein microarray technology and a three-layered microchip for one-step formation of concentration gradient and stepwise reactions. We applied the techniques on protein analysis and the study of macromolecular crowding effect on fluorescence resonance energy transfer (FRET) pairs, respectively.;In the first part, an ultralow background substrate was developed for protein microarray. The substrate was based on a fluorinated ethylene propylene (FEP) membrane. Polydopamine microspot array was fabricated on the FEP membrane, functioning as the intermediate for protein or peptide conjugation. Uniform microspot morphology was obtained without special surface treatment or printing solution. The modified FEP membrane demonstrated minimal non-specific protein adsorption, and showed its great advantage in protein and peptide microarray technology.;In the second part, a three-layered microfluidic chip was fabricated for the study of macromolecular crowding effect on FRET pairs. Two concentrating modes were performed on the microchip: gradient concentrating and constant concentrating. Sample solutions could form gradient concentrations on-chip within one step. Stepwise reactions could be performed in the microchip. Moreover, in each reagent adding step, the sample consumption was only 1 muL. Fluoro-silane vapor treatment was utilized to eliminate the adsorption of DNAs labelled with FRET pairs onto the microchip surface. Ultra-sonication was used for solution mixing in the microchip. A capillary based system was used as a positive control for the study. The results from the microchip system and capillary based system both demonstrated that the major influence on FRET signal was caused by excluded volume effect instead of the solution viscosity.