Research On The Two-dimensional Electrophoresis And Protein Preconcentration By Microfluidic Chips

Due to the complexibility of biological samples, further analysis to the samples requires separation of different components in first place. It is especilly true in proteomics research which is in great need of the the help of high performance separation technology. While traditional separation run in single modes produces limited resolution, the combination of different separation modes with low correlation could generate much higher resolution.In this work, we developed a microfluidic 2D electrophoresis system with mercury lamp induced fluorescence detection. The detection limit of this system to Fluorescein was 5×10-10 M. The RSD of the signal peaks corresponding to three consecutive injections was less than 0.5%. The results showed that our system was highly sensitive and reproducible. A double electrokinetic valve system was employed for the sample injection and the switching between separation channels. Combining MEKC and CZE for two-dimensional on-chip electrophoresis as well as optimizing electrophoresis condition,20 standard amino acids were effectively separated with 20 min with high resolution and repeatability. To our best knowledge it was the first time reported in literature. Quantitative analysis revealed linear dynamic ranges of over 3 orders of magnitudes with detection limits atμM range. To further evaluate the reliability of the system, quantitative analysis of a commercial nutrition supplement liquid was successfully demonstrated, suggesting that our system was qualified for amino acids analysis to food or medicine and health protection products. Furthermore, bovine serum albumin trypsin digest was derivatized by FITC and separated by our 2D on-chip electrophoresis system. The results showed that the peak capacity was-330.22 sample spots could be identified in the 2D electropherogram.In proteomics research, proteins were highly heterogeneously expressed both in space and time. Some low copy proteins were very difficult for detection, demanding effective preconcentration methods. In this work, we designed and fabricated two kinds of protein proconcentration chip based on different principles. One kind of protein preconcentration chip utilized high-voltage breakdown to form nanofluidic channels, another one integrated polyacrylamide gel porous membrane within the microchannels. With the optimization of concentrating conditions, FITC-labeled bovine serum albumin was used as sample protein for on-chip protein preconcentration experiments. The results showed that the two kinds of protein preconcenration chips achieve about 1000 and 300 folds of concentration increase for BSA in 15 min, respectively. In conclusion, the in-house fabricated PDMS chips were able to efficiently accumulate protein on-line, suggesting an effective guarantee for on-chip high performance protein separation and high sensitive detection.