Protein separation in plastic microfluidic devices

Conventional two-dimensional gel electrophoresis has been extensively used for proteomics research, including discovering biomarkers associated with a disease. However, the process is time consuming and labor intensive. To address the limitation, a plastic microfluidic device (1”x 3”) is developed that is capable of doing the same operation in a much shorter time with less labor. The devices are fabricated by compression molding, a similar technique for manufacturing compact discs. First two different separation mechanisms isoelectric focusing and polyacrylamide gel electrophoresis (PAGE) are separately demonstrated in microdevices. Isoelectric focusing (IEF) is optimized in terms of the applied voltage and separation medium. It is demonstrated that IEF is essentially independent of channel length, allowing miniaturization of separation apparatus. The time required for IEF is also drastically reduced when the channel length is reduced. It takes only 3-5 minutes in a 2 cm channel compared to 10-12 hours in conventional apparatus. Further integration is achieved by selective photo polymerization inside microchannels, obtaining a reliable interface that prevents one separation medium from contaminating with the other medium. PAGE takes another 5 minutes to perform. When these two separation mechanisms are integrated, it takes about 15 minutes from loading the samples to finishing the experiment whereas the conventional system takes more than a day to finish. A laser induced whole channel fluorescence imaging system is assembled for detecting the proteins separated in the devices. The sequential images obtained from the imaging system helps in understanding the dynamic nature of protein separation such as IEF. The same imaging system can be used for single point detection for two-dimensional separation. The detection limit for this system was found to be around 1 nM of fluorescien solution and 0.03 ng/µl when Green fluorescent protein is used in isoelectric focusing. Fluorescently labeled proteins are used to demonstrate the viability of the miniaturized two-dimensional protein separation system.