Two-dimensional (2D) protein separation is achieved in a plastic microfluidic device by integrating isoelectric focusing (IEF) with multi-channel polyacrylamide gel electrophoresis (PAGE). IEF (the first dimension) is carried out in a 15 mm-long channel while PAGE (the second dimension) is in 29 parallel channels of 65 mm length that are orthogonal to the IEF channel. An array of microfluidic pseudo-valves is created for introducing different separation media, without cross-contamination, in both dimensions; it also allows transfer of proteins from the first to the second dimension. Fabrication of pseudo-valves is achieved by photo-initiated, in situ gel polymerization; acrylamide and methylenebisacrylamide monomers are polymerized only in the PAGE channels whereas polymerization does not take place in the IEF channel where a mask is placed to block the UV light. IEF separation medium, carrier ampholytes, can then be introduced into the IEF channel. The presence of gel pseudo-valves does not affect the performance of IEF or PAGE when they are investigated separately. Detection in the device is achieved by using a laser induced fluorescence imaging system. Four fluorescently-labeled proteins with either similar pI values or close molecular weight are well separated, demonstrating the potential of the 2D electrophoresis device. The total separation time is less than 10 minutes for IEF and PAGE, an improvement of 2 orders of magnitude over the conventional 2D slab gel electrophoresis.

This paper describes the investigation on the effects of separation length and voltage on IEF in a plastic microfluidic device. A LIF, whole-channel imaging detection (WCID) system was developed to monitor proteins while they were moving under an electric field. IEF was carried out in a separation medium consisting of carrier ampholytes and a mixture of linear polymers (hydroxyethylcellulose and hydroxypropylcellulose). We found that the IEF separation resolution is essentially independent of separation length when the same voltage is applied, which agrees with the theory. This result supports the notion that IEF in a microfabricated device leads to more rapid analysis without sacrificing the resolving power. A higher separation voltage also brought about more rapid analysis and superior separation resolution. IEF of two proteins (green fluorescence protein and R-phycoerythrin) was achieved in 1.5 min when 500 V was applied across a 1.9-cm channel. We found that a linear relationship exists between the focusing time and the inverse of the electrical field strength. In addition, we confirmed the phenomenon in which the pH gradient was compressed to the middle of a channel, and we found that the relative amount of the gradient compression decreased with the channel length.

Among the materials used for fabricating microfluidic devices, plastics have been increasingly employed in the past few years. Although several methods for fabricating plastic devices have appeared in the literature, reports typically indicate one set of conditions that yield functional devices; little data are available detailing how results are affected by their changes in the process variables. We report in this paper a systematic study of fabrication process parameters including compression rate, molding temperature, and the force used by a hydraulic press, as well as their effects on the device properties. Using cyclic olefin copolymers as the molding material, we found that the device thickness decreased when the molding temperature and compression force increased. Fidelity in the pattern transfer from a master to a device was confirmed by the reproduction of nanostructures and channel depth/ shape. Pattern transfer fidelity appeared to be independent of the molding temperature and compression force, at least in the range of conditions we investigated. Stress whitening (or crazing) on the device surface was found to be related to the molding temperature and the cooling rate of the mold/device assembly. The bond strength between the layers of a laminated device was determined to be a function of the lamination temperature. In addition, we demonstrated the utility of a plastic microfluidic device by separating proteins.[1678]

We report our study on using hydroxyethyl cellulose (HEC) as a dynamic coating for protein separation in microfluidic devices made from cyclic olefin copolymer (COC). The coating significantly enhances hydrophilicity of COC surface, evident from the decrease in contact angle of water in a COC channel. Surface treatment of COC channels with HEC also results in a 72% drop in electroosmotic (EO) mobility and a significant reduction in protein adsorption on the channel wall. Using bovine serum albumin as a model protein, the number of theoretical plates of 1.1 9 10 4 was achieved in a separation distance of 3.3 cm using free solution electrophoresis. Hydroxyethyl cellulose dynamic coating is also found to have an effect on isoelectric focusing (IEF) of proteins. It not only prevents proteins from adsorption, but also reduces EO flow, both of which help achieve IEF of proteins with a difference of 0.1 pH values in isoelectric points (pI).

No abstract