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.

Serum albumins are the most abundant proteins in plasma. As the major soluble protein constituents of the circulatory system, they have many physiological functions. They contribute to colloid osmotic blood pressure and are chiefly responsible for the maintenance of blood pH.1) The most outstanding property of albumins is their ability to reversibly bind a large variety of endogenous and exogenous ligands. The binding involves hydrophobic, hydrophilic, and cationic substances. The knowledge about proteins has profited from the use of these different ligands. The molecular interactions are often monitored using optical techniques because these methods are sensitive and relatively easy to use. Among these, fluorescence spectroscopy is a valuable technique to study the binding of ligands to proteins. With bovine serum albumin (BSA), different compounds have been used successfully as probes such as dyes 2) and metal complexes.

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).

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The potential of a series of newly synthesized poly(N,N-dimethylacrylamide) (PDMA) grafted polyacrylamide (PAM) copolymers (P(AM-PDMA)) as a replaceable separation medium for protein analysis was studied. A comparative study with and without copolymers was performed; the separation efficiency, analysis reproducibility and protein recovery proved that the P(AM-PDMA) copolymers were efficient in suppressing the adsorption of basic proteins onto the silica capillary wall. Furthermore, the size-dependent retardation of native proteins in a representative P(AM-PDMA) copolymer was demonstrated by Ferguson analysis. The results showed that the P(AM-PDMA) copolymers combine the good coating property of PDMA and the sieving property of PAM and could be applied as a sieving matrix for the analysis of native proteins.