Publications

On-chip isoelectric focusing using photopolymerized immobilized pH gradients

Sommer, Greg J.; Singh, Anup K.; Hatch, Anson V.

We present the first successful adaptation of immobilized pH gradients (IPGs) to the microscale (μIPGs) using a new method for generating precisely defined polymer gradients on-chip. Gradients of monomer were established via diffusion along 6 mm flow-restricted channel segments. Precise control over boundary conditions and the resulting gradient is achieved by continuous flow of stock solutions through side channels flanking the gradient segment. Once the desired gradient is established, it is immobilized via photopolymerization. Precise gradient formation was verified with spatial and temporal detection of a fluorescent dye added to one of the flanking streams. Rapid (<20 min) isoelectric focusing of several fluorescent pI markers and proteins is demonstrated across pH 3.8-7.0 μIPGs using both denaturing and nondenaturing conditions, without the addition of carrier ampholytes. The μIPG format yields improved stability and comparable resolution to prominent on-chip IEF techniques. In addition to rapid, high-resolution separations, the reported μIPG format is amenable to multiplexed and multidimensional analysis via custom gradients as well as integration with other on-chip separation methods. © 2008 American Chemical Society.