| Abstract
| - We present the first successful adaptation of immobilizedpH gradients (IPGs) to the microscale (μIPGs) using anew method for generating precisely defined polymergradients on-chip. Gradients of monomer were established via diffusion along 6 mm flow-restricted channelsegments. Precise control over boundary conditions andthe resulting gradient is achieved by continuous flow ofstock solutions through side channels flanking the gradient segment. Once the desired gradient is established, itis immobilized via photopolymerization. Precise gradientformation was verified with spatial and temporal detectionof 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 pH3.8−7.0 μIPGs using both denaturing and nondenaturingconditions, without the addition of carrier ampholytes.The μIPG format yields improved stability and comparableresolution to prominent on-chip IEF techniques. In addition to rapid, high-resolution separations, the reportedμIPG format is amenable to multiplexed and multidimensional analysis via custom gradients as well as integrationwith other on-chip separation methods.
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