A previously undescribed isoelectric focusing technology allows cell signaling to be quantitatively assessed in <25 cells. Highresolution capillary isoelectric focusing allows isoforms and individual phosphorylation forms to be resolved, often to baseline, in a 400-nl capillary. Key to the method is photochemical capture of the resolved protein forms. Once immobilized, the proteins can be probed with specific antibodies flowed through the capillary. Antibodies bound to their targets are detected by chemiluminescence. Because chemiluminescent substrates are flowed through the capillary during detection, localized substrate depletion is overcome, giving excellent linearity of response across several orders of magnitude. By analyzing pan-specific antibody signals from individual resolved forms of a protein, each of these can be quantified, without the problems associated with using multiple antibodies with different binding avidities to detect individual protein forms.cell signaling ͉ immunoassay ͉ phosphorylation ͉ Western blot ͉ microfluidic

A method is described for determining the index of refraction distribution and the particle size distribution of suspended particles. The distributions are obtained by breaking down an observed volume scattering function into its contributing components. The component scattering functions are calculated using Mie theory. The component functions include all size distributions and indices of refraction that can be expected to be present. The method was applied to a volume scattering function observed in the Sargasso Sea. Forty components were used with five different indices of refraction and eight different particle size distributions. The resultant index of refraction distribution was bimodal. Components with indices of 1.05 and 1.15 dominate the calculated volume scattering function. The calculated particle size distribution falls within experimentally determined limits for the size distribution.

We describe a whole-capillary, multicolor laser-induced fluorescence scanner for microfluidic protein analysis systems. Separation of proteins is achieved by isoelectric focusing in a short length of fused-silica capillary after which the resolved proteins are immobilized to the capillary wall using photochemistry. The capillary is then evacuated, and fluorescently labeled antibodies are flowed through the capillary to bind to the immobilized proteins. This technique provides high sensitivity, the ability to spatially resolve and quantify proteins, and provides the opportunity for complete automation. Results obtained by fluorescence detection are compared to those obtained by chemiluminescence while offering enhanced resolution and signal stability.

Electrochemical detection in capillary electrophoresis requires decoupling the voltage applied to the working electrode from the separation voltage applied across the capillary. End-capillary electrochemical detection achieves this by placing the electrode just outside the ground end of the separation capillary. Obtaining adequate signal-tonoise in this arrangement requires using small inner diameter capillaries. Decreasing the inner diameter of the separation capillary, however, increases the difficulty of aligning the microelectrode with the open end of the capillary. Using scanning electrochemical microscopy (SECM), the position of the capillary opening is determined while electroactive material is continuously emerging from the end of the capillary. The SECM instrument is then used to place the electrode at the position of maximum current for subsequent separations. Subsequent measurements found that the best signal-to-noise is obtained when the detection electrode is placed directly opposite the capillary opening and just outside of the capillary opening. When the electrode is further above the opening (but still opposite the capillary opening), the signal-to-noise does not dramatically decrease until the electrode is more than 30 mm above the 10 mm inner-diameter capillary.

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