Enzyme and affinity assays by microchip capillary electrophoresis

Microchip capillary electrophoresis (CE) is the control and separation of charged solutes by electrokinetic effects within a maze of interconnecting, micrometer-sized channels. It was evaluated here for enzyme and affinity assays.; The development and optimization of an instrument designed in-house for doing microchip CE are described. The detection system was a confocal, epifluorescence microscope equipped with optics, an arc lamp for fluorescence excitation, and a photomultiplier tube (PMT) for detection. The detection system was optimized in terms of pinhole diameter, objective magnification, and PMT power. In-house designed and fabricated microchips were characterized, and optimized in terms of injection and fluid reservoirs.; An inhibition assay was developed for a model enzyme, β-glucuronidase, and its substrate, fluorescein mono-β-D-glucuronide (FMG). Microchip CE was used to separate FMG from the reaction product, fluorescein. Michaelis-Menten kinetics analysis yielded K_m, which was compared to results obtained off-chip using conventional CE. Inhibition of β-glucuronidase by D-saccharic acid 1,4 lactone was evaluated from the IC₅₀ value, obtained from a dose-response plot.; The measurement of endogenous concentrations of activated extracellular signal-regulated protein kinase (ERK) was demonstrated. ERK catalyzes the transfer of phosphate from ATP to the fluorescently labeled peptide substrate, APRTPGGRR. The phosphorylated and non-phosphorylated peptides were separated within 20 s, and the increase in phosphorylated peptide was monitored. Enzyme kinetics studies were done, and endogenous concentrations of activated ERK were determined in the lysates of endothelial cells that were stimulated for various times with a growth factor.; An aptamer-based microchip affinity probe CE assay for IgE was developed. An IgE-aptamer complex was formed reproducibly and separated from the free aptamer. IgE standard curves were obtained in buffer solution and rat serum, and it was determined that serum had a severe, negative impact on the analysis, even when it was diluted over 250-fold. The separation of free aptamer and the IgE-aptamer complex in less than 1 s was also demonstrated, suggesting that microchip CE could be used to analyze complexes with dissociation rate constants on the order of 0.1 to 1 s−1.