James J. Bao

Capillary electrophoretic immunoassays

Capillary electrophoretic immunoassay (CEIA) has recently emerged as a new analytical technique. CEIA, when combined with sensitive detection methods such as laser induced fluorescence (LIF), offers several advantages over conventional immunoassays. CEIA can perform rapid separations with high mass sensitivity, simultaneously determine multiple analytes and is compatible with automation. The objective of this review is to describe the applications of CE in antibody related studies, focussing especially on recent developments of CEIA technique. The principles for competitive and non-competitive CEIA are described with examples. Several detection methods and various applications are summarized and future developments in CEIA are speculated. CEIA has many potential applications, especially if the throughput is improved by using either multicapillary array or microchips with multiple channels.

Determination of minute enzymatic activities by means of capillary electrophoretic techniques

Capillary electrophoretic analysis of enzymes, co-enzymes, substrates and other chemical species that can be linked to an enzymatic reaction is reviewed with 80 references. Both off-line and on-line assays of minute enzymatic activities are discussed. In addition to heterogeneous on-line enzyme assays, a special emphasis is given to a newly established on-line technique called electrophoretically mediated microanalysis (EMMA). The basic principle, procedure, and various detection modes of EMMA are discussed. The recent developments in on-line determination of various enzyme substrates as well as on-line enzyme kinetic studies are also summarized. Some potential future developments in the determination of enzymatic activities by means of CE are also presented.

Fluorogenic assay for β-glucuronidase using microchip-based capillary electrophoresis

Microchip capillary electrophoresis (CE) was used with a model enzyme assay to demonstrate its potential application to combinatorial drug screening. Hydrolysis with beta-glucuronidase of the conjugated glucuronide, fluorescein mono-beta-D-glucuronide (FMG), liberated the fluorescent product, fluorescein. FMG and fluorescein were detected by fluorescence, with excitation and emission at 480 and 520 nm, respectively. Microchip CE was used to separate FMG and fluorescein. Fluorescein production was monitored to assess beta-glucuronidase activity. Michaelis-Menten enzyme kinetics analysis yielded the Km value. The results were compared with those from experiments done by conventional CE. The Km value for beta-glucuronidase with FMG is being reported for the first time as 18 microM. The inhibition of beta-glucuronidase by the competitive inhibitor D-saccharic acid-1,4-lactone (SL) was also determined using microchip CE. Reactions were done with various concentrations of inhibitor and constant beta-glucuronidase and FMG concentrations. A dose-response plot was acquired and the IC50 value for SL was determined to be 3 microM.

Effect of Age and Gender on Azimilide Pharmacokinetics After a Single Oral Dose of Azimilide Dihydrochloride

Azimilide is a new class III antiarrhythmic drug that blocks K+ channels. To determine the effects of age and gender on azimilide pharmacokinetics, a single 150‐mg oral dose was administered to 66 healthy volunteers in a 3 × 2 factorial design (age groups of 18–40, 41–64, and ≥65 years). Blood and urine were analyzed for azimilide and metabolites. The single dose was well‐tolerated. Azimilide was 94% plasma protein bound, and binding was not affected by age or gender. Age does not affect azimilide pharmacokinetics. The renal clearance of azimilide was significantly higher in women than in men (19%), but oral clearance did not differ between genders. Although the maximum azimilide concentration (Cmax) was 27% higher in women, time to maximum concentration or area under the azimilide concentration—time curve were not different from those for men. Body weight—adjusted Cmax did not differ between genders. Dosing adjustments based on either age or gender are not required. J Clin Pharmacol 1997;37:946–953.

Application of multiplexed capillary electrophoresis with laser-induced fluorescence (MCE–LIF) detection for the rapid measurement of endogenous extracellular signal-regulated protein kinase (ERK) levels in cell extracts

Multiplexed (96-lane) capillary electrophoresis with laser-induced fluorescence (MCE-LIF) detection was used for the rapid analysis of extracellular signal-regulated protein kinase (ERK) levels from in vitro cell extracts. The levels of ERK enzyme in cell extracts were determined by monitoring the conversion of a fluorescent-labeled peptide substrate to a phosphorylated fluorescent-labeled peptide product using MCE-LIF. The incorporation of a fluorescent internal standard was found to improve the precision of the analysis. The enzyme assay conditions including substrate concentration, reaction time and enzyme linear range were rapidly optimized using the MCE-LIF approach for both direct and immunoprecipitation-based ERK assays. The levels of ERK from in vitro cell extracts stimulated with angiopoietin 1 (Ang1*) were determined using the MCE-LIF approach. The advantages of MCE-LIF for developing and applying enzyme assays, as well as the figures of merit for the direct and immunoprecipitation ERK assays, are discussed.

Characterization and inhibition study of MurA enzyme by capillary electrophoresis.

A capillary electrophoresis-based enzyme assay for UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) is described. This method, based on UV detection, provides baseline separation of one of the reaction products, enolpyruvyluridine 5-diphospho-N-acetylglucosamine (EP-UDP-GlcNAc), from substrates phosphoenolpyruvate (PEP) and uridine 5-diphospho-N-acetylglucosamine (UDP-GlcNAc) within 4 min. The other product, phosphate, is not detectable by UV at 200 nm. Quantitation of individual components, substrates or product, can be accomplished based on the separated peaks. This methodology was used to determine the Michaelis constant, Km, and product formation rate constant, Kcat, for MurA. Additionally, the CE method was used to evaluate the inhibition effects on MurA using one specific compound as an example. By following similar procedures, the apparent Km values in the presence of different inhibitor concentrations were determined. The inhibition constant, Ki, can be determined from these apparent Km values. In addition, this CE method can be used to study the inhibition mechanism. The principle of this approach is generally applicable to other enzyme studies.

Optical MEMS-based fluorescence detection scheme with applications to capillary electrophoresis

A new laser-induced fluorescence detection system for capillary electrophoresis has been designed, built, and tested in this work. Multi-mode optical fibers were incorporated on-chip to couple laser light into the detection area on a borosilicate glass substrate. A metal- semiconductor-metal GaAs photodiode was fabricated and mounted over the separation capillary using a new conductive polymer flip-chip bonding approach, replacing the traditional photomultiplier tube in the detection scheme. Proper operation of the photodiode after interconnection was verified by directly impinging light into the detector using two laser sources. For both dry and filled channels, several laser source-fluorescent dye combinations were employed in order to generate a fluorescence signal (approximately few nA) detected by the photodiode. We thus demonstrate the feasibility of integrating optical MEMS into chemical micro total analysis systems ((mu) -TAS). In the future, this has the potential to improve functionality, throughput, and ease of operation of these `laboratories on chip.

Separation and quantitation of azimilide and its putative metabolites by capillary electrophoresis

Reliable methods based on capillary electrophoresis (CE) have been developed for the separation and quantitation of azimilide, an antiarrhythmic drug under development at Procter & Gamble Pharmaceuticals (P&GP). Both capillary zone electrophoresis (CZE) and micellar electrokinetic capillary chromatography (MECC) were employed in the separation of azimilide from its impurities, degradants and/or metabolites. Separation of azimilide from NE-11178, F-410, F-1054 and F-1292 was obtained by MECC at pH 9 with 50 mM sodium dodecyl sulfate (SDS). The separation of azimilide and NE-10171, a key metabolite of azimilide, was difficult because their structures differ by only a single methyl group. The best separation was achieved under acidic pH conditions with cetyltriethyl ammonium chloride (CTAC) additive in the buffer. All of the CE separations were completed within a substantially shorter time and with better resolution than the corresponding high-performance liquid chromatography (HPLC) separations. Quantitation was done with azimilide and NE-10171. Calibration curves ranging from 10 to 1000 microg/ml were obtained with R2 greater than 0.997 for both azimilide and NE-10171. The back-calculated concentrations of the calibration standards and the recoveries of the quality control (QC) samples were within the acceptance range currently used for HPLC methods. These results demonstrated the viability of CE as an alternative technique for drug metabolism studies in support of pharmaceutical development.