In-capillary nanoreactor based on transverse diffusion of laminar flow profiles for cytochrome P450 2C9 isoform reaction with diclofenac
| Authors | |
|---|---|
| Year of publication | 2010 |
| Type | Article in Proceedings |
| Conference | Book of Abstracts, 17th International Symposium on Electro- and Liquid Phase-Separation Techniques ITP 2010 |
| MU Faculty or unit | |
| Citation | |
| Field | Biochemistry |
| Keywords | CYP 2C9, diclofenac, on-line reaction, capillary electrophoresis |
| Description | A fused silica capillary as an important part of capillary electrophoresis system represents a favourable instrument not only for highly effective separations but also for creating nanoscale reaction chambers. Today commonly used methodology of electrophoretically mediated microanalysis does not allow simple changing of an incubation mixture composition, nevertheless, foreclosing in-capillary reactions from screening studies. To solve this limitation, Krylov et al. introduced the generic methodology for mixing the reactants inside the capillary based on transverse diffusion of laminar flow profiles (TDLFP) [1]. Conceptually, solutions of incubation mixture components are injected by pressure as a series of consecutive plugs which have parabolic profiles due to the laminar nature of flow inside the capillary. Resulting longitudinal interfaces between the plugs then enable the plugs mixing by transverse diffusion within minutes. In this study a method for cytochrome P450 2C9 (CYP2C9) reaction with diclofenac as a probe substrate was developed. 30 min incubation time and sandwich layout, where a plug of CYP2C9 is surrounded by two plugs of diclofenac and NADPH mixture, were selected as appropriate incubation conditions for final method within the optimizing. Although bare silica capillary was used for performing the analyses of samples consisting of enzyme preparations, strong rinsing procedure with 1 M H3PO4 and 1 M NaOH applied between following runs provides good method’s repeatability with RSD values of 1.52 % and 4.83 % (n = 6) for migration times and peak areas of 4’-hydroxydiclofenac, respectively. Since methodology of TDLFP allows simple implementation of changes in incubation mixture composition, introduced method represents a promising tool for in-capillary inhibition studies of CYP2C9. |
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