Reactants Mixing inside the In-Capillary Nanoreactor: Utilization of Longitudinal and Transverse Diffusion
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| Year of publication | 2011 |
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| Description | Today, there is ongoing trend toward miniaturization of biochemical assays. Capillary electrophoresis (CE) represents a favorable technique in this field due to fast, highly effective separations, minute sample consumption and high throughput by automation [1]. Enzyme activity assay typically constitutes two-steps (off-line) procedure when reaction mixture is incubated in a vial first and products of reaction are separated by means of CE thereafter. Since only several nl of sample are used within CE analysis, most of the reaction mixture solution is discarded. Contrary in on-line analyses, fuse silica capillary serves not only as a separation column but also as a reaction chamber and thus allows achievement of real nanoscale assays. Practical utilization of this attractive concept is constrained by lack of effective way for reagents mixing inside limited space of capillary, nevertheless. Couple of approaches based on differential electrophoretic mobilities and transverse diffusion have been already presented to deal with this limitation [2,3], however, none of them is suitable for practical implementation. For this reason, the goal of presented study was to introduce a generic methodology which enables interfusing of selectable reactants into homogenous reaction mixture inside a capillary nanoreactor. Principle based on combination of longitudinal and transverse diffusion was employed to fulfill these requirements. Conceptually, the solutions of reaction mixture components are injected by relatively low pressure as a series of consecutive narrow plugs having parabolic profiles due to laminar flow inside the capillary. Resulting character of plugs with rather longitudinal interfaces then enable creation of homogenous reaction mixture by both longitudinal and transverse diffusions within minute period. The kinetic study of probe system of cytochrome P450 2C9 with its specific substrate diclofenac was performed to prove the applicability of proposed methodology. The value of evaluated apparent Michaelis-Menten constant was in a good agreement with a data found in literature. Presented principle thus constitutes a promising tool for on-line inhibition studies of cytochrome P450 2C9 requiring frequent changes in reaction mixture composition. |
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