Application of carbon nanotubes for in-capillary incubations with cytochrome P450 enzymes

The utility of multi-walled carbon nanotubes (MWNTs) to decrease the interaction between cytochrome P450 enzymes and the capillary wall during in-capillary enzymatic incubation was investigated. First, 18 surfactants were screened to determine their MWNT-dispersing capacity. A probe sonication procedure was developed in order to attain homogeneous MWNT dispersions within a reasonable time. Next, the influence of surfactants and MWNTs on P450 activity was studied, employing verapamil and CYP3A4 as model substrate and P450 isoform, respectively. MWNTs dispersed in Brij 35 did not affect CYP3A4 activity significantly and were selected for subsequent in-capillary tests. An in-line CE assay, involving electrophoretic mixing of reagents and zero potential amplification in the thermostatted part of the capillary, was developed. In-capillary incubations without MWNTs caused adsorption of enzyme to the capillary wall and a concomitant decline of capillary lifetime, even when extensive between-run rinsing was applied. Addition of MWNTs to the enzyme solution entailed substantial improvement of migration time and peak shape repeatability. The performance of three types of MWNTs was compared.