An improved design to capture magnetic microparticles for in-line CE in a liquid based capillary coolant system
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| Year of publication | 2017 |
| Type | Conference abstract |
| Citation | |
| Description | Capillary electrophoresis as an analytical technique has emerged in recent years in the field of enzyme kinetics and inhibition. This is mainly attributed to the lower amounts of enzyme and substrate that can be used for reaction. One of the main advantages of CE is its ability to use capillary for both reaction and separation also known as in-line capillary electrophoresis. Since the introduction of this technique, a number of enzymatic reactions were performed using in-line capillary electrophoresis. In-line capillary electrophoresis is known for its lower sample consumption and organic solvents are not required to stop the reaction. Immobilizing drug-metabolizing enzymes on insoluble supports on either magnetic microparticles or capillary to develop CE based IMERs (Immobilized microenzyme reactors) have been attractive for their ability to repeated use with relatively small sample volumes. In the following work, we demonstrate the effectiveness of a new 3D printed magnet holder to hold magnets against the capillary that enables capture of magnetic microparticles in a commercially available liquid based coolant system. We discuss the design as well as the method to capture magnetic microparticles capture inside the capillary. This setup was tested at higher temperature and pH suitable for performing enzymatic reactions without any leakage of magnetic microparticles. To demonstrate its applicability in CE-IMER development, human Flavin monooxygenase 3 (hFMO3) was immobilized on amino functionalized magnetic microparticles using glutaraldehyde. In general, CYP enzymes are the major contributors of oxidative xenobiotic metabolism. However, flavin monoxygenases (FMOs) also contribute in these kinds of reactions which is less well explored than CYP counterparts. The significance of FMO should not be overlooked because FMO and CYP have overlapping substrate specificities. However, they often yield distinct metabolites with potentially significant toxicological/pharmacological consequences. hFMO3 is known to have limited stability when compared to CYP counterparts making it a suitable enzyme to develop an IMER that allows its reusability. Clozapine was used as a model substrate. This setup could be used further to employ CE-IMERs on other drug metabolic enzymes in a commercially available liquid based capillary coolant system. |
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