Analytical techniques for the determination of biological activity
An approach developed by our analytical group consists in finding active molecules in plant extracts and testing their inhibitory activity on enzymes used in cosmetic (i.e. tyrosinase) and therapeutic fields (i.e. kinase).
The novelty of this method resides in the use of frontal affinity chromatography and of the intensity ion fading mass spectrometry (IF-MS) approach. These two techniques use immobilized enzymes of interest on magnetic silica beads. In a vegetal extract, this method allows on one hand, the determination of specific interactions between the molecules of interest in the crude extract and the target enzyme, and on the other hand, the classification of ligands/inhibitors according to their affinities with the biological target.
Ion-Fading Enzyme-coupled Nanoparticle-Assisted LDI (IF-ENALDI) principle
Moreover, the Analytical Chemistry team of ICOA has an extensive experience on homogeneous enzymatic assays using capillary electrophoresis (CE) in cosmetic and therapeutic fields. The capillary can be used as nanoreactor in which the enzymatic reaction takes place. For this, low volumes of reagents (few nanoliters) are successively injected as short plugs inside the capillary and mixed by applying an electric field (EMMA) or a pressure (at-inlet or TDFLP mode) during a short period of time, few seconds, usually. Kinetic parameters (Ki and IC50) can thus be accurately determined. This methodology is also of great interest to evaluate the biological activity in cellulo in bacteria or human cells. A few cells are directly introduced and lysed inside the capillary by electroporation, and the enzymatic activity is subsequently monitored on-line. Thus the activity of the β-galactosidase as cell aging marker has been determined in bacteria and skin cells. In addition, the inhibitory activity of flavone compounds present in plant extracts (sea buckthorn) have been evaluated using CE.
Evaluation of enzyme inhibition by capillary electrophoresis (CE)
