Taking Advantage of Electric Field Induced Bacterial Aggregation for the Study of Interactions between Bacteria and Macromolecules by Capillary Electrophoresis

Abstract

The quantification of interaction stoichiometry and binding constant between bacteria (or other microorganism) and (macro)molecules remains a challenging issue for which only a few adapted methods are available. In this paper, a new methodology was developed for the determination of the interaction stoichiometry and binding constant between bacteria and (macro)molecules. The originality of this work is to take advantage of the bacterial aggregation phenomenon to directly quantify the free ligand concentration in equilibrated bacteria-ligand mixtures using frontal analysis continuous capillary electrophoresis. The described methodology does not require any sample preparation such as filtration step or centrifugation. It was applied to the study of interactions between Erwinia carotovora and different generations of dendrigraft poly-L-lysines leading to quantitative information (i.e., stoichiometry and binding site constant). High stoichiometries in the order of 10(6)-10(7) were determined between nanometric dendrimer-like ligands and the rod-shaped micrometric bacteria. The effect of the dendrimer generation on the binding constant and the stoichiometry is discussed. Stoichiometries were compared with those obtained by replacing the bacteria by polystyrene microbeads to demonstrate the internalization of the ligands inside the bacteria and the increase of the specific surface via the formation of vesicles.

Everything You Always Wanted to Know about Poly‐l‐lysine Dendrigrafts (But Were Afraid to Ask)

“…[33] Moreover,afast bacteriolytica ctivity of the second-generation DGL G2 was observed against Erwinia carotovora under low salt conditions. The interactions between DGLs and Gram-negative bacteria (i.e., Erwiniac arotovora)w ere studied by Cottet et al using capillary electrophoresis, with reported binding affinities in the micromolar range, and binding stoichiometries that decrease with increasing DGL generation (i.e.,f rom 10.2 10 6 to 1.1 10 6 for DGL G2 to G4,r espectively).…”

“…[52] Biosensing In ab iosensing purpose,athird-generation generation DGL G3,i mmobilizedo np lasma activated polypropylene filters, Figure 5. It is interesting to note that, since DGLs and bacteria also displayed high affinities based on electrostatic interactions, [33] this approachc ould be extended to the detection and/orr emoval of pathogenic bacteria from contaminated waters.R ecently,V iale tal. B) Time-dependentinv ivo fluorescence images of subcutaneousH eLa tumour-bearing mice after i.v.i njectiono fDox/duplex-Cy5.5, Dox/Mito-DGL (no AS1411) or Dox/Mito-DGL.…”

Pressure‐assisted capillary electrophoresis frontal analysis for faster binding constant determination

“…Substitute volumetric flow rate Q with the product of capillary cross section and the average linear velocity , Equation becomes …”

“…where ⌬ P,,L t ,Q and a are the pressure difference between ends of the capillary, the dynamic viscosity of the solution, the total length of the capillary, the volumetric flow rate, and the capillary inner radius, respectively. Substitute volumetric flow rate Q with the product of capillary cross section a 2 and the average linear velocitȳ v = 1 2 v 0 , Equation [13] becomes…”

“…[1][2][3] Chiral separation, [4,5] genomics, [6] metabolomics, [7,8] proteomics, [9] drug discovery [10,11] and macro molecular interaction studies are a few examples of this simple yet powerful separation technique. [12,13] In CE, charged molecules with different electrophoretic mobility are separated under a strong electric field and in some cases all species are driven by the electroosmotic flow (EOF). Compared to the flat-shaped flow profile of the EOF-driven flow, the pressure-driven laminar flow in an open tube has a parabolic flow profile.…”