Determination of binding parameters between lysozyme and its aptamer by frontal analysis continuous microchip...

An original and simple methodology based on microchip electrophoresis (MCE) in a continuous frontal analysis mode (named frontal analysis continuous microchip electrophoresis, FACMCE) was developed for the simultaneous determination of the binding parameters, i.e. ligand-site dissociation constant (k(d)) and number of binding sites on the substrate (n). This simultaneous determination was exemplified with the interaction between an aptamer and its target. The selected target is a strongly basic protein, lysozyme, as its quantification is of great interest due to its antimicrobial and allergenic properties. A glass microdevice equipped with a fluorescence detection system was coated with hydroxypropylcellulose, reducing the electroosmotic flow and adsorption onto the channel walls. This microdevice allowed the continuous electrokinetic injection of a mixture of fluorescently labelled aptamer and non-labelled lysozyme. By determining the concentration of the free fluorescently labelled aptamer thanks to its corresponding plateau height, mathematical linearization methods allowed to determine a k(d) value of 48.4±8.0 nM, consistent with reported results (31 nM), while the average number of binding sites n on lysozyme, never determined before, was 0.16±0.03. These results seem to indicate that the buffer nature and the SELEX process should influence the number and affinity of the binding sites. In parallel it has been shown that the binding between lysozyme and its aptamer presents two sites of different binding affinities.