A chemometric approach for optimizing protein covalent immobilization on magnetic core-shell nanoparticles in view of an alternative immunoassay.

A chemometric approach was developed to optimize the grafting of a bovine milk allergen: alpha-Lactalbumin (alpha-Lac) on colloidal functionalized magnetic core-shell nanoparticles (MCSNP). Such nanoparticles, functionalized with polyethyleneglycol and amino groups, exhibit a 30nm physical diameter and behave as a quasi-homogeneous system. The alpha-Lac immobilization was achieved through the covalent binding between MCSNP amino groups and alpha-Lac carboxylic moieties using the well-known tandem carbodiimide (EDC) and hydroxysulfosuccinimide (NHS). In this study, a chemometric approach was employed to highlight the parameters influencing the number of grafted proteins on the MCSNP. Three factors were evaluated: the ratio in concentration between EDC and alpha-Lac, between NHS and EDC and the concentration of alpha-Lac. After a first full factorial design to delimit the region of the space where the optimum could be located, a central composite design was then carried out to predict the best grafting conditions. It was established and experimentally confirmed that the optimum parameters are [EDC]/[alpha-Lac]=25; [NHS]/[EDC]=1.55 and alpha-Lac=24.85nmolmL(-1). In these optimal conditions, MCSNP surface was successfully saturated with alpha-Lac (34 alpha-Lac/MCSNP) with a high reproducibility (RSD=2%). The colloidal stability of MCSNP grafted with alpha-Lac as well as the immunological interactions using anti alpha-Lac antibody were then investigated in different buffers. The results emphasized that a 50mM MES buffer (pH 6) allows an efficient immune capture and a satisfying colloidal stability which provide an immunological interaction in homogeneous liquid phase.