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: α-Lactalbumin (α-Lac) on colloidal functionalized magnetic core–shell nanoparticles (MCSNP). Such nanoparticles, functionalized with polyethyleneglycol and amino groups, exhibit a 30 nm physical diameter and behave as a quasi-homogeneous system. The α-Lac immobilization was achieved through the covalent binding between MCSNP amino groups and α-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 α-Lac, between NHS and EDC and the concentration of α-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]/[α-Lac] = 25; [NHS]/[EDC] = 1.55 and α-Lac = 24.85 nmol mL−1. In these optimal conditions, MCSNP surface was successfully saturated with α-Lac (34 α-Lac/MCSNP) with a high reproducibility (RSD = 2%). The colloidal stability of MCSNP grafted with α-Lac as well as the immunological interactions using anti α-Lac antibody were then investigated in different buffers. The results emphasized that a 50 mM MES buffer (pH 6) allows an efficient immune capture and a satisfying colloidal stability which provide an immunological interaction in homogeneous liquid phase.