title: A chemometric approach for optimizing protein covalent immobilization on magnetic core-shell nanoparticles in view of an alternative immunoassay.
abstract: 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.
authors.affiliation: Physicochimie des Electrolytes, Colloïdes et Sciences Analytiques (PECSA) UMR 7195, CNRS-ESPCI-UPMC, 10 rue Vauquelin, Paris Cedex 05, France.
authors.forename: Bruno
authors.initials: B
authors.lastname: Teste
authors.forename: Jérôme
authors.initials: J
authors.lastname: Vial
authors.forename: Stéphanie
authors.initials: S
authors.lastname: Descroix
authors.forename: Thomas
authors.initials: T
authors.lastname: Georgelin
authors.forename: Jean-Michel
authors.initials: JM
authors.lastname: Siaugue
authors.forename: Jan
authors.initials: J
authors.lastname: Petr
authors.forename: Anne
authors.initials: A
authors.lastname: Varenne
authors.forename: Marie-Claire
authors.initials: MC
authors.lastname: Hennion
mesh_terms: D000485:Allergens; D000818:Animals; D002021:Buffers; D002234:Carbodiimides / Q000737:chemistry; D002417:Cattle; D002623:Chemistry Techniques, Analytical; D003102:Colloids / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007118:Immunoassay / Q000379:methods*; D007768:Lactalbumin / Q000737:chemistry; D008280:Magnetics / Q000379:methods; D008892:Milk / Q000378:metabolism; D053758:Nanoparticles / Q000737:chemistry; D011092:Polyethylene Glycols / Q000737:chemistry; D015203:Reproducibility of Results; D013388:Succinimides / Q000737:chemistry
chemical_list: D000485:Allergens; D002021:Buffers; D002234:Carbodiimides; D003102:Colloids; D013388:Succinimides; D011092:Polyethylene Glycols; D007768:Lactalbumin