Casein micelles topography and nanorheology evaluation by AFM: A novel method for casein micelles immobilization and study in their native state
Résumé
The morphology and the nanomechanical properties of single casein micelles (CMs) in their native state were investigated using AFM in liquid environment. A novel immobilization method of CMs using low energy interactions was developed and compared with a well-known chemical immobilization method involving direct covalent bonds.
For the classic method, CMs were covalently bound via carbodiimide chemistry to a gold-sputtered glass surface precoated with carboxyl groups. For the proposed method an indirect immobilization of CMs was realized via anti-human phospho-Ser/Thr/Tyr mouse monoclonal antibody covalently bound onto a gold-coated slide via carbodiimide chemistry. In both methods, immobilized CMs were then analyzed by AFM in their native environment represented by a simulated milk ultrafiltrate at room temperature.
With the classic method of immobilization, some CMs remained mobile in liquid condition during the probe sample interaction. Conversely, with the novel method, CMs were well immobilized allowing the extraction of both surface topography and elastic properties by AFM in liquid. Morphologic characteristics were obtained from 2D and 3D images (Figure1). Data analyses allowed the calculation of diameters, heights and contact angles of native CMs. They are characterized by a mean width of 148 ± 8 nm and a mean height of 42 ± 1 nm. Low forces were applied on immobilized CM to calculate force-vs-indentation curves fitted using Hertz theory in order to determine Young Moduli (E*) which represents elasticity. Their elasticity distribution exhibited a unimodal trend, with an elasticity pic centered at 269 ± 14 kPa.