Article pubs.acs.org/Langmuir
Atomic Force Microscopy Study of the Topography and Nanomechanics of Casein Micelles Captured by an Antibody Asma Bahri,† Marta Martin,‡ Csilla Gergely,‡ Martine Pugnière,§ Dominique Chevalier-Lucia,† and Sylvie Marchesseau*,† †
Université de Montpellier, UMR IATE, F-34095 Montpellier Cedex 05, France Laboratoire Charles Coulomb, Université de Montpellier, UMR 5221-CNRS, F-34095 Montpellier Cedex 05, France § IRCM-CRLC Val d’Aurelle - INSERM U896, F-34298 Montpellier Cedex 5, France ‡
ABSTRACT: Casein micelles (CMs) are colloidal phosphoprotein-mineral complexes naturally present in milk. This study used atomic force microscopy (AFM) in a liquid environment to evaluate the topography and nanomechanics of single native CMs immobilized by a novel capture method. The proposed immobilization method involves weak interactions with the antiphospho-Ser/Thr/Tyr monoclonal antibody covalently bound to a carboxylic acid self-assembled monolayer (SAM) on a gold surface. This capture strategy was compared to the commonly used covalent immobilization method of CMs via carbodiimide chemistry. With this conventional method, CMs remained mainly mobile during AFM measurements in liquid, disturbing the evaluation of their average size and elastic properties. Conversely, when captured by the specific antibody, they were successfully immobilized and their integrity was preserved during the AFM measurement. The characterization of both CM topography and elastic properties was carried out in a liquid ionic environment at native pH 6.6. The CMs’ capture efficiency via antibody was concurrently proved by surface plasmon resonance. The calculation of casein micelles’ width, height, and contact angle was carried out from the recorded 2D AFM images. CMs were characterized by a mean width of 148 ± 8 nm and a mean height of 42 ± 1 nm. Weak forces were applied to single captured CMs. The obtained force versus indentation curves were fitted using the Hertz model in order to evaluate their elastic properties. The elasticity distribution of native CMs exhibited a unimodal trend with a peak centered at 269 ± 14 kPa.
■
INTRODUCTION Casein micelles (CMs) are natural macromolecular selfassemblies with an important role in milk, assuring the stabilization and transport of essential nutrients in the context of a newborn diet.1 About 80% of milk proteins (i.e., 26 g/kg) are phosphate-specific acid proteins called caseins, present in a micellar form with high interactions with minerals, specifically calcium and phosphate. These CMs, characterized by a molecular weight of ∼1.3 × 109 Da, are composed of four types of phosphoprotein (αS1-, αS2-, β-, and κ-caseins in a molar ratio of ∼4:1:4:1.6) assembled through hydrophobic interactions and colloidal calcium phosphate bridges.2 In particular, αS- and β-caseins are mainly located in the hydrophobic micelle core surrounded by a hydrophilic surface layer attributed to the presence of the amphiphilic glycosylated κ-casein. The resulting complex structure is negatively charged at neutral pH (zeta potential ∼ −20 mV) and highly hydrated (∼4 g of water/g of casein) and forms large colloidal spherical particles (hydrodynamic diameters between 50 and 600 nm) with a median size of between 100 and 200 nm.3−5 CMs in milk are in equilibrium with soluble casein molecules and dissolved salts in serum and display rheomorphic properties characterized by an open and mobile conformation that can adapt to the environment.6 This © XXXX American Chemical Society
supramolecular structure is at the origin of milk stability and also all transformation processing. Despite the key role of the CM in dairy science, its internal structure is not yet well-known, stimulating great interest for biophysicists. Indeed, over the past few decades, a large number of studies have investigated CM structure, particularly to understand its conformational flexibility, resulting in several structural models without a general consensus.7−10 Among the physical techniques used to characterize CM structure, microscopy techniques such as scanning electron microscopy,11 field emission scanning electron microscopy,12 and cryo-transmission electron microscopy13 have allowed a better understanding of the CM morphology. However, the sample preparation required for these different methods such as dehydration, freezing, and metal coating may affect the casein structure, limiting the observation of CMs in their native state.9,12−16 Atomic force microscopy (AFM) was used in the last 10 years to investigate CMs after immobilization on a flat, solid surface in air16 or in a liquid environment.17−20 This technique offers not only the Received: January 28, 2017 Revised: March 27, 2017
A
DOI: 10.1021/acs.langmuir.7b00311 Langmuir XXXX, XXX, XXX−XXX
Article
Langmuir
industrially obtained by microfiltration and diafiltration using the milk mineral soluble phase, ensuring a native state for the prepared CMs. PC powder contained 95 g of dry solids per 100 g of powder and, on a dry basis (w/w), 86% total proteins (corresponding to 79.1% caseins), 1.6% fat, 4% lactose, and 8% minerals. Tripotassium citrate, trisodium citrate, KH2PO4, and K2SO4 were purchased from Alfa Aesar (Heysham, U.K.). K2CO3 and CaCl2 were from Amresco (Solon, OH, USA), and acetic acid, MgCl2, KCl, and KOH were from VWR BDH Prolabo (Fontenay-sous-bois, France). 11-Mercapto-1-undecanoic acid (11-MUA), N-ethyl-dimethylaminopropyl carbodiimide (EDC), and N-hydroxysuccinimide (NHS) were obtained from Sigma-Aldrich (Saint-Quentin Fallavier, France). Sodium acetate was purchased from Merck (Darmstadt, Germany). C1 sensor chip, HBS-EP buffer at pH 7.4 (10 mM 4-(2-hydroxyethyl)1-piperazineethanesulfonic acid (HEPES), 150 mM NaCl, 3 mM EDTA, and 0.005% nonionic polyoxyethylenesorbitan surfactant P20), HBS-N buffer (0.01 M HEPES, 0.15 M NaCl, pH 7.4), ethanolamine hydrochloride 1 M pH 8.5, and rabbit antimouse (RAM) antibody were purchased from GE Healthcare (Velizy-Villacoublay, France). Mouse antihuman phospho-Ser/Thr/Tyr monoclonal antibody (MAH-PSer/Thr/Tyr antibody) was from Spring Bioscience (ref E3074, Pleasanton, CA, USA). All solutions were prepared using MilliQ water (Millipore). The CM dispersion (5%, w/w) was prepared by dissolving PC powder in pH 6.6 lactose-free simulated milk ultrafiltrate (SMUF), replicating the mineral environment of native CMs.24 The dispersion was stirred at 540 rpm for 30 min at 20 °C before being stored overnight at 4 °C to improve the powder hydration. The CM dispersion was then warmed for 1 h at 40 °C and rapidly cooled to 20 °C just before the experiments to ensure complete equilibration. Methods. Photon Correlation Spectroscopy. The CM size distribution in a liquid suspension was evaluated by photon correlation spectroscopy (PCS) using a Zetasizer Nano-ZS (Malvern Instruments, Malvern, U.K.) at 25 °C in polystyrene four-sided polished cuvettes (Sarstedt, Nümbrecht, Germany). Before analysis, samples were diluted 20-fold with SMUF to avoid multiple diffusion phenomena during PCS measurements. Experimental data were assessed by the NNLS algorithm with a dispersant viscosity of 0.89 mPa·s at 25 °C and a refractive index of 1.33. Characteristics of the dispersed particles of CMs were taken as for milk proteins: 0.004 and 1.36 for the imaginary and real refractive indices, respectively.25 For each independent sample, a mean distribution curve in intensity and in number was calculated from six measurements, and the mean diameter (arithmetic mean) was obtained. CMs Immobilization Methods. All steps in CM immobilization were carried out at room temperature. Gold-sputtered glass chips (AU.0500.ALSI, Platypus Technologies LLC, Madison, WI, USA) were first cleaned twice with piranha solution (70% H2SO4 + 30% H2O2) and then three times with ethanol. The chips were immediately immersed in a 5 mM ethanolic solution of 11-MUA for 18 h to coat the surface with a self-assembled monolayer (SAM) of carboxyl groups. SAM-coated chips were then extensively rinsed with absolute ethanol and ultrapure water. Immobilization through Carbodiimide Chemistry. CMs were covalently immobilized on a SAM-coated chip using amine coupling via carbodiimide chemistry as already published.16,19,20 The SAMcoated chip was activated by carbodiimide chemistry: it was immersed and gently stirred into a freshly prepared equal-volume solution of 0.4 M EDC and 0.1 M NHS for 30 min. After ultrapure water rinsing, it was immediately covered with the CM dispersion for 1 h. The chip was finally rinsed with SMUF and equilibrated at room temperature for 4 h before the AFM experiment. Immobilization through an Antibody. The SAM-covered chip was first activated via amine coupling using carbodiimide chemistry, as described above, before being immersed for 1 h in an MAHP-Ser/ Thr/Tyr antibody solution (50 μg/mL) prepared in acetate buffer (10 mM, pH 5). The chip was then rinsed with acetate buffer (10 mM, pH 5) and with HBS-N buffer before being covered with ethanolamine solution (1 M, pH 8) for 30 min to block free binding sites. It was then immediately covered by the CM dispersion for 1 h, rinsed with SMUF
opportunity to study CM morphology, providing threedimensional images with a high power of magnification and high resolution, but also to evaluate the elastic properties of individual CMs in a native state. The immobilization of CMs before AFM investigation is a critical step because they must be strongly attached to a solid surface to avoid undesired motions during AFM analysis while maintaining their integrity and nanomechanical properties. Consequently, special attention must be paid to the selection of the substrate and the immobilization procedure.21 Until now, different solid, flat supports and immobilization methods were used to observe CMs by AFM in liquid environments. CMs were adsorbed on a mica substrate, but the disruption and dissociation of adsorbed objects were observed as a result of the mechanical force applied by the AFM tip during contact-mode imaging.18 More recently, native CMs were immobilized through carbodiimide chemistry on a gold substrate and were imaged by AFM in simulated milk ultrafiltrate (SMUF, pH 6.7) in a state close to the physiological environment.19 The surface of single CMs in a liquid environment was rough, with heterogeneities consistent with a spongelike structure. The same immobilization strategy on silicon wafers coated with titanium was used to observe CMs from freshly skimmed milk by AFM in an ultrafiltered permeate. However, the CMs diameter evaluated in this study (
