The surface properties of milk fat globules govern their interactions with the caseins: Role of homogenization and pH probed by AFM force spectroscopy

The surface of milk fat globules consists of a biological membrane rich in polar lipids and glycoproteins. However, high shear stress applied upon homogenization disrupts the membrane and leads to the adsorption of casein micelles, as the major protein fraction of milk. These changes in the interface properties could affect the interactions between native or homogenized milk fat globules and the surrounding protein matrix, at neutral pH and upon acidification. In this study, macroscale rheometry, microscopic observations, nanoscale AFM-based force spectroscopy and physico-chemical analysis were combined to examine the interfacial composition and structure of milk fat globules and to evaluate their interactions with casein micelles. We showed that the surface properties of milk fat globules (biological membrane vs. caseins) and pH govern their interactions with casein micelles. The adhesion between individual fat globules and casein micelles was higher upon homogenization, especially at acid pH where the work of adhesion increased from 3.3 x 10-18 to 14 x 10-18 J for native and homogenized fat globules, respectively. Consequently, casein-coated homogenized fat globules yield stiffer milk acid gels. These findings cast light on the importance of colloidal particle’s surface properties and pH on their connectivity with the surrounding matrix, which modulates the bulk microstructure and rheological properties with potential functional consequences, such as milk lipid digestion

Analyse quantitative et qualitative sur puce de vésicules extracellulaires en milieux complexes au sein d'une plateforme nanobioanalytique

Extracellular vesicles (EVs) are small vesicles (30 to 1000 nm) released from different cell types, upon activation or apoptosis, and present in most body fluids (Blood, Urine….). Based on the current state of knowledge of their biogenesis and biochemical properties, EVs can be devided into three distinct populations: exosomes (EXO), microparticles (MPs) and apoptotic bodies (APOb). EVs have been found to play important biological roles and are also biomarkers of different pathologies. […] The first step consists of the injection of the samples containing EVs onto the biochip surface. This step is accomplished by SPR technique that allows label-free monitoring of EVs immunocapture onto the surface of a biochip presenting different specific bioreceptors. Following the capture of EVs, a nanometrological investigation of the biochip surface by AFM is engaged to characterize the physical properties of captured vesicles (size, morphology, etc..). Owning a nanometrical resolution, AFM can discriminate between individual EVs and vesicles or protein aggregates, leading to an accurate characterization of individual vesicles. The coupling of SPR technique with AFM was adapted to offer a representative global view of each array of bioreceptors and to measure the size of thousands of individual EVs. A proteomic investigation was also engaged to characterize the proteomic compositions of the different subpopulations of EVs. Such an investigation could contribute to the understanding of EVs biogenesis, biology and pathophysiology. To evaluate the potential of our platform to detect, quantify and characterize nanoparticles, two calibration particles, which cover the lower and upper size range of EVs, were chosen: (i) virus-like particles of 50 nm of diameter, also called CP50, and (ii) protein-functionnalized synthetic beads of 920 nm of diameter, called CP920. The capture tests in SPR showed a specific capture of these two calibration particles with their specific bioreceptors, immobilized onto the biochip surface, regardless the complexity of the media in which they were diluted. Also, a positive correlation was obtained between the capture level, measured by SPR, and the particle 9Les vésicules extracellulaires (VEs) sont des nanovésicules circulantes (30 à 100nm de diamètre) libérées dans l'espace extracellulaire par la plupart des cellules humaines, suite à leur activation ou à leur apoptose. Les VEs se divisent en 3 grandes catégories ; les exosomes (Exo), les microparticules (MPs) et les corps apoptotiques (cAPO). Les VEs sont présentes à l'état physiologique dans les différents fluides biologiques du corps humain et jouent un rôle majeur dans différents processus physio-pathologiques. De nos jours, plusieurs techniques, certaines en routine, sont utilisées pour étudier les VEs. Cependant, aucune d'entre elles ne permet de déterminer à la fois leur concentration, leur taille et leurs caractéristiques biochimiques. Un consensus existe sur la nécessité de combiner des techniques pour disposer enfin d'une caractérisation fine et complète des VEs. Il est d'un intérêt majeur de développer des plateformes analytiques dédiées à ces VEs en vue d'améliorer la qualification des échantillons biologiques et de découvrir de nouveaux biomarqueurs de pathologies humaines ou de bio-indicateurs de suivi thérapeutique.Notre projet consiste à développer une plateforme NanoBioAnalytique (NBA) combinant trois techniques : l'imagerie par Résonance des Plasmons de Surface (SPRi), la Microscopie à Force Atomique (AFM) et la Spectrométrie de Masse (MS). L'enjeu est de développer une interface biopuce-instruments qui permettra d'effectuer des investigations multiphysiques et multiéchelles apportant, en une stratégie globale, les informations plus complètes sur les différentes populations de VEs.[...]Ces travaux ont montré la capacité de notre plateforme à détecter sélectivement, et simultanément, différentes sous-populations des VEs co-existantes dans un échantillon complexe tel que du plasma, en s'appuyant sur l'expression différentielle des marqueurs protéiques membranaires. Les taux de capture se sont avérés être directement corrélés à la concentration des vésicules dans l'échantillon injecté. L'analyse AFM a permis de déterminer la distribution en taille de différentes sous-populations de VEs et permettre une analyse différentielle de la distribution en taille sur la gamme 20 nm - 1000 nm. Enfin, des études protéomiques "sur-puce" ont été également engagées afin de caractériser la composition en protéines des VEs libérées sous différentes conditions. Cette analyse a permis d'établir des premiers profils protéomiques différentiels des VEs dans les échantillons étudiés.La plateforme NBA est une méthode efficace pour caractériser et quantifier les VEs, sans marquage et avec une grande sensibilité, sur une large gamme dynamique (environ 10(7) à 10(12) particules/mL) cohérente avec celle existante en fluide physiologique et sur une plage de taille couvrant 2 décades. Elle s'inscrit parmi les approches les plus prometteuses pour l'investigation des VEs en complément de la cytométrie en flux. La grande adaptabilité de cette méthode d'analyse des VEs ouvre de larges perspectives de déploiement dans les secteurs de la Santé, de l'Environnement et de l'Agro-alimentaire

On-chip analysis and nanometrology of blood microparticles with label-free detection and characterization techniques

Les vésicules extracellulaires (VEs) sont des nanovésicules circulantes (30 à 100nm de diamètre) libérées dans l'espace extracellulaire par la plupart des cellules humaines, suite à leur activation ou à leur apoptose. Les VEs se divisent en 3 grandes catégories ; les exosomes (Exo), les microparticules (MPs) et les corps apoptotiques (cAPO). Les VEs sont présentes à l'état physiologique dans les différents fluides biologiques du corps humain et jouent un rôle majeur dans différents processus physio-pathologiques. De nos jours, plusieurs techniques, certaines en routine, sont utilisées pour étudier les VEs. Cependant, aucune d'entre elles ne permet de déterminer à la fois leur concentration, leur taille et leurs caractéristiques biochimiques. Un consensus existe sur la nécessité de combiner des techniques pour disposer enfin d'une caractérisation fine et complète des VEs. Il est d'un intérêt majeur de développer des plateformes analytiques dédiées à ces VEs en vue d'améliorer la qualification des échantillons biologiques et de découvrir de nouveaux biomarqueurs de pathologies humaines ou de bio-indicateurs de suivi thérapeutique.Notre projet consiste à développer une plateforme NanoBioAnalytique (NBA) combinant trois techniques : l'imagerie par Résonance des Plasmons de Surface (SPRi), la Microscopie à Force Atomique (AFM) et la Spectrométrie de Masse (MS). L'enjeu est de développer une interface biopuce-instruments qui permettra d'effectuer des investigations multiphysiques et multiéchelles apportant, en une stratégie globale, les informations plus complètes sur les différentes populations de VEs.[...]Ces travaux ont montré la capacité de notre plateforme à détecter sélectivement, et simultanément, différentes sous-populations des VEs co-existantes dans un échantillon complexe tel que du plasma, en s'appuyant sur l'expression différentielle des marqueurs protéiques membranaires. Les taux de capture se sont avérés être directement corrélés à la concentration des vésicules dans l'échantillon injecté. L'analyse AFM a permis de déterminer la distribution en taille de différentes sous-populations de VEs et permettre une analyse différentielle de la distribution en taille sur la gamme 20 nm - 1000 nm. Enfin, des études protéomiques "sur-puce" ont été également engagées afin de caractériser la composition en protéines des VEs libérées sous différentes conditions. Cette analyse a permis d'établir des premiers profils protéomiques différentiels des VEs dans les échantillons étudiés.La plateforme NBA est une méthode efficace pour caractériser et quantifier les VEs, sans marquage et avec une grande sensibilité, sur une large gamme dynamique (environ 10(7) à 10(12) particules/mL) cohérente avec celle existante en fluide physiologique et sur une plage de taille couvrant 2 décades. Elle s'inscrit parmi les approches les plus prometteuses pour l'investigation des VEs en complément de la cytométrie en flux. La grande adaptabilité de cette méthode d'analyse des VEs ouvre de larges perspectives de déploiement dans les secteurs de la Santé, de l'Environnement et de l'Agro-alimentaire.Extracellular vesicles (EVs) are small vesicles (30 to 1000 nm) released from different cell types, upon activation or apoptosis, and present in most body fluids (Blood, Urine….). Based on the current state of knowledge of their biogenesis and biochemical properties, EVs can be devided into three distinct populations: exosomes (EXO), microparticles (MPs) and apoptotic bodies (APOb). EVs have been found to play important biological roles and are also biomarkers of different pathologies. […] The first step consists of the injection of the samples containing EVs onto the biochip surface. This step is accomplished by SPR technique that allows label-free monitoring of EVs immunocapture onto the surface of a biochip presenting different specific bioreceptors. Following the capture of EVs, a nanometrological investigation of the biochip surface by AFM is engaged to characterize the physical properties of captured vesicles (size, morphology, etc..). Owning a nanometrical resolution, AFM can discriminate between individual EVs and vesicles or protein aggregates, leading to an accurate characterization of individual vesicles. The coupling of SPR technique with AFM was adapted to offer a representative global view of each array of bioreceptors and to measure the size of thousands of individual EVs. A proteomic investigation was also engaged to characterize the proteomic compositions of the different subpopulations of EVs. Such an investigation could contribute to the understanding of EVs biogenesis, biology and pathophysiology. To evaluate the potential of our platform to detect, quantify and characterize nanoparticles, two calibration particles, which cover the lower and upper size range of EVs, were chosen: (i) virus-like particles of 50 nm of diameter, also called CP50, and (ii) protein-functionnalized synthetic beads of 920 nm of diameter, called CP920. The capture tests in SPR showed a specific capture of these two calibration particles with their specific bioreceptors, immobilized onto the biochip surface, regardless the complexity of the media in which they were diluted. Also, a positive correlation was obtained between the capture level, measured by SPR, and the particle

Atomic force microscopy of food assembly: Structural and mechanical insights at the nanoscale and potential opportunities from other fields

The atomic force microscope (AFM) has opened access to the nanoscale observation of molecular and colloidal structures in aqueous media, and of their dynamics upon environmental changes. As a miniature force scanner, it furthermore allows the correlative mapping of mechanical properties at the nanoscale or precise indentation of individual structures. Soon after its invention in 1986, the AFM rapidly found increasing applications in soft matter physics, cellular biology, polymer science and microbiology. In spite of significant successes as early as the 90s, the growth of AFM application in the field of food science has been comparatively slower. This review points to the realizations and opportunities of AFM in showing the connections between the structural and mechanical properties of food's building blocks and of their assemblies. Possible transfers from other disciplines to food science are presented as suggestions for future applications

On-chip detection, sizing and proteomics of extracellular vesicles

International audienceMicroparticles are small extracellular vesicles (EVs) (from ~100 to 1000 nm) produced by different cell types, through the budding of the plasma membrane, while exosomes (from ~30 to 120 nm) originate from the endolysosomal pathway before fusing with the plasma membrane to be released. Increased platelet-derived microparticles (PMPs) formation has been reported to contribute to the inflammatory role of blood components used for transfusion. When PMPs formation results from thrombin activation, they are able to aggregate monocyte cells in vitro. Nevertheless, the reason(s) for this EVs functionality/effect on target cells still need to be clarified, due to their high variety in size, protein composition and the potential concomitant presence of exosomes and small MPs in the analyzed samples

Selective adsorption of casein micelles onto milk polar lipids membrane bilayers: role of phase and charge states investigated using AFM

Interactions between biological membranes and proteins are crucial in biological functions such as cell signaling, trafficking or in the control of membranes’ physical stability or functional properties. Over the last decades, the nanoscale resolution of Atomic Force Microscopy (AFM) has allowed direct observation of such interactions in near-physiological conditions[1].Among biological membranes, the milk fat globule membrane (MFGM) is of special interest as the interface with the surrounding bulk of milk proteins during dairy processes or with enzymes and tissues of the gastro-intestinal tract during digestion. The MFGM is composed of polar lipids showing either zwitterionic, e.g. milk sphingomyelin (MSM) and phosphatidylcholine (PC), or anionic head groups, e.g. phosphatidylserine (PS) and phosphatidylinositol (PI). It exhibits lateral heterogeneity with microdomains of MSM in gel-phase or in liquid-ordered (Lo) phase in the presence of cholesterol, surrounded by the fluid matrix of glycerophospholipids, ie DOPC, in the liquid-disordered (Ld) phase[2,3]. In the present study, our goal was to determine how the phase and charge states of MFGM polar lipids drive interaction with the casein micelles, as the major milk protein 100-nm assembly, negatively charged at pH 6.7.For that, supported-hydrated lipid bilayers with various lipid compositions were prepared, including MFGM polar lipid extract, MSM, MSM/cholesterol, MSM/DOPC, DOPC/PS/PI. AFM topography imaging was performed before and after injection of casein micelles in the environment. Zeta-potential and Langmuir isotherms of the different polar lipids gave additional information necessary to interpret AFM observations.No interaction was observed between the casein micelles and the organized phases of MSM in gel-phase or MSM/cholesterol in Lo phase. But the casein micelles did adsorb onto the Ld phase of DOPC (figure 1), probably as a result of its larger inter-molecular distance. However, the presence of anionic polar lipids, PS and PI, in the Ld phase prevented such interaction by inducing electrostatic repulsion of the casein micelles. Thus, interaction of casein micelles with the MFGM is controlled by the phase state and the ionisation of its constitutive polar lipids.These results open perspectives for the design of emulsions and liposomes with variable capacity for protein adsorption, using milk polar lipids as food-grade ingredients

Selective adsorption of casein micelles onto milk polar lipids membrane bilayers: role of phase and charge states investigated using AFM

Interactions between biological membranes and proteins are crucial in biological functions such as cell signaling, trafficking or in the control of membranes’ physical stability or functional properties. Over the last decades, the nanoscale resolution of Atomic Force Microscopy (AFM) has allowed direct observation of such interactions in near-physiological conditions[1].Among biological membranes, the milk fat globule membrane (MFGM) is of special interest as the interface with the surrounding bulk of milk proteins during dairy processes or with enzymes and tissues of the gastro-intestinal tract during digestion. The MFGM is composed of polar lipids showing either zwitterionic, e.g. milk sphingomyelin (MSM) and phosphatidylcholine (PC), or anionic head groups, e.g. phosphatidylserine (PS) and phosphatidylinositol (PI). It exhibits lateral heterogeneity with microdomains of MSM in gel-phase or in liquid-ordered (Lo) phase in the presence of cholesterol, surrounded by the fluid matrix of glycerophospholipids, ie DOPC, in the liquid-disordered (Ld) phase[2,3]. In the present study, our goal was to determine how the phase and charge states of MFGM polar lipids drive interaction with the casein micelles, as the major milk protein 100-nm assembly, negatively charged at pH 6.7.For that, supported-hydrated lipid bilayers with various lipid compositions were prepared, including MFGM polar lipid extract, MSM, MSM/cholesterol, MSM/DOPC, DOPC/PS/PI. AFM topography imaging was performed before and after injection of casein micelles in the environment. Zeta-potential and Langmuir isotherms of the different polar lipids gave additional information necessary to interpret AFM observations.No interaction was observed between the casein micelles and the organized phases of MSM in gel-phase or MSM/cholesterol in Lo phase. But the casein micelles did adsorb onto the Ld phase of DOPC (figure 1), probably as a result of its larger inter-molecular distance. However, the presence of anionic polar lipids, PS and PI, in the Ld phase prevented such interaction by inducing electrostatic repulsion of the casein micelles. Thus, interaction of casein micelles with the MFGM is controlled by the phase state and the ionisation of its constitutive polar lipids.These results open perspectives for the design of emulsions and liposomes with variable capacity for protein adsorption, using milk polar lipids as food-grade ingredients

Selective adsorption of casein micelles onto milk polar lipids membrane bilayers: role of phase and charge states investigated using AFM

Interactions between biological membranes and proteins are crucial in biological functions such as cell signaling, trafficking or in the control of membranes’ physical stability or functional properties. Over the last decades, the nanoscale resolution of Atomic Force Microscopy (AFM) has allowed direct observation of such interactions in near-physiological conditions[1]. Among biological membranes, the milk fat globule membrane (MFGM) is of special interest as the interface with the surrounding bulk of milk proteins during dairy processes or with enzymes and tissues of the gastro-intestinal tract during digestion. The MFGM is composed of polar lipids showing either zwitterionic, e.g. milk sphingomyelin (MSM) and phosphatidylcholine (PC), or anionic head groups, e.g. phosphatidylserine (PS) and phosphatidylinositol (PI). It exhibits lateral heterogeneity with microdomains of MSM in gel-phase or in liquid-ordered (Lo) phase in the presence of cholesterol, surrounded by the fluid matrix of glycerophospholipids, ie DOPC, in the liquid-disordered (Ld) phase[2,3]. In the present study, our goal was to determine how the phase and charge states of MFGM polar lipids drive interaction with the casein micelles, as the major milk protein 100-nm assembly, negatively charged at pH 6.7. For that, supported-hydrated lipid bilayers with various lipid compositions were prepared, including MFGM polar lipid extract, MSM, MSM/cholesterol, MSM/DOPC, DOPC/PS/PI. AFM topography imaging was performed before and after injection of casein micelles in the environment. Zeta-potential and Langmuir isotherms of the different polar lipids gave additional information necessary to interpret AFM observations. No interaction was observed between the casein micelles and the organized phases of MSM in gel-phase or MSM/cholesterol in Lo phase. But the casein micelles did adsorb onto the Ld phase of DOPC (figure 1), probably as a result of its larger inter-molecular distance. However, the presence of anionic polar lipids, PS and PI, in the Ld phase prevented such interaction by inducing electrostatic repulsion of the casein micelles. Thus, interaction of casein micelles with the MFGM is controlled by the phase state and the ionisation of its constitutive polar lipids. These results open perspectives for the design of emulsions and liposomes with variable capacity for protein adsorption, using milk polar lipids as food-grade ingredients

Development of a NanoBioAnalytical platform for "on-chip" qualification and quantification of platelet-derived microparticles.

International audienceBlood microparticles (MPs) are small membrane vesicles (50-1000nm), derived from different cell types. They are known to play important roles in various biological processes and also recognized as potential biomarkers of various health disorders. Different methods are currently used for the detection and characterization of MPs, but none of these methods is capable to quantify and qualify total MPs at the same time, hence, there is a need to develop a new approach for simultaneous detection, characterization and quantification of microparticles. Here we show the potential of surface plasmon resonance (SPR) method coupled to atomic force microscopy (AFM) to quantify and qualify platelet-derived microparticles (PMPs), on the whole nano-to micro-meter scale. The different subpopulations of microparticles could be determined via their capture onto the surface using specific ligands. In order to verify the correlation between the capture level and the microparticles concentration in solution, two calibration standards were used: Virus-Like Particles (VLPs) and synthetic beads with a mean diameter of 53nm and 920nm respectively. The AFM analysis of the biochip surface allowed metrological analysis of captured PMPs and revealed that more than 95% of PMPs were smaller than 300nm. Our results suggest that our NanoBioAnalytical platform, combining SPR and AFM, is a suitable method for a sensitive, reproducible, label-free characterization and quantification of MPs over a wide concentration range (≈107 to 1012 particles/mL; with a limit of detection (LOD) in the lowest ng/µL range) which matches with their typical concentrations in blood

The phase and charge of milk polar lipid membrane bilayers govern their selective interactions with proteins as demonstrated with casein micelles

The biological membrane surrounding fat globules in milk (milk fat globule membrane; MFGM) is an interface involved in many biological functions and interactions with the surrounding proteins or lipolytic enzymes in the gastro-intestinal tract during digestion. The MFGM exhibits lateral heterogeneities resulting from the different phase states and/or head-group charge of the polar lipids, which were both hypothesized to drive interaction with the casein micelles that is the major milk protein assembly. Atomic force microscopy (AFM) imaging was used to track the interactions of casein micelles with hydrated supported lipid bilayers of different composition, phase state and charge. Zeta-potential and Langmuir isotherms of the different polar lipids offered additional information necessary to interpret AFM observations. We showed that the negatively-charged casein micelles did not interact with milk sphingomyelin in the gel or liquid-ordered phases but did interact with polar lipids in the liquid-disordered phase (unsaturated polar lipids and milk sphingomyelin above its melting point). A wide intermolecular distance between polar lipids allowed protein adsorption on the membranes. However, the presence of the anionic polar lipids phosphatidylserine and phosphatidylinositol prevented any interaction with the casein micelles, probably due to electrostatic repulsion. These results open perspectives for the preparation of tailored emulsions covered by polar lipids able to modulate the interfacial interactions with proteins