It is common knowledge that aggregation of proteins may occur in aqueous solutions under mechanical stress (shaking or high shear), even in solutions that are stable at rest. Addition of surfactants is a practical generic means to prevent this stress-induced aggregation (e.g. in formulations of therapeutic proteins), which suggests that interfaces contribute to destabilization. We studied here the role of interfacial stress by applying brief mechanical impacts on air-water interface, in presence or absence of surfactants, in solutions of immunoglobulin G (IgG), a class of proteins of high importance to the developments of new therapeutics. A variety of surfactants was tested including the neutral ones Tween80, C10-C14 foscholines, alkylaminoxide, surfactin, and two ionic ones TTAB and lauroylsarcosine sodium salt. We determined the presence of aggregates in solution by light scattering. Irrespective of the type of antibody, either human polyclonal or a monoclonal one, we show that the amount of aggregated IgG increases in proportion to the number of impacts on the interface. In absence of stress, we recorded images of oblate aggregates of IgG (ca. 12 nm height and 200-1200 nm in diameter) present at the air-water interface (fluorescence microscopy using anti-Fab or anti-Fc markers, and AFM scans after transfer on freshly cleaved mica). Our results evidence that aggregates are formed at air-water interface, and are brought in solution by transient stresses applied on the water surface. Rupture of interfacial films is an important source of aggregates in solution. Finally, the role of surface dynamics in the protection brought by surfactants is discussed based on comparison of protective efficiencies with dynamic surface tension properties (measured by the maximum bubble pressure method). Our work indicates that better protection is conferred by surfactants showing the faster interfacial dynamics, which corresponds also to conditions of faster lowering of the interfacial energy at short time scale.
When ligands and receptors are both attached on surfaces, because of the restriction of configurational freedom, their recognition kinetics may be substantially reduced as compared with freely diffusing species. In nature, this reduction may influence the efficiency of the capture and adhesion of circulating cells. Here we show that similar consequences are observed for colloids grafted with biomolecules that are used as probes for diagnostics. We exploit Brownian magnetic colloids that self-assemble into linear chains to show also that the resulting one-dimensional confinement considerably accelerates the recognition rate between grafted receptors and their ligands. We propose that because confinement significantly augments the colliding frequency, it also causes a large increase in the attempt frequency of the recognition. This work gives the basis of a rapid, homogeneous, and highly sensitive bioanalysis method.association rate ͉ bioassay ͉ magnetic colloids ͉ specific adhesion D iagnostics techniques are generally based on building a specific immunocomplex structure in which the antigen to be detected is recognized by two antibodies (1-8). Early immunochemistry was based on precipitation of large complexes made of antibodies and antigens. Following the same track, the use of Brownian particles significantly improved the detection sensitivity, because of the increase in scattered light when aggregation between grafted colloids takes place. As the most simple but very generic example, let us consider an antigen Ag having two different epitopes for two antibodies, A and B. To reveal the presence of such antigen, particles grafted with A and B antibodies are mixed with the sample to be analyzed. The formation of small clusters is then expected, at a rate depending on many factors. Change in light scattering due to the presence of these small clusters will reveal the existence of sandwich-like structures: A-Ag-B (latex agglutination immunoassay). These homogeneous assays, as opposed to heterogeneous assays in which washing steps are necessary before detection, are today by far the most simple and straightforward assays. They were introduced Ͼ40 years ago (2), and today several hundred different tests based on this principle can be found on the market, mainly for infectious disease detection and protein quantification, as long as the antigen concentration to be detected is տ1 nmol͞liter (1). Meanwhile, many diagnostic assays require sensitivity in the picomolar range. They are presently performed by using a heterogeneous approach (such as ELISA) (1). For that range of concentration, if we were still to consider the same homogeneous approach, it would in principle take too long. Indeed, decreasing the number of antigens to be detected implies also decreasing the number of particles in solution, † and thus the particles' colliding frequency. Therefore, at these very low antigen concentrations, the encounter frequency between species becomes a critical issue to consider.In this work, we demonstrate conditions that ac...
Superparamagnetic particles offer a new way to probe the kinetics of adhesive processes. Two different scenarios of physical adhesion are studied. The thermal activation of van der Waals adhesion is well described by an Arrhenius model. In contrast, it is necessary to go beyond the Arrhenius description to understand the thermal activation of bridging between colloidal particles by a polymer at equilibrium adsorbance. We show that polymer bridging requires some removal of adsorbed polymer and is strongly influenced by the proximity of a glass transition within the adsorbed polymer.
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