Alexander Wittemann

Adsorption of proteins on spherical polyelectrolyte brushes in aqueous solution

We consider the adsorption of bovine serum albumin (BSA) on spherical polyelectrolyte brushes (SPB). The SPB consist of a solid polystyrene core of 100 nm diameter onto which linear polyelectrolyte chains [poly(acrylic acid), (PAA)] are grafted. The adsorption of BSA is studied at a pH of 6.1 at different concentrations of added salt and buffer. We observe strong adsorption of BSA onto the SPB despite the effect that the particles as well as the dissolved BSA are charged negatively. The adsorption of BSA is strongest at low salt concentration and decreases drastically with increasing amounts of added salt. Virtually no adsorption takes place at salt concentration of 0.1 M. Moreover, the adsorbed protein can be washed out again by raising the ionic strength from low to high values. A major driving force for the adsorption is located at a lower pH within the brush at low ionic strength. Thus, the isoelectric point of the protein may be approached or even reached. In this case strong interaction between the SPB and the protein results. Moreover, the negative charge of the polyelectrolyte interacts with the patches of positive charges on the protein. In this way the protein becomes a multivalent counterion within the brush and monovalent counterions will be released. All results demonstrate that the SPB present a new class of colloidal carrier particles whose interaction with proteins can be tuned in a well-defined fashion.

Adsorption of β-Lactoglobulin on Spherical Polyelectrolyte Brushes: Direct Proof of Counterion Release by Isothermal Titration Calorimetry

The thermodynamics and the driving forces of the adsorption of beta-lactoglobulin on spherical polyelectrolyte brushes (SPB) are investigated by isothermal titration calorimetry (ITC). The SPB consist of a polystyrene core onto which long chains of poly(styrene sulfonate) are grafted. Adsorption isotherms are obtained from measurements by ITC. The analysis by ITC shows clearly that the adsorption process is solely driven by entropy while DeltaH > 0. This finding is in accordance with the proposed mechanism of counterion release: Patches of positive charges on the surface of the proteins become multivalent counterions of the polyelectrolyte chains, thereby releasing the counterions of the protein and the polyelectrolyte. A simple statistical-mechanical model fully corroborates the proposed mechanism. The present analysis shows clearly the fundamental importance of counterion release for protein adsorption on charged interfaces and charged polymeric layers.

Organized Plasmonic Clusters with High Coordination Number and Extraordinary Enhancement in Surface- Enhanced Raman Scattering (SERS)**

Noble metal nanoparticles exhibit optical excitations known as surface plasmons that produce large enhancement of the local light intensity under external illumination, particularly when the nanoparticles are arranged in closely spaced configurations.1 The interparticle gap distance2 plays a critical role in the generation of hotspots with high electromagnetic fields, and thus such assembled nanoparticles find application to ultrasensitive detection, for example through surface-enhanced Raman scattering3 (SERS) and nonlinear optics, among other feats.4 Controlled assembly using colloidal chemistry is an emerging and promising field for high-yield production of metal nanoparticle clusters with small interparticle gaps.5 However, most of the reported methods rely on the use of nucleic acids or other organic molecules as linking elements,6 which yield long separation distances and thus weak plasmon coupling. Additionally, only simple clusters, such as dimers and trimers, have been efficiently synthesized. Herein, we report the controlled assembly of gold nanospheres into well-defined nanoparticle clusters with large coordination numbers (up to 7) and high symmetry. We further demonstrate ultrasensitive direct and indirect SERS sensing, thus corroborating the outstanding optical performance of these clusters with robust enhancement factors that are over three orders of magnitude higher than those of single particles.

Enhanced Activity of Enzymes Immobilized in Thermoresponsive Core−Shell Microgels

We present a quantitative study of the catalytic activity of beta-d-glucosidase from almonds adsorbed on thermosensitive microgels. The core-shell particles used as a carrier system consist of a solid polystyrene core onto which a poly(N-isopropylacrylamide) (PNiPA) network is grafted. In the swollen state of this microgel, i.e., below the critical solution temperature (LCST) of PNiPA, high amounts of enzyme can be immobilized into the PNiPA network without loss of colloidal stability. The enzymatic activity of beta-d-glucosidase in its native form and in the adsorbed state was analyzed in terms of Michaelis-Menten kinetics. Moreover, the dependence of the enzymatic activity on temperature was investigated. We demonstrate that the enzymatic activity of beta-d-glucosidase adsorbed on such a core-shell microgel is increased by a factor of more than three compared to its activity in solution. This is in marked contrast to other carrier systems that usually lead to a strong decrease of enzymatic activity. Both the high loading capacity of the carrier observed and the increase of the catalytic activity of immobilized beta-d-glucosidase are traced back to the formation of strong interactions between the enzyme and microgel. Studies by Fourier-transform infrared (FT-IR) spectroscopy identify the formation of hydrogen bonds as driving forces for the adsorption. Hydrogen bonding may also be the reason for the enhanced activity.

3D Brownian diffusion of submicron-sized particle clusters.

We report on the translation and rotation of particle clusters made through the combination of spherical building blocks. These clusters present ideal model systems to study the motion of objects with complex shape. Since they could be separated into fractions of well-defined configurations on a sufficient scale and because their overall dimensions were below 300 nm, the translational and rotational diffusion coefficients of particle doublets, triplets, and tetrahedrons could be determined by a combination of polarized dynamic light scattering (DLS) and depolarized dynamic light scattering (DDLS). The use of colloidal clusters for DDLS experiments overcomes the limitation of earlier experiments on the diffusion of complex objects near surfaces because the true 3D diffusion can be studied. When the exact geometry of the complex assemblies is known, different hydrodynamic models for calculating the diffusion coefficients for objects with complex shapes could be applied. Because hydrodynamic friction must be restricted to the cluster surface, the so-called shell model, in which the surface is represented as a shell of small friction elements, was most suitable to describe the dynamics. A quantitative comparison of the predictions from theoretical modeling with the results obtained by DDLS showed an excellent agreement between experiment and theory.

Salt-induced protein resistance of polyelectrolyte brushes studied using fluorescence correlation spectroscopy and neutron reflectometry

We used two-photon excitation fluorescence correlation spectroscopy (FCS) and neutron reflectometry to study in situ the effect of salt concentration on the degree of protein binding to polyelectrolyte brushes. The binding of bovine serum albumin (BSA) to poly(acrylic acid) (PAA) brushes was characterized at neutral pH values where both the protein and the brushes carry a negative charge. Spherical PAA brush particles were used in the FCS experiments, whereas a planar PAA brush served as protein substrate in the neutron reflectometry experiments. It has been found that BSA binds strongly to both the spherical and the planar PAA brushes under electrostatic repulsion at low ionic strength. The BSA volume fraction profile, as determined from the neutron reflectivities, indicates a deep penetration of the BSA molecules into the PAA brush. However, the analysis of the FCS data reveals that the protein affinity of the spherical PAA brush particles decreases drastically when increasing the concentration of sodium chloride to a few 100 mM. This observation is in line with the measured neutron reflectivities of the planar PAA brush. The reflectivity curve obtained in the absence of protein is virtually overlapping with that measured when the PAA brush is in contact with a BSA solution but containing 500 mM sodium chloride which suggests protein resistance of the planar PAA brush at this elevated salt concentration. The results of this study provide evidence for a new kind of protein-resistant interfaces. Whereas protein binding to the PAA brush is likely to be dominated by the release of counterions, this driving force vanishes as the ionic strength of the solution is raised and protein molecules are repelled from the interface by steric interactions. In a general view, the “switching” of the protein affinity of a PAA brush by varying the ionic strength of the protein solution over a relatively small range may appear to be useful for biotechnological applications.

Quantitative analysis of polymer colloids by cryo-transmission electron microscopy.

The structure of colloidal latex particles in dilute suspension at room temperature is investigated by cryogenic transmission electron microscopy (cryo-TEM). Two types of particles are analyzed: (i) core particles made of polystyrene with a thin layer of poly(N-isopropylacrylamide) (PNIPAM) and (ii) core-shell particles consisting of core particles onto which a network of cross-linked PNIPAM is affixed. Both systems are also studied by small-angle X-ray scattering (SAXS). The radial density profile of both types of particles have been derived from the cryo-TEM micrographs by image processing and compared to the results obtained by SAXS. Full agreement is found for the core particles. There is a discrepancy between the two methods in case of the core-shell particles. The discrepancy is due to the buckling of the network affixed to the surface. The buckling is clearly visible in the cryo-TEM pictures. The overall dimensions derived from cryo-TEM agree well with the hydrodynamic radius of the particles. The comparison of these data with the analysis by SAXS shows that SAXS is only sensitive to the average radial structure as expected. All data show that cryo-TEM micrographs can be evaluated to yield quantitative information about the structure of colloidal particles.

Nanoparticle assembly by confinement in wrinkles : experiment and simulations

We created hierarchically ordered structures of nanoparticles on smooth planar hydrophilic substrates by drying colloidal dispersions in confinement under macroscopic stamps with microscopically wrinkled surfaces. Experiments were carried out with model nanoparticle suspensions that possess high colloidal stability and monodispersity. The structures ranged from single parallel lines of particles to arrays of dense prismatic ridges. The type of observed structure could be controlled by the particle concentration in the initial dispersion. Confinement between two crossed stamps led to interconnected meshes of particles. The precise morphology could be predicted in all cases by Monte Carlo computer simulations of confined hard spheres. Our findings open up possibilities for versatile nanoparticle assembly on surfaces.

Preparation of submicrometer-sized clusters from polymer spheres using ultrasonication

The fabrication of small assemblies of spherical colloidal particles is presented. Basic principles of the preparation of miniemulsions were combined with the concept originally described by Pine and co-workers for the preparation of clusters from microspheres. The application of ultrasonic emulsification limits the size distribution of the emulsion droplets and thus the statistical distribution of the number of particles per droplet. The small droplet sizes together with the use of building blocks in the submicrometer range leads to large yields of well-defined small clusters with diameters below 300 nm. Such clusters might serve to build novel promising materials that demand high complexity or special symmetries.

Glycopolymer-grafted polystyrene nanospheres

The synthesis and characterization of spherical sugar-containing polymer brushes consisting of PS cores onto which chains of sugar-containing polymers have been grafted via two different techniques are described. Photopolymerization in aqueous dispersion using the functional monomer MAGlc and crosslinked or non-crosslinked PS particles covered with a thin layer of photo-initiator yielded homogeneous glycopolymer brushes attached to spherical PS cores. As an alternative, ATRP was used to graft poly-(N-acetylglucosamine) arms from crosslinked PS cores. Deprotection of the grafted brushes led to water-soluble particles that act as carriers for catalytically active gold nanoparticles. These glycopolymer chains show a high affinity to adsorb WGA whereas no binding to BSA or PNA could be detected.