We consider the adsorption of bovine serum albumin (BSA) on spherical polyelectrolyte brushes (SPB). The SPB consist of asolid 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 weil 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 elass of colloidal carrier partieIes whose interaction with proteins can be tuned in a well-defined fashion.

We review recent experiments on the interaction of proteins with anionic polyelectrolytes in aqueous solution. Data from the literature demonstrate that proteins can form soluble complexes with linear polyelectrolytes even on the "wrong side" of the isoelectric point, that is, for pH values above the isoelectric point of the proteins under which the polyelectrolytes and the proteins are like-charged. All data published so far demonstrate that this type of adsorption becomes weaker with increasing ionic strength. A much stronger interaction is found if the polyelectrolyte chains are grafted onto solid surfaces to form polyelectrolyte brushes. Here it has been shown that spherical polyelectrolyte brushes consisting of a core of ca. 100 nm diameter and long attached polyelectrolyte chains strongly adsorb proteins at low ionic strength ("polyelectrolyte-mediated protein adsorption"; PMPA). Virtually no adsorption takes place onto the spherical polyelectrolyte brushes at high ionic strength. A critical comparison of data obtained on free polyelectrolytes and on polyelectrolyte brushes shows that both phenomena can be traced back to patches of positive charge on the surface of the proteins. Moreover, we discuss the driving force of the PMPA-process in terms of the Donnan pressure inside the brush layer. Here we find a good correlation which demonstrates that release of counterions during the process of adsorption is the main driving force.

We use small-angle x-ray scattering (SAXS) as a tool to study the binding of proteins to spherical polyelectrolyte brushes (SPB) in situ. The SPB consists of a solid core of approximately 100 nm diam onto which long polyelectrolyte chains [poly(styrene sulfonic acid, PSS) and poly(acrylic acid, PAA)] have been densely grafted. The proteins used in this investigation, Bovine Serum Albumine (BSA) and Bovine Pancreatic Ribonuclease A (RNase A), adsorb strongly to these SPB if the ionic strength is low despite their negative charge. Virtually no adsorption takes place at high ionic strength. SAXS demonstrates that both proteins are distributed within the brush. The findings reported here give further evidence that the strong adsorption of proteins to SPB is due to the "counterions release forces": The patches of positive charge on the surface of the proteins become multivalent counterions of the polyelectrolyte chains. Thus, a concomitant number of co- and counterions is thereby released and the entropy of the entire system is increased. The repulsive Coulombic interaction as well as the steric repulsion between the proteins and the brush layer are counterbalanced by this effect. The data discussed here demonstrate that the adsorption of proteins in SPB presents a new principle for the immobilization of proteins.

Summary: We report the synthesis of star‐shaped poly(acrylic acid) (PAA), with 5, 8, and 21 arms, by atom transfer radical polymerization of tert‐butyl acrylate. We employ the core‐first approach using glucose‐, saccharose‐ and cyclodextrin‐based initiators. Subsequent acidic treatment of poly(tert‐butyl acrylate) (PtBA) leads to star‐shaped poly(acrylic acid) (PAA). Alkaline cleavage of the arms enabled us to determine the initiation site efficiency. The PAA stars and arms were esterified to poly(methyl acrylate) (PMA). Molecular weight determination by means of GPC/viscosity, MALDI‐TOF MS and NMR end‐group determination showed that the initiation site efficiency is close to unity. Results from potentiometric titration of PAA arms and stars show that the apparent pKa values increase with increasing arm number, which is a direct result of increasing segment density. Osmometry measurements of aqueous solutions of the PAA stars result in osmotic coefficients between 0.05 and 0.38, indicating that most of the counterions are confined within the star. The confinement increases with arm number.

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.

The adsorption of bovine serum albumin (BSA), bovine beta-lactoglobulin, and bovine pancreatic ribonuclease A onto spherical polyelectrolyte brushes (SPB) is reported. The SPB consist of narrowly distributed poly(styrene) core particles (diameter approximately 100 nm) onto which linear chains of anionic polyelectrolytes are grafted. The polyelectrolyte shell consists of either the weak polyelectrolyte poly(acrylic acid) or the strong polyacid poly(styrenesulfonate). The SPB particles are dispersed in H(2)O at room temperature. The secondary structure of the proteins was investigated by Fourier transform infrared spectroscopy in transmission mode before and during adsorption to these colloidal brushes. The alpha-helix and beta-sheet content of the proteins was nearly fully retained in the adsorbed state for all systems. Only in the case of BSA interacting with poly(styrenesulfonic) brushes could a slight loss of alpha-helix structure be observed. As the interaction of SPB and proteins can be controlled by the ionic strength in the buffer, additional experiments were performed to release the adsorbed protein. The amount of released protein was quantified and was found to be strongly dependent on the kind of protein and brush used. The secondary structure of the released proteins could be analyzed as well. An almost full preservation of secondary structure was found. This demonstrates that SPB are well-suited to immobilize proteins. The SPB can be charged and decharged under retention of the secondary structure of the biomolecules.

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We investigate the conformation of long polyelectrolyte chains attached to colloidal latex particles by cryogenic transmission electron microscopy (cryo-TEM). The dense grafting of the polyelectrolyte chains ("polyelectrolyte brush") leads to a confinement of the counterions and a concomitantly high osmotic pressure within the polyelectrolyte layer attached to the core particles. Cryo-TEM has provided first model-independent direct proof for the strong stretching of the polyelectrolyte chains by direct visualization. If salt is added, cryo-TEM clearly shows how chains collapse because of the strong screening of the electrostatic interaction. Moreover, the analysis of interacting particles by cryo-TEM shows that the polyelectrolyte chains retract at close contact. Hence, we demonstrate how cryo-TEM can be used to analyze directly the spatial structure of polyelectrolyte brushes in situ.