Eva Bittrich

Protein adsorption on and swelling of polyelectrolyte brushes: A simultaneous ellipsometry-quartz crystal microbalance study

With a coupled spectroscopic ellipsometry-quartz crystal microbalance with dissipation (QCM-D) experimental setup, quantitative information can be obtained about the amount of buffer components (water molecules and ions) coupled to a poly(acrylic acid) (PAA) brush surface in swelling and protein adsorption processes. PAA Guiselin brushes with more than one anchoring point per single polymer chain were prepared. For the swollen brushes a high amount of buffer was found to be coupled to the brush-solution interface in addition to the content of buffer inside the brush layer. Upon adsorption of bovine serum albumin the further incorporation of buffer molecules into the protein-brush layer was monitored at overall electrostatic attractive conditions [below the protein isolectric poimt (IEP)] and electrostatic repulsive conditions (above the protein IEP), and the shear viscosity of the combined polymer-protein layer was evaluated from QCM-D data. For adsorption at the “wrong side” of the IEP an incorporation of excess buffer molecules was observed, indicating an adjustment of charges in the combined polymer-protein layer. Desorption of protein at pH 7.6 led to a very high stretching of the polymer-protein layer with additional incorporation of high amounts of buffer, reflecting the increase of negative charges on the protein molecules at this elevated pH.

In Situ Infrared Ellipsometry for Protein Adsorption Studies on Ultrathin Smart Polymer Brushes in Aqueous Environment

The protein-adsorbing and -repelling properties of various smart nanometer-thin polymer brushes containing poly(N-isopropylacrylamide) and poly(acrylic acid) with high potential for biosensing and biomedical applications are studied by in situ infrared-spectroscopic ellipsometry (IRSE). IRSE is a highly sensitive nondestructive technique that allows protein adsorption on polymer brushes to be investigated in an aqueous environment as external stimuli, such as temperature and pH, are varied. These changes are relevant to conditions for regulation of protein adsorption and desorption for biotechnology, biocatalysis, and bioanalytical applications. Here brushes are used as model surfaces for controlling protein adsorption of human serum albumin and human fibrinogen. The important finding of this work is that IRSE in the in situ experiments in protein solutions can distinguish between contributions of polymer brushes and proteins. The vibrational bands of the polymers provide insights into the hydration state of the brushes, whereas the protein-specific amide bands are related to changes of the protein secondary structure.

Adsorption of enzymes to stimuli-responsive polymer brushes: Influence of brush conformation on adsorbed amount and biocatalytic activity.

Polyelectrolyte brushes can be utilized to immobilize enzymes on macroscopic surfaces. This report investigates the influence of the pH value of the surrounding medium on the amount and the activity of enzymes adsorbed to poly(2-vinylpyridine) and poly(acrylic acid) brushes, as well as the creation of thermoresponsive biocatalytically active coatings via the adsorption of enzymes onto a mixed brush consisting of a polyelectrolyte and temperature-sensitive poly(N-isopropylacryl amide). Spectroscopic ellipsometry and attenuated total reflection-Fourier transform infrared spectroscopy are used to monitor the adsorption process. Additionally, infrared spectra are evaluated in terms of the secondary structure of the enzymes. Glucose oxidase is used as a model enzyme, where the enzymatic activity is measured after different adsorption conditions. Poly(acrylic acid) brushes generally adsorb larger amounts of enzyme, while less glucose oxidase is found on poly(2-vinylpyridine), which however exhibits higher specific activity. This difference in activity could be attributed to a difference in secondary structure of the adsorbed enzyme. For glucose oxidase adsorbed to mixed brushes, switching of enzymatic activity between an active state at 20°C and a less active state at 40°C as compared to the free enzyme in solution is observed. However, this switching is strongly depending on pH in mixed brushes of poly(acrylic acid) and poly(N-isopropylacryl amide) due to interactions between the polymers.

Polymer Brushes, Hydrogels, Polyelectrolyte Multilayers: Stimuli-Responsivity and Control of Protein Adsorption

The research field of smart polymer surfaces benefits from non-invasive ellipsometric investigations, especially in-situ measurements, monitoring the swelling of polymer films and protein adsorption processes thereon at varying ambient conditions. With ellipsometry in the VIS-range layer thickness and refractive index of the polymer layers can be evaluated. Appropriate models for in-situ measurements will be discussed and results of the influence of solution parameters summarized. In-situ IR-ellipsometry provides information about changes in the vibration band structure for swelling and adsorption processes, where optical modelling in the IR-range yields complementary information about layer thicknesses and structural properties.

Bioinspired thermoresponsive nanoscaled coatings: Tailor-made polymer brushes with bioconjugated arginine-glycine-aspartic acid-peptides

The development of bioengineered surface coatings with stimuli-responsive properties is beneficial for a number of biomedical applications. Environmentally responsive and switchable polymer brush systems have a great potential to create such smart biointerfaces. This study focuses on the bioconjugation of cell-instructive peptides, containing the arginine-glycine-aspartic acid tripeptide sequence (RGD motif), onto well-defined polymer brush films. Herein, the highly tailored end-grafted homo polymer brushes are either composed of the polyelectrolyte poly(acrylic) acid (PAA), providing the reactive carboxyl functionalities, or of the temperature-responsive poly(N-isopropylacrylamide) (PNIPAAm). Of particular interest is the preparation of grafted-to binary brushes using both polymers and their subsequent conversion to RGD-biofunctionalized PNIPAAm-PAA binary brushes by a carbodiimide conjugation method. The bioconjugation process of two linear RGD-peptides Gly-Arg-Gly-Asp-Ser and Gly-Arg-Gly-Asp-Ser-Pro-Lys and one cyclic RGD-peptide cyclo(Arg-Gly-Asp-D-Tyr-Lys) is comparatively investigated by complementary analysis methods. Both techniques, in situ attenuated total reflectance Fourier transform infrared spectroscopy measurements and the in situ spectroscopic ellipsometric analysis, describe changes of the brush surface properties due to biofunctionalization. Besides, the bound RGD-peptide amount is quantitatively evaluated by ellipsometry in comparison to high performance liquid chromatography analysis data. Additionally, molecular dynamic simulations of the RGD-peptides themselves allow a better understanding of the bioconjugation process depending on the peptide properties. The significant influence on the bioconjugation result can be derived, on the one hand, of the polymer brush composition, especially from the PNIPAAm content, and, on the other hand, of the peptide dimension and its reactivity.

In Situ Monitoring of Linear RGD-Peptide Bioconjugation with Nanoscale Polymer Brushes

Bioinspired materials mimicking the native extracellular matrix environment are promising for biotechnological applications. Particularly, modular biosurface engineering based on the functionalization of stimuli-responsive polymer brushes with peptide sequences can be used for the development of smart surfaces with biomimetic cues. The key aspect of this study is the in situ monitoring and analytical verification of the biofunctionalization process on the basis of three complementary analytical techniques. In situ spectroscopic ellipsometry was used to quantify the amount of chemisorbed GRGDS at both the homopolymer poly(acrylic acid) (PAA) brush and the binary poly(N-isopropylacrylamide) (PNIPAAm)–PAA brushes, which was finally confirmed by an acidic hydrolysis combined with a subsequent reverse-phase high-performance liquid chromatography analysis. In situ attenuated total reflection-Fourier transform infrared spectroscopy provided a step-by-step detection of the biofunctionalization process so that an optimized protocol for the bioconjugation of GRGDS could be identified. The optimized protocol was used to create a temperature-responsive binary brush with a high amount of chemisorbed GRGDS, which is a promising candidate for the temperature-sensitive control of GRGDS presentation in further cell-instructive studies.

Salt Sensitivity of the Thermoresponsive Behavior of PNIPAAm Brushes

We report investigations on the salt sensitivity of the thermoresponsive behavior of PNIPAAm brushes applying the quartz crystal microbalance coupled with spectroscopic ellipsometry technique. This approach enables a detailed study of the optical and mechanical behavior of the polymer coatings. Additional conclusions can be drawn from the difference between both techniques due to a difference in the contrast mechanism of both methods. A linear shift of the phase-transition temperature to lower temperatures with the addition of sodium chloride was found, similar to the behavior of free polymer chains in solution. The thermal hysteresis was found to be decreased by the addition of sodium chloride to the solution, hinting to the interaction of the ions with the amide groups of the polymer, whereby the formation of hydrogen bonds is hindered. The results of this study are of relevance to the application of PNIPAAm brushes in biological fluids and demonstrate the additional potential of the ion sensitivity besides the better known thermosensitivity.

Interactions of bioactive molecules with thin dendritic glycopolymer layers

The authors report on highly swellable, stable layers of spherical dendritic glycopolymers, composed of hyperbranched poly(ethylene imine) (PEI) as core and two different maltose shells (A = dense shell and B = open shell). These glycopolymers are cross-linked and attached with poly(ethylene-alt-maleic anhydride) (PEMA) or citric acid on SiOx substrates. The swelling and adsorption of biomolecules were analyzed by spectroscopic ellipsometry and quartz crystal microbalance with dissipation. The swelling degree and complexation with the drug molecule adenosine triphosphate (ATP) were found to be up to 10 times higher for dendritic glycopolymer layers cross-linked with PEMA than for layers cross-linked with citric acid. ATP complexation by electrostatic interaction with the PEI cores was confirmed by x-ray photoelectron spectroscopy analysis. Complexation led to partial collapsing, stiffening, and increase of polymer layer viscosity of the PEMA cross-linked layers. From modeling of ellipsometric data, it was deduced that ATP complexation preferably takes place at the polymer layer-solution interface. The size effect of the adsorbates was investigated by comparing ATP complexation with the adsorption of larger vitamin B12 and human serum albumin (HSA) protein. PEI-Mal A cross-linked with PEMA was found to be resistant toward B12 and HSA adsorption due to the diffusion barrier of the polymer layer. Thus, the authors present potentially biocompatible polymer surfaces for drug loading and their surface supported release.