Ralf Zimmermann

The role of the interplay between polymer architecture and bacterial surface properties on the microbial adhesion to polyoxazoline-based ultrathin films

Surface platforms were engineered from poly(L-lysine)-graft-poly(2-methyl-2-oxazoline) (PLL-g-PMOXA) copolymers to study the mechanisms involved in the non-specific adhesion of Escherichia coli (E. coli) bacteria. Copolymers with three different grafting densities α (PMOXA chains/Lysine residue of 0.09, 0.33 and 0.56) were synthesized and assembled on niobia (Nb₂O₅) surfaces. PLL-modified and bare niobia surfaces served as controls. To evaluate the impact of fimbriae expression on the bacterial adhesion, the surfaces were exposed to genetically engineered E. coli strains either lacking, or constitutively expressing type 1 fimbriae. The bacterial adhesion was strongly influenced by the presence of bacterial fimbriae. Non-fimbriated bacteria behaved like hard, charged particles whose adhesion was dependent on surface charge and ionic strength of the media. In contrast, bacteria expressing type 1 fimbriae adhered to the substrates independent of surface charge and ionic strength, and adhesion was mediated by non-specific van der Waals and hydrophobic interactions of the proteins at the fimbrial tip. Adsorbed polymer mass, average surface density of the PMOXA chains, and thickness of the copolymer films were quantified by optical waveguide lightmode spectroscopy (OWLS) and variable-angle spectroscopic ellipsometry (VASE), whereas the lateral homogeneity was probed by time-of-flight secondary ion mass spectrometry (ToF-SIMS). Streaming current measurements provided information on the charge formation of the polymer-coated and the bare niobia surfaces. The adhesion of both bacterial strains could be efficiently inhibited by the copolymer film only with a grafting density of 0.33 characterized by the highest PMOXA chain surface density and a surface potential close to zero.

Electrokinetic surface characterization of biomedical polymers — a survey

Abstract The application of streaming potential measurements in support of the development of blood compatible polymers is reviewed. Current knowledge about the relevance of interfacial charge of materials with respect to thrombogenicity of and coagulation activation by medical devices is discussed. Potentialities of streaming potential measurements for the characterization of biomedical polymers comprise the distinction between interfacial charge formation processes, the derivation of acid–base characteristics of surface functions, and the estimation of the relative contribution of known molecular surface sites to the interfacial charge density. Frequently, polymer surfaces consist of different types of surface sites. Beyond sites with Bronsted-acidic or -basic character the preferential adsorption of electrolyte ions onto indifferent surface sites may substantially contribute to the interfacial charge. Examples are given for the detection of acidic and basic properties of hemodialysis membranes of different shape made of poly(sulfone)/poly(vinylpyrrolidone) and cellulose derivates. For partially amine-modified cellulose flat membranes the percentage of basic surface sites is derived from the shift of the isoelectric point. Further, the use of streaming potential measurements is demonstrated in the optimization of surface modifications of cardiovascular implant materials by low pressure plasma treatments. The introduction of basic groups into the poly(tetrafluoroethylene) implant surfaces by ammonia plasma treatments manifests itself in the shift of the isoelectric point. A correlation between hydrophobicity and charge formation of the polymer–solution interface by preferential ion adsorption is confirmed for polymers without dissociating functions. This is based on the comparison of water contact angles for a series of poly(styrene) derivates of different hydrophobicity and the maximum zeta potential of these polymer films in potassium hydroxide solutions. Advanced applications of streaming potential measurements for the characterization of biomedical polymers are comprise the in situ characterization of protein adsorption processes onto polymer solids. This approach is demonstrated for the adsorption of the blood plasma proteins albumin and fibrinogen onto a fluorocarbon polymer substrate. The potentialities of a recently developed microslit electrokinetic setup are illustrated which permits to study simultaneously zeta potential and surface conductivity of flat polymer–liquid interfaces. Data gained by the microslit electrokinetic setup reveal the dramatic increase of the surface conductivity of a fluorocarbon polymer–solution interface caused by fibrinogen adsorption.

Aligned fibrillar collagen matrices obtained by shear flow deposition

Here we present a new technique to generate surface-bound collagen I fibril matrices with differing structural characteristics. Aligned collagen fibrils were deposited on planar substrates from collagen solutions streaming through a microfluidic channel system. Collagen solution concentration, degree of gelation, shear rate and pre-coating of the substrate were demonstrated to determine the orientation and density of the immobilized fibrils. The obtained matrices were imaged using confocal reflection microscopy and atomic force microscopy. Image analysis techniques were applied to evaluate collagen fibril orientation and coverage. As expected, the degree of collagen fibril orientation increased with increasing flow rates of the solution while the matrix density increased at higher collagen solution concentrations and on hydrophobic polymer pre-coatings. Additionally, length of the immobilized collagen fibrils increased with increasing solution concentration and gelation time.

An attempt to explain bimodal behaviour of the sapphire c-plane electrolyte interface

A tentative picture for the charging of the sapphire basal plane in dilute electrolyte solutions allows reconciliation of the available experimental observations within a dual charging model. It includes the MUltiSIte Complexation (MUSIC) model and auto-protolysis of interfacial water. The semi-empirical MUSIC model predicts protonation and deprotonation constants of individual surface functional groups based on crystal structure and bond-valence principles: on the ideal sapphire c-plane only doubly co-ordinated hydroxyl groups exist which cause quasi zero surface potential (defined as the potential in the plane of the surface hydroxyl groups) from pH 5 to 7 and rather weak charging beyond (compared to typical oxide behaviour). MUSIC predictions concur strikingly with recently published sum frequency data for the pH dependence of the so-called ice-like water band (interfacial water) and contact angle titrations. Zeta potential as well as second harmonic generation data reveal a sharp IEP of around 4 and a negative surface charge at the pristine point of zero charge predicted by the MUSIC model. New zeta-potential data corroborate (i) the low IEP and its insensitivity to salt concentration and (ii) the second harmonic results. We thus establish two groups of conflicting results arising from different techniques. A conventional model of the mineral electrolyte interface such as the MUSIC model is at odds with the negative zeta potentials in the pH range 5 to 7. Therefore an additional charging mechanism is invoked to explain all the observations. Enhanced auto-protolysis of interfacial water is the most probable candidate for this additional mechanism, in agreement with net water orientation observed with sum frequency generation and second harmonic generation. Our phenomenological explanation is further corroborated by the similarity of the zeta potential vs. pH curves of the c-plane with those of hydrophobic surfaces. Additional support comes from infrared spectroscopic data on thin water films on sapphire c-plane samples. Most stunningly, theoretical calculations on basal planes of this kind suggest a 2D water bilayer that makes such surfaces hydrophobic towards further adsorption of water. The proposed dual charging mode approach comprises the MUSIC model for protonation/deprotonation of the surface aluminols affecting the surface potential and the currently advocated enhanced auto-protolysis picture for hydrophobic surfaces controlling the zeta-potential and can explain the available information in a qualitative way. The respective contributions from the two components of this dual charging mechanism may be different for different single crystal cuts of alumina. Thus interplay between protonation/deprotonation of surface functional groups and auto-protolysis of interfacial water will cause the observed zeta potentials and isoelectric points. Repercussions of one mechanism on the other will result in the most favourable interfacial water structure, which can be followed by non-linear optic techniques like sum frequency generation.

Streaming potential and streaming current measurements at planar solid/liquid interfaces for simultaneous determination of zeta potential and surface conductivity

Abstract Strongly encouraged and supported by Stanislav S. Dukhin the authors of this article recently designed, built and tested a new device for the simultaneous determination of zeta potential and surface conductivity from streaming potential and streaming current measurements across rectangular slit channels formed between two planar samples. In this Microslit Electrokinetic Set-up (MES) the planar samples are adjusted in parallel to form a channel of variable height which can become as narrow as about 1 μm. Due to this key feature of the device electrokinetic measurements can be performed at conditions where surface conductivity can be neglected and at conditions where surface conductivity provides a substantial part of the total channel conductivity. Utilizing the novel set-up, zeta potential and surface conductivity data can be obtained for a wide variety of materials which can be prepared as thin films on top of planar, macroscopic glass carriers. In order to demonstrate the potentialities of the advanced experimental technique of electrokinetic surface characterization we discuss three examples reflecting different levels of complexity of the analysed solid/liquid interface: (1) The charge formation at unpolar polymers without dissociating surface functions is studied referring to an inert, plasma-deposited fluoropolymer layer (PDFP) in simple electrolyte solutions. An extended evaluation of the experimental data of zeta potential and surface conductivity is given; (2) Further, grafted polypetide chains bearing dissociating side groups (polyglutamic acid and polylysine) were characterized with regard to the pH-depended variation of zeta potential and surface conductivity to provide new insights into the interrelation of charge density and conformation; (3) Finally, adsorbed fibrinogen on top of plasma-deposited fluoropolymer was studied by zeta potential and surface conductivity measurements as an example for highly hydrated macromolecular adsorption layers.

On the applicability of the Brinkman equation in soft surface electrokinetics

The Stokes equation is commonly used within the field of electrokinetics of hard impermeable surfaces while the Brinkman equation is adopted for tackling hydrodynamics in the framework of soft (permeable) surface electrokinetics (SSE). The latter was initially proposed for modeling the hydrodynamics in so-called hybrid systems that consist of a porous medium and an adjacent fluid phase basically because the conventional Darcy law or Debye and Bueche model initially proposed for that purpose failed to provide the required velocity and shear stress-continuity conditions at the porous media-fluid interface. However, even though the physical background of the Brinkman equation and its boundary conditions have been discussed when applied to the hydrodynamics of hybrid systems, controversy still remains with respect to their applicability in the field of SSE. Indeed, recent experiments pointed out better agreement between shear flow into a regular array of rods oriented across the flow and the solution of the Brinkman equation for hybrid systems providing a stress-jump boundary condition is taken into account (M.F. Tachie et al., J. Fluid. Mech. 493 (2003) 319). As there is identity in the Brinkman model for hybrid systems and for SSE, the question arises whether the above discontinuity of viscous stress must be incorporated or not into SSE modeling. Recent determination of hydrodynamic penetration length lambda(o)(-1) of swollen and collapsed thermo-responsive films (J.F.L. Duval, R. Zimmermann, A.L. Cordeiro, N. Rein, C. Werner, Langmuir 25 (2009) 10691) suggests that there is no need for a cardinal revision of the Brinkman model, although further experimental investigations are required to support such a conclusion. With regard to these experiments, almost complete agreement between independent determination of lambda(o)(-1) by swelling experiments and its derivation according to Brinkman model was obtained.

Charging and structure of zwitterionic supported bilayer lipid membranes studied by streaming current measurements, fluorescence microscopy, and attenuated total reflection Fourier transform infrared spectroscopy.

The authors report on the characterization of the charge formation at supported bilayer lipid membranes (sBLMs) prepared from the zwitterionic lipid 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine on planar silicon dioxide substrates. The charging of the sBLMs was studied in KCl solutions of different ionic strengths between 0.1 and 10 mM by streaming current measurements. In addition, attenuated total reflection Fourier transform infrared spectroscopy and fluorescence microscopy were applied to determine the lipid concentration in the membrane and to study the influence of the harsh conditions (pH 9-2, shear forces) during the electrokinetic measurements on the membrane stability and the lipid diffusion coefficient. The sBLMs were found to be extremely stable. Isoelectric points of about 4 revealed that unsymmetrical adsorption of hydroxide and hydronium ions determined the charging of the outer leaflet of the membrane in the investigated pH range. The diffusion coefficients were found to be rather independent on the ionic strength at neutral and alkaline pH. However, significantly decreased lipid diffusion at pH<4 indicated a charge-induced transition of the fluidic bilayer into a gel/ordered-phase bilayer.

Interrelations between charging, structure and electrokinetics of nanometric polyelectrolyte films.

Streaming current, surface conductivity and swelling data of poly(acrylic acid) (PAA) and poly(ethylene imine) (PEI) thin films are analyzed on the basis of the theory for diffuse soft interfaces (J.F.L. Duval, R. Zimmermann, A. L. Cordeiro, N. Rein, C. Werner, Langmuir 25 (2009) 10691). Focus is put on ways to unravel the electroosmotic and migration contributions of the measured surface conductivity, which is crucial for appropriate electrokinetic analysis of films carrying high densities of dissociable groups. Results demonstrate that the osmotically-driven swelling of the PAA films with increasing pH is accompanied by an increase in diffuseness for the interphasial polymer segment density distribution. This heterogeneity is particularly marked at low ionic strength with a non-monotonous dependence of the streaming current on pH and the presence of a maximum at pH∼6.5. The analysis of the PEI films evidences heterogeneous swelling with lowering pH, i.e. upon protonation of the amine groups. The characteristic decay length in the interphasial PEI segment density distribution is found to be nearly independent of the pH, which is in line with the moderate swelling determined by ellipsometry. A critical discussion is given on the strengths and limitations of electrokinetics/surface conductivity for quantifying the coupled electrohydrodynamic and structural properties of moderately to highly swollen polyelectrolyte thin films.

Temperature dependent physicochemical properties of poly(N-isopropylacrylamide-co-N-(1-phenylethyl) acrylamide) thin films

The physicochemical properties of thermo-responsive polymer films are dynamically altered upon changes in environmental conditions. We report on the design and detailed characterization of a novel thermo-responsive polymer film with a temperature transition tuned to fit applications related to the control of marine biofouling. A copolymer consisting of poly(N-isopropylacrylamide) (PNIPAAm) and N-(1-phenylethyl) acrylamide (PEAAm) was synthesized and immobilized as a thin film onto Teflon AF surfaces using a low pressure argon plasma treatment. The temperature dependent physicochemical properties of the thermo-responsive film were thoroughly characterized and the impact of sea water on the film properties was investigated. The immobilized thermo-responsive film exhibits a reversible swelling/deswelling with temperature. Atomic force microscopy showed no morphological changes with varying temperature. Streaming current measurements performed above and below the transition temperature of the thermo-responsive hydrogel indicated that the charging of the polymer/aqueous solution interface is mainly determined by the preferential water ion adsorption at the Teflon AF surface. Inverse contact angles measured using captive air bubbles and analysed by axisymmetric drop shape analysis (ADSA) supported the intrinsic properties of the thermo-responsive film, as surface hydrophilicity decreased with increasing temperature. The advancing water contact angle decreased with increasing temperature, which may be explained by the different molecular mobility at different temperatures, allowing or hampering the re-orientation of hydrophobic segments at the solid–liquid and solid–fluid interfaces. These new films will allow investigations on the interaction of microorganisms with environmentally sensitive surfaces.

Permanent surface modification by electron-beam-induced grafting of hydrophilic polymers to PVDF membranes

Electron-beam-induced grafting of hydrophilic polymers was applied to modify PVDF membranes for biomedical applications. Grafting was performed by immersing the membrane in an aqueous solution of different hydrophilic polymers followed by electron-beam irradiation. The two polymer types are able to cross-link by recombination of adjacent radicals formed via the irradiation. Although the untreated membrane was already quite hydrophilic, the modification resulted in even lower water contact angles at the membrane surface indicating improved water wettability. The presence of different functional groups originating from the hydrophilic polymers was detected on the membrane surface by electrokinetic measurements. SEM investigations as well as porosimetry experiments showed that the grafted hydrophilic polymer layer is very thin; therefore, the membrane pore structure is not negatively affected. Soxhlet extraction revealed the stability of the modification for selected polymers: surface contact angles were comparable after extraction, and total organic carbon investigation of the extraction water revealed no significant loss of organic material. Investigated mechanical properties confirmed an increased stability due to cross-linking of the polymers. Undesired hemolysis was not detected with hemocompatibility tests, and coagulation was decreased with selected hydrophilic polymers. Because of the absence of any toxic material during surface modification and the high stability of the product, this method is believed to be suitable for the modification of membranes for medical applications, e.g. for improving the hemo- or biocompatibility.