Oswald Prucker

Enzyme containing redox polymer networks for biosensors or biofuel cells: a photochemical approach.

A photochemical approach to the generation of (microstructured) redox hydrogels with incorporated enzymes is presented and evaluated with respect to its potential in biosensor and biofuel cell applications. For this, poly(dimethylacrylamide) polymers containing both electroactive ferrocene moieties and photoreactive benzophenone groups are synthesized and deposited as thin films on electrode surfaces. Upon short irradiation with UV light, the polymer layer cross links and becomes firmly adhered to the glassy carbon electrodes. If glucose oxidase is mixed into the polymer solution prior to coating, then glucose-oxidizing electrodes with very high catalytic current responses are obtained. The influence of multivalent ions and proteins on the performance of the electrocatalytic films is studied. It is found that the interaction between bivalent HPO(4)(2-) and the oxidized redox moieties can shorten the lifetime of the redox electrodes significantly whereas the same electrodes are quite stable in the presence of monovalent ions and the reduced form of the mediator. Coating a thin, covalently attached poly(dimethylacrylamide) protective layer onto the redox polymer networks can greatly reduce the adsorption of proteins onto the surfaces and improve the long-term stability of the electrodes in physiological environments. Because the adsorption of proteins onto unprotected surfaces is one of the major causes of bioelectrode failure, this aspect is expected to contribute to the design of more biostable sensors and fuel cells.

Experimental investigation of the flow induced by artificial cilia

The fluid transport produced by rectangular shaped, magnetically actuated artificial cilia of 70 μm length and 20 μm width was determined by means of phase-locked Micro Particle Image Velocimetry (μPIV) measurements in a closed microfluidic chamber. The phase-averaged flow produced by the artificial cilia reached up to 130 μm s(-1) with an actuation cycle frequency of 10 Hz. Analysis of the measured flow data indicate that the present system is capable of achieving volume flow rates of V[combining dot above](cilia) = 14 ± 4 μl min(-1) in a micro channel of 0.5 × 5 mm(2) cross-sectional area when no back pressure is built up. This corresponds to an effective pressure gradient of 6 ± 1 Pa m(-1), which equals a pressure difference of 0.6 ± 0.1 mPa over a distance of 100 μm between two rows of cilia. These results were derived analytically from the measured velocity profile by treating the cilia as a thin boundary layer. While the cilia produce phase-averaged velocities of the order of O(10(2)μm s(-1)), time-resolved measurements showed that the flow field reverses two times during one actuation cycle inducing instantaneous velocities of up to approximately 2 mm s(-1). This shows that the flow field is dominated by fluid oscillations and flow rates are expected to increase if the beating motion of the cilia is further improved.

Simple One-Step Process for Immobilization of Biomolecules on Polymer Substrates Based on Surface-Attached Polymer Networks

For the miniaturization of biological assays, especially for the fabrication of microarrays, immobilization of biomolecules at the surfaces of the chips is the decisive factor. Accordingly, a variety of binding techniques have been developed over the years to immobilize DNA or proteins onto such substrates. Most of them require rather complex fabrication processes and sophisticated surface chemistry. Here, a comparatively simple immobilization technique is presented, which is based on the local generation of small spots of surface attached polymer networks. Immobilization is achieved in a one-step procedure: probe molecules are mixed with a photoactive copolymer in aqueous buffer, spotted onto a solid support, and cross-linked as well as bound to the substrate during brief flood exposure to UV light. The described procedure permits spatially confined surface functionalization and allows reliable binding of biological species to conventional substrates such as glass microscope slides as well as various types of plastic substrates with comparable performance. The latter also permits immobilization on structured, thermoformed substrates resulting in an all-plastic biochip platform, which is simple and cheap and seems to be promising for a variety of microdiagnostic applications.

Surface-attached PDMAA-GRGDSP hybrid polymer monolayers that promote the adhesion of living cells.

Peptide-polymer hybrid molecules are being introduced, where one part of the molecule (i.e., the peptide) promotes the adhesion of living cells, whereas the other part of the molecule (i.e., the synthetic polymer) is known to prevent cell adhesion. The hybrid copolymer, poly(dimethylacrylamide) (PDMAA)-glycine-arginine-glycine-aspartic acid-serine-proline (GRGDSP) was synthesized by first preparing an initiator-modified peptide and in a second step growing the PDMAA block directly off the peptide through atom transfer radical polymerization (ATRP). The PDMAA block length can be varied by adjusting appropriate polymerization conditions, thereby changing progressively the amount of the cell-repelling part of the molecule. The hybrid copolymer was further used to prepare surface-attached peptide-polymer monolayers at planar solid glass substrates through a photochemical immobilization process. By blending of the hybrid copolymer with PDMAA homopolymer (i.e., without peptide), the apparent peptide film concentration can be varied in a very simple manner. The adhesion of human skin fibroblast cells in serum-free medium was investigated as a function of the amount of peptide-polymer in the solution used for film preparation. Cells do not adhere to a pure PDMAA monolayer; however, already 0.02 wt % of peptide in the film is enough to induce cell adhesion, and 0.1 wt % promotes stress-fiber formation within adherent cells. Using lithographical means, chemically micropatterned peptide-polymer films were prepared that allow for a spatial control of the adhesion of living cells and thus they constitute a simple platform for the design of live-cell biochips.

Grafting of polymers to solid surfaces by using immobilized methacrylates

Abstract In this paper we present the results of studies that aim at a better understanding of the radical graft polymerizations for the formation of polymer monolayers at solid surfaces by copolymerization of monomers in solution with monomers immobilized at the surface of the substrate. Monolayers of 3-methacryloylpropyl trimethoxysilane (MPS) were deposited onto silicon wafers and polymer monolayers were attached to these surfaces by copolymerization of the immobilized double bonds with styrene using azobis isobutyronitrile (AIBN) as the initiator. In two separate sets of experiments the polymerization time and the initiator concentration were varied, while all other polymerization parameters were kept constant. It was found by varying the polymerization time that the formation of the polymer layer levels off at rather low monomer and initiator conversion, and films of 15 nm thickness are formed. It was further found, that the initiator concentration (varied over almost three orders of magnitude) does not influence the thickness of the resulting films. A model is developed that explains these findings in the light of the reaction kinetics of the surface attachment reaction.

Surface-attached polymer monolayers for the control of endothelial cell adhesion

The goal of this work is to alter the properties of glutaraldehyde modified porcine heart valves in such a way that vital endothelial cells can grow on the implant surfaces. Our approach to achieve this goal is to mask glutaraldehyde residues on the valve surfaces with a layer of hydrophilic polymers. In order to find suitable polymers for endothelial cell attachment to such surfaces, firstly model studies were carried out in which polymer monolayers were covalently attached to glass surfaces. A photochemical approach proved to be well-suited for this study. The layer thickness of the films is mostly determined by the molecular weight of the polymer and varied from about 3 to 26 nm. These layers were tested for the adhesion and the growth of endothelial cells from humans (HUVECs) and fibrocytes from animals (sheep, rat) for comparison. Some polymers were identified, e.g. poly(2-ethyl-2-oxazoline) (PEtOx), poly(ethyleneimine) (PEI), Poly(2-(methacryloyloxy)ethyl trimethylammonium chloride) (PMTA), poly(methacrylic acid) (PMAA), that promote the formation of a dense layer of healthy endothelial cells.

Printed protein microarrays on unmodified plastic substrates

A key challenge for the generation of protein microarrays is the immobilization of functional capture probe proteins at the chip surfaces. Here, a new concept for a single step production of protein microarrays to unmodified plastic substrates is presented. It is based on the printing of polymer/protein mixtures and the photochemical attachment of the obtained microstructures to the plastic chip surfaces. In the photochemical process three reactions occur simultaneously: transformation of the polymer into hydrogel dots, covalent binding of the forming gel to the substrate, and covalent immobilization of the proteins to the three-dimensional hydrogel scaffold. As an example we use anti-bovine serum albumin as a protein (anti-BSA) and a water swellable polymer network based on polydimethylacrylamide as a scaffold, which is photochemically crosslinked using benzophenone as a crosslinking agent. In one series of microarray experiments the probe density of the immobilized proteins was determined by incorporating fluorescence-labeled anti-BSA in the hydrogels. In a typical experiment, the number of immobilized probes was determined to 4 x 10(9) protein molecules per spot. In other experiments, the microarrays were brought into contact with fluorescently labeled BSA. In such analyses signal-to-noise values of more than 200 were obtained and about 9 x 10(7) antigen molecules were bound per spot. This demonstrates that in a very simple way microarrays with large amount of probes per spot can be realized and that antibodies immobilized in the printed hydrogels remain accessible and retain their functionality.

Tunable Bragg filters based on polymer swelling

We report on the optical properties of Bragg mirrors and filters fabricated from photo-cross-linked standard optical polymers. The transmittance spectra of these devices in the visible to near-infrared spectral range were measured. We demonstrate efficient tuning of the filter peak of the polymer Bragg filters over several hundred nanometers by adding organic solvents to the surrounding atmosphere of the filter. This represents what we believe to be a novel tuning principle for Bragg filters relying on the use of polymeric materials.

Influence of the Molecular Structure of Surface-Attached Poly(N-alkyl Acrylamide) Coatings on the Interaction of Surfaces with Proteins, Cells and Blood Platelets

Blood protein adsorption and blood platelet adhesion onto surface-attached poly(alkylacrylamide) networks that exhibit small and systematic variations in chemical composition are investigated. The polymer coatings are generated by depositing a thin layer of benzophenone-group-containing copolymer onto a solid substrate, followed by photo crosslinking and simultaneous surface-attachment. The correlation of the swelling of the obtained surface-attached networks with the adsorption of blood proteins and cellular adhesion is studied. The swollen surface-attached layers are inert to blood proteins and platelet cells. These results suggest that the hydrogel-coated materials are promising candidates for the generation of hemocompatible surfaces.

A polymer-based DNA biochip platform for human papilloma virus genotyping

Genotyping of the human papilloma virus (HPV) is from a clinical point of view an important diagnostic task as some genotypes play a major role in the development of cervical carcinoma. So far PCR combined with blotting or in situ labelling is known to be the most accurate and sensitive method for detection and genotyping of HPV infection in clinical samples. However, specificity, cost-efficiency and sensitivity are not always satisfactory. A novel DNA biochip is described based on a plastic substrate, onto which small polymer droplets and single-stranded DNA are printed in the form of microarrays. Immobilisation of all compounds on the chip surface is achieved by a short UV-irradiation process, inducing photochemical reactions in the polymer. The chip designed for this study contains 36 probes for determining 12 common, different HPV genotypes. After isolation of the DNA, PCR and biochip read-out, the chip allows for genotyping of the most common virus strains, which, according to current prevalence studies, cover 85-95% of all infections. Following this approach as little as 10 virus copies can be detected within a short exposure time. Even using paraffin-embedded material and 10(4) copies per PCR are sufficient to allow rapid and reliable HPV genotyping.