Advancing Glucose Sensing Through Auto-Fluorescent Polymer Brushes: From Surface Design to Nano-Arrays.

Designing smart (bio)interfaces with the capability to sense and react to changes in local environments offers intriguing possibilities for new surface-based sensing devices and technologies. Polymer brushes make ideal materials to design such adaptive and responsive interfaces given their large variety of functional and structural possibilities as well as their outstanding abilities to respond to physical, chemical, and biological stimuli. Herein, a practical sensory interface for glucose detection based on auto-fluorescent polymer brushes decorated with phenylboronic acid (PBA) receptors is presented. The glucose-responsive luminescent surfaces, which are capable of translating conformational transitions triggered by pH variations and binding events into fluorescent readouts without the need for fluorescent dyes, are grown from both nanopatterned and non-patterned substrates. Two-photon laser scanning confocal microscopy and atomic force microscopy (AFM) analyses reveal the relationship between the brush conformation and glucose concentration and confirm that the phenylboronic acid functionalized brushes can bind glucose over a range of physiologically relevant concentrations in a reversible manner. The combination of auto-fluorescent polymer brushes with synthetic receptors presents a promising avenue for designing innovative and robust sensing systems, which are essential for various biomedical applications, among other uses.

Hemocompatible Surfaces Through Surface-attached Hydrogel Coatings and their Functional Stability in a Medical Environment.

Blood compatible materials are a well-researched scientific field as such materials are required in a wide range of applications, for example, in heart-lung machines or ventricular assist devices. Surfaces coated with certain surface-bound neutral, water-swellable polymer networks have the ability to repel cells such as platelets and exhibit a significantly improved hemocompatibility. In this study, we investigate the interaction of platelets from whole blood with surfaces coated with photochemically generated surface-attached polymer networks based on polydimethyl acrylamide. As substrates medical-grade polyurethanes are used, and the networks are formed and attached to the substrate surfaces through C-H insertion reactions. The hydrogel-coated substrates are perfused with blood for extended periods of time. We show that the polymer coating prevents the adhesion of cells even at longer times of blood contact, regardless of the thickness of the coating employed. The surfaces can be sterilized following a standard autoclave procedure without any loss of function. Additionally, it is shown that the samples can be stored at least for 3 months under varying ambient conditions while retaining their functionality. The excellent blood compatibility, the possibility to coat even rather inert polymeric materials and the ability to handle the materials in an environment typical for a medical application make such coatings a promising candidate for future hemocompatible devices.

Measurements of periodically perturbed dewetting force fields and their consequences on the symmetry of the resulting patterns.

We studied the origin of breaking the symmetry for moving circular contact lines of dewetting polymer films suspended on a periodic array of pillars. There, dewetting force fields driving polymer flow were perturbed by elastic micro-pillars arranged in a regular square pattern. Elastic restoring forces of deformed pillars locally balance driving capillary forces and broke the circular symmetry of expanding dewetting holes. The observed envelope of the dewetting holes reflected the symmetry of the underlying pattern, even at sizes much larger than the characteristic period of the pillar array, demonstrating that periodic perturbations in a driving force field can establish a well-defined pattern of lower symmetry. For the presented system, we succeeded in squaring the circle.

Prevention of Ocular Tenon Adhesion to Sclera by a PDMAA Polymer to Improve Results after Glaucoma Surgery.

The authors describe a process that may eventually reduce the risk of scar formation after glaucoma surgery. For this, a thin hydrogel coating is photochemically generated and linked to the sclera surface at the surgical site. This coating is generated from a photoreactive prepolymer containing anthraquinone groups, which is administered as a thin pad to the sclera surface. Short UV irradiation leads to a reaction of the photogroups with neighboring chains via C-H insertion crosslinking, thus transforming the precursor polymer into a hydrogel. Simultaneously, a reaction between the photogroups and the underlying sclera tissue occurs, so that the hydrogel patch becomes covalently linked to the tissue. The authors show that the resulting thin coating is strongly cell repellent and hinders tenon fibroblasts to form tenon tissue at the site of the coating and is suitable for inclusion into a surgical procedure.

On the relationship of YAP and FAK in hMSCs and osteosarcoma cells: Discrimination of FAK modulation by nuclear YAP depletion or YAP silencing.

The HIPPO pathway effector YAP has been shown to be regulated by FAK-signaling. However, the existence of an inverse relationship between YAP and FAK is unknown. Here we demonstrate in hMSCs and in the human osteosarcoma derived cell line Saos that Verteporfin- or RNAi-dependent YAP depletion has opposing influence on FAK. While Verteporfin strikingly reduced cellular FAK protein and phosphorylation, RNAi led to an increase of both molecules and point on a generalizable aspect of the YAP/FAK interrelationship. YAP depletion also caused down-regulation of osteogenic genes in hMSCs, irrespective from the YAP intervention mode. Verteporfin induced topological changes in conjunction with reduced protein levels of ß1 integrin, paxillin, and zyxin of focal adhesions (FAs) in hMSCs, suggesting FAK-decrease-related alterations in FAs, which seems to be a FAK-dependent mechanism. On the cell behavioral level, YAP-FAK-interrelation involves proliferation and senescence, as indicated by proliferation inhibition and increase of ß-Galactosidase-activity in hMSCs. Our findings, derived from this dual strategy of YAP intervention, reveal a YAP-FAK relationship in conjunction with molecular and cell behavioral consequences. Moreover, they deepen the current scientific knowledge on YAP from a different scientific point of view, since this inverse YAP/FAK-relationship seems to be transferrable to other cell types, including cell entities with pathological background.

The Surface Science of Microarray Generation-A Critical Inventory.

Microarrays are powerful tools in biomedical research and have become indispensable for high-throughput multiplex analysis, especially for DNA and protein analysis. The basis for all microarray processing and fabrication is surface modification of a chip substrate and many different strategies to couple probe molecules to such substrates have been developed. We present here a critical assessment of typical biochip generation processes from a surface science point of view. While great progress has been made from a molecular biology point of view on the development of qualitative assays and impressive results have been obtained on the detection of rather low concentrations of DNA or proteins, quantitative chip-based assays are still comparably rare. We argue that lack of stable and reliable deposition chemistries has led in many cases to suboptimal quantitative reproducibility, impeded further progress in microarray development and prevented a more significant penetration of microarray technology into the diagnostic market. We suggest that surface-attached hydrogel networks might be a promising strategy to achieve highly sensitive and quantitatively reproducible microarrays.

Toward a New Generation of Smart Biomimetic Actuators for Architecture.

Motile plant structures (e.g., leaves, petals, cone scales, and capsules) are functionally highly robust and resilient concept generators for the development of biomimetic actuators for architecture. Here, a concise review of the state-of-the-art of plant movement principles and derived biomimetic devices is provided. Achieving complex and higher-dimensional shape changes and passive-hydraulic actuation at a considerable time scale, as well as mechanical robustness of the motile technical structures, is challenging. For example, almost all currently available bioinspired hydraulic actuators show similar limitations due to the poroelastic time scale. Therefore, a major challenge is increasing the system size to the meter range, with actuation times of minutes or below. This means that response speed and flow rate need significant improvement for the systems, and the long-term performance degradation issue of hygroscopic materials needs to be addressed. A theoretical concept for "escaping" the poroelastic regime is proposed, and the possibilities for enhancing the mechanical properties of passive-hydraulic bilayer actuators are discussed. Furthermore, the promising aspects for further studies to implement tropistic movement behavior are presented, i.e., movement that depends on the direction of the triggering stimulus, which can finally lead to "smart building skins" that autonomously and self-sufficiently react to changing environmental stimuli in a direction-dependent manner.

Surface-attached hydrogel coatings via C,H-insertion crosslinking for biomedical and bioanalytical applications (Review).

A novel method for the generation of surface-attached hydrogel coatings and their use in biomedical applications is discussed. This short review concentrates on surface architectures that are prepared from prepolymers carrying reactive groups suitable for crosslinking via C,H insertion reactions [C,H insertion crosslinking (CHic)]. Upon photochemical or thermal activation these groups do not only induce the crosslinking of the system, but also connect the forming gel to the surface as long as the surface itself consists of an organic material. C,H groups as the reaction partner are available in abundance at practically all types of organic surfaces such as biomaterials or polymers, rendering the technique almost universally applicable. Surface-attached gels prepared this way show unique swelling properties due to the confinement of the chains, as the obtained essentially two-dimensional gels can only swell in one dimension. This anisotropic swelling does not permit penetration of the layers by macromolecules so that the surfaces become bioinert, i.e., are strongly protein and cell repellent. It is discussed how this property can be used to control the interaction of surfaces with biological species ranging from the level of biomolecules to living cells. A combination of the CHic chemistry and microstructuring techniques opens further avenues for the study of the behavior of cells to the generation of novel bioanalytical devices.

Polymer Microstructures through Two-Photon Crosslinking.

Two-photon crosslinking of polymers (2PC) is proposed as a novel method for the fabrication of freestanding microstructures via two-photon lithography. During this process in the confocal volume, two-photon absorption leads to (formal) C,H-insertion reactions, and consequently to a strictly localized crosslinking of the polymer. To achieve this, the polymer is coated as a solvent-free (glassy) film onto an appropriate substrate, and the desired microstructure is written by 2PC into this glass. In all regions outside of the focal volume where no two-photon process occurs, the polymer remains uncrosslinked and can be washed away during a developing process. Using a self-assembled monolayer containing the same photoreactive group allows covalent attachment of the forming freestanding structures to the substrate, and thus guarantees an improved stability of these structures against shear-induced detachment. As the two photon process is carried out in the glassy state, in a simple way, multilayer structures can be used to write structures having a varying chemical composition perpendicular to the surface. As an example, the 2PC process is used to build a structure from both protein-repellent and protein-adsorbing polymers so that the resulting 3D structure exhibits spatially controlled protein adsorption.

PDMAA Hydrogel Coated U-Bend Humidity Sensor Suited for Mass-Production.

We present a full-polymer respiratory monitoring device suited for application in environments with strong magnetic fields (e.g., during an MRI measurement). The sensor is based on the well-known evanescent field method and consists of a 1 mm plastic optical fiber with a bent region where the cladding is removed and the fiber is coated with poly-dimethylacrylamide (PDMAA). The combination of materials allows for a mass-production of the device by spray-coating and enables integration in disposable medical devices like oxygen masks, which we demonstrate here. We also present results of the application of an autocorrelation-based algorithm for respiratory frequency determination that is relevant for real applications of the device.

Reduced Lateral Confinement and Its Effect on Stability in Patterned Strong Polyelectrolyte Brushes.

The stability of strong polyelectrolyte brushes (PEBs) was studied in bulk and in patterned structures. Thick PEBs of poly([(2-methacryloyloxy)ethyl]trimethylammonium chloride) with thicknesses >100 nm were synthesized using single electron transfer living radical polymerization. Brush patterning was identified using deep-ultraviolet photolithography by means of either a top-down (TD) or bottom-up (BU) method, with features as small as 200 nm. The brushes were soaked in water under a range of pH or temperature conditions, and the hydrolysis was monitored through dry-state ellipsometry and atomic force microscopy measurements. BU patterns showed reduced degrafting for smaller patterns, which was attributed to increased stress relaxation at such dimensions. In contrast to the already relaxed BU-patterned brush, a TD-patterned brush possesses perpendicular structures that result from the use of orthogonal lithography. It was found that the TD process induces cross-linking on the sidewall, which subsequently fortifies the sidewall materials. This modification of the polymer brushes hindered the stress relaxation of the patterns, and the degrafting trends became irrelevant to the pattern sizes. With proper tuning, the cross-linking on the sidewall was minimized and the degrafting trends were again relaxation-dependent.

Platelet repellent properties of hydrogel coatings on polyurethane-coated glass surfaces.

Thromboembolism is a severe complication in patients with ventricular assist devices. Interactions of platelets with the artificial surface and pathological blood flow conditions could contribute to thrombus formation. Previous studies suggested that strongly swellable polymer coatings prepared from various poly(N-alkyl acrylamides) are bioinert as they repel proteins and cells. In this study, we tested structurally similar polymer coatings with varying degrees of crosslinking and swelling to explore if the bioinert character of such coatings is also valid under whole blood perfusion and shear stress. Glass substrates coated with medical grade polyurethane (PU) were modified with surface-attached poly(N-alkyl acrylamide) layers, which differed in crosslinking and in swelling. Slides were perfused with human blood containing fluoresceinated platelets at a shear rate of 1500 s for 3 minutes. Noncoated and PU-coated glass slides served as a reference for thrombogenic surfaces. We detected severe platelet adhesion on the reference surfaces. There was no platelet adhesion on poly(N-alkyl acrylamide) coatings with a swelling factor above 1.5-2 (p < 0.001 compared with PU). A lower swelling factor resulted in severe platelet adhesion. Different degrees of crosslinking ranging from 1% to 10% did not reverse platelet repellent properties of the representative polymer tested (p < 0.001 compared with PU).

Preparation of surface-attached polymer layers by thermal or photochemical activation of α-diazoester moieties.

We report on the attachment of polymer monolayers or surface-attached, polymer networks onto SiO2 and/or polymer surfaces using thermo- and photoreactive α-diazoester groups. In the prior case, the α-diazoester groups are introduced into the system in the form of self-assembled monolayers of appropriately functionalized silanes. The monolayer decorated substrates are coated by polymer films and the α-diazoester groups in the monolayer are activated by heat or irradiation with UV-light. Upon activation, they cleave off nitrogen and the resulting carbene intermediates insert into C-H bonds of neighboring polymer chains. As a result of this binding process, surface-attached monolayers of the deposited polymer are obtained. When the polymers themselves carry such reactive moieties, the photo- or thermal activation leads to cross-linking of the polymers and thin surface-attached polymer networks result from the same process. The formation of the surface-attached layer is studied as a function of activation conditions, especially the temperature and the wavelength of the light used in the process.

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.

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.

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.

Attachment of polymer films to solid surfaces via thermal activation of self-assembled monolayers containing sulphonyl azide group.

We report on a simple and effective way to attach thin polymer films to solid surfaces. The system is based on a thermosensitive sulphonyl azide derivative that is immobilized to SiO(2) surfaces via chlorosilane anchoring group and subsequently covered with a polymer film. Upon heating the sulfonyl azide decomposes, leading to a C-H insertion reaction from the adjacent polymer chain resulting in a covalent attachment of the polymer to the surface. Any nonbound polymer can be removed by extraction. The method allows to attach a wide spectrum of polymers to solid surfaces. The film thickness of the monolayers can be tuned by adjusting the molecular weight of the polymer used and to some extent, the thermolysis conditions. The film thickness increases linearly with the radius of gyration of the polymers used for attachment. We have successfully attached thin layers of poly (styrene), poly (dimethylacryl amide) and poly (heptadecafluorodecylacrylate).

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.

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.

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.