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We report a method enabling intensity-based readout for label-free cellular assays and realize a reader device with the same footprint as a microtiter plate. For unambiguous resonance intensity measurements in resonance waveguide grating (RWG) sensors, we propose to apply resonances near the substrate cutoff wavelength. This method was validated in bulk refractive index, surface bilayer and G protein-coupled receptor (GPCR) experiments. The significantly reduced size of the reader device opens new opportunities for easy integration into incubators or liquid handling systems.

We have studied the initial phase of cell adhesion as a function of the lateral organization of individual integrin molecules with single-cell force microscopy. Nanostructures, consisting of hexagonally ordered gold dots, were prepared with diblock-copolymer micelle lithography and functionalized with arginine- glycine-aspartate peptides, thus defining integrin position with nanometer resolution. Adhesion strength was characterized with an atomic force microscope and both cell detachment forces and work of detachment showed a reinforcement of adhesion if the distance between integrin molecules was

We report a quantitative analysis of the forces acting on optically trapped single gold nanorods. Individual nanorods with diameters between 8 and 44 nm and aspect ratios between 1.7 and 5.6 were stably trapped in three dimensions using a laser wavelength exceeding their plasmon resonance wavelengths. The interaction between the electromagnetic field of an optical trap and a single gold nanorod correlated with particle polarizability, which is a function of both particle volume and aspect ratio.

In order to understand the variations in photocatalytic efficiencies of titania thin films prepared by different physical vapor deposition techniques, we studied the microstructure and resulting properties for the two widely used PVD methods, electron beam evaporation and reactive pulsed DC magnetron sputtering. In addition, we investigated the effect of oxygen vacancy defects induced by tempering in reducing atmospheres and the aging behavior. After deposition, several tempering series in oxygen, air, argon, and forming gas were carried out to control the amount of oxygen vacancy defects. The films were characterized with respect to crystallinity, texture, grain growth, grain structure, surface roughness, light transmission as well as band gap energy; the photocatalytic efficiency was measured via methylene blue degradation. The results show different nucleation and growth mechanisms between evaporated and sputtered titania thin films, resulting in severe influence on photocatalytic efficiency. The evaporated thin films exhibited homogeneous nucleation and growth, and stayed in the anatase structure even after tempering at 800 °C for 1 h. In contrast, the sputtered thin films started to form grains at the interface to the substrate and showed heterogeneous nucleation and growth. Moreover, the sputtered films already formed rutile when tempered at 600 °C for 1 h. The gain in surface area due to tempering, which promotes adsorption of photocatalytic reactants, was more pronounced in sputtered thin films (+29%) compared to evaporated thin films (+6%). Films with oxygen vacancy defects, preserved by tempering in argon or induced by tempering in forming gas, showed a large improvement in photocatalytic efficiency. However, aging of the samples over a period of 19 months lead to a progressive decline in efficiency, finally reaching the level of thin films tempered in air, which had remained stable over the same 19 month period. This strongly questions widely applied concepts based on improving the photocatalytic efficiency via oxygen vacancy defects.

Following a myocardial infarction (MI), cardiomyocytes are replaced by scar tissue, which decreases ventricular contractile function. Tissue engineering is a promising approach to regenerate such damaged cardiomyocyte tissue. Engineered cardiac patches can be fabricated by seeding a high density of cardiac cells onto a synthetic or natural porous polymer. In this study, nanocomposite scaffolds made of gold nanotubes/nanowires incorporated into biodegradable castor oil-based polyurethane were employed to make micro-porous scaffolds. H9C2 cardiomyocyte cells were cultured on the scaffolds for one day, and electrical stimulation was applied to improve cell communication and interaction in neighboring pores. Cells on scaffolds were examined by fluorescence microscopy and scanning electron microscopy, revealing that the combination of scaffold design and electrical stimulation significantly increased cell confluency of H9C2 cells on the scaffolds. Furthermore, we showed that the gene expression levels of Nkx2.5, atrial natriuretic peptide (ANF) and natriuretic peptide precursor B (NPPB), which are functional genes of the myocardium, were up-regulated by the incorporation of gold nanotubes/nanowires into the polyurethane scaffolds, in particular after electrical stimulation.

Rapid and reversible photoswitching of cell adhesion is achieved by c(RGDfK)-azobenzenes embedded in a poly(ethylene glycol) background on surfaces. The light-induced cis-trans-isomerization of the azobenzene enables switching of cell adhesion on the surface. Reversibility of switching over several consecutive switching cycles is demonstrated by single-cell force spectroscopy.

Implant materials require optimal biointegration, including strong and stable cell-material interactions from the early stages of implantation. Ti-based alloys with low elastic modulus are attracting a lot of interest for avoiding stress shielding, but their osseointegration potential is still very low. In this study, we report on how cell adhesion is influenced by linear RGD, cyclic RGD, and recombinant fibronectin fragment III8–10 coated on titanium versus a novel low-modulus TiNbHf alloy. The bioactive molecules were either physisorbed or covalently coupled to the substrates and their conformation on the surfaces was investigated with atomic force microscopy (AFM). The influence of the different bioactive coatings on the adhesion of rat mesenchymal stem cells was evaluated using cell culture assays and quantitatively analyzed at the single cell level by AFM-based single-cell force spectroscopy. Our results show that bioactive moieties, particularly fibronectin fragment III8–10, improve cell adhesion on titanium and TiNbHf and that the covalent tethering of such molecules provides the most promising strategy to biofunctionalize these materials. Therefore, the use of recombinant protein fragments is of high importance for improving the osseointegration potential of implant materials

Diblock copolymer micelle nanolithography (BCML) is a versatile and efficient method to cover large surface areas with hexagonally ordered arrays of metal nanoparticles, in which the nanoparticles are equally spaced. However, this method falls short of providing a controlled allocation of such regular nanoparticle arrays with specific spacing into micropatterns. We present here a quick and high-throughput method to generate quasi-hexagonal nanoparticle structures with well-defined interparticle spacing on segments of nanotopographic Si substrates. The topographic height of these segments plays a dominant role in dictating the spacing between the gold nanoparticles, as the nanoparticle arrangement is controlled by immersion forces and by their self-assembly within the segments. Our novel strategy of employing a single-step BCML routine is a highly promising method for the fabrication of regular gold nanopatterns in micropatterns for a wide range of applications.

Acanthamoebae are free-living protists and human pathogens, whose cellular functions and pathogenicity strongly depend on the transport of intracellular vesicles and granules through the cytosol. Using high-speed live cell imaging in combination with single-particle tracking analysis, we show here that the motion of endogenous intracellular particles in the size range from a few hundred nanometers to several micrometers in Acanthamoeba castellanii is strongly superdiffusive and influenced by cell locomotion, cytoskeletal elements and myosin II. We demonstrate that cell locomotion significantly contributes to intracellular particle motion, but is clearly not the only origin of superdiffusivity. By analyzing the contribution of microtubules, actin and myosin II motors we show that myosin II is a major driving force of intracellular motion in A. castellanii. The cytoplasm of A. castellanii is supercrowded with intracellular vesicles and granules, such that significant intracellular motion can only be achieved by actively driven motion, while purely thermally driven diffusion is negligible.

We present a handheld biosensor system for the label-free and specific multiplexed detection of several biomarkers employing a spectrometer-free imaging measurement system. A photonic crystal surface functionalized with multiple specific ligands forms the optical transducer. The photonic crystal slab is fabricated on a glass substrate by replicating a periodic grating master stamp with a period of 370 nm into a photoresist via nanoimprint lithography and deposition of a 70-nm titanium dioxide layer. Capture molecules are coupled covalently and drop-wise to the photonic crystal surface. With a simple camera and imaging optics the surface-normal transmission is detected. In the transmission spectrum guided-mode resonances are observed that shift due to protein binding. This shift is observed as an intensity change in the green color channel of the camera. Non-functionalized image sections are used for continuous elimination of background drift. In a first experiment we demonstrate the specific and time-resolved detection of 90.0 nM CD40 ligand antibody, 90.0 nM EGF antibody, and 500 nM streptavidin in parallel on one sensor chip. In a second experiment, aptamers with two different spacer lengths are used as receptor. The binding kinetics with association and dissociation of 250 nM thrombin and regeneration of the sensor surface with acidic tris-HCl-buffer (pH 5.0) is presented for two measurement cycles.

We designed and evaluated a microfluidic test chip for human blood filtration and imaging label-free detection of multiple biomarkers. The microfluidic chip has a total size of 75 mm × 25 mm × 2 mm. It is realized as an assembly of a plastic chip and a functionalized photonic crystal slab on a glass substrate. The plastic chip contains capillary channels, a filter membrane and a cavity open on one side. The photonic crystal chip is bonded with an adhesive foil to the open cavity. Human blood filtration is demonstrated. We determined that fluorescently labelled particles of diameter 3 µm or larger are filtered out by the system. Refractometric measurements are performed with the test chip in combination with a compact imaging read-out system to investigate the system response to refractive index changes. Flow dynamics in the sensor cavity are imaged for replacing water by isopropanol. Finally, the binding of 500 nm biotin dissolved in phosphate buffer saline to a photonic crystal surface locally functionalized with streptavidin is demonstrated.

Background: Mechanosensing of cells, particularly the cellular response to substrates with different elastic properties, has been discovered in recent years, but almost exclusively in mammalian cells. Much less attention has been paid to mechanosensing in other cell systems, such as in eukaryotic human pathogens.Results: We report here on the influence of substrate stiffness on the adhesion of the human pathogen Acanthamoebae castellanii (A. castellanii). By comparing the cell adhesion area of A. castellanii trophozoites on polydimethylsiloxane (PDMS) substrates with different Young’s moduli (4 kPa, 29 kPa, and 128 kPa), we find significant differences in cell adhesion area as a function of substrate stiffness. In particular, the cell adhesion area of A. castellanii increases with a decreasing Young’s modulus of the substrate.Conclusion: The dependence of A. castellanii adhesion on the elastic properties of the substrate is the first study suggesting a mechanosensory effect for a eukaryotic human pathogen. Interestingly, the main targets of A. castellanii infections in the human body are the eye and the brain, i.e., very soft environments. Thus, our study provides first hints towards the relevance of mechanical aspects for the pathogenicity of eukaryotic parasites.

There is a growing interest in using fibrils from food grade protein, e.g. β-lactoglobulin, as functional ingredients. In the present study, the functionality of fibrillar β-lactoglobulin from whey protein isolate (WPI) was compared to native WPI in terms of interfacial dilatational rheology and emulsifying activity at acidic conditions (pH 2.0 and 3.0). We report here for the first time data on microencapsulation of fish oil by spray-drying as well as oxidative stability of the oil in emulsions and microcapsules in dependence of WPI conformation. WPI fibrils exerted a significantly higher elasticity at the oil–water (o/w) interface and a better emulsifying activity at a fixed oil content compared to native WPI. Microencapsulation efficiency was also higher with fibrillar WPI (>95%) compared to native WPI (∼90%) at pH 2.0 and a total oil and protein content of 40% and 2.2%, respectively, in the final powder. The oxidative deterioration was lower in emulsions and microcapsules prepared with fibrillar than with native WPI. This was attributed to improved interfacial barrier properties provided by fibrils and antioxidative effects of coexisting unconverted monomers, particularly hydrophilic peptides.

We present a biosensor using a photonic crystal slab (PCS) surface-functionalized with aptamers (thus aptasensor) and a label-free photometric detection scheme. Resonances in the optical transmission spectrum of the PCS are highly sensitive to mass changes on the surface of the PCS. We align the resonance's spectral position to the falling edge of a light emitting diode's (LED) spectrum and use two crossed polarization filters before and after the PCS. Thus, we may detect a molecular binding process on the surface of the PCS with a simple photometric intensity measurement. Using a CMOS camera, we determine the signal and reference intensity at different positions on the PCS. The functionality of this aptasensor is demonstrated by applying a 125 nM thrombin-solution to the PCS bio-functionalized with anti-thrombin aptamers. A significant signal was obtained and the reaction kinetic was observed.

Purpose To compare the potential of different soft contact lenses to be contaminated with Acanthamoeba castellanii as a function of material parameters and cleaning procedures. Methods Different unworn soft hydrogel and silicone hydrogel contact lenses were incubated with human pathogenic A. castellanii . The adhesion of the acanthamoebae was investigated on the contact lenses and put into relation to their material parameters. The efficacy of a recommended contact lens cleaning procedure in reducing A. castellanii adhesion was investigated. Results We found that material parameters such as elastic modulus, silicone content, ionic properties and swelling do not influence the adhesion of acanthamoebae to soft contact lenses. A material parameter that influenced adhesion significantly was the water content of the lens. With increasing water content, the adhesion of acanthamoebae increased. By following the cleaning instructions of the manufacturer the contamination of the lenses with A. castellanii could be reduced to a minimum, as shown both on contact lenses and in control experiments. Conclusion With this study we show that for the tested lenses, the adhesion of A. castellanii to contact lenses is independent of the silicone content of the lens, but depends nonlinearly on the water content of the lens. Furthermore, we demonstrate that applying proper lens cleaning procedures minimizes the risk of acanthamoebae adhesion to contact lenses.

In optical microscopy the contrast of transparent objects achieved with conventional methods is often not satisfactory, for example for the automated recognition of cells. In this paper we present a nano-optical label-free approach for contrast enhancement based on photonic crystal slabs (PCS) as the specimen holder. Quasi-guided modes inside these structures cause an intrinsic color of the PCS, which strongly depends on the wavelength and the quality factor of the optical mode. Objects on the surface of the PCS experience a significant color and intensity contrast enhancement, as they change properties of the optical modes.

Cell adhesion is an essential prerequisite for survival, communication, and navigation of cells in organisms. It is maintained by the organized binding of molecules from the cell membrane to the extracellular space. This chapter focuses on direct measurements of cellular binding strength at the level of single adhesion molecules. Using atomic force microscopy-based force measurements, adhesion strength can be monitored as a function of adhesion time and environmental conditions. In this way, cellular adhesion strategies like changes in affinity and avidity of adhesion molecules (e.g., integrins) are characterized as well as the molecular arrangement of adhesion molecules in the cell membrane (e.g., molecular clusters, focal adhesion spots, and linkage to the cytoskeleton or tether). Some prominent values for the data evaluation are presented as well as constraints and preparative techniques for successful cell adhesion force experiments.

An increasing number of experimental studies employ single particle tracking to probe the physical environment in complex systems. We here propose and discuss new methods to analyze the time series of the particle traces, in particular, for subdiffusion phenomena. We discuss the statistical properties of mean maximal excursions, i.e., the maximal distance covered by a test particle up to time t. Compared to traditional methods focusing on the mean squared displacement we show that the mean maximal excursion analysis performs better in the determination of the anomalous diffusion exponent. We also demonstrate that combination of regular moments with moments of the mean maximal excursion method provides additional criteria to determine the exact physical nature of the underlying stochastic subdiffusion processes. We put the methods to test using experimental data as well as simulated time series from different models for normal and anomalous dynamics, such as diffusion on fractals, continuous time random walks, and fractional Brownian motion., 10 pages, 7 figures, 2 tables. NB: Supplementary material may be found in the downloadable source files

Combining extensive single particle tracking microscopy data of endogenous lipid granules in living fission yeast cells with analytical results we show evidence for anomalous diffusion and weak ergodicity breaking. Namely we demonstrate that at short times the granules perform subdiffusion according to the laws of continuous time random walk theory. The associated violation of ergodicity leads to a characteristic turnover between two scaling regimes of the time averaged mean squared displacement. At longer times the granule motion is consistent with fractional Brownian motion., Comment: 4 pages, 4 figures, REVTeX. Supplementary Material. Physical Review Letters, at press

One limitation on the performance of optical traps is the noise inherently present in every setup. Therefore, it is the desire of most experimentalists to minimize and possibly eliminate noise from their optical trapping experiments. A step in this direction is to quantify the actual noise in the system and to evaluate how much each particular component contributes to the overall noise. For this purpose we present Allan variance analysis as a straightforward method. In particular, it allows for judging the impact of drift which gives rise to low-frequency noise, which is extremely difficult to pinpoint by other methods. We show how to determine the optimal sampling time for calibration, the optimal number of data points for a desired experiment, and we provide measurements of how much accuracy is gained by acquiring additional data points. Allan variances of both micrometer-sized spheres and asymmetric nanometer-sized rods are considered.

SummarySporozoites are the highly motile stages of the malaria parasite injected into the host's skin during a mosquito bite. In order to navigate inside of the host, sporozoites rely on actin-dependent gliding motility. Although the major components of the gliding machinery are known, the spatiotemporal dynamics of the proteins and the underlying mechanism powering forward locomotion remain unclear. Here, we show that sporozoite motility is characterized by a continuous sequence of stick-and-slip phases. Reflection interference contrast and traction force microscopy identified the repeated turnover of discrete adhesion sites as the underlying mechanism of this substrate-dependent type of motility. Transient forces correlated with the formation and rupture of distinct substrate contact sites and were dependent on actin dynamics. Further, we show that the essential sporozoite surface protein TRAP is critical for the regulated formation and rupture of adhesion sites but is dispensable for retrograde capping.

During the cell cycle, the organization of the cytoskeletal network undergoes dramatic changes. In order to reveal possible changes of the viscoelastic properties in the intracellular space during the cell cycle we investigated the diffusion of endogenous lipid granules within the fission yeast Schizosaccharomyces Pombe using optical tweezers. The cell cycle was divided into interphase and mitotic cell division, and the mitotic cell division was further subdivided in its stages. During all stages of the cell cycle, the granules predominantly underwent subdiffusive motion, characterized by an exponent alpha that is also linked to the viscoelastic moduli of the cytoplasm. The exponent alpha was significantly smaller during interphase than during any stage of the mitotic cell division, signifying that the cytoplasm was more elastic during interphase than during division. We found no significant differences in the subdiffusive exponents from granules measured in different stages of cell division. Also, our results for the exponent displayed no significant dependence on the position of the granule within the cell. The observation that the cytoplasm is more elastic during interphase than during mitotic cell division is consistent with the fact that elastic cytoskeletal elements such as microtubules are less abundantly present during cell division than during interphase.

Spatial patterning of biochemical cues on the micro- and nanometer scale controls numerous cellular processes such as spreading, adhesion, migration, and proliferation. Using force microscopy we show that the lateral spacing of individual integrin receptor-ligand bonds determines the strength of cell adhesion. For spacings > or = 90 nm, focal contact formation was inhibited and the detachment forces as well as the stiffness of the cell body were significantly decreased compared to spacings < or = 50 nm. Analyzing cell detachment at the subcellular level revealed that rupture forces of focal contacts increase with loading rate as predicted by a theoretical model for adhesion clusters. Furthermore, we show that the weak link between the intra- and extracellular space is at the intracellular side of a focal contact. Our results show that cells can amplify small differences in adhesive cues to large differences in cell adhesion strength.

The mammalian inflammatory response significantly relies on integrin-mediated T-lymphocyte adhesion to endothelial cells. Whereas the outside-in signalling pathway of integrins in response to the proinflammatory cytokine tumor necrosis factor (TNF) has already been studied in detail, little knowledge exists about the inside-out signalling pathway of integrins in lymphocyte activation by TNF. We here show single-cell force spectroscopy (SCFS) data of T-lymphocyte (Jurkat E6-1) adhesion to fibronectin. Fibronectin is present on top of endothelial cell layers and is therefore a crucial player in mediating T-lymphocyte binding to endothelial cells. Our results show that activating integrins with TNF significantly increases the maximum adhesion force and detachment energy in Jurkat cell adhesion to fibronectin-coated surfaces. Analysis of single-molecule ruptures further proves that TNF reinforces integrin binding strength, particularly at sub-second timescales. Hence, our results provide quantitative evidence for the significant impact of TNF-induced inside-out signalling in the T-lymphocyte adhesion machinery.

Visualizing three-dimensional (3D) structures at the micrometer and nanometer scale is essential not only for characterizing materials and corrosion but also biological samples. Here, we present a material-based nano-optical method using the near-field properties of periodically nanostructured surfaces (photonic crystal slabs) to obtain 3D images. The wavelength and the quality factor of resonances in the transmission spectrum provide optical thickness information of objects on the surface, which we use for rapid topography determination of cells.

Nanoparticles are highly attractive tools for future biomedical applications. Essential steps towards using nanoparticles in living organisms include the development of strategies for a defined nanoparticle uptake by the cell, the availability of appropriate particle tracking methods and the design of nanoparticles with low cytotoxicity. Optical tweezers are versatile tools to study the interaction between particles and cells with nanometre, piconewton and microsecond resolution and they have proven capable of trapping and tracking individual nanoparticles in three dimensions. This minireview presents recent achievements for the usage of nanoparticles in in vivo studies and discusses how optical tweezers can be employed to quantify cell-nanoparticle interactions at the single particle level.

An artificial substrate system, according to the biophysical and biochemical properties of the extracellular matrix in connective tissues, has been developed. The Young's moduli EY of poly(ethylene glycol)-diacrylate (PEG-DA) based hydrogel substrates span more than four orders of magnitude between 0,6kPa and 6MPa. Since PEG-DA substrates are protein repellent, they were decorated by extended gold nanoparticle arrays, manufactured by block copolymer micellar nanolithography. To provide bioactivity in terms of cell adhesion c(RGDfK) peptide, which is specific for αVβ3 integrins, was immobilized on the nanoparticles. The interparticle spacing and, hence, spacing of integrin binding sites ΔL could be precisely tuned, independently of the substrate rigidity between 20nm and 160nm.This system was used to investigate the behavior of fibroblasts as a function of changes within two-dimensional parameters space ΔL;EY). To this end, cell spreading area and cell-substrate interaction forces were determined by phase contrast microscopy and single cell force spectroscopy (SCFS), respectively.First, the effect of variation of ligand spacing on cellular behavior was investigated on hard substrates (EY>100kPa). We could demonstrate a strong increase in detachment force and spreading area on substrates featuring low ligand spacing. Than, substrate compliance was tuned whereas the ligand spacing was kept at approximately 50nm. This reveals a significant decrease in spreading area and detachment force on soft substrates (EY

During the cell cycle, rearrangements of the cytoskeletal network play an essential role, in particular for the success of cell division. In order to quantify the influence of cytoskeletal rearrangements on the viscoelastic properties of the intracellular space, we studied the diffusion of endogenous lipid granules within single fission yeast cells in the different stages of the cell cycle. The position of the granules was tracked with optical tweezers at nanometer and sub-millisecond resolution and the data were analyzed with a power spectral analysis. We found that the majority of the lipid granules underwent subdiffusive motion during all stages of the cell cycle, i.e. the mean squared displacement of the granule is 2Dtα with α

In times of a rapid development and growing market in robotics, high-tech protheses and the personalization of medicine, biomimicking natural materials like artificial tissue are of central interest within research and industry. To fully understand the structure-function relations within living systems, comprehensive knowledge about the smallest living block, the cell, and its biomechanics are a central topic in world-wide research. However, there is so far no comprehensive technique established that can measure 3D cell forces simultaneously and quantitatively. In this project, a novel surface-integrated mechano-optical microsensor system has therefore been conceptualized, prototyped and tested, which allows for the record of pico- to micronewton traction forces in three dimensions simultaneously. First, adequate microsensor elements were designed via topology optimization and linear static finite element analysis. These designs were fabricated by micromachining processes of biocompatible thin films of nickel-titanium and amorphous silicon. Furthermore, a plasma etching process was developed to fabricate polydimethylsiloxane sensor elements. For accurate and quantitative traction force measurements, AFM cantilever based calibrations of the out-of-plane and in-plane sensor element spring constants were established. For the first time, a diamagnetic levitation force calibrator was used as an adequate pre-calibration method for the sensor elements with a high accuracy of 1 %. For the cost-efficient, simple, compact, variable and sensitive mechano-optical readout, a setting was conceptualized and tested based on the combination of digital holography and digital image correlation. To control cell adhesion, a high-throughput micro-nano structuring method was developed based on the fusion of ink-jet printing with the established method of diblock-copolymer micelle nanolithography. In Zeiten schneller Entwicklung und wachsender Märkte in der Robotik, der high-tech Prothetik und der personalisierten Medizin ist die Biomimetik natürlicher Materialien wie beispielsweise künstliche Haut von zentralem Interesse in Forschung und Industrie. Um die Struktur-Funktions-Beziehungen in lebenden Systemen umfassend zu verstehen ist die umfangreiche Wissenserweiterung hinsichtlich des kleinsten lebenden Bausteins, der Zelle, und seiner Biomechanik Gegenstand weltweiter Forschungsprojekte. Dennoch gab es bis jetzt keine Methode, die 3D Zellkräfte simultan und quantitativ messen kann. In diesem Projekt wurde ein neuartiges, oberflächen-integriertes, mechano-optisches Mikrosensorsystem konzeptioniert, prototypisiert und getestet, das die Messung piko-bis mikronewton kleiner Zugkräfte gleichzeitig in alle drei Dimensionen ermöglicht. Die Sensorelemente wurden mittels Topologieoptimierung und linear statischer Finite Elementanalyse konzipiert. Diese Designs wurden in Mikromaterialbearbeitungsprozessen aus biokompatiblen Nickel-Titan und amorphen Silizium-Dünnschschichten hergestellt. Desweiteren wurde ein Prozess entwickelt, um Polydimethylsiloxan basierte Sensorelemente herzustellen. Für genaue, quantitative Zugkraftmessungen wurden AFM-Cantilever basierte Kalibrierungen der axialen und lateralen Sensorelement-Federkonsten etabliert. Zum ersten Mal wurde dabei ein diamagnetischer Levitationskraftkalibrator mit einer Genauigkeit von 1% als geeignete Kalibrierungsmethode für die Sensorelemente genutzt. Für eine günstige, einfache, kompakte, variable und im Nanometerbereich empfindliche mechano-optische Datenauslesung wurde ein Aufbau konzeptioniert und getestet, in dem digitale Holographie und digitale Bildkorrelation kombiniert werden. Zur Zell-Adhäsionskontrolle wurde eine Hochdurchsatz-Mikro-Nanostrukturierungsmethode entwickelt, die auf der Kombination von Ink-Jet Drucken mit der etablierten Methode der Diblock-Copolymer Mizellen Nanolithographie basiert.

Magnesium (Mg) based metals have been applied in orthopaedic and cardiovascular (as stent) applications due to (e.g.) their excellent biodegradability and tissue regeneration abilities. Endothelial cells (ECs) and mesenchymal stem cells (MSCs) represent two protagonists of angiogenesis and osteogenesis. The responding of angiogenesis and the EC-MSC communication to Mg degradation products and oxygen content are vital to vascular remodelling and later fracture regeneration. Firstly, human primary ECs were exposed to various concentrations of extracellular Mg degradation products under either hypoxia or normoxia. Furthermore, in order to understand the influences of Mg degradation and oxygen conditions on the ECs-MSCs communication, a non-contacting (or transwell system) and a contacting cell culture models were established. With these models, the various stages of fracture healing could be mimicked: angiogenesis, MSCs attraction / migration and tissue modelling. In the monoculture, cell metabolism and proliferation, as well as wound healing and tube formation assays (reflecting two different stages of angiogenesis: migration and tube formation) were studied. In coculture, cells proliferation and ratio were measured by deoxyribonucleic acid (DNA) content and flow cytometry, respectively. Additionally, MSCs migration was assessed by wound healing assay. Gene regulation in monoculture and different coculture were investigated by semi-quantitative real time polymerase chain reaction (qRT-PCR). Furthermore, several angiogenic factors, such as vascular endothelial growth factor A (VEGFA) were also quantified. These results suggest that Mg extracts did not interfere with angiogenesis under hypoxia. Furthermore, Mg, hypoxia, and heterotypic contact were beneficial to stromal ECs and MSCs, providing a suitable niche environment and highlighting the application of Mg in regenerative medicine.

In the first part of this thesis research, an artificial extra-cellular matrix formed the basis for experiments with a pathogenic species of amoeba, Acanthamoeba castellanii. In order to find new therapeutic approaches to fighting diseases caused by this species, the migratory behavior of Acanthamoeba castellanii in micro-pillar structures made of silicone, which were used to mimic the confined environment of an extra-cellular matrix, was explored. For the experiments, amoebae ingested microparticles of varying shapes. While it was found that sphere-shaped particles with a diameter smaller than the distance between the micro-pillars did not impact migration behavior, absorption of larger particles by Acanthamoeba castellanii caused migration to be strongly reduced. However, in some instances, it appeared that Acanthamoeba castellanii was nonetheless able to successfully navigate the pillar structures. These observations could serve as a basis for developing methods aimed at capturing amoebae before they can enter the human body. In the second part of this thesis, the focus was on developing a material with specific mechanical properties geared at mimicking an intra-cellular effect, namely the cross-linking of fibers within the cytoskeleton as a reaction to deformation of the cell and thereby increasing its stiffness. Emulating this effect, a strain-stiffening material was developed using a flexible, slat-structured silicone. When the material is elongated, these slats touch, thereby leading to a stiffening of the material. Employing finite element analysis, the degree to which various geometrical factors of the structure and friction between the slats affect the material’s stiffening behavior was examined, and the results were verified using tensile tests. The structure that resulted from my efforts yields a stiffening behavior that is material-independent, speed-independent and reversible, and that occurs also when the material is elongated. Eine künstliche extrazelluläre Matrix war die Basis für Experimente mit der pathogenen Amöbenspezies Acanthamoeba castellanii im ersten Teil der Arbeit. Auf der Suche nach neuartigen therapeutischen Ansätzen gegen Krankheiten, die von dieser Spezies verursacht werden, wurde deren Migrationsverhalten in mikrostrukturieren Säulenstrukturen aus flexiblem Silikon, die die beengende Umgebung der extrazellulären Matrix darstellen sollten, untersucht. Im Rahmen der Experimente haben die Amöben Mikropartikel mit verschiedenen Geometrien aufgenommen. Kugelförmige Partikel mit einem Durchmesser, der kleiner als der Abstand zwischen den Säulen ist, beeinflussten die Migration in den Strukturen nicht. In den Fällen, in denen Acanthamoeba castellanii größere Partikel aufgenommen hat, war die Migration in den Strukturen stark reduziert. In einigen Fällen konnten jedoch alternative Herangehensweisen beobachtet werden, die trotzdem Migration durch die Säulenstrukturen erlaubten. Die Imitation eines intrazellulären Effektes war die Grundlage für die Entwicklung eines Materials mit definierten mechanischen Eigenschaften im zweiten Teil der Arbeit. Die Fähigkeit von Zellen, als Reaktion auf eine Deformation Fasern im Zytoskelett zu vernetzen und somit ihre Steifigkeit zu erhöhen, wurde in dehnungsversteifenden Materialien nachgestellt. Es wurde eine Struktur aus flexiblem Silikon entwickelt, die eine Lamellenstruktur enthält. Diese Lamellen berühren sich im Fall einer Elongation des Materials und führen zu einer Versteifung des Materials. Es wurde mit Hilfe von Finite Elemente Analysen der Einfluss sowohl diverser geometrische Faktoren der Struktur, als auch der Reibung zwischen den Lamellen auf das Versteifungsverhalten des Materials untersucht. Die Ergebnisse wurden mit Hilfe von realen Zugversuchen verifiziert. Die entwickelte Struktur bietet ein materialunabhängiges, geschwindigkeitsunabhängiges, reversibles Versteifungsverhalten, das zudem auch bei einer Elongation des Materials auftritt.

Labeling of biological analytes (e.g. protein, DNA) with superparamagnetic microbeads (SPM-MBs) serves as a handling tool in microfluidic lab-on-chip applications. Using an externally applied magnetic field, the separation of bio-labeled MBs on magnetic structures (MSs) of a chip is an emerging trend in biomedical research and to realize the future diagnostic tools. However, a high separation efficiency of mixed MBs populations together with a high throughput is a bottleneck, limiting the adaptability of these devices. This thesis presents manipulation techniques of MBs on soft magnetic microchips in a flow-less aqueous medium. The experimentally acquired motion patterns of MBs are phenomenologically explained by micromagnetic simulations, magnetic force microscopy measurements and magneto-optical imaging. 2-D periodic distribution of triangular MSs enables a free motion of MBs to a chosen location along the trisymmetric pathways on a chip. Selective transport of mixed populations of MBs is achievable with a separating efficiency of more than 97 %. Circular MSs in the hexasymmetric 2-D arrangement enable the driving of a single MB and its population by means of an elliptically rotating magnetic field in the chip plane. Adjusting the strength and orientation of the field can steer the moving MBs in multiple directions on the magnetic surface. Finally, to confirm the functionality of these magnetic surfaces, an example of a MB inside a Dictyostelium amoeba is shown for a selectable directed movement on triangular MSs. Ein aufkommender Trend zur Realisierung zukünftiger Diagnoseinstrumente in der biomedizinischen Forschung besteht darin, durch SPM-MBs markierte Analyte anhand von Magnetfeldern auf funktionalisierten Oberflächen gezielt zu trennen. Eine Herausforderung, die den Einsatz gegenwärtig erschwert, besteht darin, einen hohen Analytendurchsatz mit gleichzeitig hoher Trenneffizienz zu realisieren. Die Kontrolle von einzelnen frei beweglichen MBs kann z.B. dazu genutzt werden, um eine größenabhängige Trennung der Beads zu erreichen. In dieser Dissertation werden Methoden zur kontrollierten Bewegung von MBs auf weichmagnetischen strukturierten Oberflächen untersucht. Die experimentell bestimmten Bewegungsmuster werden phänomenologisch und durch mikromagnetische Simulationen, magnetische Rasterkraftmikroskopie und magneto-optische Mikroskopie erklärt. Magnetische Dreieckstrukturen auf periodisch strukturierten Oberflächen ermöglichen die gezielte Bewegung von MBs entlang der Strukturkanten der Elemente. Die gleichzeitige Bewegung unterschiedlich großer MBs in entgegengesetzten Richtungen ermöglicht die effiziente Trennung von markierten Substanzen, wie z.B. von biologischen Zellen, von Proteinen oder von DNA. Hierdurch ist die Trennung in Gruppen gleichgroßer MBs mit einer Genauigkeit von 97 % erreicht worden. Kreisrunde Strukturen in einer hexagonalen periodischen Anordnung ermöglichen die gerichtete Bewegung sowohl eines einzelnen MBs, als auch ganzer Gruppen mit Hilfe elliptisch rotierender Magnetfelder. Durch Anpassung der Feldstärke und Richtung des externen Magnetfeldes kann eine kontrollierte Bewegung der MBs entlang unterschiedlicher Richtungen auf der Chipoberfläche erreicht werden. Schlussendlich wird, zur Bestätigung der Anwendbarkeit der entwickelten Methoden am Beispiel einer mit einem MB markierten Dictyostelium Amöbe, die gezielte und gerichtete Bewegung auf magnetischen Strukturen demonstriert.