Partitioning and subsampling statistics in compartment-based quantification methods.

The precision of compartment-based quantification methods is subject to multiple effects, of which partitioning and subsampling play a major role. Partitioning is the process of aliquoting the sample liquid and consequently the contained target molecules, whereas subsampling denotes the fact that usually only a portion of a sample is analyzed. In this work, we present a detailed statistical description comprising the effects of partitioning and subsampling on the relative uncertainty of the test result. We show that the state-of-the-art binomial model does not provide accurate results for the level of subsampling present when analyzing the nucleic acid content of single specific cells. Hence, in this work we address partitioning and subsampling effects separately and subsequently combine them to derive the relative uncertainty of a test system and compare it for single cell content analysis and body fluid analysis. In point-of-care test systems the area for partitioning and detection is usually limited, which means that a trade-off between the number of partitions (related to a partitioning uncertainty) and the amount of analyzed volume (related to a subsampling uncertainty) might be inevitable. In case of low target concentration, the subsampling uncertainty is dominant whereas for high target concentration, the partitioning uncertainty increases, and a larger number of partitions is beneficial to minimize the combined uncertainty. We show, that by minimizing the subsampling uncertainty in the test system, the quantification uncertainty of low target concentrations in single cell content analysis is much smaller than in body fluid analysis. In summary, the work provides the methodological basis for a profound statistical evaluation of partitioning and subsampling effects in compartment-based quantification methods and paves the way towards an improved design of future digital quantification devices for highly accurate molecular diagnostic analysis at the point-of-care.

Influence of the Available Surface Area and Cell Elasticity on Bacterial Adhesion Forces on Highly Ordered Silicon Nanopillars.

Initial bacterial adhesion to solid surfaces is influenced by a multitude of different factors, e.g., roughness and stiffness, topography on the micro- and nanolevel, as well as chemical composition and wettability. Understanding the specific influences and possible interactive effects of all of these factors individually could lead to guidance on bacterial adhesion and prevention of unfavorable consequences like medically relevant biofilm formation. On this way, the aim of the present study was to identify the specific influence of the available surface area on the adhesion of clinically relevant bacterial strains with different membrane properties: Gram-positive Staphylococcus aureus and Gram-negative Aggregatibacter actinomycetemcomitans. As model surfaces, silicon nanopillar specimens with different spacings were fabricated using electron beam lithography and cryo-based reactive ion etching techniques. Characterization by scanning electron microscopy and contact angle measurement revealed almost defect-free highly ordered nanotopographies only varying in the available surface area. Bacterial adhesion forces to these specimens were quantified by means of single-cell force spectroscopy exploiting an atomic force microscope connected to a microfluidic setup (FluidFM). The nanotopographical features reduced bacterial adhesion strength by reducing the available surface area. In addition, the strain-specific interaction in detail depended on the bacterial cell's elasticity and deformability as well. Analyzed by confocal laser scanning microscopy, the obtained results on bacterial adhesion forces could be linked to the subsequent biofilm formation on the different topographies. By combining two cutting-edge technologies, it could be demonstrated that the overall bacterial adhesion strength is influenced by both the simple physical interaction with the underlying nanotopography and its available surface area as well as the deformability of the cell.

Enhancing the soft-tissue integration of dental implant abutments-in vitro study to reveal an optimized microgroove surface design to maximize spreading and alignment of human gingival fibroblasts.

Within this work, we demonstrate the influences of different microgrooved surface topographies on the alignment and spreading of human gingival fibroblast (HGF) cells and present the optimal parameters for an improved soft-tissue integration design for dental implant abutments for the first time. Microgrooves with lateral widths from 2.5 to 75 µm were fabricated by UV-lithography and wet etching on bulk Ti6Al4V ELI material. The microstructured surfaces were compared to polished and ground surfaces as current state of the art. The resulting microtopographies were analyzed using vertical scanning interferometry and scanning electron microscopy. Samples loaded with HGF cells were incubated for 8 and 72 hr and cell orientation, spreading, resulting area, and relative gene expression were analyzed. The effect of contact guidance occurred on all microstructured surfaces yet there is a clear preferable range for the lateral widths of the microgrooves between approx. 11.5 and 13.9 µm and depths between 1.6 and 2.4 µm for an abutment surface design, where cell orientation and spreading maximizes. For structures larger than 30 µm, cell orientation, spreading and even gene expression of intercellular adhesion molecule-1 and yes-associated protein decrease.

New Concept of Patient-specific Flow Diversion Treatment of Intracranial Aneurysms : Design Aspects and in vitro Fluid Dynamics.

PURPOSE: Current flow diverter (FD) designs limit the possibilities to achieve ideal functional parameters for intra-aneurysmal flow alteration in the implanted state. In this work, we evaluate the technical feasibility of a new patient-specific FD concept and the impact on intra-aneurysmal flow reduction compared to standard FD. METHODS: Based on a literature review, we defined functional requirements, followed by the design and manufacturing of two different prototypes, which we implanted in a patient-specific phantom model. Functional porosity distributions and contour parameters were evaluated in the implanted state and compared to standard FD. Subsequently, we carried out a series of particle image velocimetry (PIV) measurements, in order to assess the impact on intra-aneurysmal flow. RESULTS: With both patient-specific prototypes, it was possible to achieve stronger intra-aneurysmal flow reductions in terms of maximum and mean velocity and vorticity than a standard FD; however, one design showed a strong sensitivity against malpositioning. Overall, fluid dynamics parameters correlated with geometrical aspects such as the porosity and its grade of homogeneity. Beyond that, we found influences by the FD contour projection within the aneurysm, especially connected to the formation of in-jets. CONCLUSION: Our results show that there is a technically feasible concept, which enables a more specific adjustment of functional FD parameters and more effective intra-aneurysmal flow reduction. This could potentially lead to improvements in the efficacy of aneurysm occlusion in cases with challenging fluid dynamics.

Integration of digital microfluidics with whispering-gallery mode sensors for label-free detection of biomolecules.

We present a multi-sensor chip comprising an array of whispering-gallery mode (WGM) micro-goblet lasers integrated into a digital microfluidic (DMF) system. In contrast to earlier demonstrations, the lasers are fabricated from dye-doped poly-methyl methacrylate (PMMA) at low cost using spin-coating, mask-based optical lithography, wet chemical etching, and thermal reflow techniques. Pumping and read-out of the devices is accomplished via simple free-space optics, thereby allowing large-scale sensor arrays to be addressed. We demonstrate the viability of the system by bulk refractive index-sensing and by measuring the specific binding of streptavidin to a biotinylated sensor surface. This is the first time that optical cavities are used for label-free detection of biomolecules in a DMF system. This approach can be extended to a versatile detector platform that targets a wide range of clinically relevant biomolecules.

Time-resolved NMR metabolomics of plant cells based on a microfluidic chip.

The plant secondary metabolism generates numerous compounds harbouring pharmaceutical activity. In plants, these compounds are typically formed by different and specialised cell types that have to interact constituting a metabolic process chain. This interactivity impedes biotechnological production of secondary compounds, because cell differentiation is suppressed under the conditions of a batch bio-fermenter. We present a novel strategy to address this limitation using a biomimetic approach, where we simulate the situation in a real tissue by a microfluidic chamber system, where plant cells can be integrated into a process flow. We show that walled cells of the plant model tobacco BY-2 can be successfully cultivated in this system and that physiological parameters (such as cell viability, mitotic index and division synchrony) can be preserved over several days. The microfluidic design allows to resolve dynamic changes of specific metabolites over different stages of culture development. These results serve as proof-of-principle that a microfluidic organisation of cultivated plant cells can mimic the metabolic flows in a real plant tissue.

Fabrication of polymeric microfluidic devices with tunable wetting behavior for biomedical applications.

We demonstrate the fabrication of microchannels with specific fluidic behavior due to micro- and/or nanostructures on the surfaces. With a combination of hot embossing and microthermoforming it is possible to produce microchannels with specific surface properties. These surface properties are highly dependent on the micro- and nanostructures embossed into the material. Different structure sizes and geometries where examined by contact angle measurements. Here the dependency of diameter and pitch of the structures on the contact angle is examined as well as the material impact. These results enable the fabrication of highly specific surfaces tunable to an application.

Formation of a polymer surface with a gradient of pore size using a microfluidic chip.

Here we demonstrate the generation of polymer monolithic surfaces possessing a gradient of pore and polymer globule sizes from ~0.1 to ~0.5 µm defined by the composition of two polymerization mixtures injected into a microfluidic chip. To generate the gradient, we used a PDMS microfluidic chip with a cascade micromixer with a subsequent reaction chamber for the formation of a continuous gradient film. The micromixer has zigzag channels of 400 × 680 µm(2) cross section and six cascades. The chip was used with a reversible bonding connection, realized by curing agent coating. After polymerization in the microfluidic chip the reversible bond was opened, resulting in a 450 µm thick polymer film possessing the pore size gradient. The gradient formation in the microfluidic reaction chamber was studied using microscopic laser-induced fluorescence (µLIF) and different model fluids. Formation of linear gradients was shown using the fluids of the same density by both diffusive mixing at flow rates of 0.001 mL/min and in a convective mixing regime at flow rates of 20 mL/min. By using different density fluids, formation of a two-dimensional wedge-like gradient controlled by the density difference and orientation of the microfluidic chip was observed.

Diffusion- and convection-based activation of Wnt/ß-catenin signaling in a gradient generating microfluidic chip.

Stem cells and developing tissues respond to long-range signaling molecules (morphogens), by starting different nuclear programs that decide about the cell fate. Cells sense the local morphogen concentration and the shape of the gradient. We developed a two-chambered microfluidic chip to reproduce the in vivo situation under shear stress free conditions. The gradient is generated in the lower part of our device and recognized by cells grown in the upper part in the microchamber. We tested our device by activating the Wnt/ß-catenin signaling pathway in HeLa cells as proven by nuclear ß-catenin accumulation in response to the Wnt pathway activator 6-bromoindirubin-3'-oxime (BIO). Applying the same readout system to a recombinant Wnt3a and Dkk-1 bipolar gradient we demonstrate that our microfluidic chip is suitable for morphogens as well as small molecules. More interestingly, our microfluidic device is highly flexible. While the generated gradients are stable for several hours and reproducible, we can change the kind and the shape of the gradient actively on demand. We also can switch from diffusion- to convection-based transport, thus applying the morphogen gradient either in a polarized or non-polarized manner.

Microfabrication of chip-sized scaffolds for three-dimensional cell cultivation.

Using microfabrication technologies is a prerequisite to create scaffolds of reproducible geometry and constant quality for three-dimensional cell cultivation. These technologies offer a wide spectrum of advantages not only for manufacturing but also for different applications. The size and shape of formed cell clusters can be influenced by the exact and reproducible architecture of the microfabricated scaffold and, therefore, the diffusion path length of nutrients and gases can be controlled.1 This is unquestionably a useful tool to prevent apoptosis and necrosis of cells due to an insufficient nutrient and gas supply or removal of cellular metabolites. Our polymer chip, called CellChip, has the outer dimensions of 2 x 2 cm with a central microstructured area. This area is subdivided into an array of up to 1156 microcontainers with a typical dimension of 300 m edge length for the cubic design (cp- or cf-chip) or of 300 m diameter and depth for the round design (r-chip).2 So far, hot embossing or micro injection moulding (in combination with subsequent laborious machining of the parts) was used for the fabrication of the microstructured chips. Basically, micro injection moulding is one of the only polymer based replication techniques that, up to now, is capable for mass production of polymer microstructures.3 However, both techniques have certain unwanted limitations due to the processing of a viscous polymer melt with the generation of very thin walls or integrated through holes. In case of the CellChip, thin bottom layers are necessary to perforate the polymer and provide small pores of defined size to supply cells with culture medium e.g. by microfluidic perfusion of the containers. In order to overcome these limitations and to reduce the manufacturing costs we have developed a new microtechnical approach on the basis of a down-scaled thermoforming process. For the manufacturing of highly porous and thin walled polymer chips, we use a combination of heavy ion irradiation, microthermoforming and track etching. In this so called "SMART" process (Substrate Modification And Replication by Thermoforming) thin polymer films are irradiated with energetic heavy projectiles of several hundred MeV introducing so-called "latent tracks" Subsequently, the film in a rubber elastic state is formed into three dimensional parts without modifying or annealing the tracks. After the forming process, selective chemical etching finally converts the tracks into cylindrical pores of adjustable diameter.