Development of an Aptamer-Based QCM-D Biosensor for the Detection of Thrombin Using Supported Lipid Bilayers as Surface Functionalization.

Biosensors play an important role in numerous research fields. Quartz crystal microbalances with dissipation monitoring (QCM-Ds) are sensitive devices, and binding events can be observed in real-time. In combination with aptamers, they have great potential for selective and label-free detection of various targets. In this study, an alternative surface functionalization for a QCM-D-based aptasensor was developed, which mimics an artificial cell membrane and thus creates a physiologically close environment for the binding of the target to the sensor. Vesicle spreading was used to form a supported lipid bilayer (SLB) of 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphethanolamine-N-(cap biotinyl) (biotin-PE). The SLB was then coated with streptavidin followed by applying a biotinylated aptamer against thrombin. SLB formation was investigated in terms of temperature and composition. Temperatures of 25 °C and below led to incomplete SLB formation, whereas a full bilayer was built at higher temperatures. We observed only a small influence of the content of biotinylated lipids in the mixture on the further binding of streptavidin. The functionalization of the sensor surface with the thrombin aptamer and the subsequent thrombin binding were investigated at different concentrations. The sensor could be reconstituted by incubation with a 5 M urea solution, which resulted in the release of the thrombin from the sensor surface. Thereafter, it was possible to rebind thrombin. Thrombin in spiked samples of human serum was successfully detected. The developed system can be easily applied to other target analytes using the desired aptamers.

Vascular surveillance by haptotactic blood platelets in inflammation and infection.

Breakdown of vascular barriers is a major complication of inflammatory diseases. Anucleate platelets form blood-clots during thrombosis, but also play a crucial role in inflammation. While spatio-temporal dynamics of clot formation are well characterized, the cell-biological mechanisms of platelet recruitment to inflammatory micro-environments remain incompletely understood. Here we identify Arp2/3-dependent lamellipodia formation as a prominent morphological feature of immune-responsive platelets. Platelets use lamellipodia to scan for fibrin(ogen) deposited on the inflamed vasculature and to directionally spread, to polarize and to govern haptotactic migration along gradients of the adhesive ligand. Platelet-specific abrogation of Arp2/3 interferes with haptotactic repositioning of platelets to microlesions, thus impairing vascular sealing and provoking inflammatory microbleeding. During infection, haptotaxis promotes capture of bacteria and prevents hematogenic dissemination, rendering platelets gate-keepers of the inflamed microvasculature. Consequently, these findings identify haptotaxis as a key effector function of immune-responsive platelets.

Extrusion texturization of cricket flour and soy protein isolate: Influence of insect content, extrusion temperature, and moisture-level variation on textural properties.

Due to the increasing global population and unsustainable meat production, the future supply of animal-derived protein is predicted to be insufficient. Currently, edible insects are considered as a potential and "novel" source of protein in the development of palatable meat analogues. This research used high moisture extrusion cooking (HMEC), at a screw speed of 150 rpm, to produce meat analogues using full- or low-fat cricket flours (CF) and soy protein isolate (SPI). Effects of water flow rate (WFR), cooking temperature (9 and 10 ml/min; 120, 140, and 160°C, respectively), and CF inclusions levels of 0, 15, 30, and 45% were analyzed. Cooking temperature and CF inclusion had a significant effect (p < .05) on both tensile stress in parallel and perpendicular directions, while WFR had no significant effect (p = .3357 and 0.7700), respectively. The tensile stress increased with temperature but decreased with CF inclusion at both WFRs. Comparatively, the tensile stress was stronger at WFR of 9 ml/min than at 10 ml/min; however, the tensile stress in parallel was mostly greater than tensile stress in perpendicular directions. Fibrous meat analogues with high anisotropic indices (AIs) of up to 2.80 were obtained, particularly at WFR of 10 ml/min and at inclusions of 30% low-fat CF. By controlling HMEC conditions, full-/low-fat cricket flours at 15% and 30% inclusions can offer an opportunity to partially substitute SPI in manufacturing of fibrous meat analogues.

Direct Comparison of Physical Properties of Bacillus subtilis NCIB 3610 and B-1 Biofilms.

Many bacteria form surface-attached communities known as biofilms. Due to the extreme resistance of these bacterial biofilms to antibiotics and mechanical stresses, biofilms are of growing interest not only in microbiology but also in medicine and industry. Previous studies have determined the extracellular polymeric substances present in the matrix of biofilms formed by Bacillus subtilis NCIB 3610. However, studies on the physical properties of biofilms formed by this strain are just emerging. In particular, quantitative data on the contributions of biofilm matrix biopolymers to these physical properties are lacking. Here, we quantitatively investigated three physical properties of B. subtilis NCIB 3610 biofilms: the surface roughness and stiffness and the bulk viscoelasticity of these biofilms. We show how specific biomolecules constituting the biofilm matrix formed by this strain contribute to those biofilm properties. In particular, we demonstrate that the surface roughness and surface elasticity of 1-day-old NCIB 3610 biofilms are strongly affected by the surface layer protein BslA. For a second strain,B. subtilis B-1, which forms biofilms containing mainly γ-polyglutamate, we found significantly different physical biofilm properties that are also differently affected by the commonly used antibacterial agent ethanol. We show that B-1 biofilms are protected from ethanol-induced changes in the biofilm's stiffness and that this protective effect can be transferred to NCIB 3610 biofilms by the sole addition of γ-polyglutamate to growing NCIB 3610 biofilms. Together, our results demonstrate the importance of specific biofilm matrix components for the distinct physical properties of B. subtilis biofilms.

Phenomenological approaches to collective behavior in epithelial cell migration.

Collective cell migration in epithelial tissues resembles fluid-like behavior in time-lapse recordings. In the last years, hydrodynamic velocity fields in living matter have been studied intensely. The emergent properties were remarkably similar to phenomena known from active soft matter systems. Here, we review migration experiments of large cellular ensembles as well as of mesoscopic cohorts in micro-structured environments. Concepts such as diffusion, velocity correlations, swirl strength and polarization are metrics to quantify the cellular dynamics both in experiments as well as in computational simulations. We discuss challenges relating collective migration to single cell and oligocellular behavior as well as linking the phenotypic parameters to the underlying cytoskeleton dynamics and signaling networks. This article is part of a Special Issue entitled: Mechanobiology.

Marker-free detection of progenitor cell differentiation by analysis of Brownian motion in micro-wells.

The kinetics of stem and progenitor cell differentiation at the single-cell level provides essential clues to the complexity of the underlying decision-making circuits. In many hematopoietic progenitor cells, differentiation is accompanied by the expression of lineage-specific markers and by a transition from a non-adherent to an adherent state. Here, using the granulocyte-macrophage progenitor (GMP) as a model, we introduce a label-free approach that allows one to follow the course of this transition in hundreds of single cells in parallel. We trap single cells in patterned arrays of micro-wells and use phase-contrast time-lapse movies to distinguish non-adherent from adherent cells by an analysis of Brownian motion. This approach allowed us to observe the kinetics of induced differentiation of primary bone-marrow-derived GMPs into macrophages. The time lapse started 2 hours after addition of the cytokine M-CSF, and nearly 80% of the population had accomplished the transition within the first 20 h. The analysis of Brownian motion proved to be a sensitive and robust tool for monitoring the transition, and thus provides a high-throughput method for the study of cell differentiation at the single-cell level.

Flow and diffusion in channel-guided cell migration.

Collective migration of mechanically coupled cell layers is a notable feature of wound healing, embryonic development, and cancer progression. In confluent epithelial sheets, the dynamics have been found to be highly heterogeneous, exhibiting spontaneous formation of swirls, long-range correlations, and glass-like dynamic arrest as a function of cell density. In contrast, the flow-like properties of one-sided cell-sheet expansion in confining geometries are not well understood. Here, we studied the short- and long-term flow of Madin-Darby canine kidney (MDCK) cells as they moved through microchannels. Using single-cell tracking and particle image velocimetry (PIV), we found that a defined averaged stationary cell current emerged that exhibited a velocity gradient in the direction of migration and a plug-flow-like profile across the advancing sheet. The observed flow velocity can be decomposed into a constant term of directed cell migration and a diffusion-like contribution that increases with density gradient. The diffusive component is consistent with the cell-density profile and front propagation speed predicted by the Fisher-Kolmogorov equation. To connect diffusion-mediated transport to underlying cellular motility, we studied single-cell trajectories and occurrence of vorticity. We discovered that the directed large-scale cell flow altered fluctuations in cellular motion at short length scales: vorticity maps showed a reduced frequency of swirl formation in channel flow compared with resting sheets of equal cell density. Furthermore, under flow, single-cell trajectories showed persistent long-range, random-walk behavior superimposed on drift, whereas cells in resting tissue did not show significant displacements with respect to neighboring cells. Our work thus suggests that active cell migration manifests itself in an underlying, spatially uniform drift as well as in randomized bursts of short-range correlated motion that lead to a diffusion-mediated transport.

Arraying cell cultures using PEG-DMA micromolding in standard culture dishes.

A robust and effortless procedure is presented, which allows for the microstructuring of standard cell culture dishes. Cell adhesion and proliferation are controlled by three-dimensional poly(ethylene glycol)-dimethacrylate (PEG-DMA) microstructures. The spacing between microwells can be extended to millimeter size in order to enable the combination with robotic workstations. Cell arrays of microcolonies can be studied under boundary-free growth conditions by lift-off of the PEG-DMA layer in which the growth rate is accessible via the evolution of patch areas. Alternatively, PEG-DMA stencils can be used as templates for plasma-induced patterning.

Dynamic changes of acoustic load and complex impedance as reporters for the cytotoxicity of small molecule inhibitors.

Cellular motility is the major driving force of numerous biological phenomena including wound healing, immune response, embryogenesis, cancer formation, and metastasis. We studied the response of epithelial FaDu monolayers cultured on gold electrodes of an acoustic resonator (quartz crystal microbalance, QCM) and impedance sensor (electric cell-substrate impedance sensing, ECIS) to externally applied chemical stimuli interfering with cytoskeleton organization. Epithelial cell motility of confluent monolayers is characterized by subtle cell shape changes and variations in the cell-substrate as well as cell-cell distance without net directionality of individual cells. The impact of small molecules such as cytochalasin D, phalloidin, and blebbistatin as well as paclitaxel, nocodazol, and colchicin on actin and microtubules organization was quantified by conventional sensors' readouts and by comparing the noise pattern of the signals which is attributed to cellular dynamics. The responsiveness of noninvasive and label-free techniques relying on cellular dynamics is compared to classical viability assays and changes of the overall impedance of ultrasmall electrodes or acoustic loads of a thickness shear mode resonator. Depending on the agent used, a distinct sensor response was found, which can be used as a fingerprint of the cellular response. Cytoskeletal rearrangements and nuclear integrity were corroborated by fluorescence microscopy and correlated to the readouts of QCM and ECIS.

Dynamics of human cancer cell lines monitored by electrical and acoustic fluctuation analysis.

Early determination of the metastatic potential of cancer cells is a crucial step for successful oncological treatment. Besides the remarkable progress in molecular genomics- or proteomics-based diagnostics, there is a great demand for in vitro biosensor devices that allow rapid and selective detection of the invasive properties of tumor cells. Here, the classical cancer cell motility in vitro assays for migration and invasion relying on Boyden chambers are compared to a real-time biosensor that analyzes the dynamic properties of adherent cells electro-acoustically with a time resolution on the order of seconds. The sensor relies on the well-established quartz crystal microbalance technique (QCM) that measures the shift in resonance frequency and damping of an oscillating quartz crystal when adsorption, desorption or changes in material properties close to the quartz surface occur. In addition, the QCM is capable of detecting the rather subtle fluctuations of the cell bodies as an indicator for their micromotility. QCM-based micromotility readings of three different cancer cell lines (HT-29, HSC-4, FaDu) are compared with the well-known electrical cell-substrate impedance sensing (ECIS) revealing collective stochastic motion that corresponds to the malignancy of the cells.

Inhibitory effects of trans-resveratrol analogs molecules on the proliferation and the cell cycle progression of human colon tumoral cells.

Resveratrol may function as a cancer chemopreventive agent. However, few data are available on the antitumoral activities of its dimer, epsilon-viniferin, also present in human diet. So, the effects of resveratrol, epsilon-viniferin, of their acetylated forms (resveratrol triacetate, epsilon-viniferin pentaacetate) and of vineatrol (a wine grape extract) were compared on human adenocarcinoma colon cells. Resveratrol and resveratrol triacetate inhibit cell proliferation and arrest cell cycle. epsilon-Viniferin and epsilon-viniferin pentaacetate slightly reduce cell proliferation. Vineatrol inhibits cell proliferation and favors an accumulation in the S phase of the cell cycle. Consequently, resveratrol triacetate and vineatrol could constitute new putative anticancer agents on colon carcinoma.