Temperature effect on the build-up of exponentially growing polyelectrolyte multilayers. An exponential-to-linear transition point.

In this study, the effect of temperature on the build-up of exponentially growing polyelectrolyte multilayer films was investigated. It aims at understanding the multilayer growth mechanism as crucially important for the fabrication of tailor-made multilayer films. Model poly(L-lysine)/hyaluronic acid (PLL/HA) multilayers were assembled in the temperature range of 25-85 °C by layer-by-layer deposition using a dipping method. The film growth switches from the exponential to the linear regime at the transition point as a result of limited polymer diffusion into the film. With the increase of the build-up temperature the film growth rate is enhanced in both regimes; the position of the transition point shifts to a higher number of deposition steps confirming the diffusion-mediated growth mechanism. Not only the faster polymer diffusion into the film but also more porous/permeable film structure are responsible for faster film growth at higher preparation temperature. The latter mechanism is assumed from analysis of the film growth rate upon switching of the preparation temperature during the film growth. Interestingly, the as-prepared films are equilibrated and remain intact (no swelling or shrinking) during temperature variation in the range of 25-45 °C. The average activation energy for complexation between PLL and HA in the multilayers calculated from the Arrhenius plot has been found to be about 0.3 kJ mol(-1) for monomers of PLL. Finally, the following processes known to be dependent on temperature are discussed with respect to the multilayer growth: (i) polymer diffusion, (ii) polymer conformational changes, and (iii) inter-polymer interactions.

Real-time monitoring of oxygen uptake in hepatic bioreactor shows CYP450-independent mitochondrial toxicity of acetaminophen and amiodarone.

Prediction of drug-induced toxicity is complicated by the failure of animal models to extrapolate human response, especially during assessment of repeated dose toxicity for cosmetic or chronic drug treatments. In this work, we present a 3D microreactor capable of maintaining metabolically active HepG2/C3A spheroids for over 28 days in vitro under stable oxygen gradients mimicking the in vivo microenvironment. Mitochondrial respiration was monitored using two-frequency phase modulation of phosphorescent microprobes embedded in the tissue. Phase modulation is focus independent and unaffected by cell death or migration. This sensitive measurement of oxygen dynamics revealed important information on the drug mechanism of action and transient subthreshold effects. Specifically, exposure to antiarrhythmic agent, amiodarone, showed that both respiration and the time to onset of mitochondrial damage were dose dependent showing a TC50 of 425 µm. Analysis showed significant induction of both phospholipidosis and microvesicular steatosis during long-term exposure. Importantly, exposure to widely used analgesic, acetaminophen, caused an immediate, reversible, dose-dependent loss of oxygen uptake followed by a slow, irreversible, dose-independent death, with a TC50 of 12.3 mM. Transient loss of mitochondrial respiration was also detected below the threshold of acetaminophen toxicity. The phenomenon was repeated in HeLa cells that lack CYP2E1 and 3A4, and was blocked by preincubation with ascorbate and TMPD. These results mark the importance of tracing toxicity effects over time, suggesting a NAPQI-independent targeting of mitochondrial complex III might be responsible for acetaminophen toxicity in extrahepatic tissues.

Microprocessor-based integration of microfluidic control for the implementation of automated sensor monitoring and multithreaded optimization algorithms.

Microfluidic applications range from combinatorial synthesis to high throughput screening, with platforms integrating analog perfusion components, digitally controlled micro-valves and a range of sensors that demand a variety of communication protocols. Currently, discrete control units are used to regulate and monitor each component, resulting in scattered control interfaces that limit data integration and synchronization. Here, we present a microprocessor-based control unit, utilizing the MS Gadgeteer open framework that integrates all aspects of microfluidics through a high-current electronic circuit that supports and synchronizes digital and analog signals for perfusion components, pressure elements, and arbitrary sensor communication protocols using a plug-and-play interface. The control unit supports an integrated touch screen and TCP/IP interface that provides local and remote control of flow and data acquisition. To establish the ability of our control unit to integrate and synchronize complex microfluidic circuits we developed an equi-pressure combinatorial mixer. We demonstrate the generation of complex perfusion sequences, allowing the automated sampling, washing, and calibrating of an electrochemical lactate sensor continuously monitoring hepatocyte viability following exposure to the pesticide rotenone. Importantly, integration of an optical sensor allowed us to implement automated optimization protocols that require different computational challenges including: prioritized data structures in a genetic algorithm, distributed computational efforts in multiple-hill climbing searches and real-time realization of probabilistic models in simulated annealing. Our system offers a comprehensive solution for establishing optimization protocols and perfusion sequences in complex microfluidic circuits.

Measurement of surface-mediated Ca2+ transients on the single-cell level in a microfluidic lab-on-a-chip environment.

Understanding the dynamics of signal transduction processes that are induced by cell-cell or cell-surface interactions requires the physical stimulation of the cells of interest on a single-cell level and without any ill-defined contacting of their cell membrane. However, standard cell culture techniques are inapplicable for this task as they do not provide cell and particle handling at sufficiently high spatial and temporal resolution and are limited to ensemble measurements. Here, we present a novel process line for the individual stimulation of single cells with bioactive surfaces, like other cells or particles, and the simultaneous analysis of the induced cytosolic calcium signals. The method is based on a microfluidic lab-on-a-chip environment that allows for contactless cell and particle handling by dielectrophoretic forces.

Long-term microfluidic glucose and lactate monitoring in hepatic cell culture.

Monitoring cellular bioenergetic pathways provides the basis for a detailed understanding of the physiological state of a cell culture. Therefore, it is widely used as a tool amongst others in the field of in vitro toxicology. The resulting metabolic information allows for performing in vitro toxicology assays for assessing drug-induced toxicity. In this study, we demonstrate the value of a microsystem for the fully automated detection of drug-induced changes in cellular viability by continuous monitoring of the metabolic activity over several days. To this end, glucose consumption and lactate secretion of a hepatic tumor cell line were continuously measured using microfluidically addressed electrochemical sensors. Adapting enzyme-based electrochemical flat-plate sensors, originally designed for human whole-blood samples, to their use with cell culture medium supersedes the common manual and laborious colorimetric assays and off-line operated external measurement systems. The cells were exposed to different concentrations of the mitochondrial inhibitor rotenone and the cellular response was analyzed by detecting changes in the rates of the glucose and lactate metabolism. Thus, the system provides real-time information on drug-induced liver injury in vitro.

Highly controlled electrofusion of individually selected cells in dielectrophoretic field cages.

The prospect of novel therapeutic approaches has renewed the current interest in the fusion of rare cells, like stem cells or primary immune cells. While conventional techniques are only capable of mass fusion, lab-on-a-chip systems often still lack an acceptable method for making the cells available after processing. Here, we present a microfluidic approach for electrofusion on the single-cell level that offers high control over the cells both before and after fusion. For cell pairing and fusion, we employed dielectrophoresis and AC voltage pulses, respectively. Each cell has been characterized and selected before they were paired, fused and released from the fluidic system for subsequent analysis and cultivation. The successful experimental evaluation of our system was further corroborated by numerical simulations. We obtained fusion efficiencies of more than 30% for individual pairs of mouse myeloma and B cell blasts and showed the proliferating ability of the hybrid cells 3 d after fusion. Since aggregates of more than two cells can be fused, the technique could also be developed further for generating giant cells for low-noise electrophysiology in the context of semi-automated pharmaceutical screening procedures.

Dielectrophoretic stretching of cells allows for characterization of their mechanical properties.

The mechanical behavior of biological cells is mainly determined by the cytoskeleton. Its properties are closely interlinked with many cellular events, including disease-related processes, and, thus, may be exploited as potent biomarkers. We have stretched two types of cells between microelectrodes through the application of dielectrophoretic forces. Small numbers of cells of cancerous origin (MCF-7) and from related noncancerous tissue (MCF-10A) were sufficient to obtain data that allowed us to unambiguously distinguish these cells. The Maxwell tension applied has been estimated to be 56 Pa. A detailed analysis of the cells showed that the differences in the stretching response are due to cell-specific mechanical properties. Through the addition of an actin- and a microtubule-specific toxin to the cells, differences in the microtubular structures of the two cell types have been identified as the major cause for the behavior observed. Our approach shows enormous potential for parallelization and automation. Hence, it should be suitable for achieving throughputs that make it attractive for many biomedical diagnostic purposes.

Influence of medium consumption on cell elasticity.

The mechanical behavior of a living cell is highly dynamic and constantly adapts to its local environment. Changes in temperature and chemical stimuli, such as pH, may alter the structure of the cell and its mechanical response. Thus, the mechanical properties may serve as an indicator for the cellular state. We applied dielectrophoretic forces to suspension cells by means of two microelectrodes. The resultant stretching was analyzed on consecutive cultivation days with respect to the influence of medium consumption. Systematic experiments clearly showed that the medium consumption affected the viscoelastic properties of the investigated human leukemia cells HL-60. The shift in pH value and the culture medium depletion were identified as potentially responsible for the differing temporal development of the cell deformation. Both factors were investigated separately and a detailed analysis indicated that the changes observed in the cellular stiffness were primarily attributable to nutrient depletion.

Accumulation and filtering of nanoparticles in microchannels using electrohydrodynamically induced vortical flows.

We present an approach for the accumulation and filtering of nano- and microparticles in microfluidic devices that is based on the generation of electric traveling waves in the radio-frequency range. Upon application of the electric field via a microelectrode array, complex particle trajectories and particle accumulation are observed in well-defined regions in a microchannel. Through the quantitative mapping of the 3-D flow pattern using two-focus fluorescence cross-correlation spectroscopy, two vortices could be identified as one of the sources of the force field that induces the formation of particle clouds. Dielectrophoretic forces that directly act on the particles are the second source of the force field. A thorough 2-D finite element analysis identifies the electric traveling wave mechanism as the cause for the unexpected flow behavior observed. Based on these findings, strategies are discussed, first, for avoiding the vortices to optimize electrohydrodynamic micropumps and, secondly, for utilizing the vortices in the development of microdevices for efficient particle accumulation, separation, and filtering. Such devices may find numerous biomedical applications when highly diluted nano- and microsuspensions have to be processed.

The structure and functionality of contractile forisome protein aggregates.

Although they have been discovered decades ago, only in the last years forisome protein aggregates received broader attention due to their ability to convert chemical into mechanical energy. In contrast to most other motor proteins, these proteins from Fabaceae plants are independent of high-energy chemical compounds, like e.g. ATP, but undergo an anisotropic shape transition (longitudinally expanded to contracted) in response to ion concentration changes (Ca(2+), H(+), etc.), instead. We present morphological and functional data on forisomes obtained using atomic force microscopy (AFM). High-aspect ratio AFM tips allow the detailed elucidation of structural characteristics that are inaccessible with standard AFM tips. Microindentation measurements were employed to calculate the elasticity of the forisome material. Young's moduli were found to be approximately 32.7 kPa in the expanded state and approximately 2.748 kPa in the contracted state of the polymer. These results are compared to investigations where a tipless AFM cantilever was utilized to exert a load against the shape transition. In the latter experiments, an energy conversion of approximately 2.29 pJ per stroke was detected. Energetical considerations support the hypothesis that the switching process is accompanied by a change in cross-linking of the constituent subunits and allow estimating the extent of cooperativity during the pH-induced transition. Finally, useful parameters were identified and characterized that are crucial for the application of forisomes as functional elements in microfluidic chips.