Efficacy of pulsed electromagnetic fields and electromagnetic fields tuned to the ion cyclotron resonance frequency of Ca2+ on chondrogenic differentiation.

Previous studies provide strong evidence for the therapeutic effect of electromagnetic fields (EMFs) on different tissues including cartilage. Diverse exposure parameters applied in scientific reports and the unknown interacting mechanism of EMF with biological systems make EMF studies challenging. In 1985, Liboff proposed that when magnetic fields are tuned to the cyclotron resonance frequencies of critical ions, the motion of ions through cell membranes is enhanced, and thus biological effects appear. Such exposure system consists of a weak alternating magnetic field (B1 ) in the presence of a static magnetic field (B0 ) and depends on the relationship between the magnitudes of B0 and B1 and the angular frequency Ω. The purpose of the present study is to determine the chondrogenic potential of EMF with regards to pulsed EMF (PEMF) and the ion cyclotron resonance (ICR) theory. We used different stimulating systems to generate EMFs in which cells are either stimulated with ubiquitous PEMF parameters, frequently reported, or parameters tuned to satisfy the ICR for Ca2+ (including negative and positive control groups). Chondrogenesis was analysed after 3 weeks of treatment. Cell stimulation under the ICR condition showed positive results in the context of glycosaminoglycans and type II collagen synthesis. In contrast, the other electromagnetically stimulated groups showed no changes compared with the control groups. Furthermore, gene expression assays revealed an increase in the expression of chondrogenic markers (COL2A1, SOX9, and ACAN) in the ICR group. These results suggest that the Ca2+ ICR condition can be an effective factor in inducing chondrogenesis.

Cell-Imprint Surface Modification by Contact Photolithography-Based Approaches: Direct-Cell Photolithography and Optical Soft Lithography Using PDMS Cell Imprints.

New cell-imprint surface modification techniques based on direct-cell photolithography and optical soft lithography using poly(dimethylsiloxane) (PDMS) cell imprints are presented for enhanced cell-based studies. The core concept of engineering materials for cell-based studies is the material's ability to redesign the physicochemical characteristics of the cellular niche. There is a growing interest in direct molding from cells (cell imprinting). These negative copies of cell surface topographies have been shown to affect cell shape and direct mesenchymal stem cells' differentiation. Analyzing the results is however challenging as cells seeded on these substrates do not always end up in a cell pattern, which leads to decreased effectiveness and biased quantification. To gain control over cell seeding into the patterns and avoid unwanted cell population outside of the patterns, the cell-imprinted surface needs to be modified. From this perspective, the standard optical contact lithography process was modified and cells were introduced to the cleanroom. Direct-cell photolithography was used for a single-step PDMS cell-imprint (chondrocytes as the molding template) surface modification down to single-cell (approximately 5 µm in diameter) resolution. As cells come in a variety of shapes, sizes, and optical profiles, a complementary optical soft lithography-based photomask fabrication technique is also reported. The simplicity of the fabrication process makes this cell-imprint surface modification technique compatible with any adherent cell type and leads to efficient cell-based studies.

Dielectrophoresis-assisted creation of cell aggregates under flow conditions using planar electrodes.

We present a microfluidic platform allowing dielectrophoresis-assisted formation of cell aggregates of controlled size and composition under flow conditions. When specific experimental conditions are met, negative dielectrophoresis allows efficient concentration of cells towards electric field minima and subsequent aggregation. This bottom-up assembly strategy offers several advantages with respect to the targeted application: first, dielectrophoresis offers precise control of spatial cell organization, which can be adjusted by optimizing electrode design. Then, it could contribute to accelerate the establishment of cell-cell interactions by favoring close contact between neighboring cells. The trapping geometry of our chip is composed of eight electrodes arranged in a circle. Several parameters have been tested in simulations to find the best configurations for trapping in flow. Those configurations have been tested experimentally with both polystyrene beads and human embryonic kidney cells. The final design and experimental setup have been optimized to trap cells and release the created aggregates on demand.

Quantitative chemical biosensing by bacterial chemotaxis in microfluidic chips.

Whole-cell bacterial bioreporters are proposed as alternatives to chemical analysis of, for example, pollutants in environmental compartments. Commonly based on reporter gene induction, bioreporters produce a detectable signal within 30 min to a few hours after exposure to the chemical target, which is impractical for applications aiming at a fast response. In an attempt to attain faster readout but maintain flexibility of chemical targeting, we explored the concept for quantitative chemical sensing by bacterial chemotaxis. Chemotaxis was quantified from enrichment of cells across a 600 µm-wide chemical gradient stabilized by parallel flow in a microfluidic chip, further supported by transport and chemotaxis steady state and kinetic modelling. As proof-of-concept, we quantified Escherichia coli chemotaxis towards serine, aspartate and methylaspartate as a function of attractant concentration and exposure time. E. coli chemotaxis enrichment increased sharply between 0 and 10 µM serine, before saturating at 100 µM. The chemotaxis accumulation rate was maximal at 10 µM serine, leading to observable cell enrichment within 5 min. The potential application for biosensing of environmental toxicants was investigated by quantifying chemotaxis of Cupriavidus pinatubonensis JMP134 towards the herbicide 2,4-dichlorophenoxyacetate. Our results show that bacterial chemotaxis can be quantified on a scale of minutes and may be used for developing faster bioreporter assays.

An automated microreactor for semi-continuous biosensor measurements.

Living bacteria or yeast cells are frequently used as bioreporters for the detection of specific chemical analytes or conditions of sample toxicity. In particular, bacteria or yeast equipped with synthetic gene circuitry that allows the production of a reliable non-cognate signal (e.g., fluorescent protein or bioluminescence) in response to a defined target make robust and flexible analytical platforms. We report here how bacterial cells expressing a fluorescence reporter ("bactosensors"), which are mostly used for batch sample analysis, can be deployed for automated semi-continuous target analysis in a single concise biochip. Escherichia coli-based bactosensor cells were continuously grown in a 13 or 50 nanoliter-volume reactor on a two-layered polydimethylsiloxane-on-glass microfluidic chip. Physiologically active cells were directed from the nl-reactor to a dedicated sample exposure area, where they were concentrated and reacted in 40 minutes with the target chemical by localized emission of the fluorescent reporter signal. We demonstrate the functioning of the bactosensor-chip by the automated detection of 50 µgarsenite-As l(-1) in water on consecutive days and after a one-week constant operation. Best induction of the bactosensors of 6-9-fold to 50 µg l(-1) was found at an apparent dilution rate of 0.12 h(-1) in the 50 nl microreactor. The bactosensor chip principle could be widely applicable to construct automated monitoring devices for a variety of targets in different environments.

Bi-directional ACET micropump for on-chip biological applications.

The ability to control and pump high ionic strength fluids inside microchannels forms a major advantage for clinical diagnostics and drug screening processes, where high conductive biological and physiological buffers are used. Despite the known potential of AC electro-thermal (ACET) effect in different biomedical applications, comparatively little is known about controlling the velocity and direction of fluid inside the chip. Here, we proposed to discretize the conventional electrodes to form various asymmetric electrode structures in order to control the fluid direction by simple switching the appropriate electric potential applied to the discretized electrodes. The ACET pumping effect was numerically studied by solving electrical, thermal and hydrodynamic multi-physic coupled equations to optimize the geometrical dimensions of the discretized system. PBS solutions with different ionic strength were seeded with 1 µm sized fluorescent particles and electrothermally driven fluid motion was observed inside the channel for different electrode structures. Experimental analyses confirm that the proposed micropump is efficient for a conductivity range between 0.1 and 1 S/m and the efficiency improves by increasing the voltage amplitude. Behavior of the proposed electrode-electrolyte system is discussed by lumped circuit model. Frequency response of system illustrated that the optimal frequency range increases by increasing the conductivity of medium. For 0.18 S/m PBS solution, the constant pumping effect was observed at frequency range between 100 kHz and 1 MHz, while frequency range of 100 kHz to 5 MHZ was observed for 0.42 S/m. The characteristics of experimental results were in good agreement with the theoretical model.

Compact portable biosensor for arsenic detection in aqueous samples with Escherichia coli bioreporter cells.

We present a compact portable biosensor to measure arsenic As(III) concentrations in water using Escherichia coli bioreporter cells. Escherichia coli expresses green fluorescent protein in a linearly dependent manner as a function of the arsenic concentration (between 0 and 100 µg/L). The device accommodates a small polydimethylsiloxane microfluidic chip that holds the agarose-encapsulated bacteria, and a complete optical illumination/collection/detection system for automated quantitative fluorescence measurements. The device is capable of sampling water autonomously, controlling the whole measurement, storing and transmitting data over GSM networks. We demonstrate highly reproducible measurements of arsenic in drinking water at 10 and 50 µg/L within 100 and 80 min, respectively.

Polyimide/SU-8 catheter-tip MEMS gauge pressure sensor.

This paper describes the development of a polyimide/SU-8 catheter-tip MEMS gauge pressure sensor. Finite element analysis was used to investigate critical parameters, impacting on the device design and sensing characteristics. The sensing element of the device was fabricated by polyimide-based micromachining on a flexible membrane, using embedded thin-film metallic wires as piezoresistive elements. A chamber containing this flexible membrane was sealed using an adapted SU-8 bonding technique. The device was evaluated experimentally and its overall performance compared with a commercial silicon-based pressure sensor. Furthermore, the device use was demonstrated by measuring blood pressure and heart rate in vivo.

Miniaturized bacterial biosensor system for arsenic detection holds great promise for making integrated measurement device.

Combining bacterial bioreporters with microfluidics systems holds great promise for in-field detection of chemical or toxicity targets. Recently we showed how Escherichia coli cells engineered to produce a variant of green fluorescent protein after contact to arsenite and arsenate can be encapsulated in agarose beads and incorporated into a microfluidic chip to create a device for in-field detection of arsenic, a contaminant of well known toxicity and carcinogenicity in potable water both in industrialized and developing countries. Cell-beads stored in the microfluidics chip at -20°C retained inducibility up to one month and we were able to reproducibly discriminate concentrations of 10 and 50 µg arsenite per L (the drinking water standards for European countries and the United States, and for the developing countries, respectively) from the blank in less than 200 minutes. We discuss here the reasons for decreasing bioreporter signal development upon increased storage of cell beads but also show how this decrease can be reduced, leading to a faster detection and a longer lifetime of the device.

Development of a microfluidics biosensor for agarose-bead immobilized Escherichia coli bioreporter cells for arsenite detection in aqueous samples.

Contamination with arsenic is a recurring problem in both industrialized and developing countries. Drinking water supplies for large populations can have concentrations much higher than the permissible levels (for most European countries and the United States, 10 µg As per L; elsewhere, 50 µg As per L). Arsenic analysis requires high-end instruments, which are largely unavailable in developing countries. Bioassays based on genetically engineered bacteria have been proposed as suitable alternatives but such tests would profit from better standardization and direct incorporation into sensing devices. The goal of this work was to develop and test microfluidic devices in which bacterial bioreporters could be embedded, exposed and reporter signals detected, as a further step towards a complete miniaturized bacterial biosensor. The signal element in the biosensor is a nonpathogenic laboratory strain of Escherichia coli, which produces a variant of the green fluorescent protein after contact to arsenite and arsenate. E. coli bioreporter cells were encapsulated in agarose beads and incorporated into a microfluidic device where they were captured in 500 × 500 µm(2) cages and exposed to aqueous samples containing arsenic. Cell-beads frozen at -20 °C in the microfluidic chip retained inducibility for up to a month and arsenic samples with 10 or 50 µg L(-1) could be reproducibly discriminated from the blank. In the 0-50 µg L(-1) range and with an exposure time of 200 minutes, the rate of signal increase was linearly proportional to the arsenic concentration. The time needed to reliably and reproducibly detect a concentration of 50 µg L(-1) was 75-120 minutes, and 120-180 minutes for a concentration of 10 µg L(-1).

A virtual valve for smooth contamination-free flow switching.

We present a channel geometry that allows for clean switching between different inlets of a microchip without any contamination of the inlets or the downstream flow. We drive this virtual valve with a pneumatic pressure setup that minimizes disturbance of the downstream flow during the switching procedure by simultaneous variation of the pressures applied to the different inlets. We assess the efficiency of the setup by spectroscopic measurement of downstream dye concentrations, and demonstrate its practical utility by sequentially constructing multiple layers of alginate hydrogel. The method is potentially useful for a whole series of further applications, such as changing perfusion liquids for cell culture and cell analysis, metering, chemical-reaction initiation and multi-sample chromatography, to name a few.

Characterization and optimization of liquid electrodes for lateral dielectrophoresis.

Using the concept of insulator-based "electrodeless" dielectrophoresis, we present a novel geometry for shaping electric fields to achieve lateral deviation of particles in liquid flows. The field is generated by lateral planar metal electrodes and is guided along access channels to the active area in the main channel. The equipotential surfaces at the apertures of the access channels behave as vertical "liquid" electrodes injecting the current into the main channel. The field between a pair of adjacent liquid electrodes generates the lateral dielectrophoretic force necessary for particle manipulation. We use this force for high-speed deviation of particles. By adding a second pair of liquid electrodes, we focus a particle stream. The position of the focused stream can be swept across the channel by adjusting the ratio of the voltages applied to the two pairs. Based on conformal mapping, we provide an analytical model for estimating the potential at the liquid electrodes and the field distribution in the main channel. We show that the simulated particle trajectories agree with observations. Finally, we show that the model can be used to optimize the device geometry in different applications.