How fishes are able to detect trace molecules in large bodies of water is not understood. It is plausible that they use olfactory receptors to detect water-soluble compounds. How the zebra fish Danio Rerio, an organism with only 98 functional olfactory receptors, is able to selectively detect and recognize numerous compounds in water remains a puzzling phenomenon. We are interested in studying the biochemical and molecular mechanisms of olfaction in fish. Here, we report on the study of a bioengineered zebra fish olfactory receptor OR131-2, affinity-purified from a HEK293S tetracycline-inducible system. This receptor was expressed and translocated to the cell plasma membrane as revealed by confocal microscopy. Circular dichroism spectroscopy showed that the purified zebra fish receptor folded into an α-helical structure, as observed for other G-protein coupled receptors (GPCRs). Our study shows that it is possible to produce viable quantities of the zebra fish olfactory receptor. This will not only enable detailed structural and functional analyses, but also aid in the design of biosensor devices in order to detect water-soluble metabolites or its intermediates, which are associated with human health.

Micromachined nanoporous membrane for cell culture is one of the promising applications for biomolecular separation and immunoisolation, in which Silicon Nitride (SiNx) receives particular interest due to its several advantages compared to polymeric membranes. Our works focused on fabricating nanoporous SiNx layer with near zero residual stress. This paper investigated the influence of Radio Frequency (RF), power and reactant gases to the SiNx deposition rates and residual stress, and developed the optimised process to produce near zero stress SiNx layer. By applying this technique, nanoporous SiNx membranes have been fabricated and tested for cell culture. By cultivating the mouse D1 mesenchymal stem cells on top of the nanoporous membrane, the results showed that mouse D1 mesenchymal stem cells were able to grow well. This shows that the nanoporous membrane can be used as the platform for interfacing with living cells to become biocapsules for biomolecular separation and immunoisolation.

The extracellular matrix (ECM) is a rich meshwork of proteins and proteoglycans. Besides assuming a cell adhesive and structural support role, the ECM also helps to sequester and present growth factors to cells. ECM derived from tissues has been used as biological scaffolds for tissue engineering. In contrast, it has been difficult to employ ECM derived from cell lines as scaffolds due to its lack of form and structure. We have developed a mild, aqueous-based method for incorporating cell line derived ECM into biological scaffolds based on polyelectrolyte complexation, using the example of ECM from MC-3T3, a mouse preosteoblast cell line. A DNase step was incorporated in the ECM isolation procedure to further purify it of genetic material. Immunohistochemistry of fibers incorporated with MC-3T3 ECM reveal the presence of the ECM components, collagen type I, collagen type IV, fibronectin and heparan sulfate, on their surface. Reconstituted ECM scaffolds retained the cell-adhesion characteristics of the ECM, as demonstrated by ‘reseeding’ the ECM-secreting cell on the scaffolds. Human mesenchymal stem cells (hMSCs) were seeded onto the fibrous scaffolds incorporated with MC-3T3 ECM, and implanted subcutaneously into SCID mice. After 4 weeks of implantation, histological evidence showed that the hMSC seeded ECM scaffolds had induced bone formation at the ectopic site.

We have created a porous bioresorbable nanocomposite bone scaffold that chemically, structurally and mechanically matched natural bone so that it could be recognized and remodeled by natural bone. Containing collagen fibers and synthetic apatite nanocrystals, our scaffold has high strength for supporting the surrounding tissue. The foam-like scaffold has a similar microstructure as trabecular bone, with nanometer-sized and micron-sized pores. The apatitic phase of the scaffold exhibited similar chemical composition, crystalline phase and grain size as the trabecular bone apatite. The nanocomposite scaffold demonstrated excellent bioactivity for promoting cell attachment and proliferation. It was osteoconductive and successfully healed a non-union fracture in rat femur as well as a critical-sized defect in pig tibia.

The paper presents a novel microfluidic device for identification and characterization of cells in suspensions using impedance spectroscopy. The device consists of two glass wafers: a bottom wafer comprising a microfluidic channel with two electrodes added for impedance measurement, and a top glass wafer in which inlets and outlets are realized. The fact that the device is glass-based provides a few key advantages: reduced influence from parasitic components during measurements (due to the good isolation properties of the substrate), optical transparency and hydrophilic surface of the microfluidic channel. The latter feature is especially important as it enables sample suction due to capillarity forces only. Thus, no external pumping is required and only a small volume sample suffices for the measurement.The fabrication process of this device consists of three major steps. First, via-holes and inlet/outlet holes are executed in the top glass wafer by wet etching in a 49% HF solution using a low stress amorphous silicon/silicon carbide/photoresist mask. Second, the microfluidic channel is etched into the bottom wafer and Ti/Pt electrodes are then patterned on top of it using a spray coating-based lithography. The last processing step is bonding together the top and bottom glass wafers by employing a very thin adhesive intermediate layer (SU8). This adhesive layer was applied selectively only on the bottom die, from a Teflon cylinder, using a contact imprinting method.Finally, fabricated devices were successfully tested using DI water, phosphate buffer saline (PBS), and various types of both dead cells and living cells resuspended in PBS. Clear differences between dead and live cells have been observed. The impedance measurements were carried out in the frequency range 5 kHz to 10 MHz. The measured magnitude and phase were studied using different types of cells in Dulbecco's Minimal Essential medium (DMEM). The obtained impedance spectra revealed the characteristic spectra signature for each type of cell.

A field-flow dielectrophoretic separation method in a 3D filtering chip has been developed in this work. The separation method was possible due to the special configuration of the DEP filtering chip, which has a structure similar to a classical capacitor with two parallel plate electrodes (realized by using a stainless steel mesh) and a dielectric medium (defined by a suspension of 100 µm diameter silica beads in buffer solution). The dielectrophoretic phenomenon is generated by the non-uniformities of the dielectric media, which produce a gradient of the electric field and, as a consequence, a DEP force. If a suspension medium with cells flows through the filter, the DEP force can trap these cells around the contact points between the silica beads (if the cells exhibit positive DEP) or they are repelled into the space between the beads (if the cells exhibit negative DEP). It is shown that for two different cell populations, the frequency of the electric field and permittivity of the media can be tuned in such a way that one population will exhibit positive DEP and the other one negative DEP. The population that expresses negative DEP can be easily flushed out due to the hydrodynamic force which is larger at the center point between the beads. In such a way two cell populations can be separated. The working principle was verified with both live and dead yeast cells. Best results for the separation of viable and nonviable cell populations were achieved at an applied voltage of 150 V in a frequency range between 10 kHz and 20 kHz for flow rates of 0.1 ml min−1 and 0.2 ml min−1. With a few of these devices cascaded in series, higher efficiency could be achieved. As a result, this device and the associated proposed separation method can be very useful tools for bio-pharmaceutical industries since continuous flow separation at relatively high flow rates is both time and cost saving.

A novel method for fabricating recyclable hydrophilic-hydrophobic micropatterns on glass chips is presented. TiOx patterns (100-2000 mu m) were sputtered on glass chips via a through-hole mask. The patterned chips were then vapor-coated with fluoroalkylsilane, for example, (heptadecafluoro-1,1,2,2-tetrahydrodecyl)triethoxysilane (FTES) to form a hydrophobic coating layer. The fluoroalkyl chain of FTES film on TiOx patterns was photocleaved under UV irradiation, exposing the fresh hydrophilic TiOx patterns. The resulting chip could be used multiple times by repeating the coating and photocleaving processes with negligible deterioration of the hydrophobic FTES film coated on glass. If desired, bare glass patterns could also be generated by removing the TiOx patterns with KOH. The patterned glass chips have been successfully used for microarray fabrication.

The paper reports the realization of a glass-based microfluidic device for characterization of cells in suspensions using impedance spectroscopy. The device consists of two glass wafers: a bottom wafer comprising a microfluidic channel with two electrodes added for impedance measurement, and a top glass wafer in which inlets and outlets are realized. The main focus of this work is the original fabrication process of this device, which combines three key techniques: firstly, successfully applying a through-wafer wet etching method in order to pattern the inlets and outlets in the top glass wafer; secondly, patterning the electrodes not only on flat surfaces but also in the microfluidic channel etched in the bottom wafer; and thirdly, employing indirect wafer to wafer bonding using an intermediate polymer layer. No external pumping is required as capillarity enables direct suction of a sample droplet in the channel. The device can be reusable if a thorough cleaning procedure is carried out.Devices with three different electrode geometries were successfully tested in electrochemical impedance spectroscopy (EIS) measurements using DI water and HepG2 cells. Although clear differences between DI water and live HepG2 cells have been observed in all cases, different results were obtained for various electrode geometries, highlighting the critical importance of the device design in performing EIS measurements and especially when making comparisons with other reported results. (c) 2006 Elsevier B.V. All rights reserved.