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In this report electrostatic force microscopy (EFM) is used to study different peptide self-assembled structures such as tubes and particles. It is shown that not only geometrical information can be obtained using EFM, but also information about the composition of different structures. In particular we use EFM to investigate the structures of diphenylalanine peptide tubes, particles, and CSGAITIG peptide particles placed on pre-fabricated SiO2 surfaces with a backgate. We show that the cavity in the peptide tubes could be due to the presence of water residues. Additionally we show that self-assembled amyloid peptides form spherical solid structures containing the same self-assembled peptide in its interior. In both cases transmission electron microscopy is used to verify these structures. Further, the limitations of the EFM technique are discussed, especially when the observed structures become small compared with the radius of the AFM tip used. Finally, an agreement between the detected signal and the structure of the hollow peptide tubes is demonstrated. SCANNING 33: 201–207, 2011. © 2011 Wiley Periodicals, Inc.

Fluorescence in situ Hybridization (FISH) is a major cytogenetic technique for clinical genetic diagnosis of both inherited and acquired chromosomal abnormalities. Although FISH techniques have evolved and are often used together with other cytogenetic methods like CGH, PRINS and PNA-FISH, the process continues to be a manual, labour intensive, expensive and time consuming technique, often taking over 3 5 days, even in dedicated labs. We have developed a novel microFISH device to perform metaphase FISH on a chip which overcomes many shortcomings of the current laboratory protocols. This work also introduces a novel splashing device for preparing metaphase spreads on a microscope glass slide, followed by a rapid adhesive tape-based bonding protocol leading to rapid fabrication of the microFISH device. The microFISH device allows for an optimized metaphase FISH protocol on a chip with over a 20-fold reduction in the reagent volume. This is the first demonstration of metaphase FISH on a microfluidic device and offers a possibility of automation and significant cost reduction of many routine diagnostic tests of genetic anomalies.

In this paper we discuss the fabrication and characterization of three dimensional (3D) micro- and nanoelectrodes with the goal of using them for extra- and intracellular studies. Two different types of electrodes will be described: high aspect ratio microelectrodes for studying the communication between cells and ultimately for brain slice recordings and small nanoelectrodes for highly localized measurements and ultimately for intracellular studies. Electrical and electrochemical characterization of these electrodes as well as the results of PC12 cell differentiation on chip will be presented and discussed.

In this work we report a novel three dimensional electrode array for electrochemical measurements in neuronal studies. The main advantage of working with these out-of-plane structures is the enhanced sensitivity of the system in terms of measuring electrochemical changes in the environment of a cell culture in real time. In addition, the system is devised to offer a compact solution that helps to obtain a homogeneous distribution of current density among the active electrodes.

This work is dedicated to developing a novel three dimensional structure for electrochemical measurements in neuronal studies. The final prototype will allow not only for the study and culture on chip of neuronal cells, but also of brain tissue. The use of out-of-plane electrodes instead of planar ones increases the sensitivity of the system and increases the signal-to-noise ratio in the recorded signals, due to the higher availability of surface area. The main bottleneck of the out-of-plane electrode fabrication lies in the metallization process for transforming them into active electrodes, since the coverage of the side walls of almost vertical pillars is not trivial by standard processes in a clean room facility. This paper will discuss the different steps taken towards this goal and present the results that we have obtained so far.

Self-assembled amyloid peptide nanotubes (SAPNT) were manipulated and immobilized using dielectrophoresis. Micro-patterned electrodes of Au were fabricated by photolithography and lifted off on a silicon dioxide layer. SAPNT were manipulated by adjusting the amplitude and frequency of the applied voltage. The immobilized SAPNT were evaluated by SEM and atomic force microscopy. The conductivity of the immobilized SAPNT was studied by I-V characterization, for both single SAPNT and bundles. This work illustrates a way to manipulate and integrate biological nanostructures into novel bio-nanoassemblies with concrete applications, such as field-effect transistors, microprobes, microarrays, and biosensing devices.

A novel strategy to sort the cells of interest (White Blood Cells (leukocytes)) by selectively lysing the Red Blood Cells (erythrocytes) in a miniaturized microfluidic device is presented. Various methods to lyse cells on a chip exist i.e. electrical, mechanical, chemical and thermal but they need integration of electrodes, traps, reservoirs, heaters, etc which is often difficult at microscale [1 – 4]. On the other hand, FACSlyse protocol uses only osmotic pressure to lyse erythrocytes allowing further isolation of leukocytes. This motivated us to develop a novel herringbone based lyser which works on the principle of mixing whole blood with pure water in time controlled manner to lyse erythrocytes osmotically on a chip.

Networks of single-walled carbon nanotubes were assembled onto microelectrodes by dielectrophoresis. The dependence of the obtained networks on several assembly parameters such as bias voltage, field application time, frequency, electrode geometry and the nanotube solvent were investigated both structurally and electrically. Reproducible differences in morphological and electrical properties were observed for the parameters investigated. Application of a bias voltage above 10 V for more than 30 seconds with nanotubes in an SDS solution, resulted in dense networks with a relatively low resistance in the 10 kΩ regime. On the other hand, individual nanotubes and bundles were assembled with lower voltages applied for less than 10 seconds and with other nanotubes solutions. The experimental results were combined with theoretical calculations in order to find a geometry and voltage independent threshold field for the successful assembly of nanotubes between electrodes using dielectrophoresis.

Scanning conductance microscopy investigations were carried out in air on human chromosomes fixed on pre-fabricated SiO2 surfaces with a backgate. The point of the investigation was to estimate the dielectric constant of fixed human chromosomes in order to use it for microfluidic device optimization. The phase shift caused by the electrostatic forces, together with geometrical measurements of the atomic force microscopy (AFM) cantilever and the chromosomes were used to estimate a value for the dielectric constant of different human chromosomes.

Within each cell cycle, a cell must ensure that the processes of selection of replication origins (licensing) and initiation of DNA replication are well coordinated to prevent reinitiation of DNA replication from the same DNA segment during the same cell cycle. This is achieved by restricting the licensing process to G1 phase when the prereplicative complexes (preRCs) are assembled onto the origin DNA, while DNA replication is initiated only during S phase when de novo preRC assembly is blocked. Cdt1 is an important member of the preRC complex and its tight regulation through ubiquitin-dependent proteolysis and binding to its inhibitor Geminin ensure that Cdt1 will only be present in G1 phase, preventing relicensing of replication origins. We have recently reported that Cdt1 associates with chromatin in a dynamic way and recruits its inhibitor Geminin onto chromatin in vivo. Here we discuss how these dynamic Cdt1-chromatin interactions and the local recruitment of Geminin onto origins of replication by Cdt1 may provide a tight control of the licensing process in time and in space.

The ever-increasing complexity of the fabrication process of Point-of-care (POC) devices, due to high demand of functional versatility, compact size and ease-of-use, emphasizes the need of multifunctional materials that can be used to simplify this process. Polymers, currently in use for the fabrication of the often needed microfluidic channels, have limitations in terms of their physicochemical properties. Therefore, the use of a multipurpose biocompatible material with better resistance to the chemical, thermal and electrical environment, along with capability of forming closed channel microfluidics is inevitable. This paper demonstrates a novel technique of fabricating microfluidic devices using polyimide (PI) which fulfills the aforementioned properties criteria. A fabrication process to pattern microfluidic channels, using partially cured PI, has been developed by using a dry etching method. The etching parameters are optimized and compared to those used for fully cured PI. Moreover, the formation of closed microfluidic channel on wafer level by bonding two partially cured PI layers or a partially cured PI to glass with high bond strength has been demonstrated. The reproducibility in uniformity of PI is also compared to the most commonly used SU8 polymer, which is a near UV sensitive epoxy resin. The potential applications of PI processing are POC and biosensor devices integrated with microelectronics.

A surface modification of interdigitated gold microelectrodes (IDEs) with a doped polypyrrole (PPy) film for detection of dopamine released from populations of differentiated PC12 cells is presented. A thin PPy layer was potentiostatically electropolymerized from an 10 aqueous pyrrole solution onto electrode surfaces. The conducting polymer film was doped during electropolymerization by introducing counter ions in the monomer solution. Several counter ions were tested and the resulting electrode modifications were characterized electrochemically to find the optimal dopant that increases sensitivity in dopamine detection. Overoxidation of the PPy films was shown to contribute to a significant enhancement in sensitivity to dopamine. The changes caused by overoxidation in the electrochemical behavior and electrode morphology were investigated using cyclic voltammetry and SEM as well as AFM, respectively. The optimal dopant for dopamine detection was found to be polystyrenesulfonate anion (PSS-15 ). Rat pheochromocytoma (PC12) cells, a suitable model to study exocytotic dopamine release, were differentiated on IDEs functionalized with an overoxidized PSS--doped PPy film. The modified electrodes were used to amperometrically detect dopamine released by populations of cells upon triggering cellular exocytosis with an elevated K+ concentration. A comparison between the generated current on bare gold electrodes and gold electrodes modified with overoxidized doped PPy illustrates the clear advantage of the modification, yielding 2.6-fold signal amplification. The results also 20 illustrate how to use cell population based dopamine exocytosis measurements to obtain biologically significant information that can be relevant in, for instance, the study of neural stem cell differentiation into dopaminergic neurons.

More and more diseases find their cause in malfunctioning genes. There is therefore still need for rapid, low-cost and direct methods to accurately perform genetic analysis. Currently the process takes a long time to complete and is very expensive. We are proposing a system that will be able to isolate white blood cells from blood, lyse them in order to extract the chromosomes and then perform chromosome sorting on chip. As the physical properties of the chromosomes, such as size and dielectric properties, are needed for designing the chip, we have measured them using an AFM microscope.

A versatile microfluidic, multichamber cell culture and analysis system with an integrated electrode array and potentiostat suitable for electrochemical detection and microscopic imaging is presented in this paper. The system, which allows on-line electrode cleaning and modification, was developed for real-time monitoring of cellular dynamics, exemplified in this work by monitoring of redox metabolism inside living yeast cells and dopamine release from PC12 cells.

Silicon nanowire (SiNW) field effect transistor based biosensors have already been proven to be a promising tool to detect biomolecules. However, the most commonly used fabrication techniques involve expensive Silicon-On-Insulator (SOI) wafers, E-beam lithography and ion-implantation steps. In the work presented here, a top down approach to fabricate SiNW junctionless field effect biosensors using novel in-situ doped polysilicon is demonstrated. The p-type polysilicon is grown with an optimum boron concentration that gives a good metal-silicon electrical contact while maintaining the doping level at a low enough level to provide a good sensitivity for the biosensor. The silicon nanowires are patterned using standard photolithography and a wet etch method. The metal contacts are made from magnetron sputtered TiW and e-beam evaporation of gold. The passivation of electrodes has been done by sputtered Si3N4 which is patterned by a lift-off process. The characterization of the critical fabrication steps is done by Secondary Ion Mass Spectroscopy (SIMS) and by statistical analysis of the measurements made on the width of the SiNWs. The electrical characterization of the SiNW in air is done by sweeping the back gate voltage while keeping the source drain potential to a constant value and surface characterization is done by applying liquid gate in phosphate buffered saline (PBS) solution. The fabricated SiNWs sensors functionalized with (3-aminopropyl)triethoxysilane (APTES) have demonstrated good sensitivity in detecting different pH buffer solutions. Keywords: In-situ doped, Polysilicon nanowire, Field effect transistor, Biosensor

In this work, we present the development of a transparent poly(methyl methacrylate) (PMMA) based microfluidic culture system for handling long-term brain slice cultures independent of an incubator. The different stages of system development have been validated by culturing GFP producing brain slices from 8-day old (P8) mouse pups. Fluorescence microscopic monitoring of GFP was utilized as an indicator of tissue viability. The final format of the developed system, featuring ”plug-and-play” technolgy with a reusable fluidic connection board and easily changeable microfluidic chips, facilitated brain slice culturing for 16 days.

A device for concentration of nanoparticles and/or microparticles in liquid flow conditions by dielectrophoresis is disclosed in this invention.