Nick Fishelson

Cell-based screening for membranal and cytoplasmatic markers using dielectric spectroscopy

Dielectric spectroscopy (DS) of living biological cells is based on the analysis of the complex dielectric permittivity of cells suspended in a physiological medium. It provides knowledge on the polarization-relaxation response of cells to external electric field as function of the excitation frequency. This response is strongly affected by both structural and molecular properties of cells and therefore, can reveal rare insights on cell physiology and behaviour. This study demonstrates the mapping potential of DS after cytoplasmatic and membranal markers for cell-based screening analysis. The effect of membrane permittivity and cytoplasm conductivity was examined using tagged MBA and MDCK cell lines respectively. Comparing the permittivity spectra of tagged and native cell lines reveals clear differences between the analyzed suspensions. In addition, differences on the matching dielectric properties of cells were obtained. Those findings support the high distinction resolution and sensitivity of DS after fine molecular and cellular changes, and hence, highlight the high potential of DS as non invasive screening tool in cell biology research.

Theoretical examination of aggregation effect on the dielectric characteristics of spherical cellular suspension.

Dielectric dispersion analysis of cellular suspension is generally based on the analogy to equivalent periodic material made up of identical inclusions. However, under true physiological conditions, when coupling and aggregation events usually occur, this analogy can introduce severe errors when attempting to probe the dielectric characteristics of the suspended fraction. In the framework of this study, a theoretical examination of the effect of aggregation on the dielectric characteristics of spherical cellular suspension is presented. Here, small clusters of coupled and fused (gap connected) shelled spheres were used to imitate the presence of aggregates when suspended in a homogenous suspension of spherical cells. The permittivity spectra of the aggregate-cell mixtures were numerically calculated by applying computational solution of complex potential problem using 3D Boundary Element Method. The dispersion characteristics of the mixtures have been determined as function of both aggregates shape and concentration. Those reveal significant deviations in comparison to the characteristics of homogenous cellular suspension. Quantitative analyses of the induced fields and transmembrane potential gradients of the interacted cells suggest that those deviations are mainly induced due to changes occur on the polarization state of the membranes.

Dielectric dispersion of suspended cells using 3D reconstructed morphology model

In the framework of this study, novel method for dispersion analysis of cellular suspensions is presented. The method is fundamentally based on the ability to reconstruct the exact 3D morphology of a given cell with resolution accuracy of few nanometers using AFM imaging. By applying a reverse engineering approach, the morphology of the cell is constructed based on a set of measured spatial points that describes its geometry. The permittivity spectrum of the reconstructed cell is then directly derived based on computational solution of complex potential problem using 3D Boundary Element Method. The applicability of the method is demonstrated both theoretically and experimentally with tight comparison to the well known shell models. This comparison reveals significant deviations between the models, and hence, emphasises the vast effect of morphology in dispersion analysis of cellular suspensions.

The effect of irregularity on the dielectric dispersion characteristics of spherical cellular suspension

The dielectric dispersion characteristics of cellular suspensions are fundamentally determined based on the analogy to composite dielectric materials when periodically and discrete arrangement of cells is assumed. However, under native physiological conditions, when flocculation and clamping events usually occur, those assumptions are usually not valid. In the framework of this study, an examination of irregularity effect on the dispersion characteristics of spherical cellular suspensions is presented. Here, the permittivity spectra of the suspensions have been determined by both measurements of living K562 cell suspensions and finite numerical simulations. Based on the measured and simulated spectra, the dispersion characteristics of the suspensions, for several destinies and arrangements of cells, have been quantitatively analyzed using the Havriliak-Negami empirical formula. Generally, a strong correlation between the low dispersion characteristics was observed as the concentration and density of the cells was increased. In addition, all characteristics found to be significantly deviated in comparison to the characteristics of a periodically arrayed suspension. However, when low-dense arrangement was assumed, the correlation found to be much lower when all characteristics found to be less perturbated. Based on a simple model of interacting cells, it is suggested that those deviations are related to intercellular interactions between adjacent cells.

Dielectric screening of early differentiation patterns in mesenchymal stem cells induced by steroid hormones

In the framework of this study, target identification and localization of differentiation patterns by means of dielectric spectroscopy is presented. Here, a primary pre-osteoblastic bone marrow-derived MBA-15 cellular system was used to study the variations in the dielectric properties of mesenchymal stem cells while exposed to differentiation regulators. Using the fundamentals of mixed dielectric theories combined with finite numerical tools, the permittivity spectra of MBA-15 cell suspensions have been uniquely analyzed after being activated by steroid hormones to express osteogenic phenotypes. Following the spectral analysis, significant variations were revealed in the dielectric properties of the activated cells in comparison to the untreated populations. Based on the differentiation patterns of MBA-15, the electrical modifications were found to be highly correlated with the activation of specific cellular mechanisms which directly react to the hormonal inductions. In addition, by describing the dielectric dispersion in terms of transfer functions, it is shown that the spectral perturbations are well adapted to variations in the electrical characteristics of the cells. The reported findings vastly emphasize the tight correlation between the cellular and electrical state of the differentiated cells. It therefore emphasizes the vast abilities of impedance-based techniques as potential screening tools for stem cell analysis.

Electrochemical Micro Technologies for Polymeric MEMS and Biochip Applications

The overall objective of our research is to develop polymeric based MEMS technologies for medical and healthcare applications. We focus on various methods of integration of both organic and inorganic conductors on polymeric substrates. Applications are interconnects and microelectrodes on polymer substrates, low-k polymers for interconnects, organic polymers for field effect devices, and polymeric MEMS. Over the last two decades, a great deal of research was concerned with patterning of polymer films which can be used as components in molecular electronics, optical devices, etch resists, biosensors and as scaffolds for tissue engineering and fundamental studies in cell biology. In addition to their electrical properties, they offer attractive mechanical properties, such as flexibility and immune to cracking. Among the polymers, the conjugated organic polymers are especially attractive since they offer several advantages over the metals and the conventional inorganic semiconductors, such as more facile processing and ease of adjusting the conductivity in a wide range. They can be considered as potential alternatives to the metals and semiconductors as connecting wires and conductive channels, which can be used as active materials in optoelectronics, microelectronics, micro electromechanical systems (MEMS) and sensors. We developed a photolithography process for electrodes on organic substrate with conductive polymers. We study the electrochemical polymerization of polypyrrole by cyclic voltametry, first on smooth surfaces of Si covered with Cr /Au layers. Cyclic voltametry curves of the electro polymerization can be seen in Fig.1. In the reverse of the first cycle cathodic process and accordingly considerable cathodic current appears. We assume that during straight half-cycle an oxidized form of pyrrole is formed, it rehabilitates itself, thus results in a cathodic current. With the increase of the cathodic current the volume of the oxidized form increases. The thickness of the polypyrrole was also studied as a function of number of cycles, as can be seen in fig. 2. Subsequently, selective electrochemical polymerization of polypyrrole was performed inside 1μm wide trenches patterned on 15 μm thick photo-definable polymer (SU-8) on sputtered Au/Ti (100nm/5nm) on oxidized silicon. A uniform coating, on the exposed Au at the bottom of the trench, was achieved (see Fig. 3). Future extension of the research may be far reaching by its application to medical and healthcare investigations and treatment. Interfacing biological materials with MEMS devices may make it possible for microelectrodes and MEMS devices to be inserted and act as implanted. This possibility may carry a high application potential. REFERENCES 1. S. Wilson et al. , Materials Science and Engineering R 56 (2007) 1–129 2. A. Moliton and R. C Hiorns, Polymer International vol. 53, no 10 (2004) 1397–1412

Electronically Directed Integration of Whole-Cell Biosensors on Bio-Chips

Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem 91904, Israel This paper presents a whole-cell bio-chip system where viable, functioning cells are deposited onto solid surfaces that are a part of a micro-machined system. The development of such novel hybrid functional sensors depends on the cell deposition methods; in this work new approach integrating live bacterial cells on a bio-chip using electrophoretic deposition is presented. The bio-material deposition technique was characterized under various driving potential and chamber configurations. The deposited bio-mass included genetically engineered bacterial cells generating electrochemically active byproduct upon exposure to toxic materials in the aqueous solution. In this paper we present the deposition apparatus and methods, as well as the characterization results, e.g. signal vs. time and induction factor, of such chips and discussing the highlight and problems of the new deposition method.

Localization of membrane-bond OPN using force spectroscopy analysis

At this study we present molecular recognition method which is based on force spectroscopy analysis for biological markers on the whole cell level. The presented method allows recognition of specific cell surface proteins and receptor sites by nanometer accuracy level. Here we demonstrate specific recognition of membrane-bond Osteopontin (OPN) sites over a whole Preosteogenic cell membrane. By merging specific force detection map of the proteins and topography image of the cell, we create a new image (recognition image), which demonstrate the exact locations of the proteins relative to the cell membrane. The recognition results indicate on the strong affinity between the modified tip and the target molecules, therefore, it enables the use of an AFM as a remarkable nanoscale tracking tool at the whole cell level.