Electric Cell-Substrate Impedance Sensing of Cellular Effects under Hypoxic Conditions and Carbonic Anhydrase Inhibition

Tumor hypoxia provides a dynamic environment for the cancer cells to thrive and metastasize. Evaluation of cell growth, cell-cell, and cell surface interactions in hypoxic conditions is therefore highly needed in the establishment of treatment options. Electric cell-substrate impedance sensing (ECIS) has been traditionally used in the evaluation of cellular platforms as a real-time, label-free impedance-based method to study the activities of cells grown in tissue cultures, but its application for hypoxic environments is seldom reported. We present real-time evaluation of hypoxia-induced bioeffects with a focus on hypoxic pH regulation of tumor environment. To this end, multiparametric real-time bioanalytical platform using electrical impedance spectroscopy (EIS) and human colon cancer HT-29 cells is advanced. A time series of EIS data enables monitoring with high temporal resolution the alterations occurring within the cell layer, especially at the cell-substrate level. We reveal the dynamic changes of cellular processes during hypoxic conditions and in response to application of acetazolamide (AZA), a carbonic anhydrase inhibitor. Optical evaluation and pH assessment complemented the electrical analysis towards establishing a pattern of cellular changes. The proposed bioanalytical platform indicates wide applicability towards evaluation of bioeffects of hypoxia at cellular level

Electrochemical push-pull probe: from scanning electrochemical microscopy (SECM) to multimodal altering of cell microenvironment

To understand biological processes at the cellular level, a general approach is to alter the cells’ environment and to study their chemical responses. Herein, we present the implementation of an electrochemical push-pull probe, which combines a microfluidic system with a microelectrode, as a tool for locally altering the microenvironment of few adherent living cells by working in two different perturbation modes, namely electrochemical (i.e. electrochemical generation of a chemical effector compound) and microfluidic (i.e. infusion of a chemical effector compound from the pushing microchannel, while aspirating it through the pulling channel thereby focusing the flow between the channels). The effect of several parameters such as flow rate, working distance and probe inclination angle on the affected area of adherently growing cells was investigated both theoretically and experimentally. As a proof of concept, localized fluorescent labeling and pH changes were purposely introduced to validate the probe as a tool for studying adherent cancer cells through the control over the chemical composition of the extracellular space with high spatiotemporal resolution. A very good agreement between experimental and simulated results showed for instance, that the electrochemical perturbation mode enables to affect precisely only few living cells localized in a high-density cell culture

Relating membrane potential to impedance spectroscopy

Non-invasive, label-free assessment of membrane potential of living cells is still a challenging task. The theory linking membrane potential to the low frequency &alpha; dispersion exhibited by suspensions of spherical shelled particles (presenting a net charge distribution on the inner side of the shell) has been pioneered in our previous studies with emphasis on the permittivity spectra. Whereas &alpha; dispersion is related to a rather large variation exhibited by the permittivity spectrum, we report that the related decrement presented by the impedance magnitude spectrum is either extremely small, or occurs (for large cells) at very small frequencies (~mHz) explaining the lack of experimental bioimpedance data on the matter. We stress that appropriate choice of the parameters (as revealed by the microscopic model) may enable access to membrane potential as well as to other relevant parameters when investigating living cells and charged lipid vesicles. We analyse the effect on the low frequency of the permittivity and impedance spectra of: I. Parameters pertaining to cell membrane i.e. (i) membrane potential (through the amount of the net charge on the inner side of the membrane), (ii) size of the cells/vesicles, (iii) conductivity of the membrane; II. Parameters of the extra cellular medium (viscosity and conductivity). The applicability of the study has far reaching implications for basic (life) sciences (providing non-invasive access to the dynamics of relevant cell parameters) as well as for biosensing applications, e.g. assessment of cytotoxicity of a wide range of stimuli. <p><a href="http://dx.doi.org/10.5617/jeb.214" target="_blank">doi:10.5617/jeb.214 </a><br />J Electr Bioimp, vol. 2, pp. 93-97, 2011</p

Real-Time Monitoring of Cell Cyclic Progression by Dielectric Spectroscopy (INTERFACE SCIENCE-Molecular Aggregates)

A dielectric technique has been developed for monitoring of cell cycle progression in synchronized culture, which would be a promising tool for cell cycle analysis in cell biology and biotechnology

Real time SPR assessment of the structural changes of adaptive dynamic constitutional frameworks as a new route for sensing

International audienceCross linked gold-dynamic constitutional frameworks (DCFs) are functional materials of potential relevance for biosensing applications, given their adaptivity and high responsivity against various external stimuli (such as pH, temperature) or specific interactions with biomolecules (enzymes or DNA) via internal constitutional dynamics. However, characterization and assessment of their dynamic conformational changes in response to external stimuli has never been reported. This study proves the capability of Surface Plasmon Resonance (SPR) assays to analyze the adaptive structural modulation of a functional matrix encompassing 3D gold-dynamic constitutional frameworks (Au-DCFs) when exposed to pH variations, as external stimuli. We analyze Au-DCFs formed from Au nano-particles, (AuNP) connected through constitutionally dynamic polymers, dynamers, with multiple functionalities. For increased generality of this proof-ofconcept assay, Au-DCFs, involving DCFs designed from 1,3,5-benzene-tricarbaldehyde (BTA) connecting centers and polyethylene glycol (PEG) connectors, are covalently attached to standard SPR sensing chips (Au nanolayers, carboxyl terminated or with carboxymethyl dextran, CMD toplayer) and analyzed using state-of-the art SPR instrumentation. The SPR effects of the distance 24 from the Au-DCFs matrix to the Au nanolayer of the sensing chip as well as of Au-DCFs thickness 25 were investigated. This study reveals the SPR response, augmented by the AuNP, to the confor-26 mational change, i.e. shrinkage, of the Dynamer & AuNP matrix when decreasing the pH and 27 provides an unexplored insight on the sensing applicability of SPR real-time analysis of adaptive 28 functional materials

High-resolution impedance mapping using electrically activated quantitative phase imaging

Retrieving electrical impedance maps at the nanoscale rapidly via nondestructive inspection with a high signal-to noise ratio is an unmet need, likely to impact various applications from biomedicine to energy conversion. In this study, we develop a multimodal functional imaging instrument that is characterized by the dual capability of impedance mapping and phase quantitation, high spatial resolution, and low temporal noise. To achieve this, we advance a quantitative phase imaging system, referred to as epi-magnified image spatial spectrum microscopy combined with electrical actuation, to provide complementary maps of the optical path and electrical impedance. We demonstrate our system with high-resolution maps of optical path differences and electrical impedance variations that can distinguish nanosized, semi-transparent, structured coatings involving two materials with relatively similar electrical properties. We map heterogeneous interfaces corresponding to an indium tin oxide layer exposed by holes with diameters as small as ~550 nm in a titanium (dioxide) over-layer deposited on a glass support. We show that electrical modulation during the phase imaging of a macro-electrode is decisive for retrieving electrical impedance distributions with submicron spatial resolution and beyond the limitations of electrode-based technologies (surface or scanning technologies). The findings, which are substantiated by a theoretical model that fits the experimental data very well enable achieving electro-optical maps with high spatial and temporal resolutions. The virtues and limitations of the novel optoelectrochemical method that provides grounds for a wider range of electrically modulated optical methods for measuring the electric field locally are critically discussed