Analysis of the sensitivity and frequency characteristics of coplanar electrical cell-substrate impedance sensors

A PDMS-glass based micro-device was designed and fabricated with 12 coplanar impedance sensors integrated for electrical cell-substrate impedance sensing (ECIS). The sensitivity and frequency characteristics of the sensors were investigated both theoretically (equivalent circuit model) and experimentally for the commonly used micro-electrode dimension scale (20-80 microm). The experimental results matched well with the theoretical model analysis and revealed that, within this micro-electrode dimension scale, as the electrode width decreased or as the total electrode length decreased the sensitivity of sensor increased over the whole sensing frequency range, whilst electrode to electrode distance had no influence on sensitivity. Through our frequency characteristics analysis, the whole frequency range could be divided into four parts. New functions describing the dominant components in each frequency range were defined and validated experimentally, and could be used to explain the phenomenon of an ECIS sensing frequency window. The contribution to the impedance measurement of cells growing on the edges of the electrodes was determined for the first time. Finally, novel proposals for ECIS sensor design and ECIS measurements were presented.     

Influence of cell adhesion and spreading on impedance characteristics of cell-based sensors

Impedance measurements of cell-based sensors are a primary characterization route for detection and analysis of cellular responses to chemical and biological agents in real time. The detection sensitivity and limitation depend on sensor impedance characteristics and thus on cell patterning techniques. This study introduces a cell patterning approach to bind cells on microarrays of gold electrodes and demonstrates that single-cell patterning can substantially improve impedance characteristics of cell-based sensors. Mouse fibroblast cells (NIH3T3) are immobilized on electrodes through a lysine-arginine-glycine-aspartic acid (KRGD) peptide-mediated natural cell adhesion process. Electrodes are made of three sizes and immobilized with either covalently bound or physically adsorbed KRGD (c-electrodes or p-electrodes). Cells attached to c-electrodes increase the measurable electrical signal strength by 48.4%, 24.2%, and 19.0% for three electrode sizes, respectively, as compared to cells attached to p-electrodes, demonstrating that both the electrode size and surface chemistry play a key role in cell adhesion and spreading and thus the impedance characteristics of cell-based sensors. Single cells patterned on c-electrodes with dimensions comparable to cell size exhibit well-spread cell morphology and substantially outperform cells patterned on electrodes of other configurations.     

Bioelectrical impedance assay to monitor changes in cell shape during apoptosis

Apoptosis is a strictly regulated and genetically encoded cell 'suicide' that may be triggered by cytokines, depletion of growth factors or certain chemicals. It is morphologically characterized by severe alterations in cell shape like cell shrinkage and disintegration of cell-cell contacts. We applied a non-invasive electrochemical technique referred to as electric cell-substrate impedance sensing (ECIS) in order to monitor the apoptosis-induced changes in cell shape in an integral and quantitative fashion with a time resolution in the order of minutes. In ECIS the cells are grown directly on the surface of small gold-film electrodes (d = 2 mm). From readings of the electrical impedance of the cell-covered electrode, performed with non-invasive, low amplitude sensing voltages, it is possible to deduce alterations in cell-cell and cell-substrate contacts. To improve the sensitivity of this impedance assay we used endothelial cells derived from cerebral micro-vessels as cellular model systems since these are well known to express electrically tight intercellular junctions. Apoptosis was induced by cycloheximide (CHX) and verified by biochemical and cytological assays. The time course of cell shape changes was followed with unprecedented time resolution by impedance readings at 1 kHz and correlated with biochemical parameters. From impedance readings along a broad frequency range of 1-10(6) Hz we could assign the observed impedance changes to alterations on the subcellular level. We observed that disassembly of barrier-forming tight junctions precedes changes in cell-substrate contacts and correlates strongly with the time course of protease activation.     

Chronopotentiometry and Faradaic impedance spectroscopy as signal transduction methods for the biocatalytic precipitation of an insoluble product on electrode supports: routes for enzyme sensors, immunosensors and DNA sensors

The biocatalyzed precipitation of an insoluble product produced on electrode supports is used as an amplification path for biosensing. Enzyme-based electrodes, immunosensors and DNA sensors are developed using this biocatalytic precipitation route. Faradaic impedance spectroscopy and chronopotentiometry are used as transduction methods to follow the precipitation processes. While Faradaic impedance spectroscopy leads to the characterization of the electron-transfer resistance at the electrode, chronopotentiometry provides the total resistance at the interfaces of the modified electrodes. A horseradish peroxidase, HRP, monolayer-functionalized electrode is used to sense H₂O₂ by the biocatalyzed oxidation of 4-chloro-1-naphthol (1), to the insoluble product benzo-4-chlorohexadienone (2). An antigen monolayer electrode is used to sense the dinitrophenyl antibody, DNP-Ab, applying an anti-antibody–HRP conjugate as a biocatalyst for the oxidative precipitation of 1 by H₂O₂ to yield the insoluble product 2. An oligonucleotide (3) functionalized monolayer electrode is used to sense the DNA-analyte (4), that is one of the Tay–Sachs genetic disorder mutants. Association of a biotin-labeled oligonucleotide to the sensing interface, followed by the association of the avidin–HRP conjugate and the biocatalyzed precipitation of 2 leads to the amplified sensing of 4. The amount of the precipitate accumulated on the conductive support is controlled by the concentration of the respective analytes and the time intervals employed for the biocatalytic precipitation of 2. The electron-transfer resistances of the electrodes covered by the insoluble product (2) are derived from Faradaic impedance measurements, whereas the total electrode resistances are extracted from chronopotentiometric experiments. A good correlation between the total electrode resistances and the electron-transfer resistances at the conducting supports are found. Chronopotentiometry is suggested as a rapid transduction means (a few seconds). The precautions needed to apply chronopotentiometry in biosensors are discussed.     

Impedance Changes and Fibrous Tissue Growth after Cochlear Implantation Are Correlated and Can Be Reduced Using a Dexamethasone Eluting Electrode

Background

The efficiency of cochlear implants (CIs) is affected by postoperative connective tissue growth around the electrode array. This tissue formation is thought to be the cause behind post-operative increases in impedance. Dexamethasone (DEX) eluting CIs may reduce fibrous tissue growth around the electrode array subsequently moderating elevations in impedance of the electrode contacts.

Methods

For this study, DEX was incorporated into the silicone of the CI electrode arrays at 1% and 10% (w/w) concentration. Electrodes prepared by the same process but without dexamethasone served as controls. All electrodes were implanted into guinea pig cochleae though the round window membrane approach. Potential additive or synergistic effects of electrical stimulation (60 minutes) were investigated by measuring impedances before and after stimulation (days 0, 7, 28, 56 and 91). Acoustically evoked auditory brainstem responses were recorded before and after CI insertion as well as on experimental days 7, 28, 56, and 91. Additionally, histology performed on epoxy embedded samples enabled measurement of the area of scala tympani occupied with fibrous tissue.

Results

In all experimental groups, the highest levels of fibrous tissue were detected in the basal region of the cochlea in vicinity to the round window niche. Both DEX concentrations, 10% and 1% (w/w), significantly reduced fibrosis around the electrode array of the CI. Following 3 months of implantation impedance levels in both DEX-eluting groups were significantly lower compared to the control group, the 10% group producing a greater effect. The same effects were observed before and after electrical stimulation.

Conclusion

To our knowledge, this is the first study to demonstrate a correlation between the extent of new tissue growth around the electrode and impedance changes after cochlear implantation. We conclude that DEX-eluting CIs are a means to reduce this tissue reaction and improve the functional benefits of the implant by attenuating electrode impedance.     

Monitoring viral-induced cell death using electric cell-substrate impedance sensing

Using an electrical measurement known as electric cell-substrate impedance sensing (ECIS), we have recorded the dynamics of viral infections in cell culture. With this technique, cells are cultured on small gold electrodes where the measured impedance mirrors changes in attachment and morphology of cultured cells. As the cells attach and spread on the electrode, the measured impedance increases until the electrode is completely covered. Viral infection inducing cytopathic effect results in dramatic impedance changes, which are mainly due to cell death. In the current study, two different fish cell lines have been used: chinook salmonid embryonic (CHSE-214) cells infected with infectious pancreatic necrosis virus (IPNV) and epithelioma papulosum cyprini (EPC) carp cells infected with infectious hematopoeitic necrosis virus (IHNV). The impedance changes caused by cell response to virus are easily measured and converted to resistance and capacitance. An approximate linear correlation between log of viral titer and time of cell death was determined.     

Impedance analysis of MDCK cells measured by electric cell-substrate impedance sensing.

Transepithelial impedance of Madin-Darby canine kidney cell layers is measured by a new instrumental method, referred to as electric cell-substrate impedance sensing. In this method, cells are cultured on small evaporated gold electrodes, and the impedance is measured in the frequency range 20-50,000 Hz by a small probing current. A model for impedance analysis of epithelial cells measured by this method is developed. The model considers three different pathways for the current flowing from the electrode through the cell layer: (1) in through the basal and out through the apical membrane, (2) in through the lateral and out through the apical membrane, and (3) between the cells through the paracellular space. By comparing model calculation with experimental impedance data, several morphological and cellular parameters can be determined: (1) the resistivity of the cell layer, (2) the average distance between the basal cell surface and substratum, and (3) the capacitance of apical, basal, and lateral cell membranes. This model is used to analyze impedance changes on removal of Ca2+ from confluent Mardin-Darby canine kidney cell layers. The method shows that reduction of Ca2+ concentration causes junction resistance between cells to drop and the distance between the basal cell surface and substratum to increase.     

Assessment of Rapid Morphological Changes Associated with Elevated cAMP Levels in Human Orbital Fibroblasts

Orbital fibroblasts exhibit a phenotype distinct from that of other types of fibroblasts. Addition of prostaglandin E₂(PGE₂) to culture medium elicits a dramatic change in orbital fibroblast morphology. That response is mediated through the generation of cAMP. Orbital fibroblasts can generate high levels of PGE₂through induction by proinflammatory cytokines of prostaglandin endoperoxide H synthase-2 (PGHS-2). Here we compare the influence on fibroblast morphology of exogenous PGE₂, forskolin, and 8-br-cAMP to that mediated through PGHS-2 induction by a lymphocyte-derived cytokine. Within a few hours, orbital fibroblasts treated with any of these test compounds appear under phase-contrast microscopy to exhibit a stellate morphology. When these changes were assessed quantitatively by electric cell–substrate impedance sensing (ECIS), it became evident that 8-br-cAMP, forskolin, and PGE₂initiated shape changes within 30 min of addition to the culture medium, while effects of the cytokine were first evident after approximately 3.5 h. Dermal fibroblasts failed to respond to any of these compounds with regard to changes in cellular morphology. Analysis of micromotion, manifested as small impedance fluctuations, revealed that orbital fibroblasts treated with 8-br-cAMP exhibit less motion than did untreated cells. These results suggest that orbital fibroblast shape can be altered by several compounds known to alter intracellular cAMP levels. They demonstrate the utility of ECIS in the assessment of very rapid and dynamic cellular events associated with changes in cell morphology.     

Senescence-associated alterations of cytoskeleton: extraordinary production of vimentin that anchors cytoplasmic p53 in senescent human fibroblasts

The cytoskeleton of senescent cells was systematically studied using senescent and young fibroblasts. In the cell senescence, skin fibroblasts extraordinarily produced vimentin in contrast to actin and tubulin, which were down-regulated. Among the focal adhesion proteins, paxillin and c-Src decreased also. Senescent cells developed a long and dense vimentin network, long and thin actin fibers, and numerous small focal contact sites, which contrasted with young cells with short and thick actin stress fibers and prominently large focal adhesions. Noticeably, senescent fibroblasts markedly produced p53 molecules and anchored them to vimentin-cytoskeleton in the cytoplasm. The vimentin-anchored p53 was detected with antibody PAb240 that specifically recognizes a conformation variant of p53. A GFP-tagged wild type p53 cDNA was expressed by transfection and shown also to be retained in the cytoplasm in senescent cells, suggesting that p53 is structurally modified to be recognized by PAb240 and anchored to vimentin filaments. We discuss the correlation of the marked alteration of cytoskeleton and senescent cells diminished proliferation and migration, as well as the significance of cytoskeletal anchorage of tumor suppressor p53.     

Isolation of focal contact membrane using saponin

The fragments of lower cell surface remained attached to the substrate after incubation of mouse or chick fibroblasts in 0.2% saponin solution and subsequent removal of cells under the action of shearing force. These fragments corresponded exactly to the cellular focal contacts seen by interference reflection microscopy. Ultrastructurally they were membrane fragments with typical three-layered structure. No cytoskeletal components were found in saponin-isolated focal contact membranes either by immunofluorescence or electron microscopy. Only one major cell-derived protein with an apparent molecular weight (MW) of 51 kD (chick embryo fibroblasts) or 47 kD (mouse embryo fibroblasts) remained on the substrate after saponin treatment and removal of cells.     

pH sensing by FAK-His58 regulates focal adhesion remodeling

Intracellular pH (pHi) dynamics regulates diverse cellular processes, including remodeling of focal adhesions. We now report that focal adhesion kinase (FAK), a key regulator of focal adhesion remodeling, is a pH sensor responding to physiological changes in pH. The initial step in FAK activation is autophosphorylation of Tyr397, which increased with higher pHi. We used a genetically encoded biosensor to show increased pH at focal adhesions as they mature during cell spreading. We also show that cells with reduced pHi had attenuated FAK-pY397 as well as defective cell spreading and focal adhesions. Mutagenesis studies indicated FAK-His58 is critical for pH sensing and molecular dynamics simulations suggested a model in which His58 deprotonation drives conformational changes that may modulate accessibility of Tyr397 for autophosphorylation. Expression of FAK-H58A in fibroblasts was sufficient to restore defective autophosphorylation and cell spreading at low pHi. These data are relevant to understanding cancer metastasis, which is dependent on increased pHi and FAK activity.     

An endothelial cell compatible biosensor fabricated using optically thin indium tin oxide silicon nitride electrodes

This study describes the fabrication and performance of an endothelial cell compatible, optically thin, indium tin oxide (ITO) microimpedance biosensor. The biosensor was constructed by sputtering a thin insulating layer of silicon nitride (Si(3)N(4)) onto a 100 nm thick ITO layer. Indium tin oxide electrodes were formed by chemically etching 250 or 500 microm diameter holes through the Si(3)N(4) insulating layer. The exposed ITO electrode was electrically connected to an ITO counter electrode, approximately 2 cm(2) in area, via a 400 microL well containing cell culture media. A lock-in amplifier circuit monitored the impedance of porcine pulmonary artery endothelial cells (PPAECs) cultivated on the electrodes as a function of frequency, between 10 and 100 kHz, and as a function of time, at 5.62 kHz. The ITO-Si(3)N(4) microelectrodes provided consistent and repeatable impedance measurements to the attachment and spreading of PPAECs. In addition, the ITO-Si(3)N(4) electrodes were recyclable, robust, resistant to ethanol sterilization, and had a high optical transmittance. Most importantly, the ITO-Si(3)N(4) electrodes allowed optical access for dynamic cellular attachment imaging. The 5.62 kHz time dependent cellular impedance response to the drug Cytochalasin D further demonstrated the feasibility of using this electrode configuration for dynamic cellular impedance studies.     

Fabrication of a sensing module using micromachined biosensors

Micromachining is a powerful tool in constructing micro biosensors and micro systems which incorporate them. A sensing module for blood components was fabricated using the technology. The analytes include glucose, urea, uric acid, creatine, and creatinine. Transducers used to construct the corresponding sensors were a Severinghaus-type carbon dioxide electrode for the urea sensor and a Clark-type oxygen electrode for the other analytes. In these electrodes, detecting electrode patterns were formed on a glass substrate by photolithography and the micro container for the internal electrolyte solution was formed on a silicon substrate by anisotropic etching. A through-hole was formed in the sensitive area, where a silicone gas-permeable membrane was formed and an enzyme was immobilized. The sensors were characterized in terms of pH and temperature dependence and calibration curves along with detection limits. Furthermore, the sensors were incorporated in an acrylate flow cell. Simultaneous operation of these sensors was successfully conducted and distinct and stable responses were observed for respective sensors.     

A novel microfluidic impedance assay for monitoring endothelin-induced cardiomyocyte hypertrophy

Cardiac hypertrophy is an established and independent risk factor for the development of heart failure and sudden cardiac death. At the level of individual cardiac myocytes (heart muscle cells), the cell morphology alters (increase in cell size and myofibrillar re-organization) and protein synthesis is activated. In this paper, a novel cardiomyocyte-based impedance sensing system with the assistance of dielectrophoresis cell concentration is reported to monitor the dynamic process of endothelin-1-induced cardiomyocyte hypertrophy. A dielectrophoresis (DEP) microfluidic device is fabricated capable of concentrating cells from a dilute sample to form a confluent cell monolayer on the surface of microelectrodes. This device can increase the sensitivity of the impedance system and also has the potential to reduce the time for detection by a significant factor. To examine the feasibility of this impedance sensing system, cardiomyocytes are treated with endothelin-1 (ET-1), a known hypertrophic agent. ET-1 induces a continuous rise in cardiomyocyte impedance, which we interpret as strengthening of cellular attachments to the surface substrate. An equivalent circuit model is introduced to fit the impedance spectrum to fully understand the impedance sensing system.     

Targeting of cytoskeletal linker proteins to focal adhesion complexes is reduced in fibroblasts adhering to laminin-1 when compared to fibronectin

Cellular interactions with the extracellular matrix determine to a large extent cell behavior, including cell migration. These interactions take place at specialized cellular structures, the focal adhesions, which have a substrate-specific morphology. To determine the molecular and functional relevance of this observation, the composition of isolated focal adhesions developed by fibroblasts adhering to fibronectin or laminin-1 was analyzed by indirect immunofluorescence and immunoblotting with or without stabilization of the structures by cross-linking. In the absence of cross-linking, integrins, talin, vinculin and, to a lower extent, paxillin remained associated with the focal adhesions formed on both substrates, indicating a tight association of these proteins with the extracellular matrix support. By contrast, alpha-actinin, FAK, and actin were apparently loosely maintained within focal adhesions and were found associated to these structures only after stabilization by cross-linking. Interestingly, although both substrates induced clustering and aggregation of all these proteins, their relative concentration, with the exception of alpha-actinin, was lower within the focal adhesions formed on laminin-1 than in those formed on fibronectin. Moreover, as assessed in migration assays, the locomotory speed of fibroblasts was higher on laminin-1 than on fibronectin. Altogether these results indicate that integrins involved in cellular interactions with fibronectin or laminin-1 trigger the formation of focal adhesion structures which differ by molecular organization, concentration in several adhesion plaque components, and function.     

Lateral diffusion of H-2 antigens on mouse fibroblasts

We have used fluorescence photobleaching and recovery (FPR) to measure the lateral diffusion of mouse H-2 antigens, labeled with fluorescent Fab fragments, in the membrane of cl 1d fibroblasts. Diffusion coefficients, D, vary more than 20-fold from cell to cell, though they vary no more than twofold when measured at different points on a single cell. The fraction of H-2 antigens mobile, R, also varies from cell to cell, and no lateral diffusion of H-2 antigens can be detected in approximately 20% of the cells examined. Treatment of cells with NaCN + NaF, reducing their levels of ATP reduces the proportion of cells in which no lateral diffusion can be detected. The maximum values of D seen in poisoned cells are less than those in controls. Treatment of cells with the divalent inophore, {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187, greatly increases the proportion of cells in which diffusion of H-2 is rapid, D greater than 2 x 10(-9) cm2 s-1. The data obtained on diffusion by FPR can be replotted in the form of an experiment in which lateral diffusion of H- 2 antigens is measured in a population of heterokaryons. There is good agreement between this transformation and actual data on heterokaryons. Thus the two methods appear to measure the same transport process.     

Endocytosis and de novo expression of major histocompatibility complex encoded class I molecules: kinetic and ultrastructural studies

The endocytic pathway and expression of the major histocompatibility complex encoded class I molecule H-2Kk was investigated in murine fibroblasts. Internalization of H-2K molecules did not occur constitutively. Endocytosis of the molecules was induced by addition of multivalent ligands such as rabbit anti-mouse immunoglobulin serum or protein A-bearing liposomes to cells pretreated with anti-H-2Kk antibodies. The complete removal of H-2K molecules took about 5 h at 37 degrees C and was not inhibited by the lysosomotropic agent NH4Cl or the protein synthesis inhibitor cycloheximide. When targeted liposomes that contained carboxyfluorescein at a self-quenched concentration were directed against H-2K molecules, the cells became highly fluorescent after 30 min: a consequence of carboxyfluorescein release from the liposomes. This process was inhibited by NH4Cl but not by cycloheximide, suggesting internalization of H-2K molecules into acidic intracellular compartments. The endocytic pathway of liposomes directed against H-2K molecules and the subcellular compartments involved in this process were investigated with targeted liposomes containing horseradish peroxidase. By electron microscopy, the endocytic process was shown to start very rapidly (1-2 min) and involved uncoated cell surface invaginations. The cytoplasmic uncoated vesicles fused together into larger vacuoles containing concentrated liposomes and by 1 h, liposomes began to be destroyed in lysosomal compartments. Within 4 h, 90% of liposomes were lysed inside the cell. The fate of radiolabeled anti-H-2K antibody was also investigated. Degradation of the antibody occurred only when cross-linked with a second layer of antibody, beginning after 2 h and becoming more pronounced after 20 h of incubation. The original cell surface abundance of H-2K molecules was reestablished after 5 to 7 h. During this time neither NH4Cl nor cycloheximide had any effect on the cell surface expression of the molecule. However, after a second cycle of internalization, cells incubated with cycloheximide no longer expressed these molecules. These results suggested that H-2K molecules were not recycled back to the surface after internalization but were degraded in lysosomal compartments together with their ligand. Preexisting molecules, already present in intracellular pools, were expressed to replace them. By immunoprecipitation of metabolically labeled intracellular and surface H-2K molecules, we observed an intracellular pool of H-2K of about 70 to 80% of the total cellular H-2K.     

Correlated motion and oscillation of neighboring cells in vitro

It has long been realized that fibroblastic and epithelial cells establish recognizable patterns in tissue culture. This behavior implies that neighboring cells interact with one another to produce organized populations. Interaction between cells that are separated by many intervening cells is also possible and is demonstrated here using a special configuration of a biosensor referred to as electric cell-substrate impedance sensing (ECIS). Normally the electrical impedance of a single electrode covered with a confluent cell layer is measured, and the morphological changes of the cells are reflected in the impedance. In this case the cells are cultured on two closely spaced electrodes whose impedances are measured independently as a function of time, and communication between the cell populations is revealed as a correlation between these two time series. We also report for the first time another striking manifestation of dynamic cell interaction, where confluent layers of Madin-Darby canine kidney cells (MDCK) on a single electrode are observed to oscillate in synchrony with a period of approximately 2.5 h.     

Release of integrin macroaggregates as a mechanism of rear detachment during keratinocyte migration

Cell-substrate adhesion can be mediated by the relatively short-lived focal complexes and focal adhesions and by the more stable hemidesmosomes. During cell migration both types of cell-substrate adhesions must be disrupted allowing the cell rear to detach. Rear detachment has been described to be accompanied by membrane ripping and the loss of cellular material in a variety of cell types including fibroblasts and chondrocytes, but also in fast moving cells such as keratinocytes. Here we show that migrating keratinocytes leave behind "migration tracks" of cellular remnants that can be classified due to their size, distribution and molecular composition. Type I macroaggregates appeared as spherical and tubular structures with a diameter of about 50-100 nm that were arranged like "pearls on a string". These structures apparently derived from fragmentation of long tubular extensions, the retracting fibers, at the cell rear and contained high amounts of beta1 integrin and different alpha integrins that are components of fibronectin and laminin receptors in migrating keratinocytes usually found in focal adhesions. Type II macroaggregates were recognized as spherical structures with a diameter of about 30 - 50 nm that were arranged in clusters scattered over the gaps between type I, macroaggregates. In contrast to type I type II macroaggregates contained high amounts of beta4 integrin and seemed to derive from former hemidesmosomes. Both types of macroaggregates were completely membrane covered, impermeable compartments devoid of cytosolic proteins. Our observations strongly support the concept that the release of macroaggregates represents a distinct cellular mechanism of rear detachment based on the loss of adhesive receptors embedded in membrane-covered cellular remnants.