High spatial resolution impedance measurement of EIS sensors for light addressable cell adhesion monitoring

In this paper, impedance measurement of electrolyte-insulator-semiconductor (EIS) structure with high spatial resolution was proposed to monitor cell adhesion. The light addressing ability of this work overcomes the geometrical restrict of cell culture on conventional impedance detection devices such as interdigitated electrode (IDE) and electric cell-substrate impedance sensing (ECIS). Instead of studying cells on predetermined sites of IDE and ECIS, cells cultured anywhere on EIS sensor surface can be addressed and selected as target cells. Principle and primary models for high resolution impedance detection were described and tested by experiments. The EIS sensor was investigated in terms of its intrinsic characteristics, like impedance behavior, voltage characteristic, frequency dependency and photovoltaic effect. Optimized working condition was studied for cell experiments. Cell adhesion under treatment of 0.1% Triton X-100 was monitored using rat kidney cells as the source. Results showed good sensitivity (10% change of impedance) and resolution (40 μm) for cell adhesion impedance detection and suggested this work should be suitable for monitoring cell impedance. Further improvements on sensitivity, spatial resolution were discussed as well as the further applications for single cell monitoring and cell adhesion imaging.     

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.     

On-line monitoring of cell growth and cytotoxicity using electric cell-substrate impedance sensing (ECIS)

An on-line and continuous technique based on electric cell-substrate impedance sensing (ECIS) was developed for measuring the concentration and time response function of fibroblastic V79 cells exposed to mercury chloride and 1,3,5-trinitrobenzene (TNB). Attachment, spreading and proliferation of V79 fibroblastic cells cultured on a microarray of small gold electrodes precoated with fibronectin were detected as resistance changes. The response function was derived to reflect the resistance change as a result of cell attachment, spreading, mitosis and cytotoxicity effect. Exposure of V79 cells to mercury chloride or TNB led to alterations in cell behavior, and therefore, chemical cytotoxicity was easily screened by measuring the response function of the attached and spread cells in the presence of inhibitor. The half inhibition concentration, the required concentration to achieve 50% inhibition, was obtained from the response function to provide information about cytotoxicity during the course of the assay. A simple mathematical model was developed to describe the responses of ECIS that were related to the attachment, spreading, and proliferation of V79 fibroblastic cells. The novel results of this paper are mainly characterized by the systematic study of several parameters including the cell number, detection limit, sensor sensitivity, and cytotoxicity, and they may motivate further research and study of ECIS sensors.     

Automated real-time measurement of chemotactic cell motility.

We have developed a novel method, (ECIS/taxis), for monitoring cell movement in response to chemotactic and chemokinetic factors. In this system, cells migrate in an under-agarose environment, and their positions are monitored using the electric cell-substrate impedance sensor technology to measure the impedance change at a target electrode, that is lithographed onto the substrate, as the cells arrive at the target. In the studies reported here, Dictyostelium discoideum was used as a prototypical, motile eukaryotic cell. Using the ECIS/taxis system, the arrival of cells at the target electrode was proportional to the dose offolate used to stimulate the cells and could be assessed by changes in resistance at the electrode. ECIS/taxis was readily able to distinguish between wild-type cells and a mutant that is deficient in its chemotactic response. Finally, we have shown that an agent that interferes with chemotactic motility leads to the delayed arrival of cells at the target electrode. The multi-well assay configuration allows for simultaneous automated screening of many samples for chemotactic or anti-chemotactic activity. This assay system is compatible with measurements of mammalian cell movement and should be valuable in the assessment of both agonists and antagonists of cell movement.     

Recent progress on performances and mechanisms of carbon dots for gas sensing

Carbon dots (CDs), as an attractive zero-dimensional carbon nanomaterial with unique photoluminescent merits, have recently exhibited significant application potential in gas sensing as a result of their excellent optical/electronic characteristics, high chemical/thermal stability, and tunable surface states. CDs exhibit strong light absorption in the ultraviolet range and tunable photoluminescence characteristics in the visible range, which makes CDs an effective tool for optical sensing applications. Optical gas sensor based on CDs have been investigated, which generally responds to the target gas by corresponding changes in optical absorption or fluorescence. Moreover, electrical gas sensor and quartz crystal microbalance sensor whose sensing layer involves CDs have also been designed. Electrical gas sensor exhibits an increase or a decrease in electrical current, capacitance, or conductance once exposed to the target gas. Quartz crystal microbalance sensor responds to the target gas with a frequency shift. CDs greatly promote the absorption of the target gas and improve the sensitivity of both sensors. In this review, we aim to summarize different types of gas sensors involving CDs, and sensing performances of these sensors for monitoring diverse gases or vapors, as well as the mechanisms of CDs in different types of sensors. Moreover, this review provides the prospect of the potential development of CDs based gas sensors.     

Selection of a battery of rapid toxicity sensors for drinking water evaluation

Comprehensive identification of chemical contaminants in Army field water supplies can be a lengthy process, but rapid analytical methods suitable for field use are limited. A complementary approach is to directly measure toxicity instead of individual chemical constituents. Ten toxicity sensors utilizing enzymes, bacteria, or vertebrate cells were tested to determine the minimum number of sensors that could rapidly identify toxicity in water samples containing one of 12 industrial chemicals. The ideal sensor would respond at a concentration just exceeding the Military Exposure Guideline (MEG) level for the chemical (an estimated threshold for adverse effects) but below the human lethal concentration. Chemical solutions were provided to testing laboratories as blind samples. No sensors responded to deionized water blanks, and only one sensor responded to a hard water blank. No single toxicity sensor responded to more than six chemicals in the desired response range, and one chemical (nicotine) was not detected by any sensor with the desired sensitivity. A combination of three sensors (Microtox, the Electric Cell Substrate Impedance Sensing (ECIS) test, and the Hepatocyte low density lipoprotein (LDL) uptake test) responded appropriately to nine of twelve chemicals. Adding a fourth sensor (neuronal microelectrode array) to the test battery allowed detection of two additional chemicals (aldicarb and methamidophos), but the neuronal microelectrode array was overly sensitive to paraquat. Evaluating sensor performance using a standard set of chemicals and a desired sensitivity range provides a basis both for selecting among available toxicity sensors and for evaluating emerging sensor technologies. Recommendations for future toxicity sensor evaluations are discussed.     

Study of erythrocyte sedimentation behavior by piezoelectric crystal impedance sensor

Based on the impedance characteristic of erythrocytes at high frequency, the response of piezoelectric crystal impedance (PCI) sensor in the erythrocyte suspension was derived and verified experimentally. A method of using PCI sensor to investigate erythrocyte aggregation-sedimentation phenomenon was proposed. From the frequency response of the PCI sensor, the erythrocyte aggregation time and sedimentation rate could be obtained during erythrocyte aggregation and sedimentation. With the present method, the effects of the erythrocyte deformability, the osmotic pressure and the coexisting macromolecules on the erythrocyte sedimentation rate were studied. The results show that the PCI sensor possesses some advantages, such as good sensitivity, simplicity of use and no thermal effect for the impedance study of erythrocyte aggregation and sedimentation.     

Fibroblast growth and H-7 protein kinase inhibitor response monitored in microimpedance sensor arrays

Functional genomic studies and drug candidate testing both require high throughput, parallel experimentation strategies to screen for variable cellular behaviors. In this article we describe the use of an impedance sensing electrode array that is capable of sensing cell "presence" as well as the extent of cell (focal) attachment to the substrate. The signals provided by mouse fibroblasts on a sensing structure containing four different sized electrodes are reported. In the absence of cells, each electrode's impedance was found to depend as expected on electrode size and frequency. The impedance increased by several-fold when fibroblasts attached and spread out over time. More notably, the sensors also detected the cellular response to the protein kinase C inhibitor, H-7. H-7 inhibits actomyosin contractility; thereafter, the loss of focal adhesion complexes occurs. The sensors, in turn, detected an impedance decrease after H-7 addition and an increase in impedance after H-7 removal.     

The region ion sensitive field effect transistor, a novel bioelectronic nanosensor

A novel type of bioelectronic region ion sensitive field effect transistor (RISFET) nanosensor was constructed and demonstrated on two different sensor chips that could measure glucose with good linearity in the range of 0–0.6 mM and 0–0.3 mM with a limit of detection of 0.1 and 0.04 mM, respectively. The sensor is based on the principle of focusing charged reaction products with an electrical field in a region between the sensing electrodes. For glucose measurements, negatively charged gluconate ions were gathered between the sensing electrodes. The signal current response was measured using a low-noise pico ammeter (pA). Two different sizes of the RISFET sensor chips were constructed using conventional electron beam lithography. The measurements are done in partial volumes mainly restricted by the working distance between the sensing electrodes (790 and 2500 nm, respectively) and the influence of electrical fields that are concentrating the ions. The sensitivity was 28 pA/mM (2500 nm) and 830 pA/mM (790 nm), respectively. That is an increase in field strength by five times between the sensing electrodes increased the sensitivity by 30 times. The volumes expressed in this way are in low or sub femtoliter range. Preliminary studies revealed that with suitable modification and control of parameters such as the electric control signals and the chip electrode dimensions this sensor could also be used as a nanobiosensor by applying single enzyme molecule trapping. Hypotheses are given for impedance factors of the RISFET conducting channel.     

Electric cell-substrate impedance sensing (ECIS) as a noninvasive means to monitor the kinetics of cell spreading to artificial surfaces

This article describes the optimization of an experimental technique referred to as electric cell-substrate impedance sensing (ECIS) to monitor attachment and spreading of mammalian cells quantitatively and in real time. The method is based on measuring changes in AC impedance of small gold-film electrodes deposited on a culture dish and used as growth substrate. Based on experimental data and theoretical considerations we demonstrate that high-frequency capacitance measurements (f = 40 kHz) are most suited to follow the increasing surface coverage of the electrode due to cell spreading. The excellent time resolution of the method allowed an in-depth analysis of cell spreading kinetics under various experimental conditions. Using ECIS we studied the attachment and spreading of epithelial MDCK cells (strain II) on different protein coatings, and investigated the influence of divalent cations on spreading kinetics. We quantified the inhibitory effect of soluble peptides that mimic the recognition sequence of fibronectin and other extracellular matrix proteins (RGDS). We also applied the ECIS technique to monitor the detachment of confluent fibroblastic cell layers (WI38/VA-13) by means of these peptides.     

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.     

Instrumental noise estimates stabilize and quantify endothelial cell micro-impedance barrier function parameter estimates

A novel transcellular micro-impedance biosensor, referred to as the electric cell-substrate impedance sensor or ECIS, has become increasingly applied to the study and quantification of endothelial cell physiology. In principle, frequency dependent impedance measurements obtained from this sensor can be used to estimate the cell–cell and cell–matrix impedance components of endothelial cell barrier function based on simple geometric models. Few studies, however, have examined the numerical optimization of these barrier function parameters and established their error bounds. This study, therefore, illustrates the implementation of a multi-response Levenberg–Marquardt algorithm that includes instrumental noise estimates and applies it to frequency dependent porcine pulmonary artery endothelial cell impedance measurements. The stability of cell–cell, cell–matrix and membrane impedance parameter estimates based on this approach is carefully examined, and several forms of parameter instability and refinement illustrated. Including frequency dependent noise variance estimates in the numerical optimization reduced the parameter value dependence on the frequency range of measured impedances. The increased stability provided by a multi-response non-linear fit over one-dimensional algorithms indicated that both real and imaginary data should be used in the parameter optimization. Error estimates based on single fits and Monte Carlo simulations showed that the model barrier parameters were often highly correlated with each other. Independently resolving the different parameters can, therefore, present a challenge to the experimentalist and demand the use of non-linear multivariate statistical methods when comparing different sets of parameters.     

Carbon fibre composites: integrated electrochemical sensors for wound management

The applicability of employing a carbon fibre mesh as an electrochemical sensing substructure for assessing urate transformations within wound exudates is evaluated. Prototype sensor assemblies have been designed and their response characteristics towards uric acid and other common physiological components are detailed. Modification of the carbon fibre sensor through surface anodization and the application of cellulose acetate permselective barriers have been shown to lead to optimized responses and much greater sensitivity (1440% increase) and specificity. These could enable the accurate periodic monitoring of uric acid in wound fluid. The performance characteristics of the composite sensors in whole blood, serum and blister fluid have been investigated.     

Impedance analysis of adherent cells after in situ electroporation: non-invasive monitoring during intracellular manipulations

In this study adherent animal cells were grown to confluence on circular gold-film electrodes of 250 μm diameter that had been deposited on the surface of a regular culture dish. The impedance of the cell-covered electrode was measured at designated frequencies to monitor the behavior of the cells with time. This approach is referred to as electric cell-substrate impedance sensing or short ECIS in the literature. The gold-film electrodes were also used to deliver well-defined AC voltage pulses of several volts amplitude and several hundred milliseconds duration to the adherent cells in order to achieve reversible membrane electroporation (in situ electroporation=ISE). Electroporation-assisted introduction of membrane impermeable molecules into the cytoplasm was studied by using FITC-labeled dextran molecules of different molecular weights. Probes as big as 2MDa were successfully loaded into the cells residing on the electrode surface. Time-resolved impedance measurements before and immediately after the electroporation pulse revealed the kinetics of membrane resealing as well as subsequent changes in cell morphology. Cells recovered from the electroporation pulse within less than 90 min. When membrane-impermeable, bioactive compounds like N(3)(-) or bleomycin were introduced into the cells by in situ electroporation, concomitant ECIS readings sensitively reported on the associated response of the cells to these toxins as a function of time (ISE-ECIS).     

Development of an electrochemical polypyrrole-based DNA sensor and subsequent studies on the effects of probe and target length on performance

DNA sensors have a wide scope of applications in the present and emerging medical and scientific fields, such as medical diagnostics and forensic investigations. However, much research-to-date on DNA sensor development has focused on short target DNA strands as model genes. In this communication we study the effect of the length of oligonucleotide probe and target strands as a significant step towards real world applications for DNA detection. The sensor technology described uses the conducting polymer polypyrrole as both a sensing element and transducer of sensing events - namely the hybridization of complementary target oligonucleotide to probe oligonucleotide. Detection is performed using electrical impedance spectroscopy. Initially sensor development is performed, wherein we demonstrate an improvement in stability and sensitivity as well as show a reduction in non-specific DNA binding for fabricated sensors, through use of a specific dopant and post-growth treatment. Subsequently, we show that longer target DNA strands display increased response, as do sensors containing longer probe DNA strands. It is suggested that these results are a feature of the increase in negative charges associated with the longer DNA strands. The results of this comparative study are aimed to guide future design of analogous sensors.     

A computational modeling and analysis in cell biological dynamics using electric cell-substrate impedance sensing (ECIS)

In this paper, a study of computational modeling and multi-scale analysis in cell dynamics is presented. Our study aims at: (1) deriving and validating a mathematical model for cell growth, and (2) quantitatively detecting and analyzing the biological interdependencies across multiple observational scales with a variety of time and frequency resolutions. This research was conducted using the time series data practically measured from a novel on-line cell monitoring technique, referred to as electric cell-substrate impedance sensing (ECIS), which allows continuously tracking the cellular behavior such as adhesion, proliferation, spreading and micromotion. First, comparing our ECIS-based cellular growth modeling analysis results with those determined by hematocytometer measurement using different time intervals, we found that the results obtained from both experimental methods consistently agreed. However, our study demonstrated that it is much easier and more convenient to operate with the ECIS system for on-line cellular growth monitoring. Secondly, for multi-scale analysis our results showed that the proposed wavelet-based methodology can effectively quantify the fluctuations associated with cell micromotions and quantitatively capture the biological interdependencies across multiple observational scales. Note that although the wavelet method is well known, its application into the ECIS time series analysis is novel and unprecedented in computational cell biology. Our analyses indicated that the proposed study on ECIS time series could provide a hopeful start and great potentials in both modeling and elucidating the complex mechanisms of cell biological systems.     

Detection of Rev peptides with impedance-sensors--comparison of device-geometries

Two different impedance-sensor geometries have been compared for the detection of Rev peptides with a molecular weight of 2.4 kDa. Planar, two-dimensional interdigitated capacitor (IDC) sensors with electrode separations of 1.1 microm as well as three-dimensional nanogap-sensors with an electrode separation of 75 nm have been used. Both sensors have been operated at a fixed frequency of 980 MHz. We discuss the specific interaction of the Rev peptide to an immobilized RNA anti-Rev aptamer (9.2 kDa) for peptide concentrations in the range of 100 nM-2 microM. For the IDC sensor, only peptide concentrations above 500 nM gave detectable signals. For the nanogap sensor, the binding process was clearly visible for all concentrations applied. The higher sensitivity of the nanogap compared to the IDC is ascribed to the improved surface-to-volume ratio.     

Phage immobilized magnetoelastic sensor for the detection of Salmonella typhimurium

In this article, a phage-based magnetoelastic sensor for the detection of Salmonella typhimurium is reported. Filamentous bacteriophage specific to S. typhimurium was used as a biorecognition element in order to ensure specific and selective binding of bacteria onto the sensor surface. Phage was immobilized onto the surface of the sensors by physical adsorption. The phage immobilized magnetoelastic sensors were exposed to S. typhimurium cultures with different concentrations ranging from 5x10(1) to 5x10(8) cfu/ml, and the corresponding changes in resonance frequency response of the sensor were studied. It was experimentally established that the sensitivity of the magnetoelastic sensors was higher for sensors with smaller physical dimensions. An increase in sensitivity from 159 Hz/decade for a 2 mm sensor to 770 Hz/decade for a 1 mm sensor was observed. Scanning electron microscopy (SEM) analysis of previously assayed biosensors provided visual verification of frequency changes that were caused by S. typhimurium binding to phage immobilized on the sensor surface. The detection limit on the order of 10(3) cfu/ml was obtained for a sensor with dimensions 1x0.2x0.015 mm.     

Characterisation and optimisation of AC conductimetric biosensors

Urease, immobilised on interdigitated gold electrodes, is employed as a model enzyme for characterisation and optimisation of a.c. conductimetric sensors. The sensors' response is measured over a frequency range of 20 Hz to 300 kHz and an optimum operating frequency established. The activity of the urease, both in solution and immobilised states, is investigated and Km values obtained. The effect of method of immobilisation and enzyme loading on the sensors' performance are studied and urease electrodes are characterised as a function of temperature, pH and electrolyte concentration. An important finding, particularly for conductimetric sensors designed for clinical use, is that proper consideration of the effects of electrode polarisation must be taken into account in order to maintain high sensor sensitivity at physiological electrolyte concentrations. Measurements of urea concentration in untreated serum are described.