Bioelectrochemical signal monitoring of in-vitro cultured cells by means of an automated microsystem based on solid state sensor-array

In the last decade, fundamental advances in whole cell based sensors and microsystems have established the extracellular acidification rate monitoring of cell cultures as an important indicator of the global cellular metabolism. Innovative approaches adopting advanced integrated sensor array-based microsystems represent an emerging technique with numerous biomedical applications. This paper reports a cell-based microsystem, for multisite monitoring of the physiological state of cell populations. The functional components of the microsystem are an ion sensitive field effect transistor (ISFET) array-based sensor chip and a CMOS integrated circuit for signal conditioning and sensor signal multiplexing. In order to validate the microsystem capabilities for in-vitro toxicity screening applications, preliminary experimental measurements with Cheratinocytes, and CHO cells are presented. Variations in the acidification rate, imputable to the inhibitory effect of the drug on the metabolic cell activity have been monitored and cell viability during long term measurements has been also demonstrated.     

Multiplexed FRET to image multiple signaling events in live cells

We report what to our knowledge is a novel approach for simultaneous imaging of two different Förster resonance energy transfer (FRET) sensors in the same cell with minimal spectral cross talk. Previous methods based on spectral ratiometric imaging of the two FRET sensors have been limited by the availability of suitably bright acceptors for the second FRET pair and the spectral cross talk incurred when measuring in four spectral windows. In contrast to spectral ratiometric imaging, fluorescence lifetime imaging (FLIM) requires measurement of the donor fluorescence only and is independent of emission from the acceptor. By combining FLIM-FRET of the novel red-shifted TagRFP/mPlum FRET pair with spectral ratiometric imaging of an ECFP/Venus pair we were thus able to maximize the spectral separation between our chosen fluorophores while at the same time overcoming the low quantum yield of the far red acceptor mPlum. Using this technique, we could read out a TagRFP/mPlum intermolecular FRET sensor for reporting on small Ras GTP-ase activation in live cells after epidermal growth factor stimulation and an ECFP/Venus Cameleon FRET sensor for monitoring calcium transients within the same cells. The combination of spectral ratiometric imaging of ECFP/Venus and high-speed FLIM-FRET of TagRFP/mPlum can thus increase the spectral bandwidth available and provide robust imaging of multiple FRET sensors within the same cell. Furthermore, since FLIM does not require equal stoichiometries of donor and acceptor, this approach can be used to report on both unimolecular FRET biosensors and protein-protein interactions with the same cell.     

An autonomous CMOS hysteretic sensor for the detection of desorption-free DNA hybridization

This paper describes a sensor for label-free, fully electrical detection of DNA hybridization based on capacitive changes in the electrode-electrolyte interface. The sensor measures capacitive changes in real time according to a charging-discharging principle that is limited by the hysteresis window. In addition, a novel autonomous searching technique, which exclusively monitors desorption-free hybridized electrodes among electrode arrays, enhances the performance of the sensor compared with conventional capacitive measurement. The sensor system achieves a detection range of 80 dB. The integrated circuit sensor is fabricated with a 0.35 μm CMOS process. The proposed sensor offers rapid, robust and inexpensive measurement of capacitance with highly integrated detection circuitry. It also facilitates quantitative evaluations of molecular densities on a chip with distinctive impedance variations by monitoring desorption-free hybridized electrodes. Our electrical biosensor has great potential for use with bio analytical tools and point-of-care diagnosis.     

A label-free photonic crystal biosensor imaging method for detection of cancer cell cytotoxicity and proliferation

A label-free method for detecting the attachment of human cancer cells to a biosensor surface for rapid screening for biological activity is described, in which attachment of a cell results in highly localized increase of the resonant reflected wavelength of a photonic crystal narrowband reflectance filter incorporated into a standard 96-well microplate. An imaging detection instrument is used to determine the spatial distribution of attached cells by mapping the shift in reflected resonant wavelength as a function of position. The method enables monitoring of cancer cell attachment, cell proliferation, and cell detachment that is induced by exposure of the cells to drug compounds. We demonstrate the efficacy of this method as an early screening technique for the rapid quantification of the rate of cancer cell proliferation on the sensor surface, and subsequently as a means for quantifying cell detachment resulting from apoptosis that is induced by exposure of the cells to cytotoxic chemicals.     

A novel silicon patch‐clamp chip permits high‐fidelity recording of ion channel activity from functionally defined neurons

We report on a simple and high‐yield manufacturing process for silicon planar patch‐clamp chips, which allow low capacitance and series resistance from individually identified cultured neurons. Apertures are etched in a high‐quality silicon nitride film on a silicon wafer; wells are opened on the backside of the wafer by wet etching and passivated by a thick deposited silicon dioxide film to reduce the capacitance of the chip and to facilitate the formation of a high‐impedance cell to aperture seal. The chip surface is suitable for culture of neurons over a small orifice in the substrate with minimal leak current. Collectively, these features enable high‐fidelity electrophysiological recording of transmembrane currents resulting from ion channel activity in cultured neurons. Using cultured Lymnaea neurons we demonstrate whole‐cell current recordings obtained from a voltage‐clamp stimulation protocol, and in current‐clamp mode we report action potentials stimulated by membrane depolarization steps. Despite the relatively large size of these neurons, good temporal and spatial control of cell membrane voltage was evident. To our knowledge this is the first report of recording of ion channel activity and action potentials from neurons cultured directly on a planar patch‐clamp chip. This interrogation platform has enormous potential as a novel tool to readily provide high‐information content during pharmaceutical assays to investigate in vitro models of disease, as well as neuronal physiology and synaptic plasticity. Biotechnol. Bioeng. 2010;107:593–600. © 2010 Wiley Periodicals, Inc.     

Miniaturized real-time monitoring system for L-lactate and glucose using microfabricated multi-enzyme sensors.

A miniaturized on-line monitoring system for the detection of L-lactate and glucose is presented. The system is based on a microfabricated multi-enzyme silicon sensor chip with flow channels integrated on the chip. The sensors were fabricated in containment technology. They were characterized in test solutions. The cross-talking behaviour was investigated and was found to be practically negligible. The linear measurement ranges of both glucose and lactate sensors were large enough for most practical applications. As a result of the miniaturization the analyte consumption could be reduced to a few nmol min(-1). The system was equipped with a microdialysis probe whose recovery was 45% for lactate and 37% for glucose in test solutions using a flow rate of 3 microl min(-1). Lower flow rates of 0.5 microl min(-1) resulted in recoveries of over 90%. The long-term stability of the system was acceptable. Initial measurements have also been performed in vitro using human blood serum.     

Effect of Voltage Sensitive Fluorescent Proteins on Neuronal Excitability

Fluorescent protein voltage sensors are recombinant proteins that are designed as genetically encoded cellular probes of membrane potential using mechanisms of voltage-dependent modulation of fluorescence. Several such proteins, including VSFP2.3 and VSFP3.1, were recently reported with reliable function in mammalian cells. They were designed as molecular fusions of the voltage sensor of Ciona intestinalis voltage sensor containing phosphatase with a fluorescence reporter domain. Expression of these proteins in cell membranes is accompanied by additional dynamic membrane capacitance, or “sensing capacitance”, with feedback effect on the native electro-responsiveness of targeted cells. We used recordings of sensing currents and fluorescence responses of VSFP2.3 and of VSFP3.1 to derive kinetic models of the voltage-dependent signaling of these proteins. Using computational neuron simulations, we quantitatively investigated the perturbing effects of sensing capacitance on the input/output relationship in two central neuron models, a cerebellar Purkinje and a layer 5 pyramidal neuron. Probe-induced sensing capacitance manifested as time shifts of action potentials and increased synaptic input thresholds for somatic action potential initiation with linear dependence on the membrane density of the probe. Whereas the fluorescence signal/noise grows with the square root of the surface density of the probe, the growth of sensing capacitance is linear. We analyzed the trade-off between minimization of sensing capacitance and signal/noise of the optical read-out depending on kinetic properties and cellular distribution of the probe. The simulation results suggest ways to reduce capacitive effects at a given level of signal/noise. Yet, the simulations indicate that significant improvement of existing probes will still be required to report action potentials in individual neurons in mammalian brain tissue in single trials.     

Simultaneous intra- and extracellular superoxide monitoring using an integrated optical and electrochemical sensor system

A new integrated optical and electrochemical sensor system for simultaneous monitoring of intra- and extracellular superoxide (O(2)(-)) was developed using an array-based cell chip. For in vitro assays, A172 human glioblastoma cells were transferred into the cell chip and stimulated by phorbol 12-myristate 13-acetate (PMA). Intracellular O(2)(-) generation was detected via fluorescence image analysis with a dye probe, dihydrorhodamine 123 (DHR 123). Extracellular O(2)(-) was detected using an amperometric sensor constructed by immobilisation of cytochrome c using a binder, 3,3'-dithiobis(sulphosuccinimidylpropionate), to attach the redox protein onto the surface of electrodeposited Au electrodes incorporated into the optically transparent cell chip. The simultaneous intra- and extracellular production of O(2)(-) was successfully observed from PMA-stimulated A172 cells and inhibited by superoxide dismutase (SOD). The quantification of O(2)(-) concentration based on a mathematical model study and possible applications using the sensor system developed were discussed. The results confirm that there was no detectable interference or crosstalk between the optical and electrochemical assays. Feasibility of the integration of the two methods, optical and electrochemical, and the neutralisation of the intra- and extracellular O(2)(-) levels by SOD have been demonstrated.     

Electrochemical monitoring of cell behaviour in vitro: a new technology

This article describes a novel electrochemical technique for the real-time monitoring of changes in the behaviour of adherent human cells in vitro: i.e., a biosensor that combines a biological recognition mechanism with a physical transduction technique, described collectively as Oncoprobe. Confluent viable cells adherent to gold electrodes (sensors) modify the extracellular microenvironment at the cell:sensor interface to produce a change in the electrochemical potential compared to that measured in the absence of cells. The potential was measured as an open circuit potential (OCP) with respect to a saturated calomel reference in the bulk culture medium. Typical OCP values for confluent cultures of human breast carcinoma cells, 8701-BC, approximated -100 mV compared with cell-free values of approximately -15 mV. The OCP for 8701-BC cells was modified in response to temperature changes over the range 32 to 40 degrees C and also to treatments with phytohemagglutinin (PHA, 25 microg/mL), cycloheximide (30 microM) and interleukin-1 beta (IL-1, 0.5 ng/mL) over 24 h. Cultures of synovial fibroblasts also responded to the same treatments with similar responses, producing negative shifts in the OCP signal with PHA and IL-I, but a positive shift in OCP signal with cycloheximide, all relative to the untreated control cultures. From experimental data and theoretical considerations it is proposed that the cell-derived signals are mixed electrode potentials reflecting a "conditioned," more reducing environment at the cell:sensor interface. Only viable cells caused a negative shift in the OCP signal, this being lost when cells were rendered nonviable by formalin exposure. This technology appears unique in its ability to passively "listen in" on cell surface activities, suggesting numerous applications in the fields of drug discovery, chemotherapy, and cell behaviour.     

Dielectric measurement to monitor the growth and the physiological states of biological cells

Measurement of capacitance, also referred to as dielectric permittivity, is a new method of estimating the concentration of cells, monitoring the growth and detecting the physiological changes during the cultivation of organisms in various bioprocess. Several types of biological cells were studied, namely; Saccharomyces cerevisiae, Escherichia coli, Perilla frutescens (plant cells) and AFP-27 hybridoma cells. Generally, a linear correlation between cell capacitance (C) and other biomass measurement technique such as optical density (OD) and dry weight (DW) was obtained using the different types of cell suspension. Therefore, this method could be used to monitor the growth of the organism during the active growth. It could be conveniently used to make a rapid estimate of the cell concentration such as in plant cell suspension culture. The capacitance sensor could also be designed to be installed and autoclaved in-situ in a bioreactor and used for on-line monitoring of cell growth. On the other hand, distinct deviations in the capacitance value were observed in relation with the growth stage of the organism. This was observed in all the organisms studied but the type of deviation depends on the physiology of the organism. This variation in cell capacitance showed the possibility of using this method as a means to indicate changes in the physiological state of cells during cultivation. This capability would be very useful in designing control strategies that would depend on the physiological states in the bioprocess. Present address: Miles Inc., Berkeley, CA 94701 U.S.A.The authors sincerely appreciated the generosity of Dr. K. Mishima and Dr. A. Mimura of Kobe Steel Co., Japan. The useful discussions with M. Nakajima and technical assistance of J. Zhong and R. Pambayun were also acknowledged. The work in hybridoma cell culture was done through the collaboration with C. Perusich-Kussow and Prof. W. S. Hu, University of Minnesota, USA.     

The BARC biosensor applied to the detection of biological warfare agents

The Bead ARray Counter (BARC) is a multi-analyte biosensor that uses DNA hybridization, magnetic microbeads, and giant magnetoresistive (GMR) sensors to detect and identify biological warfare agents. The current prototype is a table-top instrument consisting of a microfabricated chip (solid substrate) with an array of GMR sensors, a chip carrier board with electronics for lock-in detection, a fluidics cell and cartridge, and an electromagnet. DNA probes are patterned onto the solid substrate chip directly above the GMR sensors, and sample analyte containing complementary DNA hybridizes with the probes on the surface. Labeled, micron-sized magnetic beads are then injected that specifically bind to the sample DNA. A magnetic field is applied, removing any beads that are not specifically bound to the surface. The beads remaining on the surface are detected by the GMR sensors, and the intensity and location of the signal indicate the concentration and identity of pathogens present in the sample. The current BARC chip contains a 64-element sensor array, however, with recent advances in magnetoresistive technology, chips with millions of these GMR sensors will soon be commercially available, allowing simultaneous detection of thousands of analytes. Because each GMR sensor is capable of detecting a single magnetic bead, in theory, the BARC biosensor should be able to detect the presence of a single analyte molecule.     

Phase tracking: an improved phase detection technique for cell membrane capacitance measurements.

We describe here a technique called phase tracking that greatly improves the accuracy of measurements of the membrane capacitance of single cells. We have modified the original phase detection technique to include a method for creating calibrated changes in the resistance in series with the cell. This provides a method to automate the adjustment of the phase detector to the appropriate phase angle for measuring membrane capacitance. The phase determination depends only on the cell's electrical parameters and does not require matching of the cell impedance with that of the slow capacitance cancellation circuitry of the patch-clamp amplifier. We show here that phase tracking can accurately locate the phase of the capacitance signal and can keep the detector aligned with this signal during measurements of exocytosis in mast cells, irrespective of the large drifts which occur in cell membrane resistance, membrane capacitance, or series resistance. The phase tracking technique is a valuable tool for quantifying exocytosis and endocytosis in single cells.     

An electrochemical sensor array system for the direct, simultaneous in vitro monitoring of nitric oxide and superoxide production by cultured cells

A new approach for an amperometric array sensor platform employing arrays of sensors in a 24-well cell culture plate format has been developed for simultaneous in vitro determination of nitric oxide (NO) and superoxide free radicals (O(2)(-)) produced by stimulated cells. The work reported focuses on the direct, real-time monitoring of extracellular production of these two analytes, as well as the effects of their interaction. The sensor platform was manufactured by a combination of sputtering gold electrodes and screen-printing carbon electrodes. The O(2)(-) sensor uses covalent immobilization of cytochrome c via a binder, DTSSP (3,3'-dithio-bis(sulphosuccinimidylpropionate) onto the surface of the Au electrodes, whereas the NO sensor system involves an NiTSPc (nickel tetrasulfonated phthalocyanine) film electrodeposited onto the surface of the carbon electrodes and subsequently covered with an external layer of Nafion. For in vitro demonstration of the platforms as a potential drug-screening system, A172 glioblastoma cells were cultured and transferred into the 24-well arrays. Simultaneous and direct monitoring of NO and O(2)(-) production as a response to chemicals of biomedical relevance was carried out. The results obtained demonstrated that it would be possible to envisage a drug screening platform for compounds designed to be inhibitors of nitric oxide synthase or to have an inhibitory effect on superoxide free radical production. By suitable modification of the electrodes employed it would also be possible to extend the platform to measure alternative species.     

Advanced electrochemical sensors for cell cancer monitoring

The possibility of using minimally invasive analytical instruments to monitor cancerous cells and their interactions with analytes provide great advances in cancer research and toxicology. The real success in the development of a reliable sensor for cell monitoring depends on the ability to design powerful instrumentation that will facilitate efficient signal transduction from the biological process that occurs in the cellular environment. The resulting sensor should not affect cell viability and must function as well as adapt the system to the specific conditions imposed by the cell culture. Due to their performance, electrochemical biosensors could be used as an effective instrument in cell cancer research for studying biochemical processes, cancer development and progression as well as toxicity monitoring. Current research in this direction is conducted through high-throughput, compact, portable, and easy to use sensors that enable measurement of cells' activity in their optimum environment. This paper discusses the potential of a high-throughput electrochemical multisensor system, so-called the DOX system for monitoring cancerous cells and their interaction with chemical toxins. We describe the methodology, experiments, and the operation principle of this device, and we focus on the challenges encountered in optimizing and adapting the system to the specific cell-culture conditions. The DOX system is also compared with conventional cell-culture techniques.     

Real-time monitoring of the strand displacement amplification (SDA) of human cytomegalovirus by a new SDA-piezoelectric DNA sensor system

Nucleic acid amplification has long been used in biosensor technologies, such as DNA sensors, DNA chips and microarrays, due to its advantage of high sensitivity in detecting target DNA. However, dynamic monitoring of nucleic acid amplifications with traditional DNA sensors in real-time is difficult since a constant temperature must be maintained during detection. Thus, the piezoelectric sensor, one type of traditional DNA sensor, is not applicable in real-time monitoring PCR due to the dramatic change in temperature that occurs during reaction. In this study, we introduced strand displacement amplification (SDA), an well-developed nucleic acid amplification technique that can work under conditions of constant temperature, into the development of a novel piezoelectric sensor. Using the new SDA-piezoelectric DNA sensor, we designed a stable system for liquid-phase detection, in which the crystal oscillator plate was fixed by an easily adjustable screw-threaded clamping mechanism and successfully applied the new sensor system to real-time SDA monitoring of human cytomegalovirus (HCMV). This new technique overcomes the shortcomings of traditional DNA sensors in real-time monitoring of nucleic acid amplification. The technique has proved to be a markedly simplified procedure with a number of advantages, such as higher sensitivity, better time efficiency, and the ability of dynamic real-time detection.     

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.     

Optical Sensing of Microbial Life on Surfaces

The label-free detection of microbial cells attached to a surface is an active field of research. The field is driven by the need to understand and control the growth of biofilms in a number of applications, including basic research in natural environments, industrial facilities, and clinical devices, to name a few. Despite significant progress in the ability to monitor the growth of biofilms and related living cells, the sensitivity and selectivity of such sensors are still a challenge. We believe that among the many different technologies available for monitoring biofilm growth, optical techniques are the most promising, as they afford direct imaging and offer high sensitivity and specificity. Furthermore, as each technique offers different insights into the biofilm growth mechanism, our analysis allows us to provide an overview of the biological processes at play. In addition, we use a set of key parameters to compare state-of-the-art techniques in the field, including a critical assessment of each method, to identify the most promising types of sensors. We highlight the challenges that need to be overcome to improve the characteristics of current biofilm sensor technologies and indicate where further developments are required. In addition, we provide guidelines for selecting a suitable sensor for detecting microbial cells on a surface.     

In vitro testing of a simply constructed, highly stable glucose sensor suitable for implantation in diabetic patients

We have constructed and tested in vitro a potentially implantable, needle-type amperometric enzyme electrode which is suitable for continuous monitoring of glucose concentrations in diabetic patients. The major requirements of stability during operation and ease of manufacture have been met with a sensor design which involves a simple dip-coating procedure for applying to a platinum base electrode an inner membrane of glucose oxidase immobilised in polyhydroxyethyl methacrylate (pHEMA), and an outer membrane composed of a pHEMA/polyurethane mixture. Sensors were operated at 700 mV for detection of hydrogen peroxide. Calibration curves for the sensor were linear to at least 20 mM glucose and were unaffected by a reduction in PO2 from 20 to 5 kPa. During continuous operation in 5 mM buffered glucose solutions in vitro, sensors suffered no significant loss of response over periods of up to 60 h. Such electrodes are, therefore, useful for development as in vivo glucose sensors.     

Mitogen-Induced B-Cell Proliferation Activates Chk2-Dependent G1/S Cell Cycle Arrest

B-cell activation and proliferation can be induced by a variety of extracellular stimuli. The fate of an activated B cell following mitogen stimulation can be dictated by the strength or duration of the signal, the expression of downstream signaling components necessary to promote proliferation, and the cell intrinsic sensors and regulators of the proliferative program. Previously we have identified the DNA damage response (DDR) signaling pathway as a cell intrinsic sensor that is activated upon latent infection of primary human B cells by Epstein-Barr virus (EBV). Here we have assessed the role of the DDR as a limiting factor in the proliferative response to non-viral B-cell mitogens. We report that TLR9 activation through CpG-rich oligonucleotides induced B-cell hyper-proliferation and an ATM/Chk2 downstream signaling pathway. However, B-cell activation through the CD40 pathway coupled with interleukin-4 (IL-4) promoted proliferation less robustly and only a modest DDR. These two mitogens, but not EBV, modestly induced intrinsic apoptosis that was independent from the DDR. However, all three mitogens triggered a DDR-dependent G1/S phase cell cycle arrest preventing B-cell proliferation. The extent of G1/S arrest, as evidenced by release through Chk2 inhibition, correlated with B-cell proliferation rates. These findings have implications for the regulation of extra-follicular B-cell activation as it may pertain to the development of auto-immune diseases or lymphoma.     

Simultaneous measurement of cellular respiration and acidification with a single CMOS ISFET

In vivo, the pH value and oxygen partial pressure are the most important physico-chemical parameters in the microenvironment of human tissues. In vitro, the extracellular acidification rate of cell cultures is an indicator of global cellular metabolism, while the rate of oxygen consumption is a measure of mitochondrial activity. Earlier approaches had the disadvantage that these two values had to be measured with two separate sensors at different loci within the tissue or cell culture. Furthermore, conventional Clark-type oxygen sensors are not very compatible for miniaturisation, making it impossible to measure at small cell volumes or even at the single cell level. We have, therefore, developed an ISFET based sensor structure which is able to measure both pH and oxygen partial pressure. This sensor structure was tested in vitro for simultaneous records of cellular acidification and respiration rates at the same site within the cell culture. This sensor is manufactured by a CMOS-process.