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1.
The electrical coupling of randomly migrating neurons from rat explant brain-stem slice cultures to the gates of non-metallized field-effect transistors (FETs) has been investigated. The objective of our work is the precise interpretation of extracellular recorded signal shapes in comparison to the usual patch-clamp protocols to evaluate the possible use of the extracellular recording technique in electrophysiology. The neurons from our explant cultures exhibited strong voltage-gated potassium currents through the plasma membrane. With an improved noise level of the FET set-up, it was possible to record individual extracellular responses without any signal averaging. Cells were attached by patch-clamp pipettes in voltage-clamp mode and stimulated by voltage step pulses. The point contact model, which is the basic model used to describe electrical contact between cell and transistor, has been implemented in the electrical simulation program PSpice. Voltage and current recordings and compensation values from the patch-clamp measurement have been used as input data for the simulation circuit. Extracellular responses were identified as composed of capacitive current and active potassium current inputs into the adhesion region between the cell and transistor gate. We evaluated the extracellular signal shapes by comparing the capacitive and the slower potassium signal amplitudes. Differences in amplitudes were found, which were interpreted in previous work as enhanced conductance of the attached membrane compared to the average value of the cellular membrane. Our results suggest rather that additional effects like electrodiffusion, ion sensitivity of the sensors or more detailed electronic models for the small cleft between the cell and transistor should be included in the coupling model. 相似文献
2.
Cardiomyocyte-transistor-hybrids for sensor application. 总被引:3,自引:0,他引:3
An extracellular recording system has been designed for the detection of electrical cell signals using p-channel or n-channel field-effect transistor (FET) arrays with non-metallized gates. Signals from rat heart muscle cell were recorded by these devices and the results described on the basis of an equivalent circuit. This technique is sensitive enough to detect minute changes of the extracellular membrane voltage and has potential applications in drug screening. We show that known cardiac stimulants (isoproterenol, norepinephrine) and relaxants (verapamil, carbamylcholine) have characteristic effects on the heart cells in terms of the changes of beat frequencies in the absence or presence of corresponding agents. 相似文献
3.
Meyburg S Goryll M Moers J Ingebrandt S Böcker-Meffert S Lüth H Offenhäusser A 《Biosensors & bioelectronics》2006,21(7):1037-1044
A field-effect transistor (FET) for recording extracellular signals from electrogenic cells is presented. The so-called floating gate architecture combines a complementary metal oxide semiconductor (CMOS)-type n-channel transistor with an independent sensing area. This concept allows the transistor and sensing area to be optimised separately. The devices are robust and can be reused several times. The noise level of the devices was smaller than of comparable non-metallised gate FETs. In addition to the usual drift of FET devices, we observed a long-term drift that has to be controlled for future long-term measurements. The device performance for extracellular signal recording was tested using embryonic rat cardiac myocytes cultured on fibronectin-coated chips. The extracellular cell signals were recorded before and after the addition of the cardioactive isoproterenol. The signal shapes of the measured action potentials were comparable to the non-metallised gate FETs previously used in similar experiments. The fabrication of the devices involved the process steps of standard CMOS that were necessary to create n-channel transistors. The implementation of a complete CMOS process would facilitate the integration of the logical circuits necessary for signal pre-processing on a chip, which is a prerequisite for a greater number of sensor spots in future layouts. 相似文献
4.
Recombinant maxi-K channels on transistor, a prototype of iono-electronic interfacing 总被引:5,自引:0,他引:5
We report on the direct electrical interfacing of a recombinant ion channel to a field-effect transistor on a silicon chip. The ion current through activated maxi-K(Ca) channels in human embryonic kidney (HEK293) cells gives rise to an extracellular voltage between cell and chip that controls the electronic source-drain current. A comparison with patch-clamp recording shows that the channels at the cell/chip interface are fully functional and that they are significantly accumulated there. The direct coupling of potassium channels to a semiconductor on the level of an individual cell is the prototype for an iono-electronic interface of ligand-gated or G protein-coupled ion channels and the development of screening biosensors with many transfected cells on a chip with a large array of transistors. 相似文献
5.
The release of dense-core vesicles in bovine chromaffin cells is a model for the presynaptic process in neurons. It is usually studied by microamperometry of catecholamines with carbon fibers. Here we introduce transistor recording as a tool to study vesicle release. When we stimulate a chromaffin cell placed on a field-effect transistor, the gate voltage exhibits peaks that correlate with a simultaneously performed amperometric recording. We attribute the transistor signal to a release of protons from the extruded matrix of vesicles that lowers the extracellular pH and changes the electrical surface potential of the gate oxide. The rise time of the transistor signals is similar to that of amperometric responses, whereas their duration is distinctly longer. In a model computation, the rise time is identified with the extrusion of vesicle matrix into the narrow extracellular space between cell and gate oxide, and the decay time is attributed to pH equilibration through slow diffusion in the extruded matrix. Because the transistor recording relies on protons, it can be applied to acidic vesicles with electrochemically inactive hormones or transmitters. 相似文献
6.
Recently, the ability to create bio-semiconductor hybrid devices has gained much interest for cell activity analysis. AlGaN material system has been demonstrated to be a promising cell-based biosensing platform due to a combination of unique properties, such as chemical inertness, optical transparency and low signal to noise ratios. To investigate the potential application of hybrid cell-AlGaN/GaN field effect transistor for cell electrophysiological monitoring, saos-2 human osteoblast-like cells were cultured in high density in non-metallized gate area of a transparent AlGaN/GaN heterostructure field effect transistor. We implemented and characterized the transistor recording of extracellular voltage in the cell-chip junction using the FET chip. The effect of ion channel blocker TEA on transistor signal was explored in order to test the capability of this hybrid chip for in vitro drug screening bioassay. Finally, the effect of cell adhesion on transistor signal was also studied by applying the protein kinase inhibitor H-7. 相似文献
7.
Schäfer S Eick S Hofmann B Dufaux T Stockmann R Wrobel G Offenhäusser A Ingebrandt S 《Biosensors & bioelectronics》2009,24(5):1201-1208
Electrolyte-gate field-effect transistors (EG-FETs) gained continuously more importance in the field of bioelectronics. The reasons for this are the intrinsic properties of these FETs. Binding of analysts or changes in the electrolyte composition are leading to variations of the drain-source current. Furthermore, due to the signal amplification upon voltage-to-current conversion even small extracellular signals can be detected. Here we report about impedance spectroscopy with an FET array to characterize passive components of a cell attached to the transistor gate. We developed a 16-channel readout system, which provides a simultaneous, lock-in based readout. A test signal of known amplitude and phase was applied via the reference electrode. We monitored the electronic transfer function of the FETs with the attached cell. The resulting frequency spectrum was used to investigate the surface adhesion of individual HEK293 cells. We applied different chemical treatments with either the serinpeptidase trypsin or the ionophor amphotericin B (AmpB). Binding studies can be realized by a time-dependent readout of the lock-in amplifier at a constant frequency. We observed cell detachment upon trypsin activity as well as membrane decomposition induced by AmpB. The results were interpreted in terms of an equivalent electrical circuit model of the complete system. The presented method could in future be applied to monitor more relevant biomedical manipulations of individual cells. Due to the utilization of the silicon technology, our method could be easily up-scaled to many output channels for high throughput pharmacological screening. 相似文献
8.
《Cell communication & adhesion》2013,20(4-6):393-399
Immunolabeling with antibodies against connexins 26 and 30 showed that, in the guinea pig cochlea, supporting Deiters″ cells are massively interconnected and form an orderly network within the organ of Corti. In paired patch-clamp recordings the coupling ratio (CR) of adjacent Deiters″ cells at the apex of the cochlea (~0.31) was 3-fold smaller than in isolated cell pairs due to shunting afforded by multicellular connectivity. With sinusoidal current stimuli the delay in signal propagation between adjacent cells increased with increasing frequency whereas the amplitude did not change significantly up to 200 Hz (corner frequency Fc ~220 Hz). Depolarizing voltage commands applied to an outer hair cell (OHC) elicited outward potassium currents in the OHC and inward currents in the abutting Deiters″ cells, supplying direct evidence for potassium buffering in the organ of Corti. Computational analysis indicates that electrical signals injected into a Deiters″ cell are transmitted across a network segment spanning 8 cell diameters. Thus electrical coupling in the organ of Corti is unlikely to influence the selectivity of frequency filtering performed mechanically by the mammalian cochlea. 相似文献
9.
Single-channel, macroscopic, and gating currents from sodium channels in the squid giant axon.
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Single-channel, macroscopic ionic, and macroscopic gating currents were recorded from the voltage-dependent sodium channel using patch-clamp techniques on the cut-open squid giant axon. To obtain a complete set of physiological measurements of sodium channel gating under identical conditions, and to facilitate comparison with previous work, comparison was made between currents recorded in the absence of extracellular divalent cations and in the presence of physiological concentrations of extracellular Ca2+ (10 mM) and Mg2+ (50 mM). The single-channel currents were well resolved when divalent cations were not included in the extracellular solution, but were decreased in amplitude in the presence of Ca2+ and Mg2+ ions. The instantaneous current-voltage relationship obtained from macroscopic tail current measurements similarly was depressed by divalents, and showed a negative slope-conductance region for inward current at negative potentials. Voltage dependent parameters of channel gating were shifted 9-13 mV towards depolarized potentials by external divalent cations, including the peak fraction of channels open versus voltage, the time constant of tail current decline, the prepulse inactivation versus voltage relationship, and the charge-voltage relationship for gating currents. The effects of divalent cations are consistent with open channel block by Ca2+ and Mg2+ together with divalent screening of membrane charges. 相似文献
10.
Bulai PM Molchanov PG Denisov AA Pitlik TN Cherenkevich SN 《European biophysics journal : EBJ》2012,41(3):319-327
Signals recorded from neurons with extracellular planar sensors have a wide range of waveforms and amplitudes. This variety
is a result of different physical conditions affecting the ion currents through a cellular membrane. The transmembrane currents
are often considered by macroscopic membrane models as essentially a homogeneous process. However, this assumption is doubtful,
since ions move through ion channels, which are scattered within the membrane. Accounting for this fact, the present work
proposes a theoretical model of heterogeneous membrane conductivity. The model is based on the hypothesis that both potential
and charge are distributed homogeneously on the membrane surface, concentrated near channel pores, as the direct consequence
of the inhomogeneous transmembrane current. A system of continuity equations having non-stationary and quasi-stationary forms
expresses this fact mathematically. The present work performs mathematical analysis of the proposed equations, following by
the synthesis of the equivalent electric element of a heterogeneous membrane current. This element is further used to construct
a model of the cell-surface electric junction in a form of the equivalent electrical circuit. After that a study of how the
heterogeneous membrane conductivity affects parameters of the extracellular electrical signal is performed. As the result
it was found that variation of the passive characteristics of the cell-surface junction like conductivity of the cleft and
the cleft height could lead to different shapes of the extracellular signals. 相似文献
11.
Frequency dependence of electrical coupling in Deiters' cells of the guinea pig cochlea 总被引:2,自引:0,他引:2
Immunolabeling with antibodies against connexins 26 and 30 showed that, in the guinea pig cochlea, supporting Deiters' cells are massively interconnected and form an orderly network within the organ of Corti. In paired patch-clamp recordings the coupling ratio (CR) of adjacent Deiters' cells at the apex of the cochlea (approximately 0.31) was 3-fold smaller than in isolated cell pairs due to shunting afforded by multicellular connectivity. With sinusoidal current stimuli the delay in signal propagation between adjacent cells increased with increasing frequency whereas the amplitude did not change significantly up to 200 Hz (corner frequency Fc approximately 220 Hz). Depolarizing voltage commands applied to an outer hair cell (OHC) elicited outward potassium currents in the OHC and inward currents in the abutting Deiters' cells, supplying direct evidence for potassium buffering in the organ of Corti. Computational analysis indicates that electrical signals injected into a Deiters' cell are transmitted across a network segment spanning 8 cell diameters. Thus electrical coupling in the organ of Corti is unlikely to influence the selectivity of frequency filtering performed mechanically by the mammalian cochlea. 相似文献
12.
Tomoko Okada Yuji Yamamoto Tsubasa Shibuya Hyen-Wook Kang Hirotaka Miyachi Isao Karube Hiroshi Muramatsu Jong Min Kim 《Biochemical Engineering Journal》2008,41(1):17-23
We have developed a new method for evaluating the affinity interactions between two different proteins by applying an alternating current (AC) voltage to a micro-flow channel. An AC voltage was applied to the protein-modified microspheres in the micro-flow channel, which resulted in the oscillation of the microspheres owing to their surface charges. The oscillation amplitude showed a linear relationship with the charge density of the microspheres. As an example for protein affinity measurement, the amplitude changes of a profilin-modified microsphere were measured by the addition of actin. In the same electrical condition, the oscillation amplitude of the profilin-modified microsphere increased by ≈175% by binding with actin. Similar results in the principle were obtained for the affinity interaction between biotin and streptavidin. The results showed that the higher the charge density of the microspheres induced by binding with different proteins, the higher the oscillation amplitude of the microspheres, thus, suggesting a possible application of the micro-flow channel and AC voltage on the protein property study, as well as on the biosensor application using the oscillation amplitude changes. 相似文献
13.
Relationship of calcium transients to calcium currents and charge movements in myotubes expressing skeletal and cardiac dihydropyridine receptors 总被引:12,自引:1,他引:11
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《The Journal of general physiology》1994,103(1):125-147
In both skeletal and cardiac muscle, the dihydropyridine (DHP) receptor is a critical element in excitation-contraction (e-c) coupling. However, the mechanism for calcium release is completely different in these muscles. In cardiac muscle the DHP receptor functions as a rapidly-activated calcium channel and the influx of calcium through this channel induces calcium release from the sarcoplasmic reticulum (SR). In contrast, in skeletal muscle the DHP receptor functions as a voltage sensor and as a slowly-activating calcium channel; in this case, the voltage sensor controls SR calcium release. It has been previously demonstrated that injection of dysgenic myotubes with cDNA (pCAC6) encoding the skeletal muscle DHP receptor restores the slow calcium current and skeletal type e-c coupling that does not require entry of external calcium (Tanabe, Beam, Powell, and Numa. 1988. Nature. 336:134-139). Furthermore, injection of cDNA (pCARD1) encoding the cardiac DHP receptor produces rapidly activating calcium current and cardiac type e-c coupling that does require calcium entry (Tanabe, Mikami, Numa, and Beam. 1990. Nature. 344:451-453). In this paper, we have studied the voltage dependence of, and the relationship between, charge movement, calcium transients, and calcium current in normal skeletal muscle cells in culture. In addition, we injected pCAC6 or pCARD1 into the nuclei of dysgenic myotubes and studied the relationship between the restored events and compared them with those of the normal cells. Charge movement and calcium currents were recorded with the whole cell patch-clamp technique. Calcium transients were measured with Fluo-3 introduced through the patch pipette. The kinetics and voltage dependence of the charge movement, calcium transients, and calcium current in dysgenic myotubes expressing pCAC6 were qualitatively similar to the ones elicited in normal myotubes: the calcium transient displayed a sigmoidal dependence on voltage and was still present after the addition of 0.5 mM Cd2+ + 0.1 mM La3+. In contrast, the calcium transient in dysgenic myotubes expressing pCARD1 followed the amplitude of the calcium current and thus showed a bell shaped dependence on voltage. In addition, the transient had a slower rate of rise than in pCAC6-injected myotubes and was abolished completely by the addition of Cd2+ + La3+. 相似文献
14.
Intracellular tetraethylammonium (TEA) inhibition was studied at the single-channel level in the KcsA potassium channel reconstituted in planar lipid bilayers. TEA acts as a fast blocker (resulting in decreased current amplitude) with an affinity in the 75 mM range even at high bandwidth. Studies over a wide voltage range reveal that TEA block has a complex voltage-dependence that also depends on the ionic conditions. These observations are examined in the context of permeation models to extend our understanding of the coupling between permeant ions and TEA blockade. 相似文献
15.
Human ether-à-go-go-related gene K+ channel gating probed with extracellular ca2+. Evidence for two distinct voltage sensors
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Human ether-à-go-go-related gene (HERG) encoded K+ channels were expressed in Chinese hamster ovary (CHO-K1) cells and studied by whole-cell voltage clamp in the presence of varied extracellular Ca2+ concentrations and physiological external K+. Elevation of external Ca2+ from 1.8 to 10 mM resulted in a reduction of whole-cell K+ current amplitude, slowed activation kinetics, and an increased rate of deactivation. The midpoint of the voltage dependence of activation was also shifted +22.3 +/- 2.5 mV to more depolarized potentials. In contrast, the kinetics and voltage dependence of channel inactivation were hardly affected by increased extracellular Ca2+. Neither Ca2+ screening of diffuse membrane surface charges nor open channel block could explain these changes. However, selective changes in the voltage-dependent activation, but not inactivation gating, account for the effects of Ca2+ on Human ether-à-go-go-related gene current amplitude and kinetics. The differential effects of extracellular Ca2+ on the activation and inactivation gating indicate that these processes have distinct voltage-sensing mechanisms. Thus, Ca2+ appears to directly interact with externally accessible channel residues to alter the membrane potential detected by the activation voltage sensor, yet Ca2+ binding to this site is ineffective in modifying the inactivation gating machinery. 相似文献
16.
Oleg V. Aslanidi Rakan N. Sleiman Mark R. Boyett Henggui Zhang 《Biophysical journal》2010,98(11):2420-2431
The intrinsic heterogeneity of electrical action potential (AP) properties between Purkinje fibers (PFs) and the ventricular wall, as well as within the wall, plays an important role in ensuring successful excitation of the ventricles. It can also be proarrhythmic due to nonuniform repolarization across the Purkinje-ventricular junction. However, the ionic mechanisms that underlie the marked AP differences between PFs and ventricular cells are not fully characterized. We studied such mechanisms by developing a new family of biophysically detailed AP models for rabbit PF cells and three transmural ventricular cell types. The models were based on and validated against experimental data recorded from rabbit at ionic channel, single cell, and tissue levels. They were then used to determine the functional roles of each individual ionic channel current in modulating the AP heterogeneity at the rabbit Purkinje-ventricular junction, and to identify specific currents responsible for the differential response of PFs and ventricular cells to pharmacological interventions. 相似文献
17.
F Van Goor D Zivadinovic A J Martinez-Fuentes S S Stojilkovic 《The Journal of biological chemistry》2001,276(36):33840-33846
In excitable cells, voltage-gated calcium influx provides an effective mechanism for the activation of exocytosis. In this study, we demonstrate that although rat anterior pituitary lactotrophs, somatotrophs, and gonadotrophs exhibited spontaneous and extracellular calcium-dependent electrical activity, voltage-gated calcium influx triggered secretion only in lactotrophs and somatotrophs. The lack of action potential-driven secretion in gonadotrophs was not due to the proportion of spontaneously firing cells or spike frequency. Gonadotrophs exhibited calcium signals during prolonged depolarization comparable with signals observed in somatotrophs and lactotrophs. The secretory vesicles in all three cell types also had a similar sensitivity to voltage-gated calcium influx. However, the pattern of action potential calcium influx differed among three cell types. Spontaneous activity in gonadotrophs was characterized by high amplitude, sharp spikes that had a limited capacity to promote calcium influx, whereas lactotrophs and somatotrophs fired plateau-bursting action potentials that generated high amplitude calcium signals. Furthermore, a shift in the pattern of firing from sharp spikes to plateau-like spikes in gonadotrophs triggered luteinizing hormone secretion. These results indicate that the cell type-specific action potential secretion coupling in pituitary cells is determined by the capacity of their plasma membrane oscillator to generate threshold calcium signals. 相似文献
18.
Subramanian S Aschenbach KH Evangelista JP Najjar MB Song W Gomez RD 《Biosensors & bioelectronics》2012,32(1):69-75
An electronic platform to detect very small amounts of genomic DNA from bacteria without the need for PCR amplification and molecular labeling is described. The system uses carbon nanotube field-effect transistor (FET) arrays whose electrical properties are affected by minute electrical charges localized on their active regions. Two pathogenic strains of E. coli are used to evaluate the detection properties of the transistor arrays. Described herein are the results for detection of synthetic oligomers, unpurified and highly purified genomic DNA at various concentrations and their comparison against non-specific binding. In particular, the capture of genomic DNA of E. coli O157:H7 by a specific oligonucleotide probe coated onto the transistor array results in a significant shift in the threshold (gate-source) voltage (V(th)). By contrast the signal under the same procedure using a different strain, E. coli O45 that is non-complementary to the probe remained nearly constant. This work highlights the detection sensitivity and efficacy of this biosensor without stringent requirement for DNA sample preparation. 相似文献
19.
Bing Shen Ching-On Wong On-Chai Lau Theodosia Woo Suwen Bai Yu Huang Xiaoqiang Yao 《PloS one》2015,10(4)
Mechanical forces exerted on cells impose stress on the plasma membrane. Cells sense this stress and elicit a mechanoelectric transduction cascade that initiates compensatory mechanisms. Mechanosensitive ion channels in the plasma membrane are responsible for transducing the mechanical signals to electrical signals. However, the mechanisms underlying channel activation in response to mechanical stress remain incompletely understood. Transient Receptor Potential (TRP) channels serve essential functions in several sensory modalities. These channels can also participate in mechanotransduction by either being autonomously sensitive to mechanical perturbation or by coupling to other mechanosensory components of the cell. Here, we investigated the response of a TRP family member, TRPC5, to mechanical stress. Hypoosmolarity triggers Ca2+ influx and cationic conductance through TRPC5. Importantly, for the first time we were able to record the stretch-activated TRPC5 current at single-channel level. The activation threshold for TRPC5 was found to be 240 mOsm for hypoosmotic stress and between −20 and −40 mmHg for pressure applied to membrane patch. In addition, we found that disruption of actin filaments suppresses TRPC5 response to hypoosmotic stress and patch pipette pressure, but does not prevent the activation of TRPC5 by stretch-independent mechanisms, indicating that actin cytoskeleton is an essential transduction component that confers mechanosensitivity to TRPC5. In summary, our findings establish that TRPC5 can be activated at the single-channel level when mechanical stress on the cell reaches a certain threshold. 相似文献
20.
Jordan PN Christini DJ 《American journal of physiology. Heart and circulatory physiology》2007,293(4):H2109-H2118
Experiments have provided suggestive but inconclusive insights into the relative contributions of transmembrane voltage and intracellular calcium handling to the development of cardiac electrical instabilities such as repolarization alternans. In this study, we applied a novel combination of techniques (action potential voltage clamping, calcium-transient clamping, and stability analysis) to cardiac cell models to more clearly determine the roles that voltage- and calcium-dependent coupling play in regulating action potential stability and the development of alternans subsequent to the loss of stability. Using these techniques, we are able to demonstrate that voltage- and calcium-dependent coupling exhibit varying degrees of influence on action potential stability across models. Our results indicate that cellular dynamic instabilities such as alternans may be initiated by either voltage- or calcium-dependent mechanisms or by some combination of the two. Based on these modeling results, we propose novel single-cell experiments that incorporate action-potential voltage clamping, calcium imaging, and real-time measurement of action potential stability. These experiments will make it possible to experimentally determine the relative contribution of voltage coupling to the regulation of action potential stability in real cardiac myocytes, thereby providing further insights into the mechanism of alternans. 相似文献