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Seminars in Cell & Developmental Biology 20 (2009) 41–48

Contents lists available at ScienceDirect

Seminars in Cell & Developmental Biology

journa l homepage: www.e lsev ier .com/ locate /semcdb

eview

ield-effect devices for detecting cellular signals

. Poghossiana,b, S. Ingebrandtc, A. Offenhäusserb, M.J. Schöninga,b,∗

Institute of Nano- and Biotechnologies, Aachen University of Applied Sciences, Campus Jülich, Ginsterweg 1, D-52428 Jülich, GermanyInstitute of Bio- and Nanosystems-2, Research Centre Jülich, D-52425 Jülich, GermanyDepartment of Informatics and Microsystem Technology, Kaiserslautern University of Applied Sciences,ampus Zweibrücken, Amerikastrasse 1, D-66482 Zweibrücken, Germany

r t i c l e i n f o

rticle history:vailable online 3 February 2009

a b s t r a c t

The integration of living cells together with silicon field-effect devices challenges a new generation ofbiosensors and bioelectronic devices. Cells are representing highly organised complex systems, optimised

eywords:iving cellsell-FETAPSeuron/transistor hybrid

by millions of years of evolution and offering a broad spectrum of bioanalytical receptor “tools” such asenzymes, nucleic acids proteins, etc. Their combination with semiconductor-based electronic chips allowsthe construction of functional hybrid systems with unique functional and electronic properties for bothfundamental studies and biosensoric applications. This review article summarises recent advances andtrends in research and development of cell/transistor hybrids (cell-based field-effect transistors) as well

ell acidificationction potential

as light-addressable potentiometric sensors.© 2009 Elsevier Ltd. All rights reserved.

ontents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 412. Cell/transistor hybrid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42

2.1. FETs for cellular metabolism detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 422.2. Cell/transistor hybrids for extracellular potential measurements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 432.3. Cell adhesion on Si chip . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44

3. LAPS devices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 453.1. Cellular acidification detection with LAPS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 453.2. LAPS for extracellular potential and single-cell measurements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46

4. Conclusions and outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47

. Introduction organised living microstructures containing a high concentration

One of the most challenging topics in the field of biosensors andioelectronics is the development of integrated microsystems capa-le of interactions with living cells and organisms. Cells are highly

Abbreviations: CHO, Chinese hamster ovary; CMOS, complementary metal-oxideemiconductor; EIS, electrolyte–insulator–semiconductor; FED, field-effect device;ET, field-effect transistor; HEK, human embryonic kidney; ISFET, ion-sensitive field-ffect transistor; LAPS, light-addressable potentiometric sensor; LED, light-emittingiode; RE, reference electrode; SOI, silicon-on-insulator; ID, drain current; Iph, gen-rated photocurrent; VBias, bias voltage; VDS, drain-source voltage; VG, gate voltage.∗ Corresponding author at: Institute of Nano- and Biotechnologies, Aachen Uni-

ersity of Applied Sciences, Campus Jülich, Ginsterweg 1, D-52428 Jülich, Germany.el.: +49 241 6009 53215; fax: +49 241 6009 53235.

E-mail address: m.j.schoening@fz-juelich.de (M.J. Schöning).

084-9521/$ – see front matter © 2009 Elsevier Ltd. All rights reserved.oi:10.1016/j.semcdb.2009.01.014

of chemicals, including enzymes, nucleic acids, ions, many typesof proteins and small organic molecules. They process multipleincoming information by means of a parallel activation of differ-ent signalling pathways and respond with an appropriate reactionpattern according to the type of input physical or chemical stimuli.Cell-based biosensors, where the smallest self-sustaining biologi-cal entity – whole cells – are used as a recognition element or asprimary transducer, offer the opportunity of detecting a broad spec-trum of known/unknown chemical agents. Moreover, they enableto determine the impact on human performance by monitoring thevariations in the physiological activity caused by the local environ-

ment. In spite of their limited lifetime, the most important reasonfor developing cell-based biosensors is given by the fact that onlyby using living components can one deliver direct functional infor-mation, i.e., information about the effect of an external physical or

4 & Developmental Biology 20 (2009) 41–48

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hemical stimulus on a living system [1,2]. This functional infor-ation with additional qualitative and/or quantitative analytical

nformation can be very important in molecular and cell biology,linical diagnostics, toxicology, pharmacology and drug screen-ng, environmental monitoring and biological warfare agent field.epresentative examples include the study of effects of pharmaceu-ical compounds, toxic substances and pollutants on a physiologicalystem, in particular on cellular metabolisms, electrophysiologicaleasurements, the monitoring of transmission paths of ionic chan-

els, transmission velocity of biological signals along the layer ofeurons, spatially resolved monitoring of electrical communicationithin neuronal networks, development of neuronal prostheses,

tc. Moreover, neurochips with bi-directional interfacing betweeneurons and electronic circuits would allow investigation of theynamics in neuronal networks.

Among the variety of proposed concepts, the integration of liv-ng cells together with a silicon chip consisting of an array ofbio-)chemical and/or electrophysiological transducers based onfield-effect EIS (electrolyte–insulator–semiconductor) system is

ne of the most attractive approaches [1–7]. These devices pro-ide a lot of potential advantages such as small size and weight,ast response time, high reliability, compatibility with advanced

icro- and nanofabrication technology, and the possibility of on-hip integration of transducer arrays and signal processing scheme.uch a “marriage” of living cells and silicon chips might allow notnly the creation of functional hybrid systems with new uniqueunctional and application possibilities but can also be very use-ul in fundamental research of physiological processes. This paperives a review of recent advances and current trends in researchnd development of Cell-FETs (cell-based field-effect transistors)nd LAPS (light-addressable potentiometric sensor) devices for cel-ular metabolism and extracellular potential measurements. Theaper also summarises some recent experiments realised in our

aboratories.

. Cell/transistor hybrid

The cell/transistor hybrid is obtained by direct coupling of a sin-le cell or cell system to the gate insulator of a FET. Fig. 1 showshe cell/transistor hybrid (a), the schematic of a cell attached tohe open-gate of a transistor (b), and the corresponding differen-ial interference contrast microscopic image (c) of cells (in this caseenetically modified HEK293 cells (human embryonic kidney)) onhe surface of the FET array. In the middle of the image, one indi-idual cell completely covers the sensitive gate area of the FET.

In general, (bio-)chemical sensors based on the semiconductoreld-effect are very sensitive for any kind of electrical interactiont or nearby the gate insulator/electrolyte interface. Therefore, itan be expected that nearly each chemical or electrical change athis interface, induced by coupled living cells, will modulate thepace-charge region in the silicon at the insulator–semiconductornterface, resulting in a modulation of the current in the inver-ion channel of the FET. The most critical point in informationransfer from the biological recognition part to the transducer parts the interface between these two domains (for more detailednformation concerning the operation principle of different typesf biologically modified FETs, in general, see e.g., recent reviews8–11]).

The state of a single cell or cell system can be monitored byeans of various methods, which can be distinguished into two

asic families [1]: (i) the first family of methods utilises the energyetabolism of cells and, in principle, can be extended to all cell

ypes. It is sensitive to a wide range of cellular events, like growth,oxicity, etc. Signal parameters such as changes in the extracellularH, redox potential, concentration of ions, CO2 production, oxy-

Fig. 1. Cell/transistor hybrid (a). The open-gate area of the FET is completely cov-ered by one cell as indicated in the schematics (b). The real situation as seen in thedifferential interference contrast microscopic image is shown in (c). RE: referenceelectrode; VG: gate voltage; VDS: drain-source voltage; ID: drain current.

gen consumption and other metabolic products (glucose, lactate)caused by different external stimuli (e.g., by addition of toxic agentsor drugs to the medium) can be measured by various field-effect-based single chemical sensors and biosensors, or multi-sensorsystems; (ii) the second family of cell/transistor hybrids utilises spe-cific features of electrogenic cells, like neuronal cells, muscle cells,and includes extracellular potential measurements [3–7,12–14].

2.1. FETs for cellular metabolism detection

In the last decade, fundamental advances in whole cell-basedsensors and microsystems have established the monitoring of theextracellular acidification rate of cell cultures as an important indi-cator of the global cellular metabolism. Bergveld firstly employedthe SiO2-gate ISFET (ion-sensitive FET) for an extracellular ion-concentration measurement [15]. More recently, Cell-FETs withSi3N4, Al2O3 or Ta2O5 as pH-sensitive gate insulator have beenrealised for extracellular acidification as well as respiration (oxy-gen consumption) measurements (e.g., [16–21]). In addition, itwas shown that for an adherent epithelial tumour cell line, thebasolateral acidification (i.e., amount of extruded protons) in thenano-scale cleft between the solid surface and the cell membraneis several orders of magnitude lower than the apical (space above

the cell) acidification [19].

The acidification rate is correlated to the physiological state ofthe cell. Under steady-state conditions, one cell produces about∼108 protons per second. Dependent on the size of sensitive region,up to 105–106 cells can be cultivated on a single sensor chip. After

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he external receptor stimulation, this quantity of protons can raiserom 10 to 100% depending on the cell type, the receptor and theoupling pathway [22]. The changes in the extracellular acidifica-ion rate (i.e., changes in the physiological state of the cell) causedy different external stimuli, like addition of drugs or toxic agentso the medium, can be detected using an array of Cell-FETs in aow-stop mode.

Because cells respond to external stimuli with a parallel activa-ion of different signalling pathways, the parallel and non-invasive

easurement of different parameters is very helpful for an inter-retation of different effects on living cells. Examples are giveny a cell-monitoring system combining different microsensorsncluding arrays of pH ISFETs and cell-potential FETs with differentate areas for both cell population and single-cell measurements16], and a completely automated cell-monitoring microsystem,ombining an array of 12 ISFETs for multi-site monitoring of thehysiological state of cell populations with space and time resolu-ion, having a CMOS (complementary metal-oxide-semiconductor)ntegrated circuit for signal conditioning and multiplexing [17].he experiments by monitoring changes in the extracellular acid-fication rate of Cheratinocytes and CHO (Chinese hamster ovary)ells after applying toxic agents demonstrate the capability of theeveloped microsystem for in vitro toxicity-screening applications.urthermore, a multi-sensor silicon needle consisting of two ISFETsas been realised for the simultaneous measurement of the H+-nd K+-ion concentration using myocardial tissues [23], and a Cell-ET chip with a cell-positioning system and integrated Ag/AgClseudo-reference electrode has been developed for on-line mon-

toring of the metabolic response of E. coli bacteria to glucose [24].ehmann et al. first reported the simultaneous monitoring of bothhe acidification and respiration (oxygen consumption) at the sameite in a cell culture by using a Cell-FET with a surrounding nobleetal electrode for oxygen reduction and OH−-ion generation [16].ow, such a monitoring system is commercially available (Bionas®

500 analysing system, Bionas GmbH, Rostock, Germany) [18]. Withhe Bionas® 2500 analysing system metabolically relevant data,ncluding oxygen consumption, acidification rate and adhesion (cellmpedance) of cells, can be non-invasively measured in parallel,nline and over a long period of time. The system has been alreadystablished for several cell models and cell lines [18].

It has been considered that the compounds causing cell acidifi-ation are mostly lactic acid, sodium bicarbonate and CO2 secreteduring the energy metabolism with glucose and glutamine [20]. Tovaluate the metabolic activity of cultured bovine aortic endothe-ial cells as well as rabbit aortic smooth muscle cells, a flow-throughype pH/CO2 sensor system using two ISFETs has been constructed,hat enables the measurement of the overall pH change and theH change due to CO2 around the cells [20,21]. These experimentsllow the evaluation of the cellular secretion rates of CO2, lacticcid and sodium bicarbonate during the energy metabolism withlucose and glutamine. To validate the usefulness of this system,t was applied to the quantitative analysis of metabolic switchingy the change of glucose concentration in cultured bovine arterialndothelium, human umbilical vein endothelium and rat cardiacuscle cells, respectively [21].The possible scientific applications of cell-monitoring systems

oncern all aspects of cell metabolisms, while commercial aspectsainly cover pharmacological and toxicological questions.

.2. Cell/transistor hybrids for extracellular potentialeasurements

Extracellular potential measurements from the cell membranere considered to be a reliable indicator for the determination of theresence of a specific agent in the cell’s local environment. More-ver, direct electrical interfacing of semiconductor and nerve cells

elopmental Biology 20 (2009) 41–48 43

is the physical basis for the development of hybrid neuroelectronicdevices such as neurocomputers and neuroprostheses. Althoughextracellular signals are significantly smaller than the transmem-brane voltage changes, field-effect devices enable long-term andnon-invasive recordings of extracellular signals with a large spatialand temporal resolution. Signals can be recorded from individualneurons, small networks of neurons and from large areas usingneuronal slice cultures.

The first attempt to apply an ISFET in neurophysiological mea-surements was already reported in the 70s of the last century[25,26]. More recently, the first recording from single invertebrate[27] and vertebrate neurons [28] as well as cardiac myocyte mono-layers [29] cultured onto the gate insulator of a FET was reported(see also, reviews [3,5,7]). Most recent developments are large-scaledevices such as high-resolution multi-transistor arrays with 16384(128 × 128) elements [30]. Large-scale recordings while managinga large data throughput from a multitude of sensor elements havebeen demonstrated with different systems [31–33].

Electrogenic cells generate spontaneous or triggered actionpotentials (transient changes of cell membrane potentials) that canbe monitored by a neuron/FET hybrid. The activity of a neuron leadsto ionic- and displacement currents flowing through the cell mem-brane, resulting in an extracellular voltage drop along the narrowcleft between the cell membrane and the gate insulator, whichmodulates the drain current of the Cell-FET (this current, usuallytransformed into a voltage, represents the extracellularly recordedsignals) [3–7,12–14]. Cell/transistor hybrids have been applied torecord the activation of recombinant serotonin receptors [34], vesi-cle release in chromaffin cells [35], and functional K+ [36,37], Na+

[38] and Cl− [39] channels in HEK293 cells. Moreover, the pos-sibility of creation of a bi-directional interface between neuronand transistor, which allows both a capacitive extracellular stim-ulation of an individual neuron and a capacitive recording of theneural activity of cells cultured onto the gate of a FET, has beendemonstrated in [5,40]. The mechanism of extracellular stimula-tion of nerve cells with an EIS capacitor has been discussed in[41].

For a better understanding the mechanisms of signal generationand correct interpretation of the extracellular signals, the study ofphysical phenomena in the cleft (the interface between cell andsensor surface) and at the sensor surface is mandatory. Differentapproaches have been proposed to describe the signal transfer fromelectrogenic cells to a FET and explain the recorded signal behaviour(e.g., signal shape and amplitude). A commonly used approachis the point-contact model [3,5,7]. For the interpretation of someeffects (for instance, high ionic conductance in the cleft betweencell and transistor, time delay of the extracellular signal, etc.) thathave been observed in the experiments with neuronal cells, dif-ferent extended models have been reported (see e.g., [3,5,7,36,37]and references therein). Recently, using Aplysia neurons cultured onfloating-gate FETs showed that the field-effect potential generatedby their action potential is produced by the current flow along theneuronal compartments comprising the axon, cell body and neu-rites, rather than by the flow between the membrane facing the chipsubstrate and the culture medium [42]. Moreover, the waveform ofthe signal generated by non-isopotential neurons largely dependson the morphology of the neuron [42].

In addition to neuronal cells, cardiac muscle cells are repre-senting an interesting electrically active system that processesbiological information. The spontaneous rhythmic electrical andmechanical activity of cultured myocardiac cells is widely used to

study cardiac physiology. Their beating frequency can be changedunder affects of cardiac stimulants and relaxants. As an example, abioelectronic device (array of 16 FETs) has been adapted for long-term and multi-site recording effects of different drugs on cardiacmyocytes [43,44]. The use of FET-based small extracellular sensor

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hips in pharmaceutical research holds a great promise for costeduction and less use of animals. The administration of very diluteardiac stimulants and relaxants are changing the beating rhythmsf the cell layer (“miniature heart”) similar to the reaction of a realeart [45].

The classically used configuration for the formation of a neu-on/transistor hybrid is based on culturing neurons directly onop of the exposed gate insulator of an FET, the so-called open-ate design. An alternative approach is to use floating-gate FETevices for extracellular potential measurements. Floating-gateevices can either have insulated sensing areas [30,46,47] or sens-

ng areas composed of metal electrodes [33]. The floating-gatepproach offers several advantages: the neuron’s coupling area iseparated from the channel area, the channel area can be insu-ated and shielded from the ionic solution in which the neuronsre cultured, and the parasitic photo effects can be eliminated bydding a metal-shielding layer over the transistor. Most importantlynd in contrast to the open-gate design, floating-gate FETs can beealised in an industrial CMOS process enabling large-scale devicesith on-chip logics such as shift registers and decoders. With an

ppropriate post-process [30,47] these devices can be operated in aiquid environment. Experimental tests have shown the functional-ty of floating-gate devices for recording action potentials generatedy cultured neurons [48,49]. In addition, a 16-channel backside-ontacted FET array [50], a high-resolution multi-transistor array32], and AlGaN/GaN FETs [51,52] have been developed for extra-ellular signal recording. These AlGaN/GaN heterostructures offerigh signal-to-noise ratios in combination with transparency of theevices, allowing simultaneous optical and electrical monitoringf the cellular activity. A further example represents an FET-basedensor array combining Cell-FETs for detecting the action poten-ial and ISFETs coupled with different ion-sensitive membranes forhe measurement of extracellular concentrations of Na+, K+ anda2+; here, the relationship between the membrane potential andhe influx/efflux of ions under an external stimulation and drugreatment has been investigated [53]. Generally, the advantage ofell-FETs over conventional patch-clamp technique is the possibil-

ty of real-time, non-invasive and long-term monitoring of the statef living cells.

The rapid development of nanotechnology and creation ofnew class of nano-scaled devices, like nanotubes, nanorods,

anowire transistors, etc., open new and encouraging possibilitiesor both fundamental studying of individual cells or localised bio-nvironments and pharmacological and toxicological applications.ue to their large surface-to-volume ratio and quasi-1D structure,dramatically improved sensitivity for these transducers can be

xpected. As an example, by using a SOI (silicon-on-insulator)tructure, a microneedle-type sensor with integrated siliconanowire (50 nm) tip was developed for extracellular or intracellu-

ar (bio-)chemical detection [54]. A recent intriguing report by Kimt al. [55] describes a novel way of introducing foreign materialnto cells. The authors cultured both human embryonic kidneyells and mouse embryonic stem cells on Si nanowire needle arrays6 �m long with a diameter 90 nm). Within approximately 1 hnd without application of an external force, the cells settled ontohe needle substrates. Although the nanowires were penetratinghese cells, the authors proved that the cells were still alive. Thus,anowires could be used not only as intracellular (bio-)chemicalensors to detect specific biomolecules at the cell level, but alsoor delivering genetic materials and drugs, or for in vivo electricaltimulation of cells [56]. However, before Si nanowires can be

ncorporated into new biomedical devices, their cytotoxicitynd potential adverse effects on biological systems (e.g., on celldhesion and spreading) should be thoroughly investigated [57].

Finally, the creation of two-dimensional neuronal cell net-orks coupled with networks of field-effect devices (e.g., array of

elopmental Biology 20 (2009) 41–48

nano-scaled FETs or nanowires) can open up new avenues for fun-damental study of signal transduction and propagation betweeninterconnected cells. Progress in this field will mostly dependon a successful technological realisation of large neuronal net-works, handling the large amount of data that must be processed,improvement of the signal-to-noise ratio of the sensors, as wellas understanding the complex neuronal networks’ response. As afirst step to investigate the signal propagation inside a network ofneurons, recently, high-density Si nanowire transistors have beenintegrated with individual axons and dendrites of living mam-malian neurons, where each nano-scale junction has been used for aspatially resolved detection, stimulation and inhibition of the neu-ronal signal propagation [58]. Such an array of nanowire-neuronjunctions could enable the simultaneous measurement of the rate,amplitude, and shape of signals propagating along the individualaxons and dendrites. However, because of some incorrectness inpublished experimental graphs, the results reported in [58] andtheir interpretations should be carefully evaluated.

2.3. Cell adhesion on Si chip

Cell adhesion plays an important role in biological processes, likeformation of organs, assembly of blood vessels, etc. The electricalresistance of the extracellular nanospace in brain tissue is a crucialphysical parameter which can affect the propagation of neuronalexcitation in dendrites and axons [59].

The coupling strength between a neuron and a microelectronicchip, i.e., the adhesion “quality” of neurons to the transducer surfaceis the most critical parameter that may seriously change the fea-tures of recorded extracellular signals. Therefore, the FET-neuronjunction has been modelled to explore the behaviour of the junc-tion between a patch of neuronal membrane and the FET [14].The proposed model predicts a wide range of functional couplingphenomena, ranging from a single neuron directly coupled to aFET, characterised by a weak or tight neuro-electronic coupling,to small networks of synaptically connected neurons, whose col-lective emerging electrical activity can be extracellularly recorded.In addition, the width of the cleft and the specific resistance inthe region of adhesion have been evaluated in [37,59]. Further-more, by using Aplysia neurons cultured on floating-gate FETs, ithas been observed that the adjustment of a minimal local pressureapplied to the restricted neuronal compartment increases the sealresistance and the conductance of the membrane patch that facesthe sensing gate (from 0.5 to ∼1.2 MOhm and 75–600 nS, respec-tively). Thus, this effect could be used to transform conventionalextracellular field-potential recordings (capacitive coupling) intosemi-intracellular recordings of action potentials (ohmic coupling)with amplitudes of up to 30 mV, and to improve both the signal-to-noise ratio and the quality of recordings from neurons cultured onfloating-gate FETs [60].

It is well-known that the seal resistance in the area of celladhesion determines the amplitude of the recorded extracellularvoltage. On the other hand, the resistance is an intrinsic source ofthermal voltage noise that limits the signal-to-noise ratio of theextracellular recording of neuronal excitation. However, the noiseof adhesion may also provide an excellent tool to study the natureof cell-chip adhesion without perturbing the system. For this pur-pose, Fromherz et al. have investigated spectral characteristics ofthe voltage fluctuations in the adhesion area of nerve cells from ratbrain cultured onto an oxidised silicon surface [61]. At low frequen-cies the adhesion noise was below the 1/f noise of the transistor,

whereas it exceeds the transistor noise above 2 kHz.

Recently, we have demonstrated a new approach for cell-adhesion detection based on the transfer function of the FET [62].The transfer function method is based on an impedimetric read-out circuitry using lock-in electronics [63]. Such measurements

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or fundamental exploration of the cell-sensor adhesion haveeen described earlier [64,65]. With this 16-channel FET-arrayystem operating in the transfer-function mode, simultaneous,ime-dependent adhesion and detachment assays with individualEK293 cells have been demonstrated [62].

. LAPS devices

Due to some advantages such as the selection of the measure-ent point by a scanning light beam and the spatial resolution, LAPS

as become popular in many chemical and biological applicationssee e.g., reviews [1,9–11,53,66–68] and references there). Fig. 2(a)hows the layer structure and measurement set-up of the LAPS. Asn case of the capacitive EIS sensor, a dc bias voltage is applied tohe LAPS structure via a reference electrode so that a depletion layerppears at the insulator/semiconductor interface. Electrochemical

nteractions between the transducer surface and the analyte willreate additional surface potentials, which will be added to thepplied dc bias voltage. The width and therefore, the capacitance ofhe depletion layer will vary with the gate-insulator surface poten-ial. To detect the variation of the capacitance of the depletion layer,

ig. 2. Schematic set-up of a LAPS device with living cells and light sources (a) andicroscopic picture of CHO cells adherently grown on the pH-sensitive Ta2O5-gate

urface of the LAPS structure (b). RE: reference electrode; VBias: bias voltage; Iph:enerated photocurrent.

elopmental Biology 20 (2009) 41–48 45

the LAPS is illuminated with a modulated light source using laserbeam or light-emitting diodes (LED), which induce an ac photocur-rent to be measured as the sensor signal. For different pH values, thephotocurrent–voltage curve shifts along the voltage axis. The LAPSuses this dependence of the photocurrent on voltage to sense theinterfacial potential of the field-effect structure. In the constant-photocurrent mode, by setting the photocurrent at a fixed value(at the inflection point of the photocurrent–voltage curve) using afeedback-control circuit, changes in the interfacial potentials canbe directly recorded [69]. However, in contrast to the capacitiveEIS sensor, where the obtained value of the analyte concentra-tion is an average over the whole sensing surface area in contactwith the analyte, the LAPS measurement is spatially resolved. Thelight-addressability, that is the possibility of selection of the mea-suring area on the sensing surface by positioning the light on thedesired location, is the most important feature of the LAPS princi-ple. Fig. 2(b) exemplarily depicts the growth of CHO cells on top ofthe pH-sensitive transducer structure of Ta2O5. For more details ofLAPS operation principle, see e.g. [1,9,53,66–68].

3.1. Cellular acidification detection with LAPS

The LAPS has been originally developed to detect changes in pHcaused by cells adherent to a sensor surface. To our knowledge, thefirst and only successfully commercialised system using the LAPSfor the determination of extracellular acidification of living cells isthe Cytosensor Microphysiometer system, realised in 1990s by thecompany Molecular Devices Corporation (Sunnyvale, CA) [66,67].Microphysiometry can be used to monitor many physiological pro-cesses in cells, because of their effects on the energy metabolism.The applications for these measurements have included studyingthe electrical properties of biological membranes, the detection ofbacterial growth, and the measurement of cell metabolism as wellas mechanisms of drug action on the cell physiology, etc. (see e.g.,[1,2,9,53,66–68] and references there).

The light-addressable feature of the LAPS is suitable for bothmulti-sensor and chemical imaging sensor applications. However,the commercial Cytosensor Microphysiometer does not take advan-tage of the possibility of a spatial resolution, but usually detectsthe mean acidification caused by all cells. In order to obtain thepH distribution along the LAPS sensor surface with spatial reso-lution, either multiple light pointers can be used or a laser beamcan be scanned along the surface. Therefore, by a simple modi-fication of the Cytosensor Microphysiometer, a multi-light sensorchip was developed which enables a spatial-resolved measurementby simultaneous assessing of the metabolic states of two or morecell populations co-cultured on different parts of one sensor sur-face [70]. The ability of the proposed set-up was demonstrated bydetecting the differential sensitivity to a cholinergic receptor ago-nist of two different co-cultured cellular populations. Such a devicenot only provides a rapid method of determining the cellular speci-ficity of pharmaceutical compounds but also could be useful for thenon-invasive monitoring of humoral and synaptic communicationsbetween different cell populations in co-culture. In addition, byutilising a multi-light LAPS set-up, where each light pointer is mod-ulated at a different frequency [53], a novel microphysiometer hasbeen developed for the simultaneous measurement of extracellularion concentrations of H+, Na+, K+, and Ca2+. In this microphysiome-ter, different ion-sensitive membranes are illuminated in parallelwith light sources at different frequencies. The developed micro-physiometer has been applied to study the influence of drugs on

the metabolism of suckling rat’s nephridium cells and cardiac mus-cle cells. Moreover, a chemical imaging LAPS has been appliedfor detecting the metabolic activity of microorganisms, e.g., E. colicolonies immobilised on the LAPS surface [71]. Here, by measuringthe amplitude of the photocurrent at each point while the focused

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aser beam is scanned across the surface of the semiconductor, awo-dimensional mapping (i.e., distribution) of the pH value haseen visualised.

Most of the LAPS developments suffer from the lack in terms ofon-portability, insufficient robustness, complicated handling, etc.ence, a pen-shaped miniaturised portable LAPS and a 16 spots

hip-card LAPS device with integrated signal processing unit haveeen realised by our group [68,72,73]. Such a pen-shaped LAPS cane directly dipped into the test sample to be measured. The portableevice allows a quick and uncomplicated sequential measurementn addressing of up to 16 different working spots at the same time.n further work, the authors have developed a multi-light 16 spotship-card LAPS system [68,74,75]. The LAPS card itself consists ofhe LAPS chip embedded in a standard plastic card. The chip-cardAPS system has been tested, for instance, for the detection of theetabolic activity of CHO cells and macrophages. The evaluated

verage acidification rate of CHO cells in this experiment has been0.1 pH/min.

.2. LAPS for extracellular potential and single-cell measurements

For a detailed understanding of physico-chemical phenomenat the cell/transistor interface, electrically excitable cells have to beonnected individually in a way that each cell has to be interfacedith a separate potential-sensitive device (e.g., on top of the gate

f an individual FET). However, the number of active measuringites in FET-based cell monitoring systems is limited by the numberf FETs as well as by the fact that typically many cells could notr only partially cover the gate region of the individual FET. LAPSevices overcome this problem of a limited number of active sites,ecause the scanning light-pointer allows to individually addresssingle cell: although many cells are cultured on the chip surface,nly that cell which is adhered on top of the illuminated area isnterrogated. Moreover, because the LAPS surface is usually laterallyot photolithographically patterned (in contrast to FTEs), cells candhere without any spatial restrictions. Since the LAPS respondso any change of potential in series with the external bias circuit,herefore it should be also able to record the electric activity of aingle cell.

Several attempts have been made to record action potentials ofingle cells by means of the LAPS [53,76,77]. In order to improvehe adhesion of cells to the surface and thus, to efficiently trans-er the electrical signal from the neuron to the LAPS device, a SiO2ate was covered with poly-l-ornithine- and laminine [53,77]. Themproved LAPS device was used to record the action potential of aingle rat cortical cell under affect of acetylcholine. In this exper-ment, a signal change of about ∼10 �V was observed, which haseen attributed to the action potential of a single cell.

Heavy metals are the most dangerous environmental contami-ants that present a serious threat to human health, even in traceuantities. Therefore, the LAPS has been applied to study the effectf heavy metal ions’ toxicity on the electrophysiological activity ofells [78]. After being exposed to various types of heavy metal ionsHg2+, Pb2+, Cd2+, Fe3+, Cu2+, Zn2+), cardiomyocytes show character-stic changes in terms of beating frequency, amplitude and duration.he recorded extracellular potentials of spontaneously beating car-iomyocytes were in the range of 20–40 �V. Thus, it was possibleo study acute and eventually chronic toxicities induced by heavy

etal ions in a long-term and non-invasive way using this cardiacell-based LAPS.

Furthermore, LAPS devices have been used to develop olfac-

ory and taste receptor cell-based biosensors [53,79–81]. Sense ofmell and taste as two basic senses of humans play a very impor-ant role in daily life. The mammalian olfactory system is able toecognise and discriminate thousands of distinct odours that areresent in the environment. There are 2000–3000 distinct olfac-

elopmental Biology 20 (2009) 41–48

tory receptor neurons in an animal olfactory epithelium, which canrespond to odours [79]. The great variety, specificity, high sensi-tivity and fast response of olfactory receptor neurons make theman ideal candidate for olfactory cell-based biosensors. It is sug-gested that the mechanism of signal detection and transduction inolfactory recognition is an electrophysiological process. Olfactorysensory neurons can respond to odours and convert the chemicalsignals of odour molecules into electrical signals [79,80]. Hence,the LAPS has been applied to monitor the changes of extracellu-lar potential of olfactory sensory neurons cultured on the surfaceof the chip under stimulations of the odorants or neurotransmit-ters, such as acetic acid and glutamic acid [79,80]. When glutamicacid (one of the most important neurotransmitters in the olfactorybulb) with a concentration of 25 �M has been added, extracel-lular potential signals with an amplitude of 10–25 �V have beenobserved. In addition, the LAPS has been used to detect an extra-cellular potential response of taste receptor cells to taste stimuli ofNaCl, HCl, MgSO4, sucrose and glutamate [80,81]. These preliminaryexperiments could demonstrate the future possibility of creatingan electronic nose or electronic tongue based on a hybrid systemof LAPS and olfactory/taste neurons that mimic animals’ smell andtaste by using the principle of the mammalian olfactory or tastesystem, respectively.

Although the above-described experiments have demonstratedthe principal capability of the LAPS for detecting extracellularsignals from single cells, the low signal-to-noise ratio prevents prac-tical applications so far; in all experiments the observed signalswere small, typically in the range of several tens of �V. With itspresent potential sensitivity, the LAPS is not a competitive tech-nique for extracellular signal recording as for FETs. Nevertheless,with its conceptual advantage of a free addressability of the mea-surement point with a spatial resolution in the range of a few�m, this technique is very attractive for single-cell investigations.Therefore, a further improvement of the potential sensitivity andsignal-to-noise ratio is envisaged.

Another issue is the application of LAPS devices for the detec-tion of the metabolic activity of single living cells. With the aim toimage pH gradients produced by individual cells, a scanning probepotentiometer based on a cantilever-type micro-LAPS with a spatialresolution of 10 �m has been developed in [82]. When comparedto the conventional LAPS, the active area of the micro-LAPS hasbeen reduced by a factor of 104. The measurement of the acidifi-cation rate from a single cell offers some advantages compared totechniques in which the action potential is recorded. In contrast tothe extracellular electrophysiological technique, which is applica-ble only to electrically excitable cells (i.e., neurons and muscle cells),the acidification-rate measurement can be applied to all types ofcells. In addition, acidification-rate measurements should be lessaffected by a possible change of the distance between adherentcells and the transducer surface. The main technical problem forextracellular acidification measurements with single cells is, how-ever, the necessity to confine the protons in a small defined volume[9].

4. Conclusions and outlook

The field of cell-based biosensors is one of the most exciting andmulti-disciplinary areas of research with wide application possi-bilities reaching from medicine, biotechnology and environmentalmonitoring over food and drug industries up to defence and securitypurposes including antibioterrorism and biological warfare agents

field. However, despite the intensive research and tremendousamount of published works, generally, it could be concluded thata practical realisation of cell-based field-effect devices (FED) andtheir transfer from scientific laboratories to real life remains ratherslow. Only very few of cell-monitoring systems, e.g., the LAPS-based

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ytosensor Microphysiometer system and the FET-based Bionas®

500 analysing system, have been successfully commercialised, soar. The study of the current state reveals that Cell-FEDs for energy

etabolism monitoring are at a well-developed stage, whereaseuron/transistor hybrids for extracellular potential detection aretill in the experimental stage or starting phase. Very recent devel-pments are focussing on two basic directions:

1) There is a trend in CMOS-based sensor arrays with ultimatespatial and temporal resolution integrating many thousands ofcell–sensor interfaces. Here, the data management and readoutelectronics possess major challenges.

2) There is an effort in further miniaturisation of the sensors scal-ing down to nanometer-dimensions. Also in this field, it seemsthat nano-scaled devices will boost the signal-to-noise ratio forrecording individual cells or even sub-compartments of cells.

Hence, exciting future prospects for the development of novelell-FEDs can be expected in both directions.

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