Development and Application of Cell-Based Biosensors - Springer Link

Annals of Biomedical Engineering, Vol. 27, pp. 697–711, 1999 Printed in the USA. All rights reserved.

0090-6964/99/27共6兲/697/15/$15.00 Copyright © 1999 Biomedical Engineering Society

Development and Application of Cell-Based Biosensors J. J. PANCRAZIO,1 J. P. WHELAN,2 D. A. BORKHOLDER,3 W. MA,1 and D. A. STENGER1 1

Center for Bio/Molecular Science and Engineering, Code 6910, Naval Research Laboratory, Washington, DC, 2Alexeter Scientific, 1540 North State Parkway, Chicago, IL, and 3Cepheid Corporation, 1190 Borregas Avenue, Sunnyvale, CA (Received 28 December 1998; accepted 3 September 1999)

Abstract—Biosensors incorporate a biological sensing element that converts a change in an immediate environment to signals conducive for processing. Biosensors have been implemented for a number of applications ranging from environmental pollutant detection to defense monitoring. Biosensors have two intriguing characteristics: 共1兲 they have a naturally evolved selectivity to biological or biologically active analytes; and 共2兲 biosensors have the capacity to respond to analytes in a physiologically relevant manner. In this paper, molecular biosensors, based on antibodies, enzymes, ion channels, or nucleic acids, are briefly reviewed. Moreover, cell-based biosensors are reviewed and discussed. Cell-based biosensors have been implemented using microorganisms, particularly for environmental monitoring of pollutants. Biosensors incorporating mammalian cells have a distinct advantage of responding in a manner that can offer insight into the physiological effect of an analyte. Several approaches for transduction of cellular signals are discussed; these approaches include measures of cell metabolism, impedance, intracellular potentials, and extracellular potentials. Among these approaches, networks of excitable cells cultured on microelectrode arrays are uniquely poised to provide rapid, functional classification of an analyte and ultimately constitute a potentially effective cell-based biosensor technology. Three challenges that constitute barriers to increased cell-based biosensor applications are presented: analytical methods, reproducibility, and cell sources. Possible future solutions to these challenges are discussed. © 1999 Biomedical Engineering Society. 关S0090-6964共99兲01406-X兴

Over the past 10 yr, there has been growing interest in the use of biosensors in environmental,126 medical,154 toxicological,4 and defense113 applications. For engineers and scientists involved in the development of next generation sensors, biosensors have two intriguing characteristics. First, biosensor recognition elements have a naturally evolved selectivity to biological or biologically active analytes. Second, biosensors have the capacity to respond to analytes in a physiologically relevant manner. Biosensors have the potential of providing rapid, sensitive, low-cost measurement technology for monitoring bioavailable analyte concentrations. Major issues impeding the widespread acceptance of biosensor technology have been previously identified as stability and reproducibility.94 A desirable characteristic for any biosensor implementation is the capacity for continuous monitoring or, at a minimum, multiple use ability with little sensor regeneration or renewal. Associated, but not exclusive, features of ideal biosensors include rapid response times, automation, and portability.113 Cell-based sensors face additional concerns which include analytical methods and the source of cells. In this paper, we briefly survey molecular biosensors and highlight the current status of cell-based sensors. There are at least three classes of biosensors: molecular, cellular, and tissue. Molecular biosensors utilize biomolecules such as antibodies, nucleic acids, enzymes, and ion channels. Cellular and tissue-based biosensors incorporate isolated cells or tissue derived from a wide range of plant and animal sources.

Keywords—Antibody, Environmental monitoring, Functional assay, Chemical warfare, Extracellular potential, Impedance, Microelectrode, Patterning, Stem cells.

INTRODUCTION Biosensors incorporate a biological sensing element that converts a change in an immediate environment to signals conducive for processing. While conceptually similar to the well-known canaries used by coal miners to detect lethal levels of methane gas, modern approaches to biosensors can provide detection to a wide variety of analytes over a broad range of concentrations.

ANTIBODY-BASED BIOSENSORS Antibody-based technology takes advantage of specific interactions with antigenic regions of an analyte to achieve high selectivity. Antibody-based approaches that require additional reagents for each measurement fall into the category of traditional immunoassays such as enzyme linked immunosorbant assay 共ELISA兲, colorimetric ‘‘pregnancy’’ test strips, etc. There are several approaches for detecting antigen:antibody binding rang-

Address correspondence to J. J. Pancrazio, Center for Bio/ Molecular Science and Engineering, Code 6910, Naval Research Laboratory, Washington, DC 20375. Electronic mail: [email protected]. navy.mil

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FIGURE 1. Cartoon representation of immunosensor technology for analytes of medical, toxicological, and environmental concern. „Upper panel… Flow injection sensor relies largely upon the displacement of preloaded fluorescentlylabeled antigen from antibody binding sites with antigens in the test sample. „Lower panel… Fiber optic sensor directly detects binding of an analyte via evanescent wave excitation of bound fluorescent antibodies and quantification resulting from emission from the optical fiber. A major difference between flow-injection and direct binding assays such as the fiber optic sensor is how these systems respond to agents that disrupt antibody integrity. While a flow injection immunosensor would yield a false positive as fluorescent label is liberated, the fiber optic sensor would become nonresponsive.

ing from conventional optical and piezoelectric141 to exotic methods involving antibody-modified ion channel switches.21 At the U.S. Naval Research Laboratory 共NRL兲, flow injection immunosensors have been implemented for a diverse array of analytes including insulin,81 cocaine,108 and trinitrotoluene.153 As shown in Fig. 1 共top panel兲, the flow injection sensor relies upon the displacement of preloaded fluorescently labeled antigen from antibody binding sites by antigens in the test sample. Such systems are limited by the amount of labeled antigen that can be preloaded and present particular challenges in calibration since the percentage of occupied binding sites

is constantly changing under flow conditions. Fiber optic immunosensors detect binding of an analyte via modulation of evanescent wave properties yielding rapid and specific detection.143,144 Fiber optic immunosensors have been developed for detection of cocaine,23 toxins such as Clostridium botulinum79,107 and ricin,100 and pathogens such as Yersinia pestis, the etiologic agent of plague,12 as well as food contaminants such as mycotoxins.147 Patterned arrays of antibodies have been immobilized on the surface of an optical waveguide,32 thus enabling simultaneous detection of clinically relevant analytes.128 As illustrated in Fig. 1 共lower panel兲, fiber optic sensors are particularly suited for applications where a low number of positive reactions are expected 共drug screening or environmental sampling兲 since they can be reused multiple times as long as antibody sites remain unoccupied. Once significant positive signals are measured, the consumable elements of the immunosensor must be replaced. Challenges limiting the application of immunosensors include: antibody manufacturability, inherent antibody instability, and limited reversibility of binding. Technologies exploiting genetically engineered antibodies, e.g., phage display,65 have demonstrated the most promise in addressing limitations in traditional antibody production. In addition, specific protein-binding nucleic acid sequences 共aptamers兲 have been developed which may be well suited for sensor applications that previously relied on antibodies.120 Whereas antibodies are raised in the circulation under in vivo conditions, aptamers can be evolved in the actual test media of interest. With regard to reusability, successful regeneration of immunosensors by chemical elution has been demonstrated as feasible, but not particularly practical in high throughput applications.109 Commercially available systems such as a resonant mirror-based biosensor 共Lab Systems兲 and a surface plasmon resonsance biosensor 共BIAcore兲 utilize an automated fluidic regeneration system to recycle binding surfaces. There have been reports of exploiting the natural reversibility of antibodies for ‘‘recycling’’ sensors;58 however, most immunosensors cannot operate continuously. NUCLEIC ACID-BASED BIOSENSORS Nucleic acid technology relies on the hybridization of known molecular DNA probes or sequences with complementary strands in a test sample. Development of biosensors that exploit nucleic acid binding events 共DNA sensors兲 has been more limited than antibody-based technology. Generally, nucleic acid analysis requires extensive sample preparation, amplification, hybridization, and detection. In theory, nucleic acid analysis provides a higher degree of certainty than traditional antibody technologies because antibodies occasionally exhibit cross

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they are synthesized directly onto the chip surface, in situ 共e.g., Affymetrix兲. These commercially available arrays permit samples to be tested against tens of thousands of DNA sequences where detection is accomplished by fluorescence or mass spectroscopy.123 Nucleic acid sensors constitute a sensitive approach for detection and quantification of one or more sequenced biomolecules. With these arrays, analyte-induced changes in cellular gene expression can be identified, perhaps yielding a ‘‘fingerprint’’ of the physiologic action of the analyte on the cells of interest.

ION CHANNEL BIOSENSORS

FIGURE 2. Gene arrays for monitoring analyte-induced changes in gene expression. After exposure, mRNA is extracted and reverse transcribed using primers corresponding to the cDNA sequenced genes mapped to specific, known locations on the arrays. Application of radioactively or fluorescently labeled antisense cDNA sequences to the patterned array permits hybridization to indicate the expression of genes of interest. Comparison of control and analytetreated cells may enable function-based detection of analytes based on the pattern of gene expression.

reactivity with antigens other than the analyte of interest. Near real time detection of hybridization events has been demonstrated in numerous optical119,152 or electrochemical systems.27,56,95 Further, the feasibility of sensor regeneration has been demonstrated as well in several optical-based systems, including both fiber optic46,118 and resonant mirror152 applications. In practice, however, development of nucleic acid sensor systems has been hampered by the challenges presented in sample preparation. Nucleic acid isolation remains the rate-limiting step for all of the state-of-the-art molecular analyses. Typically, cells must be mechanically or chemically disrupted and treated with enzymes to remove associated proteins before nucleic acid can be isolated for hybridization to specific probes. As a result, rapid and automatable isolation of nucleic acid is an area of intense development at present. Automated preparation and hybridization of E. coli nucleic acids from a human blood cell mixture was reported recently.15 Dielectrophoresis of the cell mixture to isolate the bacterial cells was followed by electronic lysis and proteolytic digestion on a single microfabricated biolelectronic chip. Further applications of such technologies may help overcome the difficulties in nucleic acid accessibility. Several groups have fabricated large arrays of specific DNA sequences,88,123 as depicted in Fig. 2. DNA sequences are either synthesized in solution and ‘‘printed’’ onto the chip arrays 共e.g., Incyte Pharmaceuticals兲 or

Membrane ion channels are targets of a range of transmitters, toxins, and potential pharmaceutical agents. Indeed, the finding that many ion channels and receptors could be purified and reconstituted in black lipid membranes 共BLMs兲 for studies of function and pharmacology96 spurred initial interest in the development of channel/receptor-based biosensors.84,136 Sensing strategies have been proposed using ionotropic neurotransmitter receptors for glutamate and ␥-aminobutyric acid inserted into bilayers97 or utilized in vitro as excised membrane patches.67,110 However, ion channels, particularly those relevant to mammalian physiology, cannot be considered robust in BLMs or isolated membrane patches due to the well-known property of ion channel ‘‘rundown’’ or ‘‘washout.’’ 127 In the absence of integral intracellular machinery provided by cells needed to maintain function, ion channels typical of mammalian physiology presently do not constitute practical biosensors. There are, however, promising applications of robust ion channels such as gramacidin and alpha toxin to biotechnology as signal transducers21 or nucleic acid filters.72

ENZYME-BASED BIOSENSORS Enzyme-based technology relies upon a natural 共or fortuitous兲 specificity of given enzymatic protein to react biochemically with a target substrate or substrates. Like ion channels, there are many enzymes that participate in cellular signaling and, in some cases, are targeted by compounds associated with environmental toxicity. In the medical diagnostic field, several manufacturers have marketed biosensors for measurement of common blood chemistry components including glucose, urea, lactate, and creatinine.24,35,71 In general, enzyme-based biosensors employ semipermeable membranes through which target analytes diffuse toward a solid-phase immobilized enzyme compartment. Ion selective, amperometric, or pH electrodes measure reaction components such as hydro-

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tating packaging approaches to limit degradation of biosensor performance. Packaging strategies for AChE include photocrosslinkable polymer68,89 and immobilization with polyelectrolyte and polyhydroxyl compounds known to stabilize enzymes and maintain protein tertiary structure, respectively.125 CELL-BASED BIOSENSORS

FIGURE 3. Amperometric enzyme-based biosensor for measurement of the neurotransmitter glutamate. L-glutamate oxidase is adsorped onto a lipid phosphatidylethanolamine „PEA… coated platinum electrode. The inclusion of bovine serum albumin „BSA… and polyphenylenediamine „PPD… increases the sensitivity and selectivity of the measurement, respectively. A low-noise potentiostat measures the amperometric current resulting from electro-oxidation of H2O2. Adapted from Ryan et al. „Ref. 129….

gen peroxide 共from oxidation of glucose by glucose oxidase兲 or ammonium ions 共from urease metabolism of urea兲. Rapid on-line biosensors for quantitation of neurotransmitters that are amenable for implantation have been developed. As shown in Fig. 3, glutamate oxidase, entrapped with lipid and albumin on a platinum electrode surface by electropolymerization of o-phenylenediamine, provided the basis for a biosensor for the central nervous system neurotransmitter glutamate.129 Optimal detection of acetylcholine can be achieved with a three enzyme system incorporating polymer-entrapped acetylcholinesterase 共AChE兲, choline oxidase, and horse radish peroxidase.83,104 Of particular interest for environmental monitoring is AChE, a target of organophosphate compounds comprising pesticides and several chemical warfare agents. Since many organosphosphates undergo degradation resulting in compounds that also inhibit AChE activity,82 a function-based assay using AChE can serve as a pollution or environmental monitor. Amperometric measurement of the oxidation of thiocholine, produced during hydrolysis of acetylthiocholine by AChE, has been used to detect nanomolar concentrations of organosphosphate pesticides in aqueous samples.133 Nevertheless, AChE, like many enzymes, is inherently unstable, thus necessi-

Cells express and sustain an array of potential molecular sensors. Receptors, channels, and enzymes that may be sensitive to an analyte are maintained in a physiologically relevant manner by native cellular machinery. In contrast with antibody-based approaches, cell-based biosensors should optimally only respond to functional, biologically active analytes. Cell-based biosensors have been implemented using microorganisms, particularly for environmental monitoring of pollutants. Biosensors incorporating mammalian cells have a distinct advantage of responding in a manner that can offer insight into the physiological effect of an analyte. Several approaches for transduction of cell sensor signals will be discussed; these approaches include measures of cell fluorescence, metabolism, impedance, intracellular potentials, and extracellular potentials. CELLULAR MICROORGANISM BASED BIOSENSORS Some analytes, such as pollutants, can activate microorganism pathways involved in metabolism or nonspecific cell stress, resulting in the expression of one or more genes.3 Detection of formaldehyde76 and toxicity measurements of cholanic acids10 have been performed using immobilized yeast, where changes in metabolism indicative of the analyte were detected via O2 electrode measurements or extracellular acidification rates. Recombinant E. coli have been engineered to express the enzyme organophosphate hydrolase, which generates protons during hydrolysis of organophosphate insecticides or nerve agents such as sarin, soman, and VX. Changes in pH from the effluent from these immobilized cells has been suggested as means of analyte detection.121 Another sensor approach involves genetically engineering bacteria such that a bioluminescent reporter gene is fused to the promoter sequence of a gene of the relevant pathway. Modified bacteria have served as whole cell sensor elements for the detection of napthalene and its metabolic product salicylate,59,73 benzene,1 toluene,1,9 mercury,131 and middle chain alkanes such as octane.139 However, pollutants are often mixtures, and the presence of other stimulating/inhibiting substances in a sample can affect detection integrity. While bioluminescent bacteria biosensors have been able to respond to groundwater contaminated with JP-4, a jet fuel formulation used by the

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U.S. military until recently, napthalene content was underestimated presumably due to the presence of toxic unknowns.1,59 It is possible that any alteration of a microorganism-based biosensor response is important and that insufficient selectivity actually offers advantage in providing generic detection.31 We would argue, however, that if generic detection is a goal, it might be better achieved using cell-based biosensors derived from the biological system of interest 共e.g., mammalian兲 such that it can offer functional, physiologically relevant information. FLUORESCENCE ASSAYS OF CELLULAR FUNCTION Fluorescence imaging has proven to be an invaluable tool for monitoring changes in the concentrations of ions and protein expression related to cellular signaling.26,149 More recently, fluorescence based technologies have been implemented in high-throughput screening.33 A new generation of fluorescent reagents based on the combination of molecular biology, fluorescent probe chemistry, and protein chemistry is being developed for cell-based assays. Reporter gene constructs, such as green fluorescent protein, have been implemented in genetically engineered mammalian and nonmammalian cell types157 to achieve measures of cell function rather than radioligand binding.45 In fact, the fluorometric imaging plate reader 共FLIPR™兲, commercially available through Molecular Devices, has been shown to enable high-throughput fluorometric assays of membrane potential and intracellular calcium mobilization.80,142 In spite of the obvious utility of fluorescent techniques, there are three important considerations: First, in mammalian systems, the ability to readily transfect reporter genes limits the cell types to those that are tumor derived. Second, cell loading with fluorescent dyes must be considered a potentially invasive treatment. Third, analytes of interest must be examined for autofluorescence to determine the feasibility of cellular fluorescent assays for the resolution of small effects. CELLULAR BIOSENSORS BASED ON CELL METABOLISM One category of cellular biosensors relies on the measurement of energy metabolism, a common feature of all living cells. As illustrated in Fig. 4共A兲, the Cytosensor® microphysiometer, introduced by Molecular Devices,116 makes use of a light-addressable potentiometric sensor55 to measure extracellular pH which is correlated with cellular metabolic activity.111 The microphysiometer has been used with a wide range of eukaryotic cells including primary central nervous system neurons,8,122 hepatocytes,13 neutrophils and endothelial cells,47 and

FIGURE 4. Stategies for cell-based assays. „A… Cytosensor® microphysiometer cell-based biosensor relying on metabolic rate for analyte detection. The silicon n- or p-type material, insulated with oxynitride which is pH sensitive, is photoresponsive to light produced by one or more light-emitting diodes „LEDs…. The resulting photocurrent I depends on the applied bias potential. The electrolyte–oxynitride interface provides a Nernstian response to pH or membrane potential changes. Adapted from Hafeman et al. „Ref. 55… and RaleySusman et al. „Ref. 122…. „B… Schematic of cellular impedance system for monitoring morphological changes and motility of adherent cells. Adapted from Giever and Keese „Ref. 43….

cells expressing transfected receptors.2 In fact, the microphysiometer has been shown to ‘‘detect’’ the activation of a variety of receptors in cells, regardless of the signal transduction method.112 For example, measurements of metabolism from primary neurons revealed that ␥-aminobutyric acid 共GABA兲, an inhibitory neurotransmitter, and kainic acid, an excitatory neurotransmitter, both cause an elevation in cell metabolism and a subsequent elevation in extracellular acidosis.8,122 Proper interpretation of data derived using this approach requires parallel experiments in the presence of known receptor antagonists that eliminate specific receptor responses. IMPEDANCE ASSAYS FOR CELL FUNCTION It has long been recognized that the membranes of biological materials including cells exhibit dielectric

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properties. By culturing cells over one or more electrode contacts, changes in the effective electrode impedance permits a noninvasive assay of cultured cell adhesion, spreading, and motility41,98 关Fig. 4共B兲兴. Impedance measurements rely on the observation that intact living cells are excellent electrical insulators at low signal frequencies. As cells grow or migrate to increase coverage over an electrode surface, the effective electrode impedance rises. Impedance measurements have been used to monitor the behavior of an array of nonexcitable cell types including macrophages,77 endothelial cells,148 and fibroblasts.43 Impedance techniques are theoretically capable of dynamic measurements of cellular movement at the nanometer level, a resolution above that of conventional timelapse microscopy.42,43 Impedance measurements have been used to assess the effect of nitric oxide on endothelin-induced migration of endothelial cells105 and subtle changes in cell morphology of fibroblasts treated The PhysioControlwith prostaglandin E2. 134 Microsystem®, a commercially available system from Physikalisch-Technische Studien GmbH, incorporates an interdigitated electrode structure with other microsensors for pH and O2 concentration on a planar substrate to provide continuous monitoring of cultured cells.155 Time and concentration-dependent changes in the impedance from cultured fibroblast-like kidney cells were demonstrated with administration of cadmium, indicative of potential utility in cell-based toxicity assays. From a biosensor standpoint, changes in cell migration or morphology tend to be somewhat slow; marked changes in impedance in the presence of cadmium emerged only after 2–3 h of exposure.29 CELLULAR BIOSENSORS BASED ON INTRACELLULAR POTENTIALS An important aspect of the information that can be derived from cell-based biosensors relates to the functional or physiologic significance of the analyte to the organism. To this end, bioelectric phenomena, characteristic of excitable cells, have been used to relay functional information concerning cell status.51 It is well known that membrane excitability plays a key physiologic role in neurons and myocardiocytes for the control of secretion and contraction, respectively. Thus, analytes that affect membrane excitability in excitable cells are expected to have profound effects on an organism. Furthermore, the nature of the changes in excitability can yield physiologic implications for the organism response to analytes. Direct monitoring of cell membrane potential can be achieved through the use of glass microelectrodes. Repetitively firing neurons from the visceral ganglia of the pond snail has been used to quantitatively assess the

FIGURE 5. Neuroblastoma-glioma „NG108-15… cells as electrical detectors of chemical agents. „Upper panel… Typical NG108-15 cells cultured under serum-free conditions after 21 days in vitro exhibiting neuronal phenotype. „Lower panel… Differential effects of the chemical warfare agents VX „Oethyl s-2-NN-diisopropylamin ethyl methylphosphonofluoridate… and GD „soman; O-pincolyl methylphosphonofluoridate… on spontaneous, rhythmic firing measured using standard glass microelectrodes filled with 3 M KCl, as described by Kowtha and colleagues „Ref. 78…. In contrast to GD, VX was completely reversible. Similar observations were made for 12 other cells over a range of agent concentrations. Dotted line indicates the zero membrane potential level.

concentration of a model analyte, serotonin.132 To address the feasibility of generic sensitivity of an excitable cell type to a variety of potential toxins, the utility of a neuroblastoma x glioma cell line, NG108-15, has been examined. Under serum-free media conditions, NG108-15 cells express a neuronal phenotype 共Fig. 5兲87,103 and the capability of spontaneous firing.78 Synapse formation with NG108-15 cells can be observed only under coculture conditions and at low efficiency.63 Of particular interest was whether or not cell-based sensors could be used to rapidly detect chemical warfare agents such as VX and soman 共GD兲. In bullfrog sympathetic ganglion neurons, both VX and soman have been shown to increase membrane excitability in a manner consistent with voltage-gated Ca2⫹ channel blockade.61,62 As shown in Fig. 5, spontaneous firing in NG108-15 cells was markedly affected by exposure to chemical warfare agents: GD and VX. Interestingly, the effects of

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these two organophosphate agents were distinctive; whereas GD irreversibly depolarized the resting membrane potential 共RMP兲, VX reversibly hyperpolarized the resting potential, resulting in a loss of excitability. These records illustrate the utility of excitable cells as sensors with sensitivity to chemical warfare agents; however, the invasive nature of intracellular recording significantly limits the robustness of this approach for biosensor applications. In addition, the use of a tumor-derived cell line eliminates dependence of the assay on tissue isolated from animals, but the physiologic relevance of observations from a cell line are unclear. Most importantly, excitable cells assemble into coupled networks rather than acting as isolated elements; neurons propagate information via synapses and myocardiocytes form a syncitium via gap junctions. As a result, the ability to simultaneously monitor two or more cells permits measurements of membrane excitabilty and cell coupling. EXTRACELLULAR POTENTIALS AS A BASIS FOR CELL-BASED SENSORS In recent years, the use of microfabricated extracellular electrodes to monitor electrical activity in cells has been used more frequently. Extracellular microelectrode arrays offer a noninvasive and long-term approach to the measurement of biopotentials.18 Multielectrode arrays, typically consisting of 16–64 recording sites, present a tremendous conduit for data acquisition from networks of electrically active cells. Signal propagation, via either synaptic transmission or gap junction connectivity in neurons and cardiac myocytes, respectively, offers a measure of cell coupling which is virtually inaccessible using standard intracellular recording techniques. Generating maps of interconnectivity where the number of recording sites exceeds two is beyond the capability of most electrophysiology laboratories relying on glass micropipettes. Furthermore, the invasive nature of intracellular recording, as well as voltage-sensitive dyes, limits the utility of standard electrophysiological measurements and optical approaches. As a result, planar microelectrode arrays have emerged as a powerful tool for longterm recording of network dynamics. Extracellular recordings have been achieved from dissociated cells as well; electrode arrays have permitted examination of cardiac impulse propagation from dissociated embryonic myocytes comprising a monolayer.17,66,115,146 Given the large amplitude extracellular signals characteristic of cardiac cultures, it has been suggested that this preparation may be well suited for pharmaceutical screening.22 Figure 6 illustrates a multielectrode array used in recording potentials from a chick cardiac monolayer. Simultaneously recorded extracellular cardiac potentials were multiphasic 共Fig. 6, middle

FIGURE 6. Planar multielectrode arrays permit noninvasive, simultaneous recording from excitable tissue for measurement of action potential propagation. „Upper panel… Microelectrode array fabricated at the Center for Integrated Systems, Stanford University, with 36 microelectrode sites that are 10 ␮m in diameter. Industry standard thin-film photolithographic techniques were used to implement the array of gold microelectrode sites and leads. Recording sites were platinized to reduce microelectrode impedance for optimal extracellular recording. „Lower panel… Simultaneous recording from a monolayer of embryonic day 11 chick myocardiocytes after 2–3 days in vitro. The monolayer exhibited regular beating at a rate of 1–2 Hz, where spike delays between proximal microelectrode sites allows quantification of cardiac propagation velocity.

panel兲, reminiscent of the second or third derivative of the action potential. Detailed examination of the regular activity at the recording sites showed regular phase delays 共Fig. 6, lower panel兲, consistent with propagation of the cardiac excitation wave. With this extracellular recording approach, an advantage over intracellular recording is clearly apparent in the direct measurement of propagation velocity, which in this experiment can be estimated from adjacent microelectrode sites to be approximately 100 mm/s.22,66 Brain tissue slices have been extremely useful in understanding neuronal network behavior from cells organized as in vivo .39 Planar arrays have been used to record extracellular potentials from tissue slices includ-

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FIGURE 7. Culture of spinal cord neurons for toxicological evaluation. „Upper panel… Phase contrast image of embryonic day 15 rat spinal cord neurons cultured for 18 days on a microelectrode array described in detail previously „Ref. 115…. Cells were cultured under serum-free defined media conditions on artificial self-assembled monolayer substrates of aminosilanes. Microelectrode recordings shown were all from the same site. As shown from a microelectrode contact typical of this experiment, addition of glutamate „50 ␮M… greatly augmented spike activity. Administration of an organophosphate, diisopropylfluorophosphate „DFP; 25 ␮M…, revealed a marked ablation of spontaneous firing, illustrating the utility of neurons cultured on microelectrode arrays for detection of toxic compounds.

ing the vertebrate retina93 and hippocampal organotypic slices.140 Hippocampal slices been reported to be cultured on multielectrode arrays for as long as 4 weeks,28 although the stability of long-term recording is unclear since slices undergo morphological changes including ‘‘thinning’’ and cell migration. In addition, extracellular recordings have been performed using dissociated invertebrate neurons7,38,124 and smaller mammalian neurons from superior cervical ganglia,117 mouse dorsal root ganglia,69 and spinal cord.54 Equivalent circuit models of the neuron–silicon junction have been developed to describe extracellular potentials recorded from leech neurons38 and the smaller amplitude signals from rat embryonic neurons.150,151 This work suggests that the diverse forms and time courses of neuronal extracellular potentials may be attributed to differences in the ionic conductances present in the portion of the cell membrane in contact with a recording site.37 Figure 7 共upper panel兲 shows a phase contrast image of spinal cord neurons

cultured over a microelectrode array composed of gold leads passivated with silicon nitride and platinized gold contacts 14 ␮m in diameter, as described earlier.114 Administration of glutamate 共50 ␮M兲 greatly augmented spontaneous electrical activity which was rapidly ablated by addition of the organophospate agent, diisopropylfluorophosphate 共Fig. 7, lower panel兲. More detailed work by Gross and colleagues at the University of North Texas over the past 20 yr have demonstrated the feasibility of neuronal networks for biosensor applications.50,51 In this work, transparent patterns of indium–tin–oxide conductors, 10 ␮m wide, were photoetched and passivated with a polysiloxane resin.52,53 Laser de-insulation of the resin resulted in 64 recording ‘‘craters’’ over an area of 1 mm2, suitable for sampling the neuronal ensembles achieved in culture. Indeed, neurons cultured over microelectrode arrays have shown regular electrophysiological behavior and stable pharmacological sensitivity for over 9 months.48 In fact, their precise methodological approach generates a coculture of glial support cells and randomly seeded neurons, resulting in spontaneous bioelectrical activity ranging from stochastic neuronal spiking to organized bursting and long-term oscillatory activity.50 Exhaustive surveys of neurotransmitters and neurotoxins indicate that modulation of electrophysiologic parameters, such as extracellular action potential amplitude and burst rate, are indicative of compounds that can be functionally classified as excitatory, inhibitory, disinhibitory, or cytotoxic.49 As a result, networks of excitable cells are uniquely poised to provide rapid, functional classification of an analyte and ultimately constitute a potentially useful cell-based biosensor technology. Microelectrode arrays coupled with ‘‘turnkey’’ systems for signal processing and data acquisition are now commercially available. Panasonic has introduced a multielectrode dish probe consisting of 64 platinized gold microelectrodes with dimensions of 50 ␮m by 50 ␮m on pyrex glass substrate. The multielectrode array available from Multi Channel Systems incorporates 60 titanium nitride microelectrodes with diameters of 10–30 ␮m on a glass substrate insulated with silicon nitride.28 Applications ranging from extracellular recording and stimulation of acute brain slices to cultured cardiac myocytes appear to be readily realizable with this off-the-shelf technology. In addition, we have recently demonstrated a prototype portable system capable of performing extracellular recording from excitable cells in an outdoor environment with a high signal-to-noise ratio.114 In spite of these advances, this approach for cell-based sensor implementation presently exhibits three weaknesses that constitute barriers to increased application of these types of assays: analytical methods, reproducibility, and reliance on primary, animal-derived cells.

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ANALYTICAL METHODS FOR CELL-BASED BIOSENSORS

REPRODUCIBILITY OF CELL-BASED BIOSENSORS

There is a rich history of analysis of multichannel recordings, driven by data collected from electrode array implantation experiments, that has yet to be applied to cultured systems for biosensor applications. The earliest work in this area relied on cross correlations of the time between action potentials from pairs of recording sites of an array to quantify network behavior.40 For cultured neuronal networks, cross correlations between multielectrode channels and interspike interval 共ISI兲 variance have been evaluated. Both simulation and experimental findings suggested that the ISI variance discriminated periodic bursting from asynchronous firing, whereas the cross correlation was indicative of the synchronization among the neurons in the network.5,11 As elucidated by Hampson and Deadwyler,57 there are two fundamental concerns for the application of cross correlations in multichannel data: 共1兲 cross correlations do not discriminate between connectivity and coactivity, and 共2兲 while neurons can exhibit a high degree of synaptic connectivity, cross correlations are based on pairwise calculations. Multivariate analysis, which includes linear discriminant analysis44 and principal component analysis 共PCA兲,102 has the advantages that it can take into account the activity of all the recorded neurons simultaneously and incorporate spatio–temporal features into the analysis. For example, PCA allows construction of the activity of neuron populations by the weighted addition of the integrated activity of the population, resulting in the structure underlying the collective neuronal behavior.145 Reconstruction of the first principal component from recordings of cortical, thalamic, and brain stem activity was used to more reliably track oscillatory episodes than recordings from any single neuron in the population.101 For analyte detection the analysis task is somewhat different: minimal sets of parameters, such as burst amplitude, duration, and frequency, must be determined to perform risk/threat assessment. Multichannel analysis may benefit from advances in nonlinear dynamics, which can potentially distinguish deterministic behavior from random fluctuations. For example, nonlinear time series analysis of the correlation dimension from multielectrode electroencephalograph 共EEG兲 records has been shown to yield a prognostic indicator for seizure activity.30 In addition, Schiff and colleagues, relying on the identification of unstable periodic orbits in neuronal firing to identify system deterministic behavior,135 have used EEG data to detect and treat epileptic seizures on-line.130 Such analytical techniques for rapid, on-line system characterization may benefit from the interpretation of data from multielectrode arrays.

Randomly seeded cultures of excitable cells exhibit two noteworthy advantages over tissue slices: longevity and better adhesion resulting in enhanced cell– microelectrode coupling 共Gross, G. W., personal communication兲. Nevertheless, the ‘‘cost’’ of random cultures relative to recordings from slices is the singular operational profile of each sensor–transducer combination. Without the physio–chemical cues imposed in vivo, randomly cultured neurons form multiple connections yielding a largely intractable network. Silicon fabrication of microelectrode arrays incorporating physical barriers of aluminum oxide 共700 nm兲 and poly methylmethacrylate 共1 ␮m兲 resulted in limited neurite guidance for cultured dorsal root ganglion neurons.69 Recent advances in cell patterning raise the possibility that artificial substrates can be microengineered to guide the growth of excitable cells,18 which may enable reproducible and manufacturable cell-based biosensors. One note of caution: the popular press has recently brought attention to efforts in neuronal patterning for the fabrication of ‘‘neurocomputational devices,’’ ostensibly for the introduction of a cognitive element to computation. We believe that the hyperbole-rich claims driving enthusiasm for such applications ignore many observations in neurobiology; for example, the dynamic nature of synapses, and their use is therefore doubtful. Nevertheless, neuronal patterning for the use of more reproducible biosensor arrays is certainly more likely to prove useful. Microstamping, using microfabricated polydimethylsiloxane stamps with micrometer features, has been employed by several groups to pattern cells14,99 including neurons.6 Substrates modified using laser ablation of biomolecules19 and thin-film industry standard photolithography25 have proven suitable for neuronal patterning with biomolecules16 and organosilanes.20,74,90,137 Coplanar monolayers of differentially adhesive silanes applied using photolithography can be used to direct neuronal polarity, i.e., guide axonal and dendritic neurite extensions,138 with no decrement in cellular bioelectrical characteristics.85 Photolithographic patterning of silicon substrates with the extracellular matrix protein laminin has also been shown to guide the hippocampal neurite extension91 yielding neurons on microelectronic surfaces with electrophysiological characteristics that are the same as on glass.106 However, a major technical hurdle concerning patterning is that cells, in particular neurons, do not maintain fidelity to the patterned substrates.85 Greater understanding of how exactly cells remodel their extracellular environment may be required to advance cell patterning to a useful stage.

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STEM CELLS AS BIOSENSOR ELEMENTS In the last 10 yr, there has been an increased interest in the isolation and culture of what are broadly referred to as ‘‘stem’’ cells. These are proliferating cells that remain pluripotent, i.e., capable of becoming any of multiple cell types, until they are triggered to differentiate. Stem cell types have been isolated and cultured in vitro to give rise to cells of the hemapoietic and nervous systems.64 It has been shown that the inner cell mass of early stage embryos gives rise to embyronic stems cells 共ESCs兲 that are totipotent, i.e., capable of becoming any cell type in the organism. ESCs are particularly appealing candidates for a variety of applications in biotechnology, including their use as a source of excitable cells for biosensor applications, and as a renewable source of cells for tissue engineering. In principle, ESCs have all of the potential advantages of both primary cells and tumor-derived cell lines without the inherent drawbacks of either. Formation of neuronal phenotypes from mouse D3 ESCs can be induced by exposure to retinoic acid and culture in neuronal growth media.34,36 When triggered to differentiate, they form a mixture of cell types, including neurons and myocardiocytes that spontaneously contract in culture. The major hurdle for all stem cell work relates to control of stem cell differentiation. Recent work has focussed on neuronal stem cells derived from embryonic hippocampus and telencephalon.86,92 While not totipotent, dividing neuronal stem cells are certainly multipotent; capable of giving rise to various neurons and glia. Klug et al.75 demonstrated that controlled genetic manipulation can be performed on proliferating ESCs, enabling clonal expansion of desired phenotypes. In this case, a fusion gene consisting of the alpha-cardiac myosin chain promoter and a cDNA that encoded for aminoglycoside transferase was stably incorporated into D3 ESCs, allowing cells committed to become myocardiocytes to survive selection media and form cultures having ⬎99.5% myocardiocyte composition. These cells were observed to contract spontaneously in vitro for periods of greater than 11 months 共Field, L., personal communication兲. Thus, it would appear that designed genetic modifications may be a preferred approach to the establishment of relatively pure ESC-derived phenotypes. CONCLUSION Cell-based biosensors constitute a promising field that has numerous applications ranging from pharmaceutical screening to environmental monitoring. Cells provide an array of naturally evolved receptors and pathways that can respond to an analyte in a physiologically relevant manner. Enzymes, receptors, channels, and other signaling proteins that may be targets of an analyte are main-

tained and, as necessary, regenerated by the molecular machinery present in cells. The array of signaling systems characteristic of cell-based sensors yields generic sensitivity that is a distinguishing feature in comparison to other molecular biosensor approaches. In addition, cell-based sensors offer an advantage of constituting a function-based assay that can yield insight into the physiologic action of an analyte of interest. Three important issues that constitute barriers for the use of cell-based sensors have been presented and discussed. There are certainly other areas that will require attention, as cellbased sensors move from the laboratory environment; namely, cell delivery and/or preservation technologies. As further progress is made to address fundamental challenges, cell-based biosensors and related cellular function based assays will undoubtedly become increasingly important and useful. We believe that such function-based assays will become an indispensable tool for monitoring in environmental, medical, and defense applications. Key developments will require a more complete understanding of the detection range that can be achieved with cell-based sensors. Strategies relying on a single population of excitable cells 共cardiac or neuronal兲 or a precise coculture of neurons and glia, appear most well suited for measurements of acute and direct effects of receptor agonist/antagonists. Compounds that fall within this category include ion channel modulators, metals, ligand–receptor blockers, and neurotransmitters. In fact, the detection of acute and direct effects of compounds may be sufficient and relevant for certain operational situations, such as a battlefield environment or the floor of an assembly plant, where cognitive function is absolutely critical. The prospect of detecting all physiologically activite analytes using a single cell or tissue type is improbable. It is possible that particular analytes may undergo biotransformation, resulting in a secondary or tertiary compound of substantial physiologic effect. For example, biotransformation, which is accomplished by cytochrome P450 enzymes largely expressed in the liver, of the organophosphorous insecticide parathion, results in the formation of paraoxon, a chemical warfare agent simulant.156 Clearly, the use of single populations of cells as sensors disregards the role of cell metabolism of an analyte in vivo. Thus, the development of coculture systems to broaden the operational sensitivity of excitable cells to include metabolites would enhance the present capability. There are substantial needs for function-based cellular assays utilizing immune cells. The threat of biological weapons has become a major concern to both the civilian and military populations.60 All of the present biological warfare and environmental agent rapid detection systems, in field use or under prototype development, rely on structural recognition approaches to identify anticipated agents. As discussed earlier, such assays based on anti-

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bodies and nucleic acid probes are highly specific for anticipated targets, but provide no functional information.113 As a result, there is a critical technology gap in our ability to detect unknown or engineered biological agents70 and assess the degree of threat. Due to these growing concerns, the need for the development of a generic biodetection capability for a wide range of anticipated and unknown threats is crucial. We anticipate that studies using immune cells to determine signaling pathways that may offer classification of biological agents will be of great importance in the future. ACKNOWLEDGMENTS This work was supported in part by the Office of Naval Research, the Defense Threat Reduction Agency, U.S. Marine Corps and the Defense Advanced Research Projects Agency. The authors thank Dr. H. J. Bryant 共Uniformed Services University of the Health Sciences兲 and Dr. V. C. Kowtha 共NRL兲 for providing electrophysiological records from NG108-15 cells, and Dr. P. Manos 共Gillette Corp.兲 for her contribution of spinal cord extracellular recordings. In addition, the authors extend their gratitude to Dr. G. P. Anderson and P. T. Charles 共NRL兲 for their discussions on immunosensor technology, and Dr. J. Matthew Mauro 共NRL兲 for his constructive review of this manuscript. The opinions and assertions contained herein are the private ones of the authors and are not to be construed as official or reflecting the views of the Department of the Navy.

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