JAMES J. VALDES, JOSEPH O. WALL, JR., JAMES P. CHAMBERS, and MOHYEE E. ELDEFRAWI

A RECEPTOR-BASED CAPACITIVE BIOSENSOR

The receptor-based capacitive biosensor combines the most recent advances in microelectronic tech­ nology with molecular biology and receptor biochemistry. Receptor-based biosensors have the potential for generic detection of chemical agents and toxins on the battlefield and are the best countermeasure to the threat of biotechnologically engineered agents. In addition, important spin-offs will be realized in medical diagnostics, controlled drug administration, environmental monitoring, and industrial pro­ cess control.

INTRODUCTION The production and use of chemical agents in armed lectric properties or electrical field, 2 or a chemome­ conflicts and their potential for use in low-intensity con­ chanical signal resulting from alteration of a protein's flicts pose a threat to the security of U.S. forces. The mechanical properties during binding. 3 threat is compounded by recent advances in biotechnol­ Biosensors are devices that combine biological materi­ ogy, which have blurred the distinction between tradi­ als with electronic microsensors, a merger that results tional chemical agents ahd biological organisms and in sensitivity to biological parameters. Biosensors have toxins. Toxins currently used as exotic neurobiological been designed to detect proteins, toxins, living cells, and probes in laboratory research may soon be produced in specific chemical compounds, but their applications are large quantities by advanced fermentation methods and limited because selectivity and sensitivity depend on mo­ engineered to have precise physicochemical and pharma­ lecular recognition, which is often inadequate. Flexibility cologic properties that would make them useful to a mili­ can be introduced by the addition of receptors, biological tary adversary. molecules capable of specific molecular recognition. The The detection of chemicals and toxins in combat, in biological recognition site permits a receptor-based bio­ the environment, or in body fluids has required analytical sensor to detect a compound within a complex mixture procedures that, although sensitive, are time-consum­ of ions and chemicals in air, water, or multicomponent ing and expensive. Further, these procedures require liquids. 4 This is directly analogous to the operations of some knowledge of the identity of the species to be de­ antibodies, enzymes, and receptors as they exist in situ, tected. Clearly, a new detection strategy is desirable. The awash in a complex environment of ions, hormones, lethal or incapacitating effects of many chemicals and transmitters, and other biological materials, yet respond­ toxins, the pharmacologic effects of drugs, and the ac­ ing only to their appropriate ligands. For example, a bio­ tion of viruses result from molecular recognition of these sensor that used an antibody as its recognition element agents by physiological targets known generically as re­ would be expected to detect the antigen or hapten (the ceptors. Receptors are macromolecular binding proteins part of an antigen that can be recognized by an anti­ that are vital for cellular function and can recognize and body but that is not antigenic per se) to which that anti­ respond to agents at extremely low concentrations. body was raised; a biosensor that used the acetylcholine Threat potential and pharmacologic potency are direct (ACh) receptor (AChR) would be considerably broader correlates of receptor affinity, making receptors, by na­ in its response, signaling the presence of any of the large ture, extremely efficient detectors. Receptor-based assays number of toxins and ligands that bind to it. Immobili­ are used routinely in the laboratory, but their use as de­ zation of an antibody or enzyme on a sensor generally tectors has only lately been proposed. would confer its specificity on the sensor; conversely, Recent advances in microelectronics have resulted in a receptor with a more generic recognition capability the development of sensitive, inexpensive microsensors would confer like qualities on the biosensor. that operate in real time; however, they also must be One promising microsensor development is the capaci­ made for specific applications, and they lack the recog­ tive sensor, which has been described for measuring hu­ nition capability required to detect and identify physio­ midity, 5 gases,6 and the extent of polymeric cure. 7 An logically significant compounds in a multicomponent equivalent circuit model of the capacitive sensor was de­ mixture. Several types of microsensors have been de­ veloped by partitioning the total capacitance into com­ signed, including capacitive sensors, fiber-optic wave­ ponent capacitances according to dielectric composition, guides, chemical field-effect transistors, and piezoelectric as shown in Fig. 1. Significant for the development of crystals. All rely on transduction of either an optical sig­ capacitive biosensors are the variable capacitances caused nal based on fluorescence or bioluminescence, 1 an elec­ by the biochemical layer and the aqueous film. The ex­ tronic signal resulting from a change in the sensor's die- pected change in biosensor capacitance can be computed

4 fohns Hopkins A?L Technical Digest, Volume 9, Number 1 (1988) Cs -Substrate Fingers Terminal strip

Cp-Bonding pad

nding pads

COL -Dielectric layer (passivation) COL I frt------t Ct - Transduction Passivation coating Figure 1-A simplified equivalent circuit model of the capaci­ tive sensor. Gold Plated copper (b) Chrome by a capacitance cell model in which the dielectric con­ Substrate stant of the biochemical layer is varied. For example, Figure 2-Schematic (a) and cross-sectional (b) views of the when antibody protein binds to the appropriate hapten planar capacitive sensor. immobilized on the surface of the biosensor, water is displaced, causing a change in the dielectric properties of the biochemical layer . These binding reactions are in was bound to the hapten. For receptor experiments, two dynamic equilibrium; therefore, free hapten, which is be­ radically different strategies were used. In the first recep­ ing analyzed or measured (i.e., the analyte), will cause tor configuration, a calmodulin "tether" was attached to displacement of the surface-bound antibody in relation to the derivatized silane surface, and the receptor was then the analyte concentration. electrostatically coupled to the tether. This is analogous The focus of our research is to couple biological recog­ to the antibodyIhapten configuration in that bulk dis­ nition sites (receptors) with a capacitive microsensor. The placement of the antibody or receptor occurs in the pres­ resulting biosensor would combine the sensitive and se­ ence of a competing antigen or ligand. Thus a material lective recognition capabilities of receptors with the signal with a low dielectric constant (e.g., protein) is replaced transduction and amplification characteristics of elec­ by a material with a high dielectric constant (e.g., water), tronic microsensors. The specific question we address is causing a change in capacitance that can be measured if whether the biosensor can detect receptor -specific ligands the surface is configured as a planar capacitor. In the in real time and at physiologically relevant concentra­ second receptor configuration, a lipid bilayer impregnat­ tions. Implicit in the question is whether this detection ed with receptor was deposited on the capacitive sensor capability is correlated with the in-vivo potencies of the surface. The interaction of receptor with ligands alters receptor ligands. the conformation of the receptor protein, and the move­ ment of charges is detected by the sensor. METHODS Initial work with antibody-modulated biosensors used Planar Capacitive Sensor the T-2 mycotoxin as the analyte. The silicon surface was silanized by reacting the surface silanol groups with The planar capacitive sensor used in these studies, 8 gamma-aminopropyltriethoxysilane to introduce a gam­ which was invented and patented by A. L. Newman, ma-amino group onto the surface. T -2 hemisuccinate was has a substrate composed of silicon coated with 1 JLm of then synthesized by heating T -2 in the presence of pyri­ silicon dioxide (Si02 ). The interdigitated capacitor was dine and succinic anhydride, and the T -2 hemisuccinate formed on the substrate by depositing a thin layer of was conjugated to the gamma-amino function in the aluminum etched to a photographically defined pattern. presence of a water-soluble carbodiimide. The metallization pattern consisted of 93 interdigitated Typical experiments involved measuring the capaci­ fingers, which were 1.2 cm long, 1 JLm high, 50 JLm wide, tance changes that occur when T -2-specific antibodies and spaced about 50 JLm apart. The entire pattern Was are added to a solution bathing the capacitor and when covered with a 150-nm-thick passivating layer consisting free T-2 is added. When 1 JLg/ml T-2-specific antibody of plasma-deposited silicon oxynitride. A thin layer of was added, the capacitance, measured at 1 kHz with a sputtered Si02 was then deposited to provide a surface General Radio 1657 LCR DigiBridge, dropped from for the final layer of 3-aminopropyl-triethoxy silane, the 2180 to 2120 pF. When 1 JLg/ml T-2 was added, the ca­ free amino group used for attachment to organic or bio­ pacitance returned to 2180 pF within 2 min. 9 chemical groups through standard coupling chemistries. Figure 2 shows the interdigitated and cross-sectional Immobilization of the Protein structure of the device. For antibody experiments, tpe Calcium magnesium adenosine triphosphatase hapten was immobilized on the surface and the antibody (Ca + + Mg + + ATPase) was purified from commercially fohns Hopkins APL Technical Digest, Volume 9, Number 1 (1988) 5 Valdes et at. - A Receptor-Based Capacitive Biosensor available cow brain and pharmacologically character­ the result is a bilayer impregnated with receptor layered ized.IO,11 The kinetic data were consistent with it being on the interdigitated surface of the capacitive sensor. a single synaptic membrane protein composed of the Phosphatidylserine and phosphatidylethanolamine are ATPase and either a diltiazem binding site or a change used to form the lower monolayer because the two phos­ in conformation of the associated Ca + + channel fol­ pholipids are known to predominate on the cytoplasmic lowing diltiazem administration. (inner) side of the native cell membrane. Phosphatidyl­ Ca + + Mg + + ATPase was coupled to gamma-amino­ choline and cholesterol are used to form the liposomes propyltriethoxysilane-derivatized, Si02 -coated surfaces in the second monolayer. This procedure simulates the using succinylated calmodulin in the presence of carbodi­ asymmetry found in cell membranes and thus provides imide as the linking molecule. The reaction was carried an appropriate microenvironment for receptor function. out for 2 h at 5°C, and the sensors were washed four The completed biosensor is stored in buffer until it is times with 0.01 M Tris buffer containing 16 vol. 070 gly­ tested. cerol and 0.04 vol. 070 Triton X-l00 to remove unreacted In the present experiments, we used a modified ver­ reagents. Adenosine triphosphate hydrolysis by the im­ sion of this two-step protocol. The sensor was dipped mobilized protein was then determined to ensure that it in 2070 soybean azolectins in n-hexane for 1 min, air-dried was still active. Thus the surface of the biosensor was for 1 min, and then lowered into phosphate-buffered sa­ used in an affinity-chromatographic fashion, resulting line (PBS) solution with a thin film of AChR-impreg­ in noncovalent association of the ATPase with the sur­ nated liposomes spread on the surface. The downstroke face-linked calmodulin molecule. This is an ideal situa­ ended when the sensor cleared the liposome layer, and tion because the calmodulin molecule (mol. wt. = 16 x the direction was reversed at the same speed (0.5 3 10 ) is much smaller than the ATPase moiety (mol. wt. cm/min). 3 = 140 x 10 ), such that removal of the ATPase por­ Capacitive sensors varied in their basal capacitance re­ tion results in significant changes in surface capacitance. sponse when placed into the buffered saline solutions, the Toxin challenge to the sensor was carried out with pur­ major determinant being the composition of the sensor's ified Mojave toxin from a rattlesnake, Crotalus scutula­ surface coating. Sensors covered with a borophospho­ tus scutulatus. The sensor was immersed in buffer in a silica glass (BPSG) were superior and more stable than flow chamber, and toxin was added to a final concentra­ sensors covered with 100- or 500-nm-thick Si02 films, tion of 50-nM. In the control situations, either 300 Ilg as shown in Table 1. Capacitive sensors were reusable calmodulin or buffer alone was added to the chamber. for up to six experiments if washed in warm detergent Capacitance changes were measured with the DigiBridge. solution, rinsed under running water, and dried in air. Immobilization of Nicotinic AChR RESULTS AChR was purified from the electric organ of a fish, Ca + + Mg + + ATPase Biosensor Torpedo nobiliana. To form the interface of the receptor Approximately 50 to 55 ng of calmodulin, represent­ with the capacitive sensor, a special AChR preparation ing about 5070 of the starting total, were immobilized to was made. A mixture of l-alpha-phosphatidylcholine and each sensor surface. These results are in good agreement cholesterol in chloroform (75:25), stored under nitrogen, with theoretical calculations of 25 to 75 ng, depending was evaporated to dryness to form a thin film on the on whether the calmodulin orients itself in a "lying inside of a round-bottom flask. Cholate buffer was add­ down" or "standing" configuration on the derivatized ed such that the final lipid concentration was 25 mg/ml, surface. Steric considerations are suggested, since the and the contents of the flask were sonicated to clarity. Ca + + Mg + + ATPase shows decreased ATPase activi­ The lipids were dialyzed, and the final product was desig­ ty as the amount of calmodulin is increased from 250 nated "liposome preparation" and stored at - 80°C. to 2500 ng. The general method for forming planar lipid bilayers Assay conditions and capacitance after toxin challenge on a small aperture (e.g., Ref. 12) is as follows. Flat are summarized in Table 2. A comparison of the sen­ sheets of planar bilayer form on a flat surface of the sors coated with receptors and challenged with either capacitive sensor from monolayers in the same way they calmodulin or Mojave toxin shows a 34070 increase in form on glass. The bilayer consists of two monolayers capacitance following addition of the toxin. Washing the formed in succession. Phospholipids (1 mM in n-hexane) sensors with buffer containing Ca + + Mg + + ATPase are spread on the surface of a Ringer's buffer sup­ results in a return to baseline capacitance, and the sen­ plemented with 4 mM calcium chloride. The equilibrium sors are responsive to further toxin challenge. surface pressure at the air/liquid interface drops from approximately 72 x 10 - 5 to below 35 x 10 -5 N/cm AChR Biosensor when a monolayer of lipid forms on the aqueous surface. The AChR biosensor displayed pharmacologic speci­ A capacitive sensor, already immersed in the aqueous ficity. When exposed separately to seven neurotransmit­ buffer, is slowly withdrawn (2 cm/min) by means of hy­ ters, it responded with increased capacitance readings to draulic lift, causing the deposition of a monolayer of ACh and did not respond to glutamate, gamma-amino­ phospholipids on the sensor surface. The sensor is im­ butyric acid (GABA), norepinephrine, histamine, 5-hy­ mersed in another trough containing phospholipid vesi­ droxytryptamine, or dopamine (Fig. 3). The biosensor cles, forming the second monolayer. Since the vesicles also differentiated between agonists (compounds that in the second immersion are enriched in AChR protein, bind to a receptor and mimic the actions of the natural

6 fohns Hopkins APL Technical Digest, Volume 9, Number 1 (1988) Valdes et al. - A Receptor-Based Capacitive Biosensor

Table 1-Capacitance response of three types of sensors in 19.------~ different media. _ Without ACh receptor Capacitance in _ With ACh receptor 0) Different Media (PF) 0) u:: . ~ c E 18 (5 ~ ·E 0) u co Type I Type II u 0) c ~ c a. 500-nm lOO-nm Type III 0) :§ ~ .!9(3 u 0. 0) 0) Medium Si0 film Si0 BPSG co ~ $ 0) c >-x 2 2 co c 0 c .0. ·E g. 17 ~ E -0 ·E u CD 0) co :t: co !9 0. >- !9 Air 920 845 894 :l ~ :l 0 0 I .!Q co (!) C3 z 0 JJ I n-hexane 1,030 986 995 2070 phospholipids 1,140 1,171 1,174 in n-hexane Transmitters (1 mM) 2% phospholipid 87,976 7,457 4,434 liposomes (small Figure 3-Response of the AChR biosensor to neurotrans­ unilamellar vesicles) mitters. in water Water 4,300 7,590 4,122 used as the pretreatment, as shown in Fig. 4. The sen­ Buffer (PBS) > 100,000 11,207 4,480 sor's sensitivity to agonists was dose dependent, and de­ lO-mM ACh in PBS > 100,000 14,188 9,731 tection limits were on the order of 1 nmol ACh/ ml (lIlM ACh), as shown in Fig. 5. Storage in buffer for several lO-mM d-TC in PBS 95,040 8,627 4,390 hours after receptor immobilization did not reduce the Lipid liposomes, 1- 71,725 7,516 4,437 sensitivity of the biosensor to ACh. mg/ml Ca + + -free Control biosensors made of immobilized lipids only, Ringer's solution without AChR protein, did not respond to any of the from 2% cholate test drugs and were used to establish baseline capacitance AChR-liposomes 73,229 6,817 4,415 measurements for each sensor. Biosensors with immobi­ in 2% cholate ex- lized AChR and acetylcholinesterase displayed sensitivity tract with I-mg/ ml to both receptor-active drugs and anticholinesterases. The lipids [azolectin + organophosphate diisopropyl fluorophosphate (DFP) in­ cholesterol (6: 1)] hibited acetylcholinesterase irreversibly, and the high concentration of unhydrolyzed ACh stimulated the AChR and increased capacitance (Table 3). Table 2-Response of the Ca + + Mg + + ATPase capacitive Finally, biosensors constructed from protein extracted sensors (all plates contained immobilized from rat brain, which is multi synaptic (i.e., contains nu­ Ca + + Mg + + ATPase Ca + + channel complex). merous receptor types), responded positively to all seven transmitter substances, as predicted. Baseline Challenge Capacitance Capacitance DISCUSSION Assay Condition (pF) (pF) The data given here represent proof of the concept of No additions 28,382 a receptor-based capacitive biosensor. Significantly, a re­ sponsive system can be made using two qualitatively dif­ Buffer + 50 J11 Ca + + (4 J1M) 10,538 10,168 ferent receptor types and two different immobilization Buffer + 50 J11 Ca + + (4 J1M) 5,821 5,692 approaches. However, the actual events at the surface + calmodulin (300 J1g) of the biosensor that lead to transduction of a signal Buffer + 50 J11 Ca + + (4 J1M) 6,763 7,642 from the receptor binding event remain speculative. + Mojave toxin (180 J1g) In the Ca + + channel studies, two mutually exclusive hypotheses may be considered. First, the binding of the toxin to the receptor may displace the receptor from the ligand for that receptor) and antagonists (compounds calmodulin, either by direct competition or by inducing a that bind to a receptor and block the action of the nat­ conformational change in the receptor. Alternatively, the ural ligand for that receptor); agonists such as ACh and receptor may remain attached to the calmodulin and the carbamylcholine increased capacitance, but antagonists conformational changes that follow binding may alter such as d-tubocurare (d-TC) and the alpha-neurotoxin the configuration of charges at the surface. Resolution of cobra (Naja naja) venom did not. Further, when the of this issue will require deposition studies using either biosensor was pretreated with the reversible antagonist radiolabeled or fluorescently tagged receptors. d-TC and then slowly perfused with ACh containing Experiments using AChR embedded in a lipid bilayer buffer, it recovered its response to ACh. This did not suggest that the salient event is the opening of the channel occur when the irreversible antagonist Naja toxin was caused by agonist binding and subsequent ion transloca-

Johns Hopkins APL Technical Digest, Volume 9, Number I (1988) 7 Valdes et aI. - A Receptor-Based Capacitive Biosensor interact with the allosteric sites in the ion channel. (Al­ 1.2r----.-----.-----.----.----,.---~ losteric sites are binding sites on a receptor, other than the "active site," which binds the natural ligand. These Control 1.0 binding sites may be associated with an ion channel cou­ • ••••••••••••••••••••••••• pled to the receptor and may modulate the binding of u: ligands to the active site.) Specifically, it will be necessary c:: • • to design a system with some intrinsic level of receptor Q) 0.8 () c:: activity such that an agonist or antagonist will trigger ~ '(3 Pretreated with a signal by, respectively, increasing or decreasing ion (I:Jg. 0.6 d-TC (100 /L M) translocation. () •• ••• • •• While these studies support the concept of a receptor­ ro ••• based biosensor, they remain preliminary laboratory 'E • •• ~ 0.4 •••• demonstrations with at least two major, albeit implicit, ••••• unresolved issues: receptors must be produced in quanti­ i:S ••••• •••••••••••••••••••••••••••• • ties sufficient for practical applications and they must be 0.2 Pretreated with immobilized and stabilized in an interfacial system com­ Naja a-neurotoxin patible with microsensors. (1 /LM) Production of large (i.e., gram) quantities of purified o~--~----~----~----~--~----~ receptor proteins requires cloning the gen~s that encode o 10 20 30 for the subunits and reassembling them into functional Time (min) receptors in an expression vector that can be scaled up Figure 4-Response of the AChR sensor to reversible (d-TC) to production levels. (Expression vectors are cell lines and irreversible (Naja alpha-neurotoxin) antagonists. in cultures, used to produce or express proteins for which a cloned gene codes.) Several such systems are promising candidates. 13- 16 Alternatively, artificial receptors might ACh 1 /LM 10/LM 100 /LM be produced using macrocyclic compounds to which re­ 30r-----~----~------,------,----~ ceptor-active site fragments are attached 17 or by raising antiidiotypic antibodies to the receptor. 18 (Antiidiotypic u: antibodies are antibodies raised against another antibody E 20 to mimic the antigenic site to which the original anti­ Q) () c:: body was raised.) Such artificial technology sacrifices the ~ ability of the native receptor to recognize classes of com­ '(3 (I:J •••••• pounds, but may be useful in limited applications and g. 10 u •• • • ••••••••••• may enhance biosensor stability. A discussion of im­ mobilization (e.g., lipid-protein interactions, biomateri­ 2~----~----~------~----~----~ als, surface chemistry, biomedical polymers) is beyond o 5 10 15 20 25 the scope of this article; however, several excellent texts Time (min) are available on the subject. 19,20 Figure 5-Dose-dependent response of the AChR sensor to If the foregoing technical issues can be resolved, re­ ACh (filled circles). Triangles = control. ceptor-based biosensors will be able to meet battlefield detection requirements, but military applications repre­ sent only a small fraction of their potential use. The devel­ Table 3-Response of Type II and Type III biosensors to ACh opment of flexible receptor- and enzyme-based biosen­ and d-TC in the presence and absence of DFP. sors will have profound implications for basic research, Type II Type III medicine, industry, and environmental health. Specific applications include microbioassays for mutagens; 21 -DFP +DFP -DFP +DFP chemical analyses for toxins, drugs, and chemicals in bio­ logical fluids; 22 process control and fermentation; 23 in­ PBS 20,153 20,404 23,428 24,573 dustrial monitoring of the workplace and of toxic waste (±490) (± 1503) (± 1561) (±3) dumps; 24 veterinary medicine; and toxicological screen­ 25 PBS 24,225 24,562 25,573 30,242 ing. Despite powerful economic incentives to develop + 100nM ACh (±290) (±440) (±3273) (±580) biosensor technology, the lack of reliable biosensors for ACh 4,102 4,158 2,145 5,669 many industrial applications has precluded automation, resulting in increased costs. PBS 18,268 19,015 17,041 The development of a biosensor that uses biological + 100mM d-TC recognition sites to detect compounds of physiological significance represents a qualitative advance that could tion. This is indicated by the ability of receptor antag­ lead to true generic sensing. Long-term applications are onists to block ion channel opening, a situation that leads limited only by the imagination. Ultimately, it will be pos­ to problems in designing a biosensor that is responsive to sible to design intelligent drug-administration devices that both agonists and antagonists as well as compounds that use the biosensor to monitor serum levels of drugs and

8 Johns Hopkins APL Technical Digest, Volume 9, Number 1 (1988) Valdes et al. - A Receptor-Based Capacitive Biosensor

hormones and dispense drugs in appropriate amounts, 141 . P . Burand, M. D. Summers, and G. E. Smith, " Transfection with Baculovirus DNA," Virology 101, 286-290 (1980). and to build biosensors that detect carcinogenic markers 15C. Miyamoto, G. E. Smith, 1. Farrell-Towt, R. Chizzonite, M. D. Sum­ while they are still in the precancerous state. mers, and G. 1u, "Production of Human c-myc Protein in Insect Cells Infected with a Baculovirus Expression Vector," Mol. Cell. Bioi. 5, 2860-2865 (1985). REFERENCES 16y. Matsuura, R. D. Possee, H. A. Overton, and D. H. Bishop, "Baculovi­ rus Expression Vectors: The Requirements for High Level Expression of IV. A. Kratasyuk and 1. 1. Gitelzon, "Bacterial Bioluminescence and Bio­ Proteins, Including Glycoproteins," J. Gen. Virol. 68, 1233-1250 (1987). luminescent Analysis," Biofizika 27,937-952 (1982). 17M. U. Hosseini, 1. M. Lehn, S. R. Duff, K. Gu, and M. P. Mertes, "Syn­ 2G. Guilbault, "Analysis of Environmental Pollutants Using a Piezoelec­ thesis of Monofunctionalized and Difunctionalized Ditopic (24)N602 Mac­ tric Crystal Detector," Int. J. Environ. Anal. Chern. 10,89- 98 (1981). rocyclic Receptor Molecules," J. Org. Chern. 52, 1662- 1666 (1987). 3V. N. Morozov and T. Y. Morozova, "Mechanical Properties for Globu­ 18 1. L. Marx, "Making Antibodies Without Antigens," Science 228, 162-165 lar Proteins," Mol. Bioi. (Moscow) 17, 577-586 (1983). (1985). 4G. R. Ivanitskiy, "Biological Microdevices," trans. of "Biologicheskiye 19 1. D. Andrade, ed., Surface and Interfacial Aspects of Biomedical Poly­ mikroustroystva," Vestn. Akad. Nauk SSSR 3, 118-128 (1984). mers. Protein Adsorption, Vol. 2, Plenum Press, New York (1985). 5D. D. Denton, in Proc. International Con/. on Solid State Sensors and 20G. W. Hastings and P. Ducheyne, Macromolecular Biomaterials, CRC Actuators, Philadelphia, p. 202 (1985). Press, Boca Raton (1984). 6E. C. M. Hermans, "CO, CO2, CH4 and H20 Sensing by Polymer Cov­ 21 F. Mizutani, K. Sasaki, and Y. Shimura, "Sequential Determination of ered Interdigitated Electrode Structures," Sensor. Actuator. 5, 181-186 I-Lactate and Lactate Dehydrogenase with Immobilized Enzyme Elec­ (1984). trode," Anal. Chern. 55, 35-38 (1983). 7N. F. Sheppard, D. R. Day, H . L. Lee, and S. D. Senturia, "Microdi­ 22T. C. Pinkerton and B. L. Lawson, "Analytical Problems Facing the De­ electrometry," Sensor. Actuator. 2, 263-274 (1982). velopment of Electrochemical Transducers for In Vivo Drug Monitoring: 8A. L. Newman, JHU/APL Technical Memo S36, pp. 86-147. An Overview," Clin. Chern . (Winston-Salem, N.c.) 28,1946- 1955 (1982). 9A. L. Newman, W. D. Stanbro, K. W. Hunter, and A. Andreou, "De­ 23M. Hikuma, T. Yasuda, I. Karube, and S. Suzuki, "Applications of velopment of an Antibody-Modulated Planar Capacitive Sensor," in Proc. Microbial Sensors to the Fermentation Process," Ann. N. Y. A cad. Sci. IEEE Con/. Synthetic Microstructures in Biological Research, Airlie, Va., 369, 307-319 (1981). pp. 45 - 49 (1986). 24M. Hikuma, H. Suzuki, T. Yasuda, 1. Karube, and S. Suzuki, "Ampero­ 1°1. P. Chambers, M. 1. Wayner, 1. Dungan, E. D. Rael, and 1.1. Valdes, metric Estimation of BOD by Using Living Immobilized Yeast," Eur. J. "The Effects of Purified Mojave Toxin on Rat Synaptic Membrane Appl. Microbiol. Biotechnol. 8, 289 (1979). Ca + + Mg + + ATPase and the Dihydropyridine Receptor," Brain Res. 25p. Ross, "Biosensors: A New Analytical Technology," BioFeature 1, Bull. 16, 639-643 (1986). 204-207 (1983). 111. P. Chambers, M. 1. Wayner, E. Rizopoulos, M. L. Gonzales, R. B. Taylor, and 1.1. Valdes, "Partial Characterization of Two Ca + + Mg + + Dependent ATPase Activities from Bovine Brain Synaptic Membrane ACKNOWLEDGMENTS-The success achieved in this program is due, in Homogenates," Brain Res. Bull. 18, 99-107 (1987). large part, to the collaborative efforts of A. L. Newman, W. D. Stanbro, and 12M. Montal, "Formation of Biomolecular Membranes from Lipid K. Hunter of Biotronics Systems Corp., N. Blum of APL, and A. Andreou of Monolayers," Methods Enzymol. 32, 545-554 (1974). The 10hns Hopkins University. The authors also thank E. Rael of the University l3S. A. Parent, C. M. Fenimore, and K. A. Bostian, "Vector Systems for of Texas, EI Paso, who donated the Mojave toxin for our research, as well as the Expression, Analysis and Cloning of DNA Sequences in S. cerevisiae," F. P. Ward of the U.S. Army Chemical Research, Development, and Engineer­ Yeast 1, 83-138 (1985). ing Center for his leadership and support.

THE AUTHORS JOSEPH G. WALL, Jr., was born in Wilkes-Barre, Pa., in 1932 and earned a B.S.E.E. at the Universi­ ty of Idaho in 1961. After 4 years JAMES J. VALDES received his with Westinghouse Electric, he went Ph.D. in 1979 from the Chemistry to the Space Systems Division of of Behavior Program at Texas Chris­ Fairchild Hiller Corp. as a senior en­ tian University and worked as a gineer working in the evaluation and postdoctoral fellow in neurotoxicol­ design of digital data systems. In ogy at The Johns Hopkins Univer­ 1965, he joined APL as a specialist sity until 1982. He is Biosensor Pro­ in digital design, test, and evalua­ gram Manager and director of the tion. Mr. Wall has been program neuropharmacology laboratory at manager for the Miniature-Ship­ the U.S. Army Chemical Research board Automatic Data Recording Development and Engineering Cen­ System, the Coast Guard Precision ter and is an adjunct associate pro­ Intercoastal Loran Translocator, the fessor of environmental health sci­ GEOSAT-A Radar Altimeter, and ences at The Johns Hopkins Univer­ the Portable Loran Assist Device. He supervised the Space Depart­ sity. Dr. Valdes is the author of ment Planning and Operations Office and is program manager for the more than 50 scientific papers; his Navy SATRACK II Program, the Coast Guard Automated Aids to research interests include receptor Navigation Positioning System, the Bird-Borne Transmitter Program, mechanisms and toxicology. and the receptor-based capacitive biosensor.

Johns Hopkins APL Technical Digest, Volume 9, Number 1 (1988) 9 Valdes et at. - A Receptor-Based Capacitive Biosensor

JAMES P. CHAMBERS received MOHYEE E. ELDEFRAWI re­ his Ph.D. in biochemistry in 1975 ceived his Ph.D. in toxicology in from the University of Texas Med­ 1960 from the University of Califor­ ical School at San Antonio. He took nia, Berkeley. He held faculty posi­ postdoctoral training in biochemis­ tions at the University of Alexandria try at the University of Pittsburgh (1960-68) and Cornell University School of Medicine (1976-78) and (1968-76) before joining the faculty in human genetics at Washington at the University of Maryland School University (1978-79) before joining of Medicine, where he is currently the faculty at the University of Tex­ professor of pharmacology. Dr. El­ as Health Sciences Center, Houston defrawi, a consultant for the Unit­ , (1979-84). Dr. Chambers, currently ed Nations, the World Bank, and associate professor at the Universi­ the State Department, is the author ty of Texas at San Antonio, is the of more than 180 scientific papers author of more than 40 scientific in the areas of receptor biochemis­ papers in the areas of enzymology try and toxicology. and glycoprotein characterization.

10 fohns Hopkins APL Technical Digest, Volume 9, Number 1 (1988)