d
R. Michael Buch and Gany A. Rechnltz Department of Chemistry and Biochemistry University of Delaware Newark, DE 19716
Chemoreceptors are among the newest tools being employed by bioanalytical chemists because their unique binding properties enable them to be used as highly selective molecular recognition elements. Chemoreceptors are being used in affinity chromatographic methods as well as in novel assay techniques such as the enzyme-amplified receptor assay (I).Difficulties associated with isolation, stabilization, and immobilization arise, however, when attempts are made to incorporate chemoreceptors into electrodes to produce biosensors. Our approach to receptor-based biosensors, or “receptrodes” (Z),has been to use the intact chemosensing structures (antennules) of Caffinectes sapidus, the blue crab. This approach allows the receptors to remain in their native environment, which presumably has already been optimized by nature. The antennular chemosensing cells serve not only as highly selective and sensitive molecular recognition elements, but also as biological transducers, converting chemical information into electrical impulses in a matter of milliseconds. Physiologists have studied the mechanisms involved in these processes for 0003-2700/89/0361-533A/$O 1.50/0 0 1989 American Chemical Society
years. Both Case (3)and Laverack (4) demonstrated that decapod receptor systems could be analyzed electrophysiologically. Since that time, many electrophysiologists have investigated the transduction processes occurring in a variety of crustaceans, particularly lobsters, shrimp, and crayfish. Such studies provided the impetus for our research-the application of intact chemosensing systems to the determination of relevant chemical stimuli. The ongoing development of these antennular receptrodes bas been described in several articles (5-8).This REPORT will review progress in the development of antennular receptrodes, describe the procedures involved, and examine possible analytical uses. Physiology ot antermlar CheIllWC@On
The blue crab, C. sapidus (see Figure l),is found along the East Coast of the United States. Because the crab t y p cally inhabits turbid waters where eyesight is of little use, it has developed highly sensitive chemical senses. Crabs use these senses, known as gustation and olfaction, to detect various chemicals in their environment (e.g., to locate food, find mates, detect potential danger, and recognize territory). The antennules (olfactory organs) and the periopod dactyls and mouthparts (gustatory organs) are the major chemosensory organs of C . sapidus. Because olfaction and gustation occur in the same aqueous environment for
C. sapidus (unlike with our own senses, where gustation is aqueous and olfaction is gaseous),an arbitrary parameter of distance is used to distinguish between the two chemical senses. If a particular sensing organ responds to chemical cues whose source is distant, the organ is termed an olfactory organ. If, on the other hand, a chemosensing organ responds to chemical cues originating a short distance away, the organ
REPOR7 is referred to as a gustatory organ. Because the antennules respond to chemical cues originating from distant sources, they are inherently more sensitive than the gustatory organs. This sensitivity in aqueous solutions and the ease with which the antennules can be removed from the crab made them appropriate for use in the development of neuronal biosensors. The blue crab has two jointed antennules, each about 1cm long and 1mm in diameter, on the front of its shell between the eyestalks. The first joint connects the coxa segment of the antennule to the shell, and the second joint conneck the basis segment to the coxa segment. The tip of the basis segment (the distal end of the antennule) Flgure 1. Callinectes sapidus, the blue
crab.
ANALYTICAL CHEMISTRY. VOL. 61, NO. 8. APRIL 15. 1989
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(odoranta) to directly contact the underlying chemoreceptors (1I). When no odoranta are present, the chemcaensitive neuron is in the resting state. As with all neurons, a stimulus is required to change the state of the chemoreceptor cell. During the resting state, the concentration of sodium ions is about 10times greater in the external medium than in the internal medium (axoplasm) of the cell, while the concentration of potassium ions is about 40 times greater internally than extern d y . The relative ion activities are such that a transmembrane potential of -90 mV is established during the resting state. This nonequilibrium condition is maintained by an ATP/ ATPase-driven “sodium pump.” Although potassium ions can traverse the membrane freely, the internal electronegativity resulting from the exit of sodium ions causes the potassium ions to concentrate within the axoplasm (12). When binding murs at a receptor, the permeability of the neuromembrane is altered, allowing sodium ions to enter the cell. Because this influx of sodium ions causes the transmembrane potential to ground to zero, a membrane depolarization occurs, and the neuron is transformed from the resting state to the active state. Each axon has a threshold (a particular number of binding eventa occurring within a particular time window) that must be exceeded before a change of state occurs. If the number of binding events occurring within a given amount of time exceeds the axon’s threshold, membrane depolarization will occur. As shown in Figure 3, the ionic nux does not bring the membrane potential
-
Figure 2. Scanning electron micrograph of the antennule tip showing a Mt of aek thetascs on the endopod (40X).
has two branches, the larger of which is the endopod and the smaller of which is the exopod. A tuft of several hundred hairlike sensillae, known as aesthetasca, is located on the endopod (see Figure 2). Electron microscopy studies reveal the structure of the aesthetascs to be that of thin-walled, open-ended tubules protruding from the flagella (9). The dendrites of many sensory neurons (chemoreceptor cells) stimulate these aesthetasca, which contain an even greater number of chemoreceptors (IO). The chemoreceptors are actually protein molecules embedded in the cellular membranes of the dendrites. These chemoreceptor proteins usually contain one or more specific binding sites for particular chemical compounds. In the C B B ~of olfactory receptors, the compounds are referred to as odorants. Hence the chemoreceptor serves as a target molecule for a specific odorant. Physiologists have divided the chemoreceptor-containing dendrites into t h e general regions: basal, transitional, and distal. The basal region, which contains some cytoplasmic inclusions such as mitochondria, vesicles, and microtubules, is the region c l w t to the cell body. It also contains rootlets and basal bodies, each of which provides the terminus for a ciliary substructure. The transitional region begins at the base of the cilia, where the cilia split into branches, each of which contains one microtubule. The distal region is 534A
the region beyond the branching that contains the extremities of the dendrites. In this region, the dendrites straighten and run directly to the tips of the aesthetascs, where they terminate. The chemoreceptors are presumed to be located in this region (although some may be located in other regions) because of the proximity of the distal reeion to the wall of the aesthetasc.&J penetration tests using crystal violet indicate that the aestbetascs are permeable to liquids, thus allowing
Flgure 3. The membrane depolarization process as it appears at one location along the antennular nerve.
ANALYTICAL CtEMISTRY, VOL. 81, NO. 8, APRIL 15. 1989
process in order to g guarantees thalI Modified manufacturing process ensures minimum column bleed. A comprehensive "Grob" type test mix is analyzed isothermally and column performance indicators determined. A three point linearity plot is obtained for Pentachlorophenol, 2,CDinitrophenol, N-Nitrosodiphenylamine and Benzidine.
Relative response for the four polar check compounds is calculated.
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immediately to ground, but overshoots it, making the outside of the membrane negative with respect to the inside. This negative afterpotential is followed by an overshoot in the opposite direction, creating a positive afterpotential. After several milliseconds, the original resting state is restored at a particular cross section of the membrane via the sodium pump mechanism. The voltage pulse, together with its negative and positive afterpotentials, is known as the action potential. and the large initial pulse is termed the spike potential. The localized depolarization of the membrane caused by the binding event causes depolarization of the neighboring membrane regions, and the action potential is propagated in both directions along the length of the axon. Because this propagation is an active process driven by cellular metabolism, no attenuation of the signal occurs. The mons of the cell bodies that lie directly below the aesthetascs converge and run proximally to form the antennular nerve, which extends through the coxa and hasis segments of the antennule. It is this nerve to which electrodes can be attached to intercept the neural messages being generated by the chemoreceptive cells. Nerves can be classified as either efferent (carrying information away from the brain) or afferent (carrying information to the brain) even though action potentials are propagated in both directions along the axon. This directional character is achieved through a very small (-1 pm) gap between adjacent neurons, known as the synapse. The end of the presynaptic fiber (the axon) is shaped like a knob and has vesicles containing transmitter molecules, the best known of which is acetylcholine. The postsynaptic (dendrite) surface contains receptors that are specific for the transmitter compounds. The depolarization of the distal end of the presynaptic fiber (arrival of the action potential) triggers the release of the transmitter compounds. Although the mechanism of this process is not thoroughly understood, an influx of calcium ions probably results in fusion of the vesicular membranes with the axoplasmic membrane, allowing transmitter compounds to be discharged from the cell via exocitosis (13). The transmitter compounds diffuse across the synaptic gap in less than a millisecond and bind at the receptors in the postsynaptic region. These binding events, like the binding events in the antennular chemoreceptor cells, generate an action potential. The transmitter compounds are then inactivated so that the next pulse can be transmitted. 5MA
In the case of acetylcholine, this inactivation is accomplished by acetylcbolinesterase. In summary, odorant molecules permeate the cuticular walls of the aesthetascs and bind with receptor proteins located within the dendritic membranes of the underlying neurons (the chemoreceptor cells). If enough of these binding events occur within a given period of time to overcome the threshold values of the chemoreceptor cells, action potentials are produced and sent to the brain via the afferent neurons of the antennular nerve. Chemical signals received from the environment are thus converted into electrical signals. Because the action potential is propagated along the length of the antennu-
lar nerve by an active procesa (cellular metabolism), the signal does not decay with distance from the point of origin and electrode contact can be made anywhere along the length of the antennular nerve. This process always produces signals of the same amplitude, as determined by the transmembrane charge gradient, and therefore the signal amplitude does not contain any information about the concentrations of odorants. The frequencies, however, are not constant. Because chemical binding events trigger the membrane depolarization (which ultimately results in the production of the action potential) and odorant concentration relates statistically with the frequency of the binding events, the action potential's frequency
I-
Figure 4. The flow c%iiwith mounted antennule and the various electrode connections: (a) side view and (b) top view.
ANALYTICAL CHEMISTRY, VOL. 61, NO. 8. APRIL 15, 1989
is directly proportional to the concentration of stimulant (odorant). The relationship between a cell's response ( R ) and the concentration of odorant (C) is given by the following equation (2):
R=
Rm,
1
+ (K/C)"
where ,R is the maximum response frequency, n is the Hill coefficient (a cooperativity factor between receptors), and K is a constant. For the most simple case, assuming identical receptors where n = 1,the equation reduces to the more familiar Michaelis-Menten equation for enzyme kinetics. The dose-response relationship between frequency and stimulant concentration (14) can be used to obtain analytical data from the receptrode system. Theneuonalbiosensor The dissected antennule is mounted in aspecially designed flow cell consisting of several chambers, as shown in Figure 4. The rhemosensing tip of the antennule is inserted into a tubular chamber through which artificial seawater (ASW) ran be pumped (15). Adjacent to this seawater rhamber and connected by a small mounting hole is another chamber in which the exposed antennular neurons are bathed in an artificial isotonic intercellular fluid (Panulirus saline solution) (16). This chamber also provides holes through which
ground and reference wires can be inserted. As shown in Figure 5, the flow cell is mounted on the stage of a binocular dissecting microscope, allowing the exposed neurons to be viewed during the experimental procedures. A four-port, two-way sample injection valve is used to introduce an analyte into the ASW carrier stream. A sample injection loop is used to provide a constant stimulant concentration for approximately 10 s. Responses to these stimulants are received by a glass suction pickup electrode with an internal tip diameter of -50 pm. The leads from this electrode as well as the leads from the ground and reference wires are fed into a neurological preamplifier, and the output of this amplifier is then divided and fed to several devices. One lead provides viewing of the amplified action potentials on a storage oscilloscope,while another feeds the signal to a window discriminator, the output of which is sent to a digital event counter. Adjustment of the window discriminator enables spikes of a particular amplitude to be selectively counted from a group 01 spikes of varying amplitudes. Signals are also fed into one channel of a stereo cassette tape recorder for data storage. Outputs from the cassette recorder are fed into the same devices that receive input from the preamplifier, enabling the stored data to be analyzed at a latex time. A microphone allows an oral ac. count of each experiment to be record.
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Flgure 5. The antennular receptrode mounted on the microscope stage. ANALYTICAL
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REPORT ed on the second channel of the stereo tape. Both channels are output individually to separate amplified audio speakers. This arrangement provides real-time audio output of the action potentials as well as an audio output of the research narrative. After a baseline is established, demonstrating that the particular antennule employed is active, various stimulant solutions are passed over the chemosensing tip. If no response is observed to a particular stimulant. another compound is injected. If no responses are observed for any of the various stimulant solutions, the pickup electrode is repositioned along the antennular nerve and the procedure repeated. This searching procedure is necessary because we do not yet possess the ability to recognize particular locations along the antennular nerve as coding information from particular receptor sib. After a chemoresponse is found, a selectivity study can be performed. Usually, the system is quite specific, responding to only one of the compounds b i n g tested. After the specificity tests are completed, various concentrations of the particular analyte are introduced into the carrier to generate a dose-response curve. Additions are always made from lowest concentration to highest, and the system is allowed to flush with ASW for 5 min hetween each addition to minimize any adaptation of the chemoreceptivecells to the analyte. Dose-response curves are generated by playing recorded data (action potentials) through a digital event counter for a given period of time. The
13
Flgure 6. Dose-response 530 A
number of spikes counted is then divided by the counting time to obtain response frequencies with the units of spikes per second. These frequencies are plotted against the analyte concentrations to generate a dose-response mrve that can he used as a calibration curve for the antennular receptrode.
Typical rerults Because the antennule is an olfactory organ, one would expect the system to respond to compoundsthat occur naturally within the crab’s native environment. Our initial experiments, therefore, dealt with the determination of amino acids, which are components of all of the crab’s food sources. One of the earliest successful experiments with this system resulted in the dose-response curve for isoleucine depicted in Figure 6. At the time this experiment was performed, the concentration range of the receptrode system was assumed to be relatively narrow, and only a narrow stimulant concentration range was tested. This assumption was based on the physiological work of Atema and eo-workers (10,which indicated that the overall range of a receptor cell was about 2 or 3 orders of magnitude. The fact that the system possessed a much broader dynamic range did not become apparent until subsequent experiments were performed. Our original, rather naive assumption that each observed signal was generated hy a single receptor cell did not explain this wider dynamic range. We now believe that the responses ohserved are produced by a population of chemoreceptor cells whose narrow individual response ranges are summed
0
C U N ~for
isoklucine.
ANALYTCAL CHEMISTRY. VOL. 81, NO. 8, APRIL 15, 1989
to produce a wide overall dynamic range (6). Because of the large number of cells and the relatively large size of the pickup electrode, a number of neurons are usually sampled simultaneously. The neurons that are in position to best contact the pickup electrode appear to produce signals with the largest amplitudes, whereas the neurons forming slightly poorer contacts with the pickup electrode appear to generate signals of smaller amplitude. When a signal contains spikes of many amplitudes (thus indicating contact with many different neurons), the signal is termed a multi-unit signal. When a signal consists of spikes of equal amplitudes, the signal is termed a single-unit signal (see Figure 7). Because the multi-unit signal is produced by an array of chemoreceptor cells, it contains more information than a single-unit response, is much more complex, and is therefore more difficult to analyze. If fewer than four different amplitudes are present and the relative difference in amplitudes is large, the signal is called a distinguishable multiunit signal (because the various action potentials are easily distinguished from each other). These signals can be isolated by the window discriminator and counted as single units. Because of the difficulties associated with deciphering the multi-unit data, we have concentrated our efforts on the singleunit data. We believe, however, that development of a multi-unit analysis technique would be useful because it would allow the vast array of cbemoreceptors to be used. Among the amino acids, glutamic acid seems to be the strongest stimulant. This is not surprising, because the antennular chemosensing system is at least in part a food-locating system, and glutamic acid is a major constituent of a wide variety of proteins found in many food sources. For example, glutamic acid comprises 17.9% of insulin, 12.4%of ribonuclease, and 13.0% of cytochrome (18). Because the ASW carrier stream is buffered to a pH of 8.0, the glutamic acid actually exists as its conjugate base, the glutamate anion. The relationship between response and glutamate concentration is linear over 6 orders of magnitude, with an apM. parent limit of approximately At a nanomolar concentration, the response frequency (67.2spikes per second) is still well above the baseline frequency of 20.1 spikes per second, but the sensitivity of the system has decreased significantly. The system also responds analytically to glutamine hut the response frequencies are not as high, and the overall sensitivity (slope
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of the doseresponse curve) to glutamine is not as good (7). Response to all 20 amino acids (with the exceptions of tryptophan, arginine, and cysteine) have been measured with antennular receptrcdes. Each of these responses is specific to the particular compound in concentrations from 10-2 to 10-7 M. Purines (adenine and adenosine nucleotides) can also be measured-with antennular receptrodes. The importance of these compounds as biochemical modulators or transmitters has been extensively reviewed (19-22),and several researchers have p r o p o d the idea that certain internal newtrans-
mitters, neuromodulators, and newt active agents evolved from more prim tive structuresthat may originally ha! been external chemoreceptors (23,24 Behavioral studies conducted by Ca and Thompson (25)demonstrated th a marine shrimp, Palaemonetespugi was attracted to 5’-adenosine moni phosphate (5’-AMp), thereby indica ing the existence of some type of purh detection system on a marine crust cean. Because C. sapidur is a relative primitive animal, we assumed that r rine receptors might exist on the ane nules of this organism, and we were I entirely surprised by the responses I duced by several purine compounds.
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The doseresponse curve generated from 5'-AMP is depicted in Figure 8a. It is linear over 6 orders of magnitude in concentration and has a detection limit in the nanomolar range. The purinergic response frequencies generally were lower than the amino acid response frequencies. In addition, the purinergic responses are unaffected by any of the amino acid solutions, indicating purinergic specificity. The responses are not totally specific for one purine compound, however. Antennular receptrodes responding to 5'-AMP also respond analytically to adenosine diphosphate (ADP). The responses to ADP are smaller and of a narrower analytical range than those for 5'-AMP, as shown in Figure 8b. No responses, however, are observed for adenosine triphosphate (ATP). Another interesting class of compounds that may be analyzed with the antennular receptrode system is the hormones. Numerous examples of hormonal chemodetection have already been described in the physiology literature. For example, the male silk moth Bornbyx mori can detect incredibly minute quantities (as few as 200 molecules) of the female sex pheromone bombykol. Behavioral studies indicate that male blue crabs respond to a pheromone released by females of the species (26). These studies further indicate that the chemoreceptors responsible for the detection of this pheromone are located within the aesthetascs of the antennules. This particular pheromone has not yet been isolated but some other crustacean hormones such as 20-hydroxyecdysone are commercially available. Although the ecdysones are generally considered to be insect molting hormones, many also appear in crustaceans. Male crabs should be able to detect molting females because crabs can only mate immediately after molting (when the exoskeleton is pliable). The analytical proportionality between response frequency and ecdysone concentrations ranging from 2 x M to 2 X M have been determined. The 20-hydroxyecdysone (8-ecdysone) system does not respond to amino acids, purines, or a-ecdysone, a commonly occurring isomer. Ecdysones and related compounds are becoming increasingly important because their biological actions may enable them to be used as pest control compounds (27,28).The antennular receptrode system may respond to other bormones; work in this area is progressing. Experimentation to test the ability of the antennular receptrode to detect other, more common pesticides is also under way.
I 2
0
ryure 8. Comparison of the dose responses induced by (a)5'-adenosine monophosphate and (b) adenosine diphosphate.
Advantages and disadvantagesot neuOnalMosenwrs
The antennular receptrode system has many advantages, the most important of which is ita extremely short response time. This response time is limited by the time required by the neurons to generate the action potential after the odorant-receptor binding event, which occurs on a millisecond time scale. Therefore the response time of the antennular receptrode is on the order of milliseconds. Another obvious advantage is the high degree of specificity that can he attained. The specificity of the system can he adjusted by changing the pickup electrodeherve contact. In addition, a vast array of chemoreceptors could he used as an array detection system (contingent on the development of an adequate data analysis system) by employing a larger pickup electrode and therehy contacting a greater number of afferent neurons in the antennular nerve. Yet another advantage is the broad analytical range and sensitivity of the antennular receptrode. The system routinely responds to analytes over concentration ranges of at least 6 orders of magnitude, and responses to nanomolar concentrations of analytes are typical. The limit of detection (threshold) for many analytes may be as IOW as 10-15 M. A major disadvantage of the antennular receptrode is its relatively short lifetime. Under optimal conditions, the biological components remain viable for about 48 h, with an average lifetime closer to 12 h. Design changes in the flow cell and pickup electrode have extended the lifetime of the system by
more than 6GQ% over the prototype system of Belli and Rechnitz (5),but 48 h is not long enough for an analytically useful device and we are currently working to further extend the lifetime of the system. Decapod motor axons have been reported to remain physiologically active in vivo for 250 days after being severed from their cell bodies (29).If the in vivo conditions could be duplicated in vitro, the neurons could remain active for extended periods of time. Another difficulty associated with the antennular receptrode arises because of the presence of tactile receptors in the antennule. These receptors respond to changes in pressure and can, therefore, interfere with the analytical signal. The design of the flow cell and injection loop minimizes pressure changes by providing a smooth flow of the ASW carrier that introduces the analyte to the detector. We are currently investigating methods by which tactile responses can be distinguished and ultimately eliminated from the chemoresponses. Like all biological sensory systems, the antennular receptrode is subject to adaptation. When receptor binding sites become saturated, the system no longer responds to changes in analyte concentration. Our studies indicate that flushing the cell with ASW for at least 2 min is usually required to eliminate any adaptational phenomena, and it is this recovery time that limits the rate of data collection. Further studies are necessary to better characterize the adaptational effects on the system and thereby provide guidelines by which throughput can he maximized. In fact, such studies should lead to the e l i i n a tion or optimization of most of these
difficulties. Neuronal biosensors differ greatly from other biosensors. Most biosensors consist of two components: a molecular recognition element, which provides and a transducer, whch converts chemically eoded information received at the molecular recognition element into electrical or optical signals that can he easily measured. In contrast to most hiosensors, in which only the molecular recognition is biological, the antennular receptrode uses biological components as both molecular recognition elements and transducers. The antennular receptrode possesses characteristics that are most desirable in a biosensor: a short response time, high degrees of specificity and sensitivity, a broad dynamic response range, and the ability to respond to a wide variety of analytes. These attributes, combined with the fact that the sensing tip is of conveniently small dimensions (approximately 1 1x11119, make the antennular receptrode a promising new biosensor. Although this device cannot yet be used as a practical sensor, as a model system the antennular receptrode demonstrates the potential of chemoreceptor-based biosensors. If the difficulties associated with the system can be circumvented, the device may prove to be a valuable tool for analytical chemists in the future. References (1) Hallowell. S. F.: Rechnitz. G. A. Analvtl r d Lorrers.1987; 200(12), 1929-49. (2) Belli, S. L.;Rechnitz,G.A. Fredenrus 2. Anal. Chem. 1987,331,439-47. (3) 1961. . . Case.J.:Cwilliam.G.F.Biol.Bull. 127,42&46. (4) Laverack, M.S. Comp. Bioehem. Physiol. 1964.13,310-21.
ANALYTICAL CHEMISTRY, VOL. 61. NO. 8, APRIL 15. 1989
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REPOR7 Wu, P. Prog. Neurobiol. 1981,16,197-239. (21) Stone, T.Newoscience 1981,6, 52355. (22) Su, C. Annu. Rev. Pharmacol. Toxicol. 1983,23,397411. (23) Boyd, C. J . Theor. Biol. 1984,76,41& 17. (24) Lenhoff, H.; H e w , W. Annu. Reu. Phrmocol. Toxieol. 1977, I 17.24h58. (25) Carr, W.; Thompson, H. J. Cornp. (20) Phillip, J.;
Physiol. 1983,153,4733. (25) Gleesou, R.; Adsma, M.; Smith, A. Biol. Bull. 1987,172,14, (27) Wing, K. D.; Slawecki, R A,; Cmhu, G. R Science 1989,5i1,470-72. (28) Wing,K. D. Science 1989,241,46749. (29) Kennedy, D.; Bittner, G. Cell Tissue Res. 1974,148,97-110. Support of this research by the National Science Foundation isgratefullysctnowledged,
R. Michael Buch earned a bachelor’s degree in chemistry f r o m Franklin and Marshall College in 1984 and a m u ter’s degree in chemistryfrom the Uniuersity of Delaware in 1987.He is currently pursuing a Ph.D. in chemistry
Uniuersity of Delaware. His research on sensors has resulted in 285publications and has prouided research experience for more than 125 pre- and
at the Uniuersity of Delaware.
postdoctoral students.
Carry A. Rechnitz is Unidel Professor of Chemistry and Biotechnology at the
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