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Anal. Chem. 1999, 65,3202-3200
This Research Contribution is in Commemoration of the Life and Science of I . M. Kolthoff (1894-1993).
Biomagnetic Neurosensors D6nal Leech and Garry A. Rechnitz' Hawaii Biosensor Laboratory, Department of Chemistry, University of Hawaii, Honolulu, Hawaii 96822
In this report we demonstrate the first analytical application of biomagneticfield detectionat nerve fibers for biosensing purposes. A ferrite core toroid surrounding the nerve, coupled to a lownoise, low-input-impedanceamplifier, is used to inductively detect the compound action current (CAC) in crayfish giant axons upon stimulation of nerve firing. Detection of the local anesthetic lidocaine, which blocks neuronal conduction by binding in the ion channel of the voltage-gated sodium channel receptor, is achieved by monitoring the disappearanceof theCAC. The application of this novel detection principle to the screening of neurotoxic and neuromodulatory drugs and natural product extracts is proposed.
INTRODUCTION In this study, we report on the first analytical use of biomagnetic field detection in the design of neuronal biosensors. This technique permits noninvasive monitoring of ion currents in intact nerve fibers and their response to analytical binding events resulting from the introduction of channel-blocking analytes. This research is made possible by the availability of a new instrument that can detect the magnetic field surrounding a single axon upon firing of an action potential event14 without the need for elaborate shielding or liquid helium temperatures required for the superconducting quantum interference device (SQUID)magnetometer.4 The biomagnetic current probe6 employed consists of a low-noise, low-input-impedance, room-temperature amplifier coupled to a toroidal pickup coil surrounding the nerve that can inductively detect the action current in the nerve fiber. Our preliminary study demonstrates the applicability of the biomagnetic current detection system for the assay and detection of the local anesthetic (LA) and antiarrhythmic agent lidocaine. LAs can block the propagation of the action potential/current by binding in the ion channel of the voltagegated sodium channel receptor, thus blocking the influx of sodium ions responsible for neuronal conduction. Recently we have demonstrated a novel application of intact neuronal structures for the detection of conduction blockers and other neuromodulatory agents using the sodium channel (1) Wikswo, J.P., Jr.;Samson,P. C.;Giffard,R.P.IEEE Trans.Biomed. E w . BME-30, 215-221. (2) Wikswo, J. P., Jr.; Henry, W. P.; Samson, P. C. In Biomagnetism: Applications and Theory; Weinberg, H., Stroink, G., Katila, T., Eds.; Pergamon Press: New York, 1986; pp 83-87. (3) Gielen, F. L. H.; Roth, B. J.;Wikswo, J. P., Jr. IEEE Trans.Biomed. Eng. 1986, BME-33,910-920. (4) Wikewo, J. P., Jr.; Barach, J. P.; Freeman, J. A. Science 1980,208, 53-55. (5) Vanderbilt BiomagneticCurrent Probe isa trademark of Vanderbilt University. 0003-2700/93/0365-3262$04.00/0
receptors located on the axons of crayfish walking leg nerves for detection purposes.6.7 The procedure was demonstrated for the detection of quaternary ammonium local anesthetics as model compounds and consisted of the use of electrophysiological microelectrode techniques to monitor disappearance of the action potential event upon application of LA to the cell. Simultaneous measurement of the action potential using electrophysiological microelectrodes is used in the present report to confirm the biomagnetic detection of the action potential event. Detection of the biomagnetic current of the nerve fiber offers an alternative to the electrophysiological methods normally used for nerve-firing detection. Progress in the area of biomagnetism promises to lead to the noninvasive monitoring of brain activity and other neuronal processes to aid in the diagnosisand the treatment of neurologicaldisorders.a#Q The analytical application of this noninvasive technique may lead to longer lifetimes of the neuronal biosensors under investigation in our 697310-12 and otherla laboratories and is a promising new direction for the study of intact receptorbinding events.
EXPERIMENTAL SECTION Description and Operation of the Biomagnetic Current Probe. The Vanderbilt BiomagneticCurrent Probe6 measures the action currents associated with the propagation of the action potential in nerve fibers. This is achieved by sensing of the induced current in a toroidal coil surrounding the nerve in response to the magnetic field produced by the action currents flowing through the nerve. The entire sensing system coneista of three portions: the pickup coil, the amplifier, and the control electronics. A schematic of the instrument is depicted in Figure 1. The amplifier and control electronics are housed together in a Model LSP-3 biomagnetic current probe (purchased from Vanderbilt University). The toroidal probe consistsof a ferrite core of high permeability (type 40401, Magnetics,Butler, PA) and with dimensionsof 4.83mm outer diameter,2.29-mm inner diameter and 1.2%" width. This core is wound with copper wire (40gauge) and electrically insulated from the saline bath by a thin layer of epoxy coating. A second single winding of copper wire around the toroid is also (6) Leech, D.; Rechnitz, G. A. Anal. Chim. Acta 1993,274,25-35. (7) Leech, D.; Rechnitz, G. A. Anal. Lett., in presa. (8) Adoanceain Biomagnetism; Williamson, S. J., Hoke, M., Stroink, G., Kotani, M., Ede.; Plenum Press: New York, 1989. (9) Biomagnetism: An Interdisciplinary Approach; Williamson, S. J., Romani, G.-L., Kaufman, L., Mdena, I., Eds.; Plenum Press: New York, 1983. (10) (a) Belli, S. L.; Rechnitz, G. A. Anal. Lett. 1986,19,403-416. (b) Buch, R. M.; Rechnitz, G. A. Anal. Lett. 1989,22,268&2702. (c) Buch, R. M.; Barker, T. Q.; Rechnitz, G. A. Anal. Chim. Acta 1991,243,157166. (11) (a) Wijeeuriya, D.; Rechnitz, G. A. And. Chim. Acta 1992,256, 39-46. (b) Wijeeuriya, D.; Rechnitz, G. A. Anal. Chim. Acta 1992,264, 189-196. (12) (a) Buch, R. M.; Rechnitz, G. A. Anal. Chem. 1989, 61, 533A542A. (b) Leech, D.; Rechnitz, G. A. Ekctroanaiysis 1993,5,103-111. (13) (a) Skwn, R. S:; Kisaalita, W. 5.;Van Wie, B. J.; Fung, S. J.; Barnes, C. D. Baosen. Bzoelectron. 1990,5,491-510. (b) Skeen, R. S.;Van Wie,B. J.;Fung,S. J.;Barnes,C. D. Biosens. Bioelectron. 1992,7,91-101. 0 1993 American Chemlcal Society
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I Flguro 2. Schematicrepresentation of a cross section of a nerve flber showingthe electric current (thln lines)and the correspondingmagnetlc field (wide lines) associated wlth the propagating action potential.
Flgurr 1. Schematic diagram of the Vanderblk Biomagnetlc Current Probe instrument.
present and is used to apply calibration current pulses to the coil. The induced current flowing through the coil in response to both the calibration pulse and the magnetic field associated with the action current is detected by a low-noise, low-inputimpedance, room-temperature amplifier. The amplifier circuitry also contains a low-frequency-compensationcapability that can shift the high pass cutoff frequency of the toroid-amplifier system to a frequency between 2 Hz and the characteristic toroid frequency, which is typically around 100 Hz.14 The control electronics of the amplifier system can generate a trigger pulse (which can also be externally applied) to initiate measurement, a stimulus phase, and a calibration pulse. The action current detection can be clamped at an adjustable time after the trigger pulse to ensure that the measured trace is on the same position on the oscilloscope screen for each trace. The instrument also contains a switched integrator that can compute the transmembrane action potential from the measured action current by use of a core conductor appro~imation.'~ Action currents in physiological systems are carried by ions, which flow in response to both concentration and potential gradients.16 Inside the fiber the current flowsin an axial direction with the return paths extending into the extracellular space, as depicted schematically in Figure 2. The corresponding magnetic field produced by these currents is shown by the large arrow8 surrounding the nerve in Figure 2. There are two widely used models for the computation of the electric field produced in a single nerve fiber: the volume conductor model" and the core conductor model.15J8 The application of these models to biomagnetismcan be accomplished by incorporation of the Biot-Savart law to calculate the magnetic field. Barach et al.16 have shown how the core conductor model can be used to relate magnetic measurements of a single nerve axon to the transmembrane action potential, the membrane currenta, and the intracellular conductivity. The more rigorous ~~
(14) Vanderbilt Biomcagnetic Current Probe Model LSP-3; usem manual. Vanderbilt University,Nashville, TN. (16) Barach, J. P.:h t h , B. J.; Wikewo, J. P., Jr.ZEEE Tmns.Biomed. EM. 1986, BME-32,136-140. (16) Wikewo, J. P., Jr. In Advances in Biomognetiem; Williamson,S . J., Hoke, M., Stroink,G., Kotani, M., Me.; Plenum Press: New York, 1 9 8 9: BD 1-18. ----.rr (17) (a) Clark,J.;Plonsey,R. Biophys. J. 1968,8,842-864. (b) Plonaey, R. Proc. ZEEE 1977,65,601-611. (18) F t d , W. In Handbook of Physiology; Kandel, E., Ed.;American Physiological Society Betheeda, MD, 1977; Vol. 1, pp 74-76.
volume conductor model has been used to account for errors in the assumptions for the core conductor model.l9P The advantages of measuring biomagnetic currents in research and clinicalapplications are several.14 A magnetic measurement can quantitatively determine the intracellular action current, which cannot be done with electric methods. Magnetic and electric measurements, when combined, offer information on the intracellular conductivity of tissue. Finally, the opportunity to monitor nerve firing noninvasively reduces the risk of damage to the tissue, which is important both for clinical applications and for applicationof biomagnetic current detection to biosensing. Reagents. The saline solution used was of a modified van Harreveld (MVH)formula consisting of 205 mM NaC1,13.5 mM CaC12, 2.6 mM MgC12, 5.4 mM KC1, and 10 mM Tris-maleate buffered to pH 7.4. The local anesthetic lidocaine hydrochloride was obtained from Sigma Chemical Co., and standard solutions were prepared in the MVH saline daily or as required. A dye solution of 2,6-dichlorophenolindophenol (DCPIP, Aldrich) was used for tracer studies of the flow cell. Apparatus. A block diagram of the experimental apparatus is shown in Figure 3. The specially designed flow cell described previously' consisted of two interconnecting chambers separated by a 2-mm-wide slot raised 7 mm above the chamber bottom. The bottom of the cellwas lined with a Sylgard 184(Dow Coming) elastomer to allow pinning of the nerve in the cell. The cell was mounted on a Fisher Scientific Stereomaster I1 microscope, operated at low magnification in order to view both chambers simultaneously. Flow into and out of the cell to both chambers was controlled by a multichannel peristaltic pump (Technicon autoanalyzer), with the outlet tubing of a larger diameter than the inlet tubing to ensure a constant chamber volume. A threeway switching valve located upstream of the pump allowed switching between saline and test solutions. One chamber of the cell contained the stimulating electrodes (Ag/AgCl) connected via a stimulus isolator (Model A360D, World Precision instruments) to the stimulus portion of the Vanderbilt Biomagnetic CurrentProbe (ModelLSP-3, Vanderbilt University). The other chamber contained the toroidal pickup coil, the pickup glass suction microelectrode (Ag/AgCl), and ground and reference copper wire electrodes. The stimulus and pickup extracellularmicroelectrodes consistedof borosilicate glass capillarytubes with a 10-25~mfire-polishedtapered end, pulled using a Narishige PP-83 microelectrode puller. These electrodes were positioned in holders (E. W. Wright), which allowed suction to be applied to the back of the electrode. The microelectrodes and the toroidalcoil were placed in micromanipulators (Narishige) for positioning. Output from the detection electrodes was amplified with a Grass P-15 physiological preamplifier, while the toroidal signal was fed to the biomagnetic current probe amplifier. The detected waveforms were viewed with a Sony Tektronix 314 digital storage oscilloscopeand were captured and digitized at a sampling rate of 50 kHz using an assembled IBM(19) (a) Woosley, J. K.; Roth, B. J.; Wikewo, J. P., Jr. Math. Biosci. 1985,76,1-36. (b) Roth, B. J.; Wikswo, J. P., Jr. Math. Biosci. 1985,76, 37-57. (20) Roth, B. J.; Wikewo, J. P., Jr. Biophys. J. 1985,48, 93-109.
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PC 386 clone equipped with a DAS-16 data acquisition board and Streamersoftware(Keithley-Metrabyte). The stored ASCII fiies were imported into the KaleidaGraphdata analysis/graphica applicationon a Macintosh SE/30 computer for further analysis. Procedures. The freshwater crayfish Procambarus c l a ~ k i i were captured at the nearby Manoa stream and stored in holding tanks. Prior to dissection the animals were cooled on ice for immobilization purposes. The immobilized crayfishwere pinned ventral side up in oxygenated chilled MVH saliie and all appendages removed. The cuticlecovering the abdominalportion of the animal was removed, and all the roots to the six abdominal ganglia were severed. The nerve cord was then cut above the sixth abdominalganglion and at the tailfan, removed, and pinned ventral side up in the Sylgard l i e d flow through cell. The nerve cord was stretched across the slot separating the two chambers in the cell such that the thoracic end of the cord was pinned in the stimulus chamber while the tailfan end was pinned in the detecting chamber. This setup helped alleviate the stimulus artifact detected from the stimulus electrodes. The tailfan end of the nerve cord was threaded through the toroidal pickup coil up to the fourth abdominal ganglion, and oxygenatedsaline flow through the cell was initiated. Extracellularmonopolar stimulus and detection of action potential firing in the giant axom was achieved by placing the glass suction microelectrodes close to the nerve fiber and applying gentle suction to the back of the electrode. Stimulation of action potential firing was achieved by gradually increasingthe square wave current pulse, of 0.5-me duration (0.5-Hz stimulus frequency) until threshold level was reached. The stimulus electrode was applied to the nerve approximately 1cm away from the toroidal detection coil in the stimulus chamber. Electrical detection of the action potential event was monitored with a suction microelectrode placed approximately 0.5 cm away from the toroidal coil on the tailfan side in the detection chamber. Test solutionsof the local anesthetic lidocaine were introduced into the cell by switching the valve to a flow of the standard solution. The effect of various concentrations of the LA on the detected action potential at the microelectrode and the biomagnetic current at the toroid was monitored, with the time taken to achieve complete blockage of neuronal conduction and the duration of the block noted for each concentration. Tracer studies of the flow cell were performed by switching to a flow of a 1 X lo-' M solution of the dye DCPIP in deionized
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Figure 4. Tracer study of the tlme course of the injection and washout characteristics of the flow-throughcell. The response Isthe normalized absorbance at 600-nm wavelength following Switching at t = 0 to a solution of 1 X lo4 M dye (DCPIP). The response observed in the stimulus chamber is lndlcated by 0 while that In the recording chamber is indlcated by +. The flow was c h a n g d back to deionlzed water after 180 8, as indlcated by the arrow In the figure.
water and taking periodic samples of 50 p L from both chambers of the cell. The time course in the change in dye concentration in the cell was thus established by monitoring the increase in absorbance of the dye samples of 6Wnm wavelength using a Milton Roy Spectronics 1201 spectrophotometer.
RESULTS AND DISCUSSION Tracer Studies. Tracer studies of the flow cell were performed in order to investigate the mixing process in the cell and to determine the time required to reach the equilibrium concentration in the cell and the washout time of the cell. The response profiie upon switching to a flow of 1 X l(r M DCPIP is shown in Figure 4. Sampling of both chambers was from a point where the ends of the nerve cord would be pinned. The lag time between switching to the dye solution and the increase in response can be attributed to the void volume of the connecting tubing between the switching valve and the cell. From Figure 4, the time required to reach the equilibrium concentration of the injected solution is estimated to be 160 8. A washout time of a minimum of 180s is shown to be required to clear the cell of the injected dye. The protocol adopted for the detection of lidocaine blockage of the nerve firing thus entailed application of a flow of lidocaine solution into the cell for a period of 5 min, resulting in a the disappearance of the compound action potential (CAP) and compound action current (CAC), followed by changing the flow back to to MVH saline and monitoring the time taken for the reappearance of the CAP/CAC. Figure 4 also illustrates the similarity between the time profiles observed for both cell chambers, with little variation observed between the stimulus and detection chambers. Biomagnetic Detection of Nerve Firing. Firing of the nerve may be induced by a depolarizing stimulus. This may be a chemical stimulus6 or an electrical stimulus.7 Stimulus of nerve fiing in the present case is induced by the application of an electrical current of 0.5 ms duration using a monopolar cathodic Ag/AgCl glass suction microelectrode. The detected waveforms are compound waveforms consisting of a sum of the firing in all of the nerves stimulated by the electrodes and are referred to as the compound action potential and the compound action current. The signal-averaged CAC (n = 20) detected at the toroidal current probe in response to the stimulus is shown in Figure 6. The stimulus artifact is visible at approximately 2 ms after triggering of detection. The biomagnetic current detected in the giant mons is preceded by a l-ms calibration pulse of 0.2 PA, which is utilized to calibrate both the low-frequency compensation of the am-
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plifier and the detected action currents. Figure 5 illustrates the signal-to-noiseratio achievablewith biomagnetic recording of the action currents and with a limited amount of signal averaging. In this experiment,the stimulatingelectrodes were positioned 0.9 cm from the toroid. Thus, a propagation speed for the action potential of approximately 22 ms-1 can be calculated. From experiments conducted on four different preparations, an average propagation speed of 18 ms-1 was calculated, which compares well to previously published results from crayfish giant axons.20 Detection of Lidocaine Using the Biomagnetic Current Probe. For successful analytical application of biomagnetic current measurements, it is imperative that single recordings of the CAC can be viewed with good signal-tonoise ratios and without the need for signal averaging. This is possible with the large-diametergiant axons and using the dimensions of the toroid in this study, as is illustrated in Figure 6, which depicts a single recording of the biomagnetic current detected at the toroid upon stimulus with the glass microelectrode. Blockage of the CAC is also illustrated in Figure 6 upon application of a solution of 50 mM lidocaine to the cell for 5 min. It is obvious from this trace that no
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action current can be detected. The disappearance of the action current correlates with the disappearance of the electrically detected CAP, illustrated in Figure 7 before and after application of 50 mM lidocaine to the cell. The time taken to achieve complete blockage of the CAC upon switching to a flow of a standard solution of lidocaine can be used to construct dose-response curves for this drug. A dose-response curve for liodcaine plotted in this way is depicted in Figure 8. The data were normalized by plotting the time taken to achieve complete conduction block at each concentration, expressed as a percentage of the time taken upon application of the minimum concentration of lidocaine required to achieve complete conduction block (C,). Alternative dose-response curves for lidocaine can be generated by plotting the duration of the conduction block vs the concentrationof lidocaine applied to the cell. A dose-response curve for lidocaine plotted in this way is illustrated in Figure 9. In both the dose-responsecurves the error bars represent
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the standard deviation from the mean of four experiments conductedwith different nerve preparations. The large errors may be attributed to the fact that the concentration required for complete conduction block of the CAP in nerve fibers with local anesthetics has been shown to be dependent on the diameter of the nerve fibers,21with the large diameter fibers requiring higher concentrations to achieve block. Thus the variability in the dose-responsecurves for lidocainemay reflect the variability of the nerve fiber diameter between preparations. The experimentallyuseful range of the dose-response curvesforlidocaine in this case is from 5mM to a concentration of approximately 50 mM. Above this concentration the time taken for recovery from block is the limiting factor. The useful concentration range obtained for lidocaine at the giant axons of the crayfish is 1 order of magnitude greater than that found in a previous study using crayfish walking leg nerves and electrical detection of the CAP.' The average diameter of the peripheral walking leg nerves is approximately (21)Staiman, A.; Seeman, P.Can. J. Pharmacol. 1974,52, 535-550.
10 pm while that of the giant axons is in the order of 100-200 pm, thus explaining the positive shift in the dose-response curves. These results demonstrate the application of a novel detection principle to neuronal biosensing. The detection of the biomagnetic field surrounding the nerve in response to action potential propagation along the nerve fibers has been utilized for the assay and detection of the local anesthetic lidocaine. This assay procedurehas the potential to be applied to the determination of other conduction-blocking agents and of neuromodulatory drugs and toxins. It is postulated that this assay procedure would be useful for the screening of the neurological potency of various agents which are capable of blocking or modulating neuronal conduction in nerve fibers. These include the local anesthetics, narcotics (including cocaine, originally introduced as a local anesthetic), and neurotoxins such as tetrodotoxin and saxitoxin, as well as important compounds such as alcohols, barbiturates, antiepileptics,and other drugs. The application of this procedure as a nonisotopicassay for the preliminary screening of natural product extracts of plants and animals for neurotoxic or neuromodulatory activity of novel neurotoxins and drugs is also envisaged. Owing to the noninvasive nature of the biomagnetic detection technique, it is expected that the useful lifetime of neuronal biosensors might be extended over previous designs.
ACKNOWLEDGMENT The authors gratefully acknowledge National Science Foundation Grant CHE-92 16304. Scientific Parentage of the Author. G. A. Rechnitz: Ph.D. under H. A. Laitinen, Ph.D. under I. M. Kolthoff. RECEIVEDfor review June 1, 1993. Accepted August 26, 1993.'
* Abstract published in Advance ACS Abstracts, October 1, 1993.
