Anal. Chem. 1994,66, 3193-3197
Biomagnetic Neurosensors. 2. Magnetically Stimulated Sensors David R. Coon, Christopher W. Babb, and Gary A. Rechnttz' Hawaii Biosensor Laboratory, Department of Chemistry, University of Hawaii, Honolulu, Hawaii 96822
We report the generationof action potentials in crayfiihneurons by magnetic pulses for analytical purposes. A copper wire toroid, containing a ferrite core, was placed around the nerve bundle, and square wave current pulses were sent through the wire to generate a magnetic field. The magneticfield generated an action potential in the neuron that was detected downlield by a pickup microelectrode. The biosensor was used to detect local anestheticsby monitoringthe time necessary for complete blockage of the action potential. Techniques for improving the efficiency and lifetimes of neural biosensors are discussed. One approach in the development of new biosensors is the use of living biological tissues in combination with standard analytical instrumentation to quantify an analyte. It is hoped that the tissue, with its intact biochemical processes, will produce a measurable signal in response to the analyte of interest. Measurement of this signal must be as innocuous to the tissue as possible so that the sensor response is not affected or the viable lifetime shortened. Biomagnetic monitoring is of great interest due to its numerous applications and noninvasive nature.' The medical application of biomagnetically monitoring nerves intraoperatively holds much potential. Biomagnetic measurements would be able to check nerve continuity at an injury site more easily and with less damage to the nerves than electrical technique^.^^^ These measurements have also been used to study functioning of the brain, heart, lungs, and, even, chick embryonic deve1opment.l The superconductingquantum interference device (SQUID) magnetometer is used by many researchers for biomagnetic This instrument requires shielding and very low temperatures for operation. We have recently reported on the biomagnetic detection of action currents from electrically stimulated crustacean nerve tissue using the Vanderbilt Biomagnetic Current Probe, which operates at room temperature with a high degree of sensitivity.6 Neural biosensors have been studied by our group and others for the last several years.' Neural tissue is extremely (1) Aduances in Eiomagnetism; Williamson, S. J., Hoke, M.,Stroink, G., Kotani, M.,Eds.; The Seventh International Conference on Biomagnetism; Plenum Press: New York, 1989; Vol. 1. (2) Wikswo, J.P., Jr.;Abraham,G.S.;Hentz,V.R.InEiomagnctism: Applications & Theory; Weinbcrg, H.,Stroink, G.,Katila, T., Us.; The Fifth World Conference on Biomagnetism; Pergamon Pres: Vancouver, Canada, 1984; Vol. 1, pp 88-92. (3) Wikswo, J. P., Jr.; Henry, W. P., et al. In Aduances in Eiomagnetism; Williamson, S. J., Hoke, M.,Stroink, G.,Kotani, M.,Eds.; The Seventh International Conference on Biomagnetism; Plenum Press: New York, 1989; pp 137-140. (4) SuperconductingDevicesandTheir Applications;Koch, H., Lubbig, H., Eds.; The 4th International Conference SQUID '91; Springer-Verlag: Berlin, Germany, 1991; Vol. 64. ( 5 ) Tavrin, Y.; Zhang, Y.; et al. Appl. Phys. Lett. 1993, 62, 1824-1826. (6) Leech, D.;Rechnitz, G. A. Anal. Chem. 1993.65, 3262-3266.
0003-2700/94/0366-3193$04.50/0 0 1994 American Chemical Society
sensitive to a wide assortment of different neuromodulatory chemicals. Our approach has been to use the intact nerve tissue of crustaceans in the fabrication of biosensors. This methodology leaves the chemoreceptors on the nerves in their optimized state. The sensing ability of these biosensors is well d o c ~ m e n t e d The . ~ ~ response ~ of nerve tissue to different analytes, in the form of a compound action potential or action current, can be measured electrically or biomagnetically. We report here on the use of magnetic pulses, generated by a toroid with a ferrite core, to stimulate, rather than detect, action currents in crayfish ganglia. This noninvasive method of action current stimulation avoids direct electrical stimulation and may be less damaging to the nerve tissue. We have demonstrated an application of this technique with the monitoring of local anesthetics. Action potential height as generated by the magnetic pulse varies with the concentration of the anesthetic present in solution. Time for complete conduction block of the nerve tissue signal is used as a parameter to establish dosage-response curves to local anesthetics. Magnetic hobe Background. Current has a magnetic component that is in the perpendicular direction to the axis of conduction and can be qualitatively envisioned by using the right-hand rule. A magnetic field from a current-carrying wire decays as l/r, where r is the distance from the carrier. The Biot-Savart law can be used to solve for the dependence and is given by eq 1. B(r) = p i / 2 m
(1)
Equation 1 shows that the magnetic field (B) (in tesla) is proportional to the current (i) being passed through the wire. The permeability of free space is given by p, and r is the radial distance from the wire. A magnetic field produced by the action currents in a nerve fiber also falls off as 1 /r at small distances from the nerve.1° This magnetic field is very weak but has been measured and compared to theoretical predictions.' l I l 2 The Vanderbilt current probe is capable of measuring very small biomagnetic currents at room temperature.'3J4 It (7) Kurdikar, D. L.; Skecn, R. S.; et al. Whole Cell Eiosensors: A Brief Uvewiew and Presentation of A Now1 Neuron-Based Approach; Frontiers in Bioprocasing 11; American Chemical Society: Boulder, CO, 1990; pp 126-137. (8) Buch, R. M.;Rechnitz. G.A. Anal. Chem. 1989.61, 533-542. (9) Leech, D.; Rcchnita, G. A. Electroanalysis 1993, 5, 103-1 11. (10) Wilcswo, J. P., Jr. J . Appl. Phys. 1981, 52, 2554-2559. (1 1) Barach, J. P.; Roth, B. J.; Wikswo, J. P., Jr. IEEE Trans. Eiomed. Eng. 1985, 32, 136-140. (12) Roth. B. J.; Wikswo, J. P., Jr. Eiophys. J . 1985, 48, 93-109. (13) Gielen, F.;Roth, B. J.; Wikswo, J. P., Jr. IEEE Trans. Eiomed. Eng. 1986, 33,910-921. (14) van Egeraat, J. M.;Wikswo, J. P., Jr. In Eiomagnetism: Clinical Aspects; Hoke, M., et al., Eds.;The Eighth International Conference on Biomagnetism; Elsevier Science Publishers: New York, 1992; pp 895499.
Analytical Chemism, Voi. 66, No. 19, October 1, 1994 3103
I-'
A
4 Flgure 1. Solenoid depicting the magnetic lines of force as a current flows through the wire.
consists of three basic parts: control electronics, amplifier circuitry, and interchangeable toroidal current probes. The diameter of the toroids is purposefully small so that the biomagnetic signal is maximized.15 A solenoid is a wire that is twisted into a coil. Figure 1 is a drawing of a solenoid and the magnetic fields generated as current flows through the wire. The magnetic field of the solenoid is greatest in the lumen of the wire and is easily calculable using Ampere's law. If the solenoid is bent into a toroid, the resulting magnetic field is given by
In eq 2, iw is equal to the current flowing in the solenoid and n is equal to the number of wire turns per unit length (Nl27rr) of the toroid. The permeability of the substance the wire encloses is given by p. Many materials, such as ferrite, are able to concentratethe magneticlines of flux, greatly increasing the magnetic field. In previous work, we used biomagnetic detection of compound action currents in crayfish nerve tissue to develop a neural biosensor. To detect biomagnetic signals in nerve tissue using the Vanderbilt probe, we threaded the ganglion through the toroid and then electrically stimulated the tissue.6 When an action current passed down the ganglion, the biomagnetic field associated with it induced a current in the toroid winding that was detected by the supporting instrumentation. In the present work, we pass a current through the copper wire to induce a magnetic field in the toroid. The strength of the magnetic field is increased by increasing the current through the copper wire until an action current is generated in the nerve tissue. This typically requires 2-3 mA of current passing through the wire and, neglecting imperfections,should generate a magnetic field close to 0.25 T inside the toroid. The compound action potential produced in the nerve tissue is detected by a pickup electrode placed no more than 2-3 mm downfield from the toroid. EXPERIMENTAL SECTION Apparatus. The experimental setup is shown in Figure 2. The Vanderbilt Biomagnetic Current Probe has been described previo~sly.~JThe electronicsof the biomagneticprobe sends two signals through the toroid. The first is a stimulus pulse which, when connected to a pickup electrode, is used to electrically stimulate the nerve tissue at a minimum frequency (15) VanderbiltBiomagneticCurrent Probe Model U P - 3 UsersManual;Vanderbilt
University: Nashville, TN, 1990.
3194
AnalvticaIChemistry, Vol. 66, No. 19, October 7, 7994
B
J
u
I
\
U
A:, U
'
Flgure 2. Experimental arrangement for biomagnetic sensors. The flow cell at the base of the dissecting microscope is connected to the flow lines. The stimulatingtoroid and the pickup electrodeare posrtioned above the cell, clasped by micromanipulators: (A) Vanderbilt Biomagnetic Current Probe; (B) stimulus isolator: (C) physiological preamplifier;(D) oscilloscope; (E) IBM-PC clone;(F) analyte connected to three-way switching valve.
of 0.5 Hz. The second pulse is of low current and is used for calibration of the toroid during biomagnetic detection measurements. The toroid consists of a low-reluctance ferrite core (W40200-TC; Magnetics, Butler, PA). The core has an outer diameter of 2.54 mm, an inner diameter of 1.27 mm, and a width of 1.27 mm. The core is wound with 44-gauge ( 5 1 pm) copper wire and insulated from the saline bath by electrovarnish and an epoxy coating decreasing the inner diameter of the encapsulated toroid to 0.6 mm. The resistance between the toroid and the saline bath is 20 MQ or greater.15 The flow cell used in the experiments is made in-house out of Plexiglas. The internal chamber dimensions are 3.17 cm X 1.27 cm X 1.91 cm high. The diameter of the inlet is 0.086 cm, and the outlet is 0.476 cm. The bottom 0.635 cm of the cell is lined with Sylgard 184 SiliconeElastomer (Dow Corning Co., Midland, MI). This gives the cell a surface soft enough to pin the nerve down while not allowing saline to leak through. The volume capacity of the cell is 5.12 mL, but the actual volume of solution flowing through the cell during a typical experiment was maintained between 0.8 and 1.5 mL. This volume was enough to cover the nerve and the top of the toroid. Three platinum wire leads were secured inside the cell with nylon screws. The flow cell with the connected nervous tissue is depicted in more detail in Figure 3. Two multichannel peristaltic pumps (Rainin Instrument Co.,
\\
.-m5
100
' ' ' '
I
CI
.'' ..
.:.'.'.I.'"*'
' ' '
' ' '
.'
'
'
'
'
-I
1
"I t 1 , .-X
Figwe 3. Side view of inside of the Row cell. The arrangement of
the toroid and the electrode as they contact the ganglia is shown.
E
20
8
0
L,,
I , ,
0
100
'.
I
,
1
,
200
I
I
1
1
,
300
I
1
1
1
:*,:'-A
400
500
time (s) Figure 4. Concentratbn-time profile for the anesthetic dibucaine In
Woburn, MA) were used to control inflow and outflow, respectively,to ensure a constant volume in the flow chamber. A three-way switching valve was attached to the inflow line in order to introduce analyte into the flow stream. The detection electrode consisted of a chloridized Ag wire enclosed in a glass capillary tube. The tube end was pulled to a final orifice diameter of approximately 10&200 pm using a Narishige PP-83 glass microelectrode puller and then fire polished to remove any rough edges. The capillary was held over the Ag/AgCl wire by an E. W. Wright electrode holder, allowinggentlesuction to be applied to the backoftheelectrode holding the neural membrane firmly against the capillary. Electrode positioning was accomplished using a Narishige micromanipulator. The stimulus signal was fed into a constant current stimulus isolator (WPI, Sarasota, FL) in order to gradually increase the current to the toroid. The signal from the detection electrodewas fed into a physiological preamplifier with a cutoff filter (Grass Instruments, Quincy, MA) and then into a storage oscilloscope (Tektronix, Beaverton, OR) for viewing. The oscilloscope was connected to an IBM-PC 386 clone computer with a DAS-16 data acquisition board and Streamer software (Keithley-Metrabyte, Taunton, MA). Data were captured at a sampling rate of 50 kHz on this computer and then transferred into KaleidaGraph software on a Macintosh SEI30 for analysis. The dibucaineabsorbance was monitored at 320 nm using a Milton Roy Spectronic 1201 spectrophotometer. Procedures. Crayfish were easily captured in stream-fed taro fields close to the university and stored in freshwater holding tanks until needed. Prior to use, a crayfish was placed in a freezer for approximately 60 s to anesthetize it. Initially, all appendages were removed to facilitate dissection. The ventral cuticle covering the abdominal section was then cut off, exposingthe largest lateral nerve that extends to the tailfan. After cutting its connections to other abdominal ganglia, the nerve was severed at the tailfan and againjust below the thorax. The excised ganglion was carefully placed inside the flow cell that contained oxygenated saline. The flow cell was positioned under the stereomicroscope, and the flow lines were attached to allow modified Van Harreveld's solution to flow over the nerve. The nerve bundle was pinned to the bottom of the cell so that the tailfan end of the ganglion was downfield of the stimulating toroid. The nerve tissue was threaded through the toroid and the pins securing each end of the tissue to the cell were positioned to ensure an unadulterated section of nerve tissue between the toroid and the detection electrode for unobstructed conduction of the compound action current. The
the flow cell. fhe concentratlon of the drug was measured spectrophotometricallyby removing 10-& aliquots from the cell over the course of the experlment. The dibucaine flow (4.7 mL/min) was switched on at time, zero, and the arrow lndlcates when the flow of dibucaine was switched off.
recording electrode was then attached 2-3" downfield from the toroid. The toroid was connected to the stimulus output of the biomagnetic probe and the equipment turned on. Current through the toroid was gradually increased by use of the stimulus isolator and held constant when a stable, singleunit neural response appeared on the oscilloscope. Analytes could then be introduced into the carrier stream through the three-way switching valve. The stimulator was turned off between runs to prevent overstimulationof the nervous tissue. The average cell temperature was 19 OC as determined by periodic measurements. Reagents. The modified van Harreveld (MVH) solution (approximately 17.17 mS/cm) consisted of 205 mM NaCl, 5.4 mM KCl, 13.5 mM CaCb2.6 mM MgC12, 10 mM Tris, and maleic acid buffered to pH 7.5. Pure oxygen was bubbled through the MVH solution during the course of all experiments, and the solution was refrigerated overnight. Lidocaine, tetracaine, and dibucaine were purchased from Sigma Chemical Co., and all anesthetic solutions were prepared on the day of use with oxygenated MVH solution. The highpurity silver and platinum wires used for electrode fabrication were purchased from Aldrich.
RESULTS AND DISCUSSION Flow Cell Characteristics. Figure 4 shows how the concentration of a drug changes with time in the flow cell. A 10 mM solution of the anesthetic dibucaine (A = 320 nm, e = 8200) was introduced into the carrier stream for a period of 5 min. At specific intervals, 20-pL aliquots of buffer were taken from inside the flow cell where the nerve tissue would normally be located. Drug concentration in the cell was monitored spectrophotometrically. The equilibrium concentration of the drug lasts for approximately 4 min and is reached 2 min after switching on the anesthetic flow. This results from the dead volume in the influx line between the sample loop and the cell inlet. Dibucaine concentrations in the cell do not begin to change for close to 60 s after the flow of the anesthetic is terminated for the same reason. The time to block as reported in the figuresbelow incorporates this interval. Biomagnetic Stimulationof Nerve Tissue. Action currents in nerve tissue have been induced in different ways. In our work, chemicalI6 and electrical" means have been used. In AnalyticalChemlsby, Vol. 66, No. 19, October 1, 1994
3195
700
i
600
'
-100
0
'
1 " " J
' " l ' " ' r
1 "
1
2
3
4
5
time (ms)
Figure 5. Blomagnetlcally stimulated trace of the action potential of a crustacean neuron. The actlon potential occurs 1 ms downflekl from the stimulus spike. The current through the torold was 2.31 mA.
the following study, action currents in nerves were generated magnetically and then electrically detected. Figure 5 is a typical action potential response of the tissue using this method I -200 of stimulation. The action potential occurs 1 ms downfield 0 0.2 0.4 0.6 0.8 1.0 from the stimulus spike. The conduction velocity down the time (ms) nervous tissue is calculated to be 2 m/s. One action potential Flgure 8. Effect of a local anesthetic on the action potential of the spike is present in Figure 5 because the magnetic field was neurons: (A) magnetlcally stimulated action potential In the buffer kept as low as possible. An increase in the magnetic field can solution; (B)3 mln after the Introduction of 0.075 M lldocalne Into the easily generate multiple action potential events in the ganglion, flow stream; (C) signal completely blocked after 3 mln and 11 s. The current through the toroid was 2.38 mA. which is also the case when electrical stimulation is used. Single-unit responses are best suited for analytical purposes 1250 ' ' ' ' since they are the most easily monitored. 1000 : Detection of Local Anesthetics. Local anesthetics inhibit neural conduction by binding to the sodium channels along 2 750 the axon of a nerve.'* As the ion channels become blocked by the drug, the nerve can no longer pass current down its membrane and the action potential diminishes. Figure 6 is 250 0 0 a graph of lidocaine effect on the action potential in crayfish nerve tissue. Trace A is the action potential as seen before 0 anesthetic is added to the cell. As the drug binds to the sodium 0 0.02 0.04 0.06 0.08 0.1 channels, the waveform decreases in strength and conduction Lidocaine Concentration (M) velocity down the membrane begins to decrease. Trace B in Flgure 7. Time for complete action potential block (0)and the tlme Figure 6 shows the action potential several seconds before it needed for recovery (0)versus the concentratlon of lidocaine used. was completely blocked. The trace is slightly shifted to the The current through the torold was 3.5 mA. The average recovery interval between samples was 13 mln. right of the previous trace and the amplitude is diminished. This behavior is an indicator that conduction blockis imminent. Trace C, taken 4 s later, shows the complete absence of any method to use for two reasons. First, this parameter is easily action potential. measured, requiring the least amount of equipment or data There are many parameters that can be correlated with treatment. Second, it allows the stimulation to be discontinued drug concentration: the time for complete conduction block, for a 10-20-min period after the conduction block has been the time needed for recovery, the width at half-height of the achieved. We find that this recovery period greatly increases action current, or the frequency of the action p o t e n t i a l ~ . ~ J ~ , ~the ~ lifetime of the nerve tissue. Figure 7 is a graph of the time needed for complete action Figure 8 is a more detailed study of dose-response time for potential block and time needed for recovery of the action local anesthetics. Varying concentrations of tetracaine were potential plotted against varying concentrations of lidocaine. introduced into the flow stream at a rate of 4.7 mL/min. The time needed for block becomes extended as the anesthetic Changes in tetracaine concentration between solutions were concentration decreases. During this period, the nerve tissue kept small in order to demonstrate the ability of the sensor must be constantly stimulated in order to produce action to detect small differences in concentration. The shape of the potentials. Continued long-term stimulation has deleterious curve is a typical result when local anesthetics are tested. effects on the tissue. We find that the time for complete Repeatability of the sensor response was examined by block of a single-unit action potential is the most convenient subjecting a ganglion to repeated injections of 7.5 mM tetracaine. After eight injections and recovery periods, the (16) Leech, D.; Rechnitz, G. A. Anal. Chim. Acra 1993, 274, 25-35. average time for complete conduction block was 118.9 f 7.5 (17) Leech, D.; Rcchnitz, G. A. Anal. Leu. 1993, 26, 1259-1279. (18) Lechat, P. Local Anesthetics; Pergamon Press: New York, 1971. s. This indicates that the results from each individual neural (19) Skccn, R. S.;Van Wie, B. J., et al. Biosenr. Bioelecrron. 1992, 7, 91-101. preparation are very repeatable. However, there is wide (20) Wijesuriya, D.; Rechnitz, G . A. Anal. Chim. Aero 1992, 264, 189-196.
'
-
8
A
1.
3196
AnalyticalChemlstry, Vol. 66,No. 19, October 1, 1994
I
I
I
I
I
I
I
'
'
- 1 ' t cn
Y
loo0
0
8oo 600
.
i
o""'~"""""'"'""'' 0
0.005
0.01
0.015
0.02 0.025
Tetracaine Concentratlon (M)
Flgure 8. Typlcai dose-response curve for the drug tetracaine. The time for completed conduction block Is used to calibrate the sensor against the concentratlonof the drug. The flow rate was 4.7 mL/min, and the currentthrough the torokl was 2.65 mA. The average recovery period between samples was 20 mln.
variability between individual crayfish in response to a particular drug. This is due, in part, to differing diameters of the ganglia, with larger preparations requiring higher analyte dosages for complete conduction block. Different neural preparations also require very different magnetic fields in order to stimulate firing. As the stimulation strength increases, the amount of drug needed to block compound action potentials also increases. This is presumably due to stimulation of nerve fibers buried deeper within the ganglion that are harder for thedrug to reach. Due to this effect, all experiments were run as close to the firing threshold as possible in an attempt to standardize results from different neural sensors.
However, variability from sensor to sensor was still too great for results from different sensors to be combined. At the present time, each neurosensor has to be individually calibrated, but some standardization methods are currently under investigation. One possibility is the accurate measurement of ganglia dimensions and their relationship tostimulus response. CONCLUSIONS These experiments demonstrate the stimulation of action currents in crayfish neurons by magnetic pulses. This technique is preferable to electrical stimulation since it is noninvasive to the ganglion. Such noninvasive approaches may be useful in lengthening the viable lifetime of neural biosensors. We foresee an even greater lifetime for sensors by further decreasing the stimulus frequency below 0.5 Hz and combining the techniques of biomagnetic stimulation and detection in a single experiment. Such sensors may prove useful in screening samples which might contain agents that affect ion channels in neural membranes. The method is capable of assessing the potency of many neuromodulatory chemicals such as narcotics and neurotoxins. ACKNOWLEDGMENT Support from National Science Foundation Grant CHE92 16304is gratefully acknowledged. The authors also thank David Kempton for the efficient machining of the flow cell. Recelved for review March 15, 1994. Accepted June 21, 1994.' *Abstract published in Advance ACS Abstracts, August 1, 1994.
Ana!yticalChemistry, Vol. 66,No. 19, October 1, 1994
3197
