Anal. Chem. 1996, 68, 1671-1675
Biomagnetic Neurosensors. 4. Design and Optimization for Analytical Use David R. Coon, Christopher W. Babb, and Garry A. Rechnitz*
Hawaii Biosensor Laboratory, Department of Chemistry, University of Hawaii, Honolulu, Hawaii 96822
Biomagnetic neurosensors based on magnetic stimulation and magnetic detection of neural events depend critically upon the effective matching of the magnetic transducers and the neural tissue employed. Although the properties of wire-wrapped ferrite core transducers can be predicted from electromagnetic fundamentals, meaningful analytical measurements using real nerves as molecular recognition elements require additional calibration and optimization steps in order to achieve good system response and lifetimes. This note provides some design guidelines and experimental test procedures to enable potential users to employ biomagnetic neurosensors in other laboratories. In a previous report from this laboratory, we described a new technique useful for analytical biosensing.1 This technique is based on a room temperature biomagnetic transducing system2 able to detect action currents in nerve tissue placed through the lumen of a wire-wrapped ferrite core. Such instrumentation had been successfully applied to study action currents during surgical procedures3,4 and toward the elucidation of various problems in neurology.5,6 We have also used magnetic stimulation to elicit action potentials in excised crustacean nerve tissue.7 The combination of magnetic stimulation and biomagnetic detection composes a noninvasive transducer system, in that there is no physical contact with the excised nerve.8 Drugs, venoms, and other ion-channel modulatory analytes can be quantified by the change in the frequency and intensity of the action current spikes in the nerve tissue. Magnetic neural stimulation was achieved by sending current pulses through the windings of a toroid encircling the nerve tissue, creating a variable magnetic field. A changing magnetic field, cutting through a closed conducting loop, is able to produce an electromotive force in that loop. In much the same way, the magnetic field lines resulting from the action current passing through the nerve tissue induce a current in a second (detection) toroid. The small induced current, on the order of several nanoamperes, is amplified to an observable signal. The stimulus signal and biomagnetic recording and amplification are controlled by the Vanderbilt Biomagnetic Probe, an instrument purchased from Vanderbilt University. Unit characteristics and capabilities are described elsewhere.2,9 Ferrite core (1) Leech, D.; Rechnitz, G. A. Electroanalysis 1993, 5, 103-111. (2) van Egeraat, J. M.; Wikswo, J. In Biomagnetism: Clinical Aspects; Hoke, M., et al., Eds.; Proceedings of the 8th International Conference on Biomagnetism; Elsevier Science: Germany, 1991; pp 895-899. (3) Gielen, F.; Friedman, R.; Wikswo, J. J. Gen. Physiol. 1991, 98, 1043-1061. (4) Wikswo, J.; van Egeraat, J. M. J. Clin. Neurophysiol. 1991, 8 (2), 170-188. (5) Reference 2, pp 385-388. (6) van Egeraat; Stassaski, R.; et al. Biophys. J. 1993, 64, 1299-1305. (7) Coon, D.; Babb, C.; Rechnitz, G. Anal. Chem. 1994, 66, 3193-3197. (8) Babb, C.; Coon, D.; Rechnitz, G. Anal. Chem. 1995, 67, 763-769. 0003-2700/96/0368-1671$12.00/0
© 1996 American Chemical Society
toroids, which attach to the output (for magnetic stimulation) and input (for biomagnetic detection) of the main unit, were constructed in our laboratory using a procedure slightly modified from that outlined in the probe manual.10 Nevertheless, the response characteristics of the toroids themselves have been previously explicated. The low-reluctance ferrite cores provide excellent frequency response characteristics when wrapped with 60-70 turns of small-diameter insulated copper wire. The core wrapping and annealing process can be completed in less than a week with a very high success rate. The toroids designed for stimulation are able to elicit current flow in copper wires having a load comparable to that of nerve tissue with high repeatability. The strength and intensity of the action potentials depend on many factors. Of prime importance is the strength of the stimulating signal that is sent to the tissue. If this stimulus is too high, it overtaxes the nerve and causes fatigue, from which the tissue is slow to recover. Such stimuli cause a rapid disappearance of all resultant action potentials and death of the excised tissue. Moreover, maintenance of the flow rate, pH, and buffer composition at optimal levels ensures maximal nerve lifetimes. While the nerves do remain viable over a pH range, use of an optimum pH permits the user to obtain the largest possible number of sample points. The temperature of the cell should be kept below room temperature to further ensure against premature tissue death. Trauma to the nerve tissue itself, resulting from the excision from the crustacean host and placement into the transducer system, must be kept to a minimum. Finally, the health and vitality of the crustacean host has to be sound at the time of dissection in order to provide a good signal response. In this note, we examine the requirements and optimization of various aspects of analysis using the fully magnetic system for stimulating and deteecting currents in nerve tissue. It is our intent to present uncomplicated and easily repeatable testing procedures to assess the analytical performance level of the magnetic system during trouble-shooting, before or after an experimental run, or after the installation of new system components. A thorough physical characterization of the toroid amplifier system has been presented by the originators of the system.9 The considerations presented here are relatively simple procedures that have been developed in our laboratory in optimizing the response of the analytical system. EXPERIMENTAL SECTION Magnetic Probes. The construction of the toroid-shaped probes used for magnetic stimulation has been described in detail elsewhere.8 In short, a 40 gauge (79 µm) copper wire is wrapped (9) Gielen, F.; Roth, B. J.; Wikswo, J. IEEE Trans. Biomed. Eng. 1986, 33, 910-921.
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Figure 1. (A) Block diagram of the experimental setup showing the flow of information between system components. Path 1 represents the magnetic stimulus supplied to the nerve tissue. Path 2 represents the biomagnetic collection. Path 3 represents the connections for electrical detection. (B) Block diagram showing the support components for the biological tissue.
∼70 times around a highly permeable ferrite core. Even though the wire is coated, it is further insulated by several layers of electrovarnish before being encapsulated by epoxy and attached to a firm supporting rod. The construction of the probes used for detection of biomagnetic signals is similar and has also been described elsewhere.10 The main difference lies in the detection probes having a second, separate, several-turn wire wrapped around the ferrite core. This independent wrap is used for calibration purposes. Magnetic Probe Apparatus. The connections between the different experimental components are depicted schematically in Figure 1A. The Vanderbilt Biomagnetic Probe puts out a 5 V square wave pulse at the signal output. The frequency of this pulse can be changed from 0.5 to 30 Hz, and its width can be adjusted from 0.01 to 1.0 ms. For magnetic neural tissue stimulation, this pulse is fed into a stimulus isolator (WPI, Sarasota, FL), which uses the pulse as a gating signal to produce an output current of adjustable strength as specified by the user. This output current is fed into the BNC connector of the stimulating toroid. When detecting biomagnetic signals, the current produced in the detection toroid from the magnetic component of the neural current is sent into the signal input of the Vanderbilt probe unit. (10) Vanderbilt Biomagnetic Current Probe Model LSP-3 Users Manual; Vanderbilt University, Nashville, TN, 1990.
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There it is amplified, and the output is sent to a Hewlett Packard 54603B 60 MHz digital oscilloscope, connected by an HP RS-232 interface to a IBM 486DX computer and controlled by Hewlett Packard benchlink software. The oscilloscope is externally triggered by the biomagnetic probe unit. The probe unit also sends a voltage pulse, used for calibration, through a second, separate winding around the core of the detection toroid. This pulse, the strength of which can be varied, produces an observable signal on the oscilloscope output that can then be used to determine the strength of the magnetic signals originating from the biological tissue. Experimental Preparation of the Nerve Tissue. Large crayfish were captured in streams that flow near the University of Hawaii. To begin an experiment, a crayfish was placed in a freezer (-20 °C) for ∼60 s to anesthetize it. The ventral tail carapace was then removed to expose the ganglion. This ganglion was severed below the thorax and again as it entered the lower tail, giving a length of ∼2 cm to the excised portion. This portion was placed in oxygenated buffered saline in a flow cell (Figure 1B). The nerve was then threaded through the stimulating and detection toroids, and the two outer ends were pinned to the bottom of the flow cell. Buffered, oxygenated saline was passed over the nerve section at a rate of 4 mL/min. The inflow and outflow were independently controlled by two peristaltic pumps (Rainin Rabbit).
Figure 2. Input signal to the stimulating toroid (A) and the voltage response produced across the 3.3 kΩ resistor (B).
Reagents. Lidocaine and prilocaine were purchased from Sigma Chemical Co. (St. Louis, MO). Mepivacaine hydrochloride was purchased from Spectrum Chemical Co. (Gardena, CA). All reagents were prepared on the day of use using oxygenated buffered saline. The saline was a modified van Harreveld (MVH) solution, the composition of which has been previously reported.7 RESULTS AND DISCUSSION Optimization of the Stimulation Toroid. The mutual inductance between the stimulation toroid and a wire in its lumen is given by eq 1. In eq 1, µr, the relative permeability of the ferrite
M)
µrNpNsw b ln 2π a
()
(1)
core, is 10000µ0. Np and Ns are the number of wraps in the primary (70) and secondary coils (1), respectively, b is the outer radius of the toroid, w is the toroid width, and a is the inner radius. The mutual inductance calculated using eq 1 for an average 70wrap toroid is ∼0.25 mH. During a test of the stimulation strength, copper wire (d ) 0.22 mm) was looped through the stimulating toroid’s lumen and connected to two ends of a 3.3 kΩ resistor (0.5% accuracy). The voltage drop across the resistor was monitored on the oscilloscope. Figure 2 shows the voltage gate signal that the Vanderbilt probe uses to trigger the stimulus isolator. The shape of the induced voltage across the secondary resistor is characteristic of magnetic induction. A voltage is induced in the secondary winding only when the current in the primary winding is changing. The square wave signal output from the Vanderbilt Biomagnetic Probe has an average rise time of 33 ns and an average fall time of 102 ns. However, the rise and fall time of the stimulus isolator is, on average, 10 µs. Using these values, we experimentally find the induced resistor voltage to be within 30-50 mV of the predicted value when using a precisely constructed stimulating toroid. Between the rise and fall of the pulse, the current is constant and the induced voltage falls to zero. When the square wave pulse is used as the primary voltage gate source, as it was in our earlier biomagnetic neurosensor experiments, the secondary winding (copper wire or nerve) is stimulated twice, with the time difference between stimulation events being roughly equivalent to the pulse width (set at ∼10 µs). This period of time is well below the refractory period of nerve tissue. Further, when circuits were designed to shape the voltage gate signal to produce a continually changing current through the primary winding, the nerve response showed no detectable difference from the present system.
Figure 3. Change in the stimulated voltage across several different resistor loads, R. If 5 mA or more current is supplied to the windings of the primary coil in the toroid, there is no further increase in the stimulated voltage.
Alternate circuits were constructed to elevate the current output of the stimulus isolator from its maximum of 10-50 mA. As the induced magnetic field in the ferrite core is proportional to the current, higher current values would increase the field. Figure 3 shows the change in the current supplied to the primary winding in the toroid as compared to the voltage induced across a resistor acting as a secondary winding. It can be seen that, at a certain level of input, the induced voltage remains constant. Although more current can be placed across the windings of the stimulating toroid, there is no further increase in the magnetic field due to saturation of the ferrite core. This saturation current is a constant for each individual toroid. Figure 3 also shows this test using different resistance loads. The resistor values chosen correspond to those of biological tissue. As can be seen, the saturation current does not change with a change in the resistance of the load. Therefore, the saturation current value for each toroid can be used as a set-point during an experiment to magnetically stimulate nerve tissue. If the desired nerve stimulation is not seen, a different toroid needs to be chosen. Optimization of the Detection Toroids. Several different types of magnetic probe designs are depicted in Figure 4. The multiturn solenoid (Figure 4B) is the only design devoid of a ferrite core. A solenoid is able to concentrate magnetic flux in the space enclosed by the loops. This design does produce a voltage in a resistance load placed in its lumen. The number of wire turns needed to produce an action potential in an excised nerve, however, is on the order of 1000 or more, and this amount of wire increases the total outside diameter of the probe to ∼910 mm, making the probe design too unwieldy. The horseshoeshaped (Figure 4C) and the collapsible probe (Figure 4D) designs are very interesting, as their shapes do not necessitate total excision of nerves from the crustacean host. Both of these probes produce a voltage across a resistance load when used for stimulation. The toroids for magnetic detection can be evaluated in terms of their ability to detect a current pulse sent across a 3.3 kΩ load placed through the ferrite core lumen. Current pulses were produced in the load by connecting it across the output of the stimulus isolator. The toroidal response was sent into the Vanderbilt probe, amplified there, and then sent to the oscilloscope. The enclosed ring toroid (Figure 4A) is able to detect very small currents in the wire. In fact, as the current in the wire reaches ∼50 µA, the ferrite core on the detection toroid saturates. It is no longer capable of detecting any increase in current. The Analytical Chemistry, Vol. 68, No. 9, May 1, 1996
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Table 1. Comparison of the Blocking Times of the Local Anesthetic Mepivacaine (M)a in Solutions of Varying pH pH
[M]/[HM+] time to block (s) nb
6.0
7.6
9.2
0.025 765 3
1 480 3
39.8 280 4
a The mepivacaine concentration (pK ) 7.6) was 50 mM for every a experimental trial. b Denotes the number of sample points.
Figure 4. Stimulating toroid geometries. (A) Standard toroid; core used had an o.d. ) 2.73 mm, i.d. ) 1.27 mm, W ) 2.73 mm. (B) Solenoid; no core present, but lumen diameter is 1 mm. (C) Horseshoe toroid; core is a standard ferrite toroid with a 20° section cutout. (D) Collapsible toroid; core employs a standard toroid cut into two equal halves.
collapsible toroid, an experimental design, is still not very sensitive to small currents. It begins to register a response only after 1 mA is flowing through the wire, and it does not saturate, even at 10 mA. This is due to the inherent design of the toroid. As it is not a continuous ring, the ferrite core cannot concentrate magnetic flux as well as the full ring toroid. We are currently investigating methods of cutting and positioning the two halves of collapsible toroids so that the ring geometry is restored when encircling a nerve (or wire). Optimization of the Analytical Signal. When incorporating living biological tissue into the magnetic stimulation and recording instrumentation, it is important to remember that nerve tissue does not behave like a wire. Action potential events have a threshold voltage response level. Moreover, if the voltage delivered to the tissue is too high, the response of the nerve varies with time, and the tissue may die. Easily measurable parameters, such as the width or the diameter of the ganglion, were investigated in an attempt to qualitatively predict the response and input characteristics of the sensor. The criterion used for evaluation of the response was the input current to the stimulation toroid necessary to produce a 40 mV or greater action potential in the nerve tissue. An action potential of this magnitude is usually sufficient for an analytical response. The diameter of the nerve showed the greatest correlation to the input current: the smaller the nerve tissue, the more current necessary to achieve an action potential. However, a more stringent numerical rule between observable parameters and the input/output characteristics of the neuromagnetic sensor was not achievable. Every nerve tissue and, hence, every neuromagnetic sensor is different. Although the excised 1674 Analytical Chemistry, Vol. 68, No. 9, May 1, 1996
length and approximate diameter of the tissue can be controlled, the stimulation requirements and the signal output strengths still remain largely unpredictable, except for rules-of-thumb. On the other hand, very few neuromagnetic sensors fail to work. While we cannot predict at the outset the required stimulus strength, it is almost always within the capabilities of the stimulus isolator and easily found by adjusting a dial. Observable analytical action signals can be easily established by detection toroid repositioning along the nerve tissue axis until optimum output is achieved. Buffer Composition. The lifetimes of the tissue preparation are longest around physiological pH and decrease with changes in pH in either direction. The useful range for the sensor system is between pH 5 and 10. A lifetime-pH curve can be very useful when testing for analytes whose ionization is pH dependent, as only one form of the analyte usually determines how much analyte is needed to achieve a block or otherwise observable analytical signal on the recording instrumentation. An estimate of analyte pKa can determine the usefulness of the neuromagnetic technique as a probe of the respective potency of ionized and unionized forms of an analyte. For example, a drug with a pKa of 10 or higher would not work very well, as the pH needed to achieve a high concentration of the neutral form would also be quickly lethal to the nerve tissue. We chose to try mepivacaine (pKa ) 7.6) to demonstrate the effect of the pH environment on the potency of the analyte. The data for three different pH environments on three different nerve tissue sections are given in Table 1. If pH 7.6 is taken as normal, a 94% increase in the concentration of the neutral (M) form results in a 60% decrease in the time to block. Conversely, a 95.2% decrease in the concentration of the cationic (MH+) form results in a 62% rise in the blocking time. Above pH 9 and at pH 6, the lifetime of the nerve tissue was only 60% of the lifetime at optimum pH. However, several points of blockage were still achieved for each pH on the same nerve, enabling a comparison to be made between the behavior of the ionized and unionized forms of the drug. As can be seen, a judicious choice of buffer pH is important when assaying unknown analytes. In our laboratory, we run unknown natural product extracts in three different pH environments: acidic, neutral, and alkali. Once the most effective environment is found, dose-response curves can be prepared at a pH that balances tissue lifetime against blocking time. Such considerations become even more important when the sample size is limited. Lateral Placement of the Toroids. Figure 5 shows the effect on the amplitude of the action potential as the source of stimulation is moved increasingly farther away from the point of detection. The kinks in the curves probably correspond to nodal regions where the nerve tissue is nonuniform or, perhaps, damaged during the excision procedure. As graphically depicted, the amplitude
Figure 5. Change in the amplitude (‚‚‚) and displacement (s) of the nerve signal response potential as the stimulating toroid is moved farther away from the point of detection. In a usual experiment, 1-2 mm is the easiest experimental distance to maintain.
is fairly constant and not dependent upon the placement of the detection toroid except at the point of zero separation between the stimulation and detection probes. At this point, the observable voltage is usually very high (off the scale in Figure 5) but unreliable. The normal distance between the stimulation and detection toroids is ∼1-2 mm. This distance is the easiest to maintain experimentally in a cell of tight dimensions. Figure 5 also shows the time displacement of the action potential as the stimulation source is moved farther away from the point of detection. Due to a temporarily stable conduction velocity, here calculated to be ∼7 m/s, the action potential is displaced in time with increasing distances to the point of detection. With extended experimental time, or the introduction of drugs or anesthetics into the flow cell, the conduction velocity can change greatly from its initial value. When these changes are due to drug action, we have used them as an indicator of imminent conduction block. CONCLUSIONS The magnetically based sensing technique described above offers a new way of monitoring certain analytically useful data from
biological tissues. The magnetic technique is able to elicit the same information from nerve tissue as the analogous electrophysiological techniques but is less damaging to the tissue. Magnetic measurements are fast and nondisruptive to the biological tissue and can be very sensitive, depending on the instrumentation employed. Such instrumentation can be purchased for less than the cost of many standard pieces of laboratory equipment. Furthermore, the capabilities of such instrumentation can often be augmented through the in-house construction of simple electronic circuits. General disadvantages of magnetically based neural biosensors are the degree of experimental difficulty and the short tissue lifetimes. We realize that short tissue lifetime, a limitation found in many tissue-based sensors, will tend to preclude this technique from routine use. However, the advantages of this type of biosensor in the analytical laboratory far outweigh the experimental setbacks. For example, the on-site ability to screen natural product extracts for neurologic activity is an attractive option for the analytical chemist. We have employed the system for this purpose on many extracts from aquatic and land-based Hawaiian species and have been able to compare the potency of the extracted chemicals with those of other chemicals or plant extracts within a few minutes. The magnetic system’s efficiency in generating such useful analytical data warrant its consideration as an analytical technique under appropriate circumstances. ACKNOWLEDGMENT The authors gratefully acknowledge support from National Science Foundation Grant CHE-9216304. We also thank Don Cole for assistance and discussion in electronics.
Received for review September 21, 1995. February 20, 1996.X
Accepted
AC9509418 X
Abstract published in Advance ACS Abstracts, April 1, 1996.
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