Anal. Chem. 1994,66, 2621-2629
Quantitation of Choline in the Extracellular Fluid of Brain Tissue with Amperometric Microsensors Michael 0. Gargullo and Adrian C. Michael' Department of Chemistry, Universiw of Pittsburgh, Pittsburgh, Pennsylvania 15260
Amperometric microsensors for the detection of choline in the extracellular fluid of brain tissue have been prepared by immobilizing horseradish peroxidase and choline oxidase onto carbon fiber microcylinder electrodes with a cross-linkable redox polymer. The microcylinders have diameters of 7 or 10 pm and lengths of 200-400 pm. To detect choline, the microsensors are operated at an applied potential of -0.1 V vs SCE. At this potential, ascorbate and other easily oxidizable interferant molecules present in brain tissue are not detected by the electrode. Ascorbate, however, can interfere with the response to choline by acting as a reducingagent in the enzymecontaining polymer film. So, a Nafion overlayer is required in order to reliably detect choline in the presence of physiologically relevant concentrations of ascorbate ( 200 pM). The Nafion-coatedmicrosensors have a detectionlimit of -5 pM choline and give a linear response beyond 100 pM when calibrated in vitro at 37 O C . Exposure of the microsensors to brain tissue for several hours causes less than a 10%loss in redox polymer surface coverage and less than a 25%loss in sensitivity to choline. To assess the ability of the microsensors to monitor choline levels in brain tissue, small volumes of a choline solution were injected into brain tissue at a site about 1 mm away from a microsensor. The current arising at the microsensor was converted to choline concentration by calibrating the sensor following the in vivo experiment. The resultant choline concentrations were in excellent agreement with those predicted by appropriate diffusion equations. N
Amperometric sensors based on enzymes and redox mediators can be used to detect a variety of substances.'-l0 Self-contained sensors constructed by attaching a redox mediator to a cross-linkable polymer that is also used to immobilize the enzymes onto an electrode have recently been reported."-15 These sensors are potentially suitable for use in delicate environments, such as the tissues of a living animal,
because they can be miniaturized16J7and because the leaching of the enzymes and the redox mediator into the surroundings is minimal. In a previous paper,ll we reported on the development of prototype choline (Ch) sensors which were based on conventional glassy carbon electrodes with millimeter dimensions. This paper concerns the development of selfcontained microsensors suitable for monitoring Ch in the extracellular fluid (ECF) of brain tissue. These microsensors are valuable not only because of the biochemical importance of Ch in its own right18J9but also because they are envisioned to be forerunners to microsensors suitable for the in vivo monitoring of acetylcholine,' which is an important neurotransmitter in the mammalian brain.20 Scheme 1 shows that the detection of Ch is based on the enzymatic oxidation of Ch by immobilized choline oxidase (ChOx), which leads to the generation of H202. The use of ChOx in the sensor demands that 0 2 be used as the oxidant for Ch because ChOx exhibits a very high specificity for this cosubstrate. A detailed investigation of the impact of 0 2 on the measurement of Ch has not yet been performed because, as will be shown herein, preliminary data suggest that the availability of 0 2 in brain ECF is sufficient for reliable operation of these sensors. The H202 is reduced to water by immobilized horseradish peroxidase (HRP), which is in turn reduced by the redox polymer. The redox polymer consists of a poly(viny1pyridine) backbone with pendant osmium-based redox groups, indicated as 0s" and Os111in Scheme 1. The redox groups consist of osmium ions that form octahedral complexes with a pair of 2,2'-bipyridine ligands, a chloride ion, and a pyridine unit of the polymer. Approximately one of every four pyridine units of the polymer carries a redox group.'' Charge transport between H R P and the electrode occurs by electron hopping2] through the redox polymer. Finally, reduction of the mediator at the electrode surface produces the amperometric signal. As Scheme 1 shows, the amperometric signal arises from the electrocatalytic reduction of H202.22123 This approach
(1) Ruiz, B. L.; Dempsey, E.;Hua, C.;Smyth, M. R.; Wang, J. Anal. Chim. Acta 1993, 273, 425430. (2) Kulys, J.; Schmid, R. D. Eiosens. Eioelecrron. 1991, 6, 43-48. (3) Casclla, I. G.; Dcsimoni, E.; Salvi, A. M. AMI. Chim. Acra 1991,243,61-63. (14) (4) Hale,P.D.;Boguslavsky,L.I.;Inagaki,T.;Karan,H.I.;Lee,H.S.;Skotheim, (15) T. A.; Okamoto, Y. Anal. Chem. 1991,63,677-682. (16) (5) Hale, P. D.; Inagaki, T.;Lee, H. S.; Karan, H. I.; Okamoto, Y.; Skotheim, (17) T.A. Anal. Chim. Acra 1990, 228, 31-37. (18) (6) Kulys, J. J.; Bilitewski, U.; Schmid, R. D. Anal. Le??.1991.24(2), 181-189. (7) Tatsuma, T.;Watanabc, T. Anal. Chim. Acra 1991, 242, 85-89. (19) (8) Hale, P. D.; InagaLi, T.; Lee,H. S.; Karan, H. I.; Okamoto, Y.; Skothcim, (20) T.A. Anal. Chim. Acra 1990, 228, 31-37. (9) Frcw, J. E.; Hill, H. A. 0. Anal. Chem. 1987, 59, 933A-944A. (IO) Gorton, L. J. Chem. Soc., Faraday Trans. I 1986,82, 1245-1258. (21) (1 1) Garguilo, M. G.; Huynh, N.; Proctor, A.; Michael, A. C. Anal. Chem. 1993, 6.9, 523-528. (12) Ye, L.; HHmmerle, M.; Olsthoorn, A. J. J.; Schuhmann, W.; Schmidt, H.-L.; Duine, J. A.; Hcller, A. Anal. Chem. 1993, 65, 238-241. (13) Vrcckc, M.; Maidan, R.; Hcller, A. Anal. Chem. 1992, 64, 30843090.
0003-2700/94/03662621$04.50/0 0 1994 American Chemical Society
~
~
~
~~~~
Katakis, 1.; Hellcr, A. AMI. Chem. 1992, 64, 1008-1013. Gregg, B. A.; Hellcr, A. J. Phys. Chem. 1991.95, 5976-5980. Wang, D. L.; Heller, A. Anal. Chem. 1993.65, 1069-1073. F'ishko, M. V.; Michael, A. C.; Hcllcr, A. Anal. Chem. 1991.63.2268-2272. Ulus, I. H.; Wurtman, R. J.; Mauron, C.; Blusztajn, J. K. Erain Res. 1989,
484, 217-227. Blusztajn, J. K.; Wurtman, R. J. Science 1983, 221, 614-620. Cooper, J. R.; Bloom, F. E.; Roth, R. H. The Biochemical Busis of Neuropharmacology, 6th 4.;Oxford University Press: New York, 1991; Chapter 8. Dalton, E. F.; Surridge, N. A.; Jemigan, J. C.; Wilboum. K. 0.;Facci, J. S.; Murray, R. W. Chem. Phys. 1990,141, 143-157. (22) CsBregi, E.; Gorton, L.; Marko-Varga, G. Anal. Chim. Acra 1993,273.59-
70.
(23) Wollenbcrger, U.; Bogdanovskaya, V.; Bobrin, S.; Schcllcr, F.; Taraacvich, M. Anal. Le??.1990, 23(10), 1795-1808.
Analytical Chemism, Vol. 66, No. 17, September 1, lQQ4 2621
Scheme 1. Schematic Representation of the Sequentlai Reactlons That Occur at a Bienzyme Sensor for Ch (solld Ilnes) and of the Mechanism of Ascorbate (AA) Interference (dashed lines).
*Horseradish peroxklase (HRP) and choline oxldase (ChOx) are immobilized within a cross-linkable, redox polymer deposited on carbon electrodes.
offers two important advantages over the more conventional use of the direct oxidation of H202 at platinum and carbon e l e ~ t r o d e s . ~First, ~ J ~since the sensor is operated at potentials negative with respect to the half-wave potential of the mediator (Ell2 = -0.3 V vs SCE), the oxidation of other compounds present in brain ECF, most notably ascorbate, is greatly diminished. Second, HRP in the membrane prevents H202 from simply diffusing away from the sensor before being detected. This is important because diffusion of H202 away from the sensors would diminish the Ch signal and would be toxic to surrounding tissue. We have shown before, for example, that immobilized HRP is highly effective at reducing the peroxide formed by coimmobilized glucose oxidasell and provide evidence to the same effect in this paper for the case of coimmobilized ChOx. Scheme 1 also shows that, despite being operated at negative applied potentials, the response of these sensors to Ch can still suffer from interference by ascorbate. Ascorbate can reduce the redox mediator as well as peroxide and is also a known cosubstrate for peroxidases.*6J7 The oxidation of ascorbate (AA) to dehydroascorbate (DHA) is chemically irreversible, so redox equivalents consumed by these interfering reactions cannot be recovered. For these reasons, the Ch sensors require a Nafion overlayer in order to function in the presence of ascorbate. Nafion is permselective for cations and limits the permeation of ascorbate into the sensing membrane.28-33 Miniaturized Ch sensors have been developed by using the cross-linkable redox polymer to immobilize HRP and ChOx onto carbon fiber microelectrodes. The performance of the microsensors, in terms of detection limits and current densities, is very similar to that obtained with the prototype sensors. So, miniaturization does not lead to a sacrifice of analytical (24) Rouillon, R.; Mionetto, N.; Marty, J.-L. Anal. Chim. Aero 1992, 268, 347350. (25) Navera, E. N.; Sode, K.; Tamiya, E.; Karube, I. Biosens. Bioelecrron. 1991, 6 , 675-680.
(26) Lowry, J. P.; ONeill, R. D. Anal. Chem. 1992, 64, 456-459. (27) Stryer, L. Biochemistry; Freeman: New York, 1988; p 422. (28) Navera, E. N.;Suzuki, M.;Tamiya, E.;Takeuchi, T.;Karube, I. Electroumlysis 1993, 5, 17-22. (29) Fan, Z.; Harrison, D. J. Anal. Chem. 1992, 64, 1304-1311. (30) Rice, M. E.; Nicholson, C. Anal. Chem. 1989, 61, 1805-1810. (31) Kristensen, E. W.; Kuhr, W. G.; Wightman, R. M. Anal. Chem. 1987, 59,
1752-1757.
W.;Ghatak-Roy, A. R.; Moore, R. B.; Penner, R. M.; Szentirmay, M. N.; Martin, C. R. J. Chem. SOC.,Faraday Trans. 1 1986,82,
capabilities. The performance of the microsensors in vivo has been assessed with measurements in the brain of anesthetized rats under well-defined conditions. The microsensors were used to monitor the diffusion of Ch from a microinjection pipet. The diffusion equations that describe this process are well-known and have been used before to determine the diffusion coefficients of a variety of molecules in brain tissue.34 After calibration, the signals arising at the microsensors are in excellent temporal and quantitative agreement with the diffusion theory predictions. EXPERIMENTAL SECTION Reagents. Choline oxidase (Alcaligenes species), peroxidase type I1 (horseradish), catalase (Aspergillus niger),choline chloride, L-ascorbicacid (sodium salt), and 2-bromcethylamine hydrobromine were obtained from Sigma (St. Louis, MO). Nafion (5% solution, 1100equiv wt) was obtained from Aldrich (Milwaukee, WI). K20sC16 was obtained from Johnson Matthey (Ward Hill, MA). Poly(4-vinylpyridine) (PVP, MW 50 000) and poly(ethy1ene glycol 400 diglycidyl ether) (PEGDGE) were purchased from Polysciences (Warrington, PA). The abovechemicals were used as received. Phosphatebuffered saline (PBS), pH 7.4, was prepared with ultrapure water (Nanopure) and used for all electrochemical and UVvisible experiments and for the preparation of standard solutions. The cross-linkable redox polymer (PVP-Os(bpy)~Cl;bpy = 2,2'-bipyridine) was synthesized according to the published procedure.35 The final chloride form of the redox polymer was additionally washed with acetone. Immobilization of HRP and ChOx on Glass. Glass microscope slides ( 2 5 X 75 X 1 mm) were cleaned with methanol and rinsed with ultrapure water. The glass was modified (see Results section for detailed recipes) by spreading five aliquots of 10 p L each of an aqueous mixture containing the redox polymer, cross-linking agent, and appropriate enzymes over a 10 X 25 mm section of the slides. The films were cured in air for at least 48 h, soaked in ultrapure water for 15 min, and redried in air for at least 75 min before use. The coated ends of the slides were immersed in PBS. Following addition of enzyme substrate, small aliquots of the solution were removed and colorimetrically assayed for hydrogen peroxide. The aliquot was combined with 20 pg of HRP and 0.26 mM o-dianisidine and allowed to react for 2 min. The quantity of H202 in the aliquot was then determined by measuring the absorbance due to the oxidized form of o-dianisidine at 460 nm (e = 1.13 X lo4 M-' cm-1). ElectrodeModification. Microcylinder electrodes, 200400 pm in length, were made with 10- (P-55) or 7-pm-(T300) diameter carbon fibers (Union Carbide, New York) sealed with Spurr epoxy (Polysciences) into pulled glass capillary tubes.36 Microsensors were prepared by modifying the microcylinder electrodes with a 2-pL aliquot of an aqueous mixture of the redox polymer, cross-linker (PEGDGE), and appropriateenzymes (see Result sections for detailed recipes). A droplet of the solution was formed at the tip of a micropipet and held in contact with the microelectrode until the solution
-
(32) Espenscheid, M. 1051-1070.
(33) Martin, C. R.; Rubinstein, I.; Bard, A. J . J. Am. Chem. SOC.1982, 104, 4817-4824.
2622
AnaiyticaiChemistry, Vol. 66, No. 17, September 1, 1994
(34) Rice, M. E.; Gerhardt, G. A.; Hierl, P. M.; Nagy, G.; Adams, R. N. Neuroscience 1985, 15, 891-902. (35) Gregg, B. A.; Heller, A. J. Phys. Chem. 1991, 95, 5970-5975. (36) Michael, A. C.; Justice, J. B., Jr. AMI. Chem. 1987.59, 405-410.
became sufficiently viscous to remain on the carbon fiber. The microsensors were cured in air for at least 48 h, following which they were soaked in ultrapure water for 15 min and redried in air for at least 1 h before use. Nafion-coated microsensors were prepared by a dip-coating procedure. The sensors were first dipped in a 0.5% Nafion solution, prepared by diluting the as-received solution with 2-propanol, and dried in air for 10 min. The sensors were dipped into the 0.5% solution five times for 10 s each time with a drying period of 20 s between each dip. Finally, the sensors were dipped in the as-received 5% Nafion solution and dried in air for at least 3 h before use. The sensors were dipped into the 5% solution five times for 20 s each time with a drying period of 40 s between each dip. Electrochemistry. Cyclic voltammetry and constant potential amperometry were performed with apparatus controlled by an 80386-based personal computer (Twinhead SS-600/ 25C) with an analog interface (Labmaster DMA, Scientific Solutions, Solon, OH) and software developed in-house. A locally constructed potentiostat interface and a picoammeter (Keithly 427, Cleveland, OH) were used to control the applied potential and to amplify the electrode current, respectively. Amperometric measurements were made in a flow-injection analysis (FIA) system equipped with a pneumatically actuated sample injectionvalve (Rheodyne 50/5701, Cotati, CA) which was controlled by the computer via a locally constructed relay interface. The microsensors were mounted in the outlet of a custom-made flow cell. A flow of 1.2 mL/min through the system was created and maintained by gravity feed from an elevated reservoir containing air-equilibrated PBS. Cyclic voltammetry was also performed in the air-equilibrated PBS. All electrochemical experiments were performed with a saturated calomel reference electrode (SCE, Fisher) and with a Faraday cage. Experimental measurements at 25 and 37 OC were made with the custom-made flow cell immersed in a temperature-controlled water bath. The temperature controller (Thermomix 1441,B. Braun, Melsungen,Germany) was placed in a secondary water bath outside the Faraday cage in order to avoid 60-Hz noise. Water was circulated between the two baths via plastic tubing (Nalgene). Temperature was monitored with a thermocouple positioned as near as possible to the microsensor. Animal Surgery. Male Sprague-Dawley rats, 250-350 g, were anesthetized with chloral hydrate (400 mg/mL). The animals were placed in a stereotaxic frame (Kopf 1430 frame assembly, Tujunga, CA), kept unconscious with additional doses of chloral hydrate, and kept at a constant body temperature of 37 OC with a homeothermic blanket. Small holes were drilled in the skull to allow insertion of the microsensors and micropipets into the striatal region of the brain. A salt bridge fashioned from a plastic pipet tip plugged with tissue provided electrical contact with the reference electrode. Local Injection Experiments. A microsensor and a micropipet (both made using l -"-diameter glass tubes, Sutter Instruments, Novato, CA) filled with a 100 mM Ch solution were mounted side by side with their tips separated by 1 mm and were lowered into the striatal region of the brain. The micropipet was connected by PTFE tubing to a 50-pL gastight syringe mounted in a syringedriver (Sutter Instruments,
-
-
ChOx + HRP
0
15
30
45
time (min) Figure 1. Amount of H202released from glass slides. "edwlth polymer films containingChOx wlth and wlthout HRP, exposed to 1 mM Ch solutions.
Model NA- 1). The signal from the microsensorwas monitored following the injection of 300-500-nL droplets of the choline solution into the brain. Scanning Electron Microscopy (SEM). Micrographs of the microsensors with and without Nafion overlayers were obtained with a JEOL JSM-35C scanningelectron microscope. The microsensors were mounted on a -1-in. diameter conductive stand and were coated with gold using a Polaron E-5 100 sputterer. Micrographs were then obtained with acceleration voltages and magnification ranges shown in Figures 2 and 4. RESULTS AND DISCUSSION Catalase Activity of HRP. In a previous paper, we coimmobilized HRP and glucose oxidase in redox polymer films. Since both HRP and glucose oxidase undergo electron transfer with the redox polymer, it was possible toshow directly that HRP did not limit the response of the sensor to glucose when the sensor was operated at negative potentials." The chemistry of glucose oxidase and ChOx are significantly different, in that ChOx apparently does not undergo electron transfer with the redox polymer. ChOx is well-known, in fact, to exhibit high selectivity for oxygen, which is its natural cosubstrate. So, in order to confirm the ability of immobilized HRP to efficiently collect the peroxide formed by coimmobilized ChOx, the escape of H202 from polymer layers containing ChOx with and without HRP was measured spectrophotometrically. Figure 1 shows the amount H202 released into the bathing solution from glass slides modified with 37 pg of redox polymer, 11 pg of cross-linker, and 55 pg of ChOx, with and without 28 pg of HRP. The bathing solution contained 1 mM Ch. The amount of H202 in the bathing solution increased continuously with time when the slide without HRP was used. The amount of H202 that escaped from the slide with HRP was greatly diminished. It should be pointed out that even though a redox polymer was used to immobilize the enzymes, there is presumably no reduction of H202 occurring during these experiments since there is no electrode present. Thus, in this case, it is apparently the catalase-type activity of HRP which prevents the escape of H202 from the polymer layer. These data show, therefore, that HRP will serve to protect tissue surrounding a biosensor from peroxide-induced toxicity regardless of the efficiency of the electrical communication between HRP and the substrate electrode. Analvtlml Chemlshy, Vol. 66, No. 17, September 1, 1994
2623
Miniaturizationof ChSensors. Ample evidence now exists that microelectrodes constructed from carbon fibers are suitable for monitoring neurotransmitters and related substances in the extracellular space of the mammalian brain. Thus, we have sought to develop carbon fiber based microsensors for Ch. Previously, we carried out a series of characterizationexperiments with conventionalglassy carbon electrodes to formulate an optimized electrode modification recipe for the preparation of prototype Ch sensors. The optimized recipe produced sensors which had a detection limit of 1 pM Ch and a sensitivity, over the low micromolar range, of 5.3 f 1.9 mA cm-2 M-I or 0.38 f 0.13 nA pM-I (n = 9, r > 0.99).11 Now we report on the behavior of carbon fiber microcylinder electrodes modified with the same optimized recipe (an aqueous mixture containing 0.37 mg/mL ChOx, HRP, and redox polymer and 0.22 mg/mL cross-linker). As with the prototype sensors, the microsensors were calibrated in the FIA system. The microsensors were held at a constant applied potential of 4 . 1 V vs SCE, and the amperometric signal was recorded during the injection of a 30-s bolus of a choline solution into the flow stream via the sample injection valve. For the purposes of reporting detection limits and sensitivities,the amperometricsignal was recorded at the end of the sample bolus. The microsensors had a detection limit of 1 pM Ch (S/N = 4) and a sensitivity of 12.5 i 6.7 mA cm-2 M-I or 0.72 i 0.18 pA pM-I (n = 4, r > 0.99). Thus, the detection limit and current density values obtained with the microsensors are very similar to those obtained with the prototype sensors; i.e., miniaturization has been successfully accomplished. SEMs of Ch Microsem" Figure 2 shows scanning electron micrographs (SEMs) of a 7-pm-diameter carbon fiber microcylinder electrode modified according to the optimized recipe for Ch sensors. The top micrograph shows that the film applied to the carbon surface is -2-3 pm thick but is uneven and does not completely cover the underlying electrode. It must be remembered that the film is a hydrogel and probably suffers some damage due to dehydration in the vacuum chamber of the microscope. Nevertheless, the appearance of these films is of lower quality than previously reported for filmscontaining glucose oxidase, which appeared smooth and uniform under SEM. The nonuniformity of these ChOxcontaining films is due to a brown, grainy precipitate which rapidly forms in solutions containing ChOx and the redox polymer. Thus, the Ch sensors are prepared with a heterogeneous mixture of the polymer and enzyme, in contrast to glucose sensors which are prepared with a homogeneous solution. ChOx forms an electrostatic complex with the polymer which can be solubilized by the addition of acid to the solution. Unfortunately, sensors prepared with the acidtreated solution do not respond to Ch. Presumably, the acid destroys the enzyme. So, the results reported below were obtained with these nonuniform films. Film uniformity will be addressed in the future. Nafion-Coated Ch Microsensors. Since the ECF of the brain contains high levels of ascorbate?' the impact of ascorbate on the response of the microsensors to Ch was evaluated by introducing sample solutions containing both 50 (37) Ghasemzedah, B.; Cammack, J.; Adams, R. M Bruin Res. 1991,517, 162166.
2624 Ana&thICh8mbiry, Vol. 66, No. 17, September 1, 1994
f
Flgure2. Scanning electron micrographs of a Ch microsensor based on a 7-pm carbon fiber microcylinder electrode -200 pm in length.
pM Ch and various concentrations of ascorbate ( W O O pM) into the FIA system. As before, the sensors were placed in the outlet of the FIA system and were operated at a constant applied potential of 4 . 1 V vs SCE at room temperature; the current was recorded at the end of a 30-s sample bolus. To eliminate the possibility of detecting H202 produced from the air oxidation of ascorbate, 1 pg/mL catalase or 5 pg/mL HRP was added to the standard solutions. Figure 3 shows that as the ascorbate concentration is increased, the Ch response obtained with a microsensor which lacked a Nafion overlayer diminishes significantly. In fact, even at the low applied potential employed, a slight net oxidation of ascorbate was observed. This type of interference occurs because ascorbate is a strong reducing agent and can reduce the oxidized form of the mediator in competition with reduction of the mediator at the electrode surface. Likewise, ascorbate can reduce the oxidized form of HRP in competition with reduction of the enzyme by the mediator. Finally, ascorbate can also reduce peroxide in competition with its reduction by the enzyme. These reactions are illustrated in Scheme 1. Figure 3 also shows, however, that as the ascorbate concentration is increased, the Ch response observed with a microsensor with a Nafion overlayer remains constant. The Nafion overlayer eliminatesthe ascorbate interference because it is permselectivefor cations and, therefore, prevents ascorbate from reaching the enzyme-containing film. The Ch response
9.0 W
I1 \\\
25 "C
6.0
c,
c L
3
0.0
without Nafion
0
100 200 300 400
ascorbate (pM) Flgure 3. Comparison of ascorbate interferenceat Ch microsensors wfth and without a Nafion overlayer. Data were collected at the end of a 30-9sample bolus containing50 pM Ch and 0-400 pM ascorbate in the FIA system. The microsensorswere heM at a constant potential Of -0.1 V vs SCE.
of the Nafion-coated sensor in the absence of ascorbate is decreased in comparison to the sensor without Nafion, suggesting the Nafion also interferes with the ability of Ch to permeate the enzyme layer. This is something of a surprise, sincethe sensitivityof these sensors is far below (by -5 orders of magnitude) the mass transport limit. This implies that the decreased sensitivityof the Nafion-coated microsensors results from a passivation of the enzyme layer. The decreased Ch response in the absence of ascorbate does not appear to be due to a change in the performance of the redox polymer, however, since Nafion had no significanteffect on cyclicvoltammograms of the osmium redox couple obtained in the absence of enzyme substrate. The decrease in Ch response caused by the Nafion overlayer led to an increase in the detection limit of the sensor to 10 p M and a decrease in sensitivity to 0.096 0.029 PA pM-l (n = 1 1). The Nafion overlayer allows the microsensors to detect Ch in the presence of a wide range of ascorbate concentrations. Ascorbate concentrations in brain ECF rarely fluctuate over the wide range covered in Figure 3 since the ascorbate in brain ECF is under homeostatic control.38 Furthermore, fluctuations in extracellular ascorbate can be monitored with carbon fiber mi~roelectrodes,3~ if necessary, to examine the extent to which they might affect signals measured with the choline microsensors. Also, the data show that Nafion-coated microsensors can be used in different regions of the brain where different levels of ascorbate exist. In the long run, it will be desirable to decrease the detection limit of the Nafion-coated sensors since existing estimates place the level of Ch in brain ECF at -10 pM,39 Le., near the detection limits established so far. First, however, it is worthwhile to know whether the performance characteristics of the sensors revealed by these calibration experiments reflect how the microsensors will operate in the brain environment. This issue will be addressed below. SEMsof Nafion-Coated ChMicrosensom. Figure 4 shows SEMs of a Nafion-coated microsensor based on a 7-pmdiameter carbon fiber microcylinder electrode. The figure shows that the dipcoating method described above produced a Nafion overlayer with a thickness of -4-6 pm. This is probably more Nafion than is actually required to prevent
-
*
(38) Schenk, J. 0.;Miller, E.; Gaddis, R.; Adam, R. N. Bruin Res. 1982, 253, 353-356. (39) Brehm, R.; Lindmar, R.; Uffelholz, K. J. Neuruchem. 1987,48,1480-1485.
Figure 4. Scanning electron micrographs of Ch microsensors, with a Nafionoverlayer,basedon 7-pm carbon fiber microcylinder electrodes -200-pm in length.
ascorbate interference. The large amount of Nafion that is required on these microsensors is likely a consequence of the unevenness of the underlying polymer film (Figure2). Figure 4 raises the concern that the thick Nafion overlayer might degrade the response time of the sensor. We have found, however, that this is not the case. We have previously shown that Nafion-coated microsensors exhibit a rapid response to Ch.40 Furthermore, we have not noticed that Nafion-coated microsensorsexhibit a slower response to Ch than microsensors without a Nafion overlayer. So, it appears that the response time of Nafion-coated sensors is controlled by the properties of the sensing membrane itself. Unfortunately, direct comparisons of the response time of individualmicrosensors before and after Nafion coating have not yet been possible, since we have had little success with coating microsensors after they have been used for Ch sensing. The reason behind this observation is not clear. The cracks in the polymer film which are visible in Figure 4 are most likely due to dehydration of the film under thevacuum conditionsof the microscope. There are usually only a few of these cracks on each microsensor, but they are shown in the SEM because they proved to be useful as focal points for the microscope. Preliminary in Vivo Testingof ChMicrosensom. For initial in vivo tests of the Nafion-coated microsensors, microsensors (40) Garguilo, M.G.;Michae1,A.C.J. Am.Chem.Suc. 1993,115,12218-12219.
Anal)McalChemisby, Vol. 66, No. 17, September I, 1994
2625
-
n
ac
c,
'0
c
g3 0
-10
-
. .-
.. ,, .., ,.
-20 -
I
600
0
5
10 15 20 25 30
time (min) Fme 1. Traces of cwent measureddurlng localInjectionexperiments using Naflon-coatedmicrosensors implanted In the rat striatum. Trace A was obtained with a Ch microsensor after the local injection of 375 nLofa 100mMChsdution.TraceBwasobtalnedwkhaChmkrosensor after the local lnjectlon of 375 nL of a Ch-free solution (Pes only). Trace C was obtained with a microsensor lacklng ChOx after the local lnjectlon of 375 nL of the 100 mM Ch solution. The mlcrosensors were operated at a constant potential of -0.1 V vs SCE.
and micropipets were mounted side by side and were implanted in the brain tissue of an anesthetized rat. The amperometric signal from the microsensor was monitored after small volumes of solution were injected into the brain tissue with the micropipet, which was positioned about 1 mm away from the microsensor. Figure 5 shows the response of three different microsensors, each held at a constant potential of -0.1 V vs SCE, to the injection of Ch and Ch-free solutions into brain tissue. Trace A in Figure 5 is the response obtained with a Ch microsensor following the injection of 375 nL of a 100 mM Ch solution. The data were used to determine the diffusion coefficientof Ch in brain tissue. ad am^^^ has shown that the diffusion coefficient of injected material, D,can be calculated by using a simple point source expression, D = &/6t,,,, where d is the spacing between the microsensor and micropipet and tmx is the time required for the response to reach a maximum. The diffusion coefficient of choline measured with these data is (2.8 f 0.5) X 10-6 cm2/s (X f SD,n = 18 observations from six animals). As discussed in detail below, this value is an overestimate of the diffusion coefficient since the injected droplet of choline does not constitute a point source. Nevertheless, the value is in excellent agreement with diffusion coefficients reported for a number of other small molecules in the rat brai11.3~ This confirms that the Nafion-coated microsensors can provide reliable temporal information about Ch levels in vivo. Figure 5 also shows two other traces. Trace B shows the response obtained with a Nafion-coated Ch microsensor after the injection of 375 nL of a Ch-free solution (PBS only) into brain tissue. Trace C shows the response obtained with a microsensor lacking ChOx after the injection of 375 nL of a 100 mM Ch solution into brain tissue. These two control experiments confirm that the response in trace A is due to injected Ch and is not due to artifact. Effect of Temperature on Cyclic Voltammograms of the Redox Polymer. In order to quantify the concentration of Ch reaching the microsensor following the injection of Ch into brain tissue (trace A, Figure 5), comparisons were made of the performance characteristics of the microsensors in vivo 2626
AnaiytlcaIChemlstry, Vol. 66, No. 17, September 1, 1994
25'C
*'
0-
-
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and in vitro. Since the operational characteristics of the microsensors are dependent on the performance of the redox mediator, cyclicvoltammograms of the mediator were recorded in vivo and in vitro for comparison. Figure 6 shows voltammograms of a Nafion-coated Ch microsensor recorded at 5 mV/s in the brain ECF of an anesthetized rat (dashed line) and invitro at 25 and 37 OC (solid lines). Thevoltammograms obtained in vitro were recorded from a sensor that had been previously used in brain tissue. The voltammogram obtained in vivo is essentially identical to that obtained in vitro at 37 OC. Figure 6 shows that the redox mediator behaves similarly in vivo and in vitro and also shows that temperature has a major influence on the performance of the sensor. Effect of Temperature on HzOz and Ch Response. Figure 7 shows H202 and Ch calibration curves obtained in vitro with a Nafion-coated Ch microsensor operated at an applied potential of-O.l V vs SCE. The calibration data werecollected in the FIA system at the end of a 30-ssample bolus of H202 or Ch. The error bars represent the standard deviation for three replicate injections made in random order. Figure 7A shows that the sensitivityof the microsensor for H202 increases from 2 to 7 pApM-', while Figure 7B showsthat the sensitivity for Ch increases from 0.05 to 0.12 pA pM-l, when the temperature increases from 25 to 37 OC. Also, when the temperature increases from 25 to 37 'C, the detection limits of the microsensor decrease. So, in order to accurately calculate the concentration of Ch measured by themicrosensor during local injection experiments, postcalibration data must be obtained at 37 "C. Effect of Temperature on Ascorbate Interference. Since it has been shown that temperature has an effect on the overall performance of the Nafion-coated Ch microsensors, it is necessary to evaluate the response of the sensor to sample solutions containing both Ch and ascorbate at 37 OC. Figure 8 shows the response obtained with Ch microsensors with and without a Nafion overlayer. The microsensors were operated at an applied potential of -0.1 V vs SCE in the FIA system, and calibration data were collected at the end of a 30-ssample bolus containing 1 mM Ch and 0 to 400 pM ascorbate. In Figure 8, the error bars represent the standard deviation of three replicate injections. In these experiments, a rather high concentration of Ch was used because the Ch solution injected into brain tissue is expected to be diluted by a factor of -20 before it reaches the micro~ensor.~~ This suggests that, during
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the in vivo microinjection experiments, the Ch reaching the microsensor will be in the millimolar concentration range. Figure 8A shows that the Ch response obtained with a microsensor without a Nafion overlayer decreases as the concentration of ascorbate increases. This occurs with the microsensor at both room temperature (25 "C) and 37 OC. However, the effect of ascorbate on the Ch response is more severe at the higher temperature. Apparently, the higher temperature promotes electron transfer between ascorbate and the various redox components of the sensing membrane. Figure 8B shows the data obtained with a Nafion-coated Ch microsensor. As the concentration of ascorbate increases, the Ch response remains unchanged at room temperature (25 "C) but decreases at 37 OC when the ascorbate concentration exceeds 100 pM. So, some ascorbate interference occurs at 37 OC even with Nafion-coated microsensors. It should be noted, however, that the interference is not so severe that these sensors cannot be used in vivo. For example, since a response to Ch can be measured in the presence of up to 400 pM ascorbate, the microsensors can still monitor Ch fluctuations in the brain ECF, as Figure 5 demonstrates. Clearly, though, the effect of ascorbate must be taken into account if the signal shown in trace A of Figure 5 is to be converted from current to Ch concentration. It should also be noted that the calibration experiments reported in Figure 8 span a very wide concentration of ascorbate. The level of ascorbate in brain ECF, which is under homeostatic regulation, is typically found to be -200 pM,37Le., in the middle of the concentration range covered by Figure 8. Although in the face of certain pharmacological manipulations the ascorbate level may change, the extent of those changes is usually found to be only
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ascorbate (pM) Figure 8. Comparison of ascorbate Interference at 25 and 37 OC. Data were collected with Ch bienzyme microsensors (B) with and (A) without a Nafion overlayer and at the end of a 30-s sample bolus containing 1 mM Ch and 0-400 pM ascorbate in the FIA system. The microsensors were operated at an applied potentialof -0.1 V vs SCE.
a small percentage of the resting ascorbate level. Figure 8B shows that changes in the ascorbate level by up to -&50% do not lead to statistically significant changes in the Ch signal. Furthermore, Figure 8B also makes it clear that an increase in ascorbate leads to a decreasing response to Ch, so an increase in ascorbate levels cannot be misidentified as an increase in Ch. Microsensorsafter Exposure to Brain Tissue. Often, when sensors are placed in direct contact with biological samples, fouling of the sensor surface by proteins and other biomolecules leads to a change in the sensitivity of the sensor. The impact of exposing Ch microsensors to brain tissue has been assessed by inspecting both the cyclic voltammetry of the redox polymer and the amperometric response to Ch. The cyclic voltammograms of the redox polymer, recorded in the absence of enzyme substrate, are essentially identical before and after exposure of the microsensor to brain tissue for up to 5 h. The surface coverage of the redox mediator, as measured by integration of a slow-scan cyclic voltammogram, decreases by about 10%. Similar results were obtained with sensors without a Nafion overlayer. Thus, the redox mediator remains well attached to the microsensor, which alleviates concern about the potentially toxic redox complex leaching from the sensor into the surrounding tissue. These data also confirm that Nafion servesonly as a selectivity layer, but is not required as a containment membrane for the mediator. The sensitivity of the microsensors to Ch has not been observed to decrease by more than 25% upon exposure to brain tissue for 0.5-5 h. Since the sensitivity of these microsensors decreases during the first 0.5 h following insertion into brain tissue and then remains stable for several hours, postcalibration of the sensors may be used for the quantitative interpretation of signals recorded in vivo. Ana@icalChemisi?y, Vol. 66, No. 17, September 1, 1994
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Theoretical Evaluation of in Vivo Data. The results presented in the previous sections of this paper reveal that temperature, exposure to brain tissue, and level of ascorbate present in a sample impact the sensitivityof thesemicrosensors toward Ch. The question exists, therefore, as to whether or not it is possible to quantitate signals recorded in vivo by taking these factors into account. In order to address this question, microsensors were used to again monitor Ch levels in brain ECF following injection of Ch into the tissue at a site nearby the sensor. This experiment is convenient because the relevant diffusion equations, which are readily available from the literature (see eqs 1 and 2, below), provide a theoretical prediction which can be used as a guideline for the purposes of comparison with the experimental outcome. Such a comparison is discussed next. Following in vivo experiments, sensors were removed from the brain of the rat, were returned to the FIA apparatus, and were postcalibrated at 37 OC with Ch standard solutions containing 200 p M ascorbate. The calibration data soobtained were used to convert the amperometric signals recorded in vivo into Ch concentrations. Since the extracellular space in brain amounts to -20% of the total tissue volume,“l the Ch concentrations were multiplied by 5 to arrive at a final estimate of the in vivo Ch concentration. In this work, we have not further adjusted the estimated in vivo Ch concentrations to account for the tortuosity of the extracellular This is because, as mentioned above, the response of these Ch sensors is not determined by the rate of diffusion of Ch in the surroundings. The currents measured are - 5 orders of magnitude smaller than expected for such diffusion control. The solid lines in Figure 9 show the outcome of this calibration procedure for two different volumes of injected Ch solution. These data were collected at a sampling rate of one point per second and were subjected to a 33-point moving average before being plotted. The noise component removed by the smoothing algorithm was induced by the breathing of the animal and was likely a motion artifact. Figure 9 also compares the experimental estimates of the in vivo Ch concentrations with predictions based on the diffusion theories presented in the appendix of ref 34. That reference presents two diffusion equations, each based on slightly different underlying assumptions. Since the experiments performed do not rigorously meet the assumptions of either equations, predictions based on both versions of the theory are used here for the sake of completeness. In one version of the theory, the droplet of solution injected into brain tissue is treated as a point source of diffusing material. In that case, the concentration of material diffusing away from the point source, C(r,t), is C(r,t) =
M 8a(rot)3’2
where Mis the amount of material injected expressed in moles, a is the volume fraction of the extracellular space (assumed herein to be 0.2),D is the diffusion coefficient, r is the distance from the point source to the microsensor (nominally 1 mm), (41) Nicholson, C. Can. J . Physiol. Pharmacol. 1992, 70, S 3 1 4 4 3 2 2 . (42) Bcnvcnistc, H. J. Neurochem. 1989, 52, 1667-1679.
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Analytical Chemkby, Vol. 66, No. 17, September 1, 1994
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time (s) Figure 9. Comparison of experimental data (solid Ilnes), obtalned In brain ECF of the living rat using a NaHoncoeted Ch microsensor set at a constant potentiel of -0.1 V vs S a , with Predictions based on diffusion eqs 1 (dottedlines) and 2 (dashed4otted lines). See text for detalls. The experimental data were collected after local n lJ” of (A) 375 and (e)500 nL of a 100 mM Ch solution and converted from measured currents to Ch concentrations using postcallbration data obtained at the end of 30-5 sample bolus containing mllllmolar concentratlons of Ch and 200 pM ascorbate In the FIA system. The experimental In vivo Ch concentrations were muMpHed by a factor of 5 to take Into account the 20% volume fraction of the extracellular space of brain tlssue.
and t is the time since the microinjection. To employ eq 1, described above. Equation 1 does not take into account the effects of the finite size of the injected droplet, however, which in these experiments is quitelarge(3OG500nL). When thesizeoftheinjecteddroplet must be taken into account, the appropriate equation is
D was first determined from t,,as
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where COis the concentration of Ch in the injected solution and b is given by (3V/4?ra)l13,where Vis the volume of the solutionejected from the micropipet. Parameter b is the radius of the injected droplet, taking into account the 20% volume fraction of the extracellular space, a. To employ eq 2, the diffusion coefficient determined from tmx in the manner described above was first adjusted according to eq A7 of ref 34. This correction takes into account the decreased distance that the Ch must diffuse to reach the microsensor due to the contribution of the radius of the injected droplet, b. Figure 9 compares the predictions of eqs 1 and 2 with the experimentally estimated in vivo Ch concentrations. Given the extent of approximation in both the experimental estimation of the Ch concentration and the theoretical predictions,the level of agreement between them is remarkable. Both eqs 1 and 2 involve a series of assumptions, discussed more fully in refs 34 and 41, that are not rigorously obeyed by the experiment. Equation 1 treats the injected droplet as
an ideal point source while eq 2 treats it as an ideal spherical source. The actual situation is likely somewhere between the two. Furthermore, both equations rigorously apply only when a well-defined distance, r, separates the source and the sensor. Since the sensors employed here are cylindrical with a length of 200-400 pm, the separation distance is not well-defined in these experiments. Also, both equations assume spherical symmetry and isotropic diffusion through tissue. Because of the number of assumptions involved, both equations have been used here only as guidelines in predicting experimental outcome. Nevertheless, the level of agreement between the experiments and theoretical predictions provides strong evidence that these sensors operate reliably in the complex extracellular environment of brain tissue. Finally, it should also be noted that neither equation takes into consideration the possibility that the concentration of Ch in brain ECF is influenced by neurochemical processes. Cholinergic neurons present in the brain region where these experiments were performed are known to exhibit Ch uptake, a process which is extremely important in regulating the levels of acetylcholine in the tissue. In these experiments, however, we have attempted to minimize the impact of Ch uptake, which has a Km of 1-5 pM,*O by injecting a very high concentration of Ch. It is interesting to note, however, that in both panels of Figure 9 the rate of disappearance of the Ch signal appears to be more rapid than either diffusion theory predicts. Whether or not this is a reflection of Ch uptake by cells in the vicinity of the microsensors will require further testing. Furthermore, all cells utilize Ch for the production of phosphatidylcholine, an important constituent of cell membranes, and the brain also has the unique ability to synthesize Ch”9 The experimental protocol used here may well prove to be a useful new way to study these phenomena in vivo.
CONCLUSIONS We have developed microsensors for Ch that have dimensions suitable for measurements in brain tissue. The goal of
this initial characterization of these microsensors was to identify the factors that influence the quantitative interpretation of amperometric signals recorded in vivo. The injection of a known amount of Ch at a site a known distance away from the microsensor was adopted as a method because the resulting concentration at the microsensor can be predicted. In vitro calibration experiments have shown that temperature, changes in sensitivity of the microsensor upon exposure to brain tissue, and presence of ascorbate are three important factors that influence the in vivo performance of the sensors. Taking these factors into account yields excellent agreement between measured signals and predicted concentrations. In addition, the excellent agreement seems to indicate that the availability of oxygen in brain tissue is adequate for the reliable operation of these sensors. If this had not been the case, then the high level of agreement between the measurements and predictions would not have been possible. Oxygen availability is expected to become even less of an issue when the sensors are used to measure more physiologically relevant concentrations of Ch, which are substantially lower than the concentration involved in these local injection experiments. The results reported in this paper motivate further efforts aimed toward reducing the Ch detection limit and interference by ascorbate.
ACKNOWLEDGMENT We acknowledge the University of Pittsburgh and the National Institute of Neurological Disorders and Stroke (Grant NS19608)for their financial support of this research. We also thank Dr. Stephen G. Weber for the use of his temperature controller.
Received for review April 29, 1994. Accepted June 30, 1994.O
.Abstract published in Aduance ACS Abstracts, August I , 1994.
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