Anal. Chem. 2004, 76, 1098-1106
Acetylcholine and Choline Amperometric Enzyme Sensors Characterized in Vitro and in Vivo Kim M. Mitchell*
Center for Neurobiology and Immunology Research and Department of Pharmacology and Toxicology, University of Kansas, Lawrence, Kansas 66047, and Higuchi Biosciences Center, 2099 Constant Avenue, Lawrence, Kansas 66047
Acetylcholine (ACh) and choline (Ch) are important neuroactive molecules, yet detection of these substances in vivo presents significant analytical challenges. New multienzyme amperometric biosensors are presented here with measurement of physiologically relevant levels of ACh and Ch in vivo. Poly(m-(1,3)-phenylenediamine) (pmPD) electropolymerized on a platinum iridium wire (Pt) served as a template for immobilization of enzymes. A multienzyme layer containing choline oxidase (ChOx) and ascorbic acid oxidase (AAO) for a Ch sensor or ChOx, acetylcholinesterase (AChE), and AAO for a ACh/Ch sensor was immobilized with bovine serum albumin by cross-linking with glutaraldeyhyde. The pmPD enzyme sensors displayed enhanced sensitivity, stability, and selectivity compared to the same multienzyme systems immobilized to solvent cast Nafion and cellulose acetatemodified Pt. Sensor response was linear up to 100 µM ACh or Ch. Detection limits were 0.66 ( 0.46 µM ACh and 0.33 ( 0.09 µM Ch, and response times were 33:1d***
228:1 ((61)*** nd, >71:1***
a Abbreviations given in text. Asterisks indicate significant difference by t-test compared to Nafion/CA sensor: * p < 0.01, ** p < 0.05, and *** p < 0.001. b Sensitivity as slope for 5-100 µM Ch and correlation coefficient, r2. Data given as mean ( SD, n ) 7. c Calculated as ratio of the Ch response to the extrapolated response for the interference at a concentration equal to that of Ch (10 µM). Data given as mean ( SD (n ) 4 or 5). d nd, >, interference was not detected, minimum possible ratio for a hypothetical interference response at the limit of detection (for a S/N ) 3).
to substrate tested from 5 to 100 µM. The Nafion/CA Ch sensors gave a significantly lower sensitivity to Ch compared to pmPD Ch sensors (p < 0.05) or pmPD ACh/Ch sensors (p < 0.001) as shown in Table 1. Surprisingly, the pmPD ACh/Ch sensor response to Ch was significantly greater than the pmPD Ch sensor response (p < 0.05) to Ch. However, an increase in sensitivity with time for sensors stored in buffer was greater for the pmPD Ch sensors than for pmPD ACh/Ch sensors such that after 2 weeks the sensitivities of the Ch sensors (0.16 ( 0.05 nC/µM Ch, mean ( SD, n ) 7) and ACh/Ch sensors (0.20 ( 0.11 nC/ µM Ch, mean ( SD, n ) 7) were comparable as discussed further below. The detection limits were determined from the slope for a signal-to-noise ratio (S/N) of 3. The choline sensor had a detection limit of 0.33 ( 0.09 µM choline (mean ( SD, n ) 7). The ACh/ choline sensor had detection limits of 0.55 ( 0.41 µM choline and 0.66 ( 0.46 µM ACh (mean ( SD, n ) 7). For an initial comparison between sensor types, the selectivity for enzyme substrate relative to endogenous electroactive species, AA and dopamine (DA), was determined for the Nafion/CA and pmPD sensor types as shown in Table 1. The pmPD sensors had significantly improved sensitivity and selectivity compared to the Nafion/CA sensors. The Nafion/CA sensors were not examined further, and the remainder of this work addresses characterization of pmPD Ch and ACh/Ch sensors. These sensors will be referred to as Ch or ACh/Ch sensors for the remainder of this report. Dynamics of Sensor Response. Hydrodynamic measurement of the sensor response to substrate up to a saturating concentration was performed in stirred aECF at 37 °C. Analysis of these data to determine the apparent Michaelis-Menton constant, Km′, was performed by two methods as described previously in the Experimental Section. The Km′ of the pmPD Ch sensor was 0.80 ( 0.16 mM Ch (mean ( SD, n ) 4) as determined by nonlinear regression analysis or 0.69 ( 0.12 mM Ch (mean ( SD, n ) 4) as determined by the slope of the Eadie-Hofstee plot, i.e., nC versus nC/[Ch]. The Km′ values determined by either Analytical Chemistry, Vol. 76, No. 4, February 15, 2004
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method were comparable, and only nonlinear regression analysis was subsequently used. The Km′ of the pmPD ACh/Ch sensor was 0.65 ( 0.46 mM for substrate Ch and 0.61 ( 0.41 mM for substrate ACh (mean ( SD, n ) 5). These Km′ values for the pmPD sensors were comparable to the reported Km for choline oxidase in solution of 0.87 mM Ch.41 The values indicate that the enzyme activity remained highly functional in the immobilized state. In addition, the values obtained for Km′ suggest that the enzymatic reaction is the rate-determining event for the sensor response, since the Eadie-Hofstee plot is linear with a negative slope of Km′. If the response depended primarily on diffusion, the Km′ would be considerably larger than the Km of the free enzyme and the Eadie-Hofstee plot would be nonlinear.42 ACh/Ch sensors generally had a high collection efficiency by immobilized ChOx for Ch produced by hydrolysis of ACh by coimmobilized AChE as demonstrated by several results. The sensitivity to ACh was nearly as great as that for Ch. In addition, the Km’s were similar for the ACh/Ch sensor response to Ch and ACh indicates that AChE kinetics were not the limiting factor for the ACh/Ch sensor response to ACh but rather the response rate was limited by ChOx activity. This is not surprising as the activity of AChE at 1000-2000 activity units min-1 mg-1 is so much greater than that of ChOx at 10.6 units min-1 mg-1. The effect of changes in O2 levels on the sensor response should be considered since ChOx requires O2 as a cofactor. Although the effect of different levels of O2 was not determined in the present study, the sensor response to Ch or ACh in vivo should not be greatly influenced by fluctuations in oxygen tension since the Km′ is much greater than physiological concentrations of Ch and ACh in brain ECF. In addition, changes in Ch and ACh concentrations are anticipated to be much lower than O2 concentration reported at ∼50 µM at the basal level43 and increased with evoked neuronal activity by as much as 60 µM.44 Further, the present results demonstrated a robust response of the Ch sensors in the rat hippocampus in vivo upon local pressure ejection of the enzyme substrates, ACh and Ch, as discussed below. Operational and Storage Stability. The effects of lifetime and storage conditions on pmPD sensor sensitivity were examined with results as shown in Table 2. Possible changes that would affect the sensitivity include changes in enzyme activity or permeability to H2O2. Thus, sensitivity to substrate and to H2O2 over time was determined and expressed as percent response compared to the initial sensitivity for each sensor determined on day 1, i.e., day of fabrication. The Ch sensors stored dry at room temperature (∼25 °C) had significantly decreased sensitivity to Ch and H2O2 by day 7. In contrast, Ch and ACh/Ch sensors stored in PBS buffer at 4 °C had significantly increased sensitivity to H2O2 after a period of 7 days after the initial test on day 1 (p < 0.05). The increase in sensitivity with time to Ch for Ch sensors stored in buffer may have been due to an increase in H2O2 permeability in the pmPD films due to film swelling. This phenomenon was previously reported as a significant increase in sensitivity to H2O2 by day 2 and the continued slight increase thereafter for Pt (41) Ohta-Fukuyama, M.; Miyake, Y.; Emi, S.; Yamano, T. J. Biochem. 1980, 88, 197-203. (42) Goldstein, L. Methods Enyzmol. 1976, 44, 397-450. (43) Nair, P. K.; Buerk, D. G.; Halsey, J. H., Jr. Stroke 1987, 18, 616-622. (44) Zimmerman, J. B.; Kennedy, R. T.; Wightman, R. M. J. Cereb. Blood Flow Metab. 1992, 12, 629-637.
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Table 2. Stability of Ch Sensor and ACh/Ch Sensor Response to Enzyme Substrate and H2O2 with Lifetime and Storage Conditionsa sensor storage day 3 day 7 day 17 day 3 day 7 day 17
Ch dry
Ch PBS
ACh PBS
% of Initial Sensor Response to Substrate 81 ((11) 97 ((16) 52 ((4)** 187 ((120)* 154 ((52) 208 ((117)* 147 ((54) % of Initial Sensor Response to H2O2 94 ((10) 63 ((31) 48 ((17)* 144 ((64)* 191 ((205)
87 ((31) 191 ((97)* 163 ((93)
a Data shown as mean ( SD, n ) 3 for dry storage at 25 °C, n ) 9 or 10 for storage in PBS 4 °C. Asterisk indicates significantly different from value at day 1: * p < 0.05 and ** p < 0.001.
Table 3. Selectivity Ratioa for Pmpd/ACh/Ch and Pmpd/ Ch Sensor Response to ACh or Ch versus Interference and Changes with Time Stored in PBS at 4 °C interference (µM tested) AA (200) DA (10) NE (10) 5-HT (10) DOPAC (50) 5-HIAA (25) L-Cys (10) urate (10)
ACh/Ch sensor ACh substrate day 1 day 7 164:1 ((79) nd,b >94:1 nd, >94:1 nd, >94:1 nd, >471:1 nd, >236:1 nd, >94:1 57:1 ((16)
50:1 ((15) nd, >70:1 nd, >69:1 nd, >71:1 nd, >397:1 nd, >199:1 nd, >74:1 33:1 ((12)
Ch sensor Ch substrate day 1 day 7 161:1 ((94) nd, >37:1 nd, >37:1 nd, >45:1 nd, >196:1 nd, >101: 1 nd, >45:1 54:1 ((25)
68:1 ((31) nd, >58:1 nd, >58:1 nd, >58: 1 nd, >292:1 nd, >146:1 nd, >58:1 33:1 ((12)
a Calculated as ratio of the Ch response to the extrapolated response for the interference at a concentration equal to that of Ch (10 µM). Data given as mean ( SD (n ) 7 or 8). Abbreviations given in text. b nd, >, interference was not detected, minimum possible ratio for a hypothetical interference response at the limit of detection (for a S/N ) 3).
electrodes with electropolymerized PD films stored in buffer.38 The present results suggest that pmPD Ch and ACh/Ch sensors retained enzyme activity when sensors were stored in buffer over the 17-day period examined. The results also suggest that the change in sensitivity of Ch sensors stored dry may result from decreased permeability to H2O2, although a decrease in enzyme activity could not be ruled out. Selectivity. The selectivity of the Ch and ACh/Ch sensors relative to a variety of potential interferences known to be present in the extracellular milieu as well as a variety of pharmacological agents commonly used in the examination of cholinergic physiology was tested in vitro in aECF at 37 °C. The dependence of selectivity with sensor lifetime was examined as some films are reported to provide stable selectivity over time, e.g., Nafion and CA,24 while others lose selectivity over time, e.g., pPD.38 The sensor selectivity was examined over time for Ch and ACh/Ch sensors stored in PBS at 4 °C after initial testing on day 1 with results shown in Table 3. The selectivity ratios for sensor response to Ch and ACh versus AA, urate and neurotransmitters, DA, norepinephrine (NE), serotonin (5-HT), and electroactive metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and 5-hydroxyindolacetic acid (5-HIAA) were determined. Other endogenous electroactive species, L-tyrosine, L-cysteine, L-tryptophan, and
L-glutathione,
were tested at 10 µM and only L-cysteine was detectable. The concentrations of potential interferences tested were at levels that occur in brain ECF. Some of the potential interferences were not detectable (nd) at the concentrations tested and the measured sensor sensitivity to enzyme substrate, Ch or ACh, was used to determine the minimum selectivity ratio for a hypothetical response to an interference at the detection limit for a S/N ) 3. The electroactive neurotransmitters, DA, NE, and 5-HT, are typically present at concentrations much lower than 10 µM and therefore should have little effect on sensor response in vivo when not detected at this level in vitro. The endogenous electroactive species AA and urate are usually those that present the greatest potential interference. The typical ECF concentrations of AA fluctuate in the range 100-300 µM.33 Urate has been demonstrated to vary in concentration from basal levels of ∼5 µM that may increase to 50 µM under pathological conditions.45 The effect of some pharmacological agents commonly used to examine ACh and Ch neurochemistry was tested at concentrations typically applied in physiological studies for both a direct sensor response and a possible effect on the sensor response to enzyme substrate. The AChE inhibitors, physostigmine and neostigmine, and the Ch uptake inhibitor, hemicholinium-3, were tested at 1 µM. The nicotinic antagonists, dihydro-β-erythroidine (1 µM), methyllycaconitine (1 µM), and mecamylamine (10 µM), and nicotinic agonist, nicotine (10 µM), were also tested for a potential effect on the sensor response. The muscarinic agonist, acetyl-βmethylcholine chloride (10 µM), muscarinic antagonist, atropine (1 µM), and the sodium channel blocker, tetrodotoxin (1 µM), were also tested. None of the these pharmacological agents produced a sensor response directly or any change in sensor response to the ACh or Ch. Nicotine produced no direct response from the sensors, whereas at high concentrations (100 µM), it slightly diminished the sensor response to Ch and ACh. The reason for this effect may be attributable to interference with the active site of the enzymes.46 Similarly, the AChE inhibitor, physostigmine, did not produce a direct sensor response but at higher concentration (2.5 µM) produced a slightly diminished sensor response to ACh as discussed further below. Sensors generally retained selectivity for up to 1 week with some selectivity loss for substrate relative to AA and urate. Sensors selected for use in vivo should be those with highest selectivity. Sensors should be calibrated after use in vivo for sensitivity and selectivity. The conditions of the in vivo environment should be taken into account; e.g., the selectivity for sensor substrate relative to AA should be of particular concern under pathological conditions such as ischemia where large changes in AA occur.33 Temperature Dependence. The temperature dependence of the Ch sensor the ACh/Ch sensor response for Ch and ACh, respectively, was examined at 25 and 37 °C with typical results shown in Figure 2. The sensitivity of the sensors was enhanced with an increase in temperature. Linearity of response from 0 to 100 µM was observed at both temperatures. The results demonstrate that accurate correlation of sensor response in vivo to calibration response in vitro requires measurement at 37 °C. pH Dependence. The pH dependence of the Ch sensor response to Ch was determined for every 0.5 pH unit over the (45) O’Neill, R. D.; Lowry, J. P. Behav. Brain Res. 1995, 71, 33-49. (46) Nwosu, T. N.; Palleschi, G.; Mascini, M. Anal. Lett. 1992, 25, 821-835.
Figure 2. Temperature dependence of sensor response. The response to ACh for the ACh/Ch sensor (upper panel) and the response to Ch for the Ch sensor (lower panel) are shown at 25 (circle b) and 37 °C (square 9). Table 4. pH Dependence of Ch Sensor Response to Ch Percent of Sensor Response Compared to pH 7.4a pH
6.5
7.0
7.2
7.3
7.5
7.6
8.0
8.5
%
31 ((6)
62 ((8)
76 ((12)
88 ((17)
101 ((3)
102 ((11)
148 ((5)
177 ((15)
a
Data given as mean ( SD (n ) 3).
pH range of 6.5-8.5 and for every 0.1 pH unit from pH 7.2-7.6 in PBS with results as shown in Table 4. The optimum pH for the free enzymes is between 7.0 and 8.0 for ChOx41 and between 8.0 and 9.0 for AChE.47 The pH in the ECF is maintained near pH 7.4 with some variation such as that associated with neuronal activity.48 The sensor response varied to a small extent with pH changes in the physiological range and should be considered when conditions in vivo would be expected to produce large changes, e.g., during ischemia, that correspond to several tenths of a pH unit.48 Response Time. The sensor response times were determined by FIA as the time to reach 90% of the maximal response (t90) and were 0.45 s for H2O2 (100 µM), 0.50 s for Ch (500 µM), and 0.75 s for ACh (500 µM). These values demonstrated a relatively rapid response time well suited to monitor chemical changes in the ECF. Effect of AA on Sensor Response. Interference by AA due to direct oxidation at the Pt surface was minimized by AAO included in the immobilized enzyme layer. Another potential source of interference by AA is the reduction of H2O2 produced by the enzyme reaction,34 in competition with oxidation at the Pt surface. The immobilized AAO served to minimize that interference from AA as demonstrated by several experiments. First, it was demonstrated that AA in solution at a concentration similar to endogenous levels affected the sensor response to H2O2 added to solution. The ratio for Ch sensor response to added H2O2 (5 (47) Nachmansohn, D.; Wilson, I. B. In Advances in Enzymology; Nord, F. F., Ed.; Interscience Publisher: New York, 1991; Vol. 12, p 259. (48) Chesler, M.; Kaila, K. Trends Neurosci. 1992, 15, 396-402.
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µM) in the presence of 200 µM AA to the response to added H2O2 without AA in the buffer was 0.47 ( 0.06 (n ) 4). Thus, this level of AA effectively diminished solution H2O2 at a concentration near to that estimated to be produced by immobilized ChOx in response to basal endogenous levels of Ch. Next, different concentrations of AA typical of changes in ECF were tested for an effect on the sensor response to enzyme substrate. The ratio of response of the Ch sensor to 10 µM Ch in the presence of different concentrations of AA compared to the response without AA present was for concentrations of AA of 100, 200, and 300 µM, 1.14 ( 0.05, 1.13 ( 0.1, and 1.18 ( 0.14, respectively (mean ( SD, n ) 3). Further, addition of catalase (200 U), which catalyzes the conversion of H2O2 to H2O, during the in vitro calibration had no effect on Ch or ACh response but eliminated the response to H2O2 that was added to the buffer. These results indicate that the H2O2 generated by the immobilized ChOx enzyme was effectively oxidized at the surface of the electrode prior to diffusion away from the sensor or reduction by AA in solution. Effect of Tissue Exposure on Sensor Response. The sensitivity of the sensors to the enzyme substrates was determined prior to and after use in vivo to account for any changes in sensor response following tissue exposure, e.g., due to adsorption of protein. The sensitivity to the enzyme substrates was diminished during the initial 30 min of exposure to tissue and remained at a constant level thereafter as determined by intermittent in vitro calibrations performed over the course of in vivo experiments. The fraction of the initial sensor sensitivity obtained in vitro after tissue exposure relative to sensitivity prior to tissue implantation was 76 ( 14% (mean ( SD, n ) 5) for Ch sensor response to Ch and 71 ( 1% (mean ( SD, n ) 3) for ACh/Ch sensor response to ACh. The time course and magnitude of the changes in the ACh/ Ch and Ch sensor response with tissue exposure are similar to those measured previously for other electrochemical sensors used in vivo.49-51 Since changes in sensor sensitivity are rapid and stable thereafter indicates that the sensors can provide a reliable measure of in situ chemical changes that should be correlated with in vitro calibration after tissue exposure. The effect of ACh on Ch sensor response after tissue exposure was tested since a previous study demonstrated that other Ch oxidase sensors developed sensitivity to ACh after exposure to brain tissue ostensibly due to adsorption of endogenous AChE.11 In the present study, some Ch sensors also developed sensitivity to ACh after exposure to brain tissue as demonstrated by simultaneous in vitro calibration of ACh/Ch and Ch sensor response to ACh and Ch after ∼3 h in vivo as shown in Figure 3. The acquired sensitivity to ACh was ∼12% of the sensitivity to Ch for this particular Ch sensor. A total of 11 sensors each implanted in a different rat brain in DG for several hours resulted in 8 sensors that showed no response to ACh (10 µM) and 3 sensors that acquired sensitivity to ACh that was 12 ( 6% of the sensitivity to Ch (mean ( SD; n ) 3). The hypothesis that AChE adsorption to the sensor surface was responsible for the acquired sensitivity of the Ch sensor to ACh was tested. Calibration of the (49) Capella, P.; Ghasemzadeh, B.; Mitchell, K.; Adams, R. N. Electroanalysis 1990, 2, 175-182. (50) Logman, M. J.; Budygin, E. A.; Gainetdinov, R. R.; Wightman, R. M. J Neurosci. Methods 2000, 95, 95-102. (51) Kulagina, N. V.; Shankar, L.; Michael, A. C. Anal. Chem. 1999, 71, 50935100.
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Figure 3. In vitro calibration of Ch and ACh/Ch sensors after exposure to brain tissue. Time of addition of small-volume aliquots of ACh or Ch standards to a final concentration of 10 µM in aECF at 37 °C is shown by the arrowheads.
Ch sensor in vitro after in vivo use showed that an acquired response to ACh was eliminated after the addition of physostigmine (to total 2.5 µM) to the buffer. The effect of physostigmine on the ACh/Ch sensor was determined to be a small decrease in response to ACh by 14.8 ( 3.3% (mean ( SD, n ) 3). The effect of physostigmine on the ACh/Ch sensor and the Ch sensor with AChE adsorbed was slow to develop (20-min exposure for maximal effect) and was reversible after 12-h immersion in fresh buffer. The possibility that ACh sensitivity acquired in vivo might be reduced during calibration, e.g., AChE adsorbed might desorb, was also examined. Calibration performed immediately, i.e., within a few minutes after removal of the sensors from brain tissue or after immersion in aECF overnight, showed no change in sensitivity to ACh, suggesting that desorption of AChE from the sensor surface during calibration was unlikely. The ACh sensitivity acquired with brain tissue exposure is a serious impediment to use of a differential measurement between adjacent ACh/Ch and Ch sensors to evaluate in situ changes in ACh. However, the majority of Ch sensors showed no detectable sensitivity to ACh (10 µM), and for those that did, the fraction of the acquired ACh sensitivity of sensors relative to Ch sensitivity was relatively small. In this work, experimental results in vivo were excluded if the Ch sensor demonstrated any sensitivity to 10 µM ACh on calibration. The preliminary evaluation of the sensors in vivo included differential measurements as discussed further below. Since adsorption of endogenous AChE was shown to cause an acquired ACh sensitivity by some Ch sensors, it should be less of a problem in physiological systems that contain little to no AChE. Potential interference by acquired ACh sensitivity could possibly be minimized by addition of an outer membrane such as polyurethane to minimize protein adsorption on the sensor surface.52 In Vivo Measurement. The initial examination of sensor performance in vivo was conducted with consideration of characteristics that are significant for reliable and interpretable measurements in the physiological system. The sensor characteristics demonstrated included the following: sensitivity sufficient to measure physiologically relevant changes in Ch and ACh in (52) Cha, G. S.; Liu, D.; Meyerhoff, M. E.; Cantor, H. C.; Midgley, A. R.; Goldberg, H. D.; Brown, R. B. Anal. Chem. 1991, 63, 1666-1672.
Figure 4. Ch sensor response in vivo to exogenous Ch or ACh (10 mM) applied locally by pressure ejection (2 s, 20 psi) from a pipet located 350 µm from the Ch sensor at the time shown by the bar.
the in vivo environment; reliable estimation of in vivo concentration by in vitro calibration; adequacy of levels of endogenous O2, cosubstrate for ChOx, for sensor operation; and adequacy of sensor selectivity in the complex in vivo environment. These issues were addressed with in vivo measurements of exogenous Ch and ACh applied by pressure ejection and of basal, depolarization-evoked, and pharmacologically modified endogenous Ch and ACh. The sensor response measured in vivo was correlated to concentration determined by calibration in aECF at 37 °C after tissue exposure. Exogenous ACh and Ch applied by pressure ejection was monitored in the anesthetized rat DG. The Ch sensor located 350 µm distant from a multibarrel pipet detected ACh and Ch applied by pressure ejection as shown in Figure 4. Ch applied by pressure ejection (10 mM, 2 s, 20 psi) was detected almost immediately by the Ch sensor and increased rapidly to the peak response equivalent to 99 µM Ch. For comparison, pressure ejection of ACh (10 mM, 2 s, 20 psi) was detected as Ch at a later time following hydrolysis of ACh by extracellular AChE and yielded a peak concentration of 85 µM Ch. The data shown in Figure 4 are the average of three responses to Ch or ACh. The temporal differences in the onset and peak of the sensor response presumably reflect the time required for hydrolysis of ACh to Ch in vivo. The present results are in general agreement with those obtained with other Ch electrochemical sensors in vivo.10 This result suggests that endogenous O2 was sufficient to act as the cosubstrate for immobilized ChOx necessary to sensor performance. The effect of endogenous changes in O2 on sensor response should be addressed in greater detail in future work. Endogenous basal levels of [Ch] in the ECF were determined in the present study by obtaining the difference between a sensor for Ch and a “blank” sensor, i.e., identical construction minus ChOx. The Ch and blank sensor tips were located at 100-µm distance from one another in vivo. Basal Ch was determined as 7.3 ( 3.2 µM (n ) 5). The present results are comparable to previously reported determination of basal Ch in ECF of 6.7 µM Ch53 and in the range 6.7-13.3 µM Ch54 by HPLC/EC and of 6.6 ( 2.9 µM (n ) 5) by electrochemical detection in rat brain based on the differential measurement between adjacent sensors with (53) Klein, J.; Gonzalez, R.; Koppen, A.; Loffelholz, K. Neurochem. Int. 1993, 22, 293-300. (54) Nilsson, O. G.; Kalen, P.; Rosengren, E.; Bjorklund, A. Neuroscience 1990, 36, 325-338.
Figure 5. Changes in basal ACh and Ch in vivo with AChE inhibition. Responses are shown for (a) ACh/Ch and (b) Ch sensors located within 1 mm of a dialysis probe. The AChE inhibitor, neostigmine (10 µM), was added to the perfusate for the time shown by the bar. Subtraction of the Ch component from the ACh/Ch sensor response represents the change in ACh.
and without immobilized ChOx.10 Basal ACh levels reported previously have been mostly obtained by microdialysis with an inhibitor of AChE in the perfusate. In the presence of AChE inhibitor, neostigmine (10 µM), basal ACh in rat hippocampus has been reported to be ∼0.72 µM54 while basal ACh without AChE inhibition was reported at ∼0.06 µM.54 In the same study, basal Ch decreased by an average 25-35% upon addition of neostigmine from a basal range of 6.7-13.3 µM Ch prior to AChE inhibition.54 Basal extracellular levels of ACh without AChE inhibition are below the detection limit of the present ACh/Ch sensors. To examine basal ACh, the AChE inhibitor, neostigmine (10 µM), was delivered by microdialysis perfusion into the brain parenchyma at ∼1-mm distance from Ch and ACh/Ch sensors. The change in basal ACh was determined by subtraction of the response attributable to Ch as determined with the Ch sensor. The change in basal ACh and Ch upon dialysis perfusion with 10 µM neostigmine was an increase of ∼1 µM ACh and a decrease by ∼6 µM Ch as shown in Figure 5. The calculations were performed as follows.
Ch sensor: δ nCin vivo ÷ nC/µM Chin vitro ) δ µM Chin vivo δ µM Chin vivo × nC/µM Chin vitro at ACh/Ch sensor ) δ nC at ACh/Ch sensor due to change in µM Chin vivo ACh/Ch sensor: δ nC δ nC at ACh/Ch sensor due to change in µM Chin vivo ) δ nC at ACh/Ch sensor due to change in µM ACh in vivo δ nC at ACh/Ch sensor due to change in µM ACh × nC/µM AChin vitro at ACh/Ch sensor ) δ µM ACh in vivo
Stimulated increases of endogenous ACh and Ch in the ECF evoked by application of the general depolarizing agent, K+, were recorded from adjacent ACh/Ch and Ch sensors in the DG as shown in Figure 6. K+ was administered by microinjection (200 nL) from a microsyringe located ∼400 µm distant from the sensors. The response at the ACh/Ch sensor was due to a Analytical Chemistry, Vol. 76, No. 4, February 15, 2004
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by other investigators produced results comparable to those obtained here, although AChE inhibition was employed in the former. Addition of KCl (100 mM) to the perfusion fluid in the presence of 10 µM neostigmine resulted in a 3-fold increase in extracellular ACh and an elevation of extracellular Ch by ∼50%.54
Figure 6. In vivo measurement of K+-evoked increases in endogenous ACh and Ch. Responses to microinjection of 100 mM K+ (200 nL) in the hippocampal dentate gyrus obtained from adjacent sensors for ACh/Ch and Ch are shown in (A). The concentrations of Ch and ACh shown in (B) were determined from the data in (A) and calibration data where ACh was obtained as a differential measurement. See text for method of calculation.
contribution from both ACh and Ch. The portion of the response of the ACh/Ch sensor due to ACh was estimated by subtraction of the Ch response using the sensitivity of the ACh/Ch sensor to Ch and the Ch concentration determined by the Ch sensor response in vivo. Similar results were obtained in three animals with an average increase of 26 ( 8 µM ACh and 15 ( 10 µM Ch (mean ( SD) in response to microinjection of K+. These changes in extracellular ACh and Ch with K+ depolarization were obtained without AChE inhibition. Microdialysis studies in rat hippocampus
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CONCLUSIONS Microsensors for Ch and ACh/Ch were developed for use in the physiological system and were extensively characterized in vitro. Preliminary characterization of sensor performance in vivo demonstrated measurement of physiologically relevant concentration changes. The present method of electropolymerization of a nonconducting polymer, pmPD, onto a Pt surface and subsequent enzyme immobilization by chemical cross-linking with glutaraldehyde produced sensors with rapid response time (
