Polymer−Enzyme Composite Biosensor with High Glutamate

Examples of unfiltered data recorded amperometrically (+700 mV vs SCE) at 10 Hz with ... Inset: closeup of the region 0−50 μM Glu for the cylin...
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Anal. Chem. 2005, 77, 1196-1199

Polymer-Enzyme Composite Biosensor with High Glutamate Sensitivity and Low Oxygen Dependence Colm P. McMahon and Robert D. O’Neill*

Chemistry Department, University College Dublin, Belfield, Dublin 4, Ireland

Two first-generation glutamate (Glu) biosensors are described: one based on a Pt cylinder (125-µm diameter, 1-mm length); the other on a Pt disk (125-µm diameter) with a 30 times smaller surface area. Both designs incorporated the enzyme Glu-oxidase in a polymer (polyo-phenylenediamine) matrix deposited on the Pt surface. Surprisingly, the smaller disk biosensor displayed a combination of higher Glu current density and lower oxygen dependence compared with the cylinder design. An analysis to estimate the oxygen interference in the Glu signal showed that 90% of the disk biosensor current for 10 µM Glu remains on changing the dissolved oxygen concentration from 200 to 5 µM. These results indicate that brain Glu monitoring in vivo using this design, combined with an enzyme-inactive sensor for differential elimination of electroactive interference, can now be explored without significant influence by fluctuating tissue pO2. The development of devices for monitoring L-glutamate (Glu) on-line has become an vibrant research area due to the important role this amino acid plays in a range of complex matrixes, including food processing,1,2 cell cultures,3-5 tissue slices ex vivo,6,7 and intact brain in vivo.8-13 As an excitatory amino acid, Glu is * Author to whom correspondence should be addressed. Fax: +353-1-7162127. Tel: +353-1-7162314. E-mail: [email protected]. (1) Moser, I.; Jobst, G.; Urban, G. A. Biosens. Bioelectron. 2002, 17, 297-302. (2) Nakorn, P. N.; Suphantharika, M.; Udomsopagit, S.; Surareungchai, W. World J. Microbiol. Biotechnol. 2003, 19, 479-85. (3) Kurita, R.; Hayashi, K.; Torimitsu, K.; Niwa, O. Anal. Sci. 2003, 19, 15815. (4) O’Neill, R. D.; Chang, S. C.; Lowry, J. P.; McNeil, C. J. Biosens. Bioelectron. 2004, 19, 1521-8. (5) Mikeladze, E.; Schulte, A.; Mosbach, M.; Blochl, A.; Csoregi, E.; Solomonia, R.; Schumann, W. Electroanalysis 2002, 14, 393-9. (6) Qhobosheane, M.; Wu, D. H.; Gu, G. R.; Tan, W. H. J. Neurosci. Methods 2004, 135, 71-8. (7) Isobe, Y.; Nishihara, K. Brain Res. Bull. 2002, 58, 401-4. (8) Burmeister, J. J.; Gerhardt, G. A. Trends Anal. Chem. 2003, 22, 498-502. (9) Matsushita, Y.; Shima, K.; Nawashiro, H.; Wada, K. J. Neurotrauma 2000, 17, 143-53. (10) Hu, Y.; Mitchell, K. M.; Albahadily, F. N.; Michaelis, E. K.; Wilson, G. S. Brain Res. 1994, 659, 117-25. (11) Burmeister, J. J.; Pomerleau, F.; Palmer, M.; Day, B. K.; Huettl, P.; Gerhardt, G. A. J. Neurosci. Methods 2002, 119, 163-71. (12) Cui, J.; Kulagina, N. V.; Michael, A. C. J. Neurosci. Methods 2001, 104, 183-9. (13) Kulagina, N. V.; Shankar, L.; Michael, A. C. Anal. Chem. 1999, 71, 5093100.

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the most widespread neurotransmitter in the mammalian CNS,14 plays a major role in a wide range of brain functions, and has been implicated in a number of neurological disorders.15 Systems for monitoring Glu in brain extracellular fluid (ECF) have therefore been a key goal in the analytical and neurobiological sciences in recent years. There have been two main approaches to the detection and quantification of brain ECF Glu concentrations: those using microdialysis perfusion followed by ex situ analysis16-19 and, less commonly, those involving direct detection of Glu in the ECF using implanted amperometric biosensors.10,11,13 The relative advantages and drawbacks of these two approaches have been reviewed,20,21 but clearly, the high spatial and temporal resolutions provided by electrochemical sensors is particularly appealing for studies of the neurochemical correlates of behavior.22-26 However, although considerable effort has gone into minimizing contamination of biosensor signals by endogenous electroactive species present in brain ECF,20,27,28 the problem of interference by fluctuating levels of the cosubstate (molecular oxygen, reaction 2) of the most commonly used enzyme in Glu biosensors, L-glutamate oxidase (GluOx), has not been addressed in detail. Normally, air-equilibrated buffer is used as a calibration medium,4,13 and dissolved oxygen levels above 40 µM have been identified as being adequate for oxygen-independent signals.10,29 (14) Orrego, F.; Villanueva, S. Neuroscience 1993, 56, 539-55. (15) Belsham, B. Hum. Pharmacol. Clin. Exp. 2001, 16, 139-46. (16) Hutchinson, P. J.; O’Connell, M. T.; Kirkpatrick, P. J.; Pickard, J. D. Physiol. Meas. 2002, 23, R75-109. (17) Westerink, B. H. C.; Timmerman, W. Anal. Chim. Acta 1999, 379, 26374. (18) Fillenz, M. Behav. Brain Res. 1995, 71, 51-67. (19) Benveniste, H. J. Neurochem. 1989, 52, 1667-79. (20) O’Neill, R. D.; Lowry, J. P.; Mas, M. Crit. Rev. Neurobiol. 1998, 12, 69127. (21) Khan, A. S.; Michael, A. C. Trends Anal. Chem. 2003, 22, 503-8. (22) Lowry, J. P.; McAteer, K.; El Atrash, S. S.; Duff, A.; O’Neill, R. D. Anal. Chem. 1994, 66, 1754-61. (23) Lowry, J. P.; Fillenz, M. Bioelectrochemistry 2001, 54, 39-47. (24) Garris, P. A.; Budygin, E. A.; Phillips, P. E. M.; Venton, B. J.; Robinson, D. L.; Bergstrom, B. P.; Rebec, G. V.; Wightman, R. M. Neuroscience 2003, 118, 819-29. (25) Robinson, D. L.; Phillips, P. E. M.; Budygin, E. A.; Trafton, B. J.; Garris, P. A.; Wightman, R. M. Neuroreport 2001, 12, 2549-52. (26) Roitman, M. F.; Stuber, G. D.; Phillips, P. E. M.; Wightman, R. M.; Carelli, R. M. J. Neurosci. 2004, 24, 1265-71. (27) Voltammetric Methods in Brain Systems, Humana Press: Totowa, NJ, 1995. (28) Pantano, P.; Kuhr, W. G. Electroanalysis 1995, 7, 405-16. (29) Kenausis, G.; Chen, Q.; Heller, A. Anal. Chem. 1997, 69, 1054-60. 10.1021/ac048686r CCC: $30.25

© 2005 American Chemical Society Published on Web 01/11/2005

The oxidative deamination of Glu, catalyzed by GluOx,30 can be represented by the following steps: L-glutamate

+ H2O + GluOx/FAD f

GluOx-based first-generation biosensor in terms of an apparent Michaelis-Menten constant for oxygen, KM(O2), to determine whether this design is suitable for Glu monitoring in media with low dissolved oxygen levels (eq 4). Surprisingly, miniaturization

R-ketoglutarate + NH3 + GluOx/FADH2 (1) GluOx/FADH2 + O2 f GluOx/FAD + H2O2

(2)

The H2O2 produced in reaction 2 can be oxidized, usually amperometrically, either directly on the electrode surface at relatively high applied potentials (reaction 3)31,32 or catalytically at lower potentials.5,13

H2O2 f O2 + 2H+ + 2e

IGlu )

IGlumax 1 + KM(O2)/[O2]

(4)

of Pt/GluOx/PPD-BSA electrodes led to increased Glu current density coupled with a lower oxygen dependence that provides improved performance for Glu monitoring in oxygen-challenged systems.

(3)

The oxygen dependence of a “first-generation” biosensor designed for monitoring brain glucose has been characterized in vitro and in vivo.33 Based on Pt modified with a polymer-protein composite layer consisting of glucose oxidase (GOx), poly(o-phenylenediamine) (PPD), and an albumin (BSA), this Pt/GOx/PPD-BSA biosensor was shown not to suffer from significant oxygen interference for glucose concentrations relevant to brain ECF in vivo. Biosensors for Glu of a similar design (Pt/GluOx/PPDBSA)32 have shown promising responses in monitoring brain Glu in alert rats implanted with probes of moderate sensitivity (0.02 µA cm-2 µM-1),34 whereas sensors of lower sensitivity (0.01 µA cm-2 µM-1) failed to detect Glu changes associated with mild behavioral stimulation (10-s tail pinch). 35 Moreover, that latter study and others36,37 have indicated that brain H2O2 may represent a potential interference for first-generation biosensors targeting analytes with low ECF concentrations, such as Glu. It is clear, therefore, that successful monitoring of brain Glu using amperometric biosensors in vivo should involve the use of coupled “blank” (enzyme inactive) sensors for differential measurements, a strategy already employed in a number of biosensor applications to minimize interference from electroactive species reacting directly on the electrode surface.11,13,31,38 Such “difference” Glu signals may, however, be susceptible to interference by limitations in oxygen availability. Even “second-generation” biosensors incorporating a redox mediator to replace dioxygen in reaction 2 may be prone to oxygen interference due to competition between the mediator and solution oxygen for the FADH2 moiety.39 The work presented here, therefore, aims to quantify the oxygen dependence of a (30) Kusakabe, H.; Midorikawa, Y.; Fujishima, T.; Kuninaka, A.; Yoshino, H. Agric. Biol. Chem. 1983, 47, 1323-8. (31) Cosnier, S.; Innocent, C.; Allien, L.; Poitry, S.; Tsacopoulos, M. Anal. Chem. 1997, 69, 968-71. (32) Ryan, M. R.; Lowry, J. P.; O’Neill, R. D. Analyst 1997, 122, 1419-24. (33) Dixon, B. M.; Lowry, J. P.; O’Neill, R. D. J. Neurosci. Methods 2002, 119, 135-42. (34) Lowry, J. P.; Ryan, M. R.; O’Neill, R. D. Anal. Commun. 1998, 35, 87-9. (35) Lowry, J. P.; Ryan, M. R.; O’Neill, R. D. Monitoring Molecules in Neuroscience, O’Connor, W. T.; Lowry, J. P.; O’Connor, J. J. O’Neill, R. D., Eds.; National University of Ireland: Dublin, 2001. (36) Kulagina, N. V.; Michael, A. C. Anal. Chem. 2003, 75, 4875-81. (37) Chen, B. T.; Avshalumov, M. V.; Rice, M. E. J. Neurophysiol. 2002, 87, 1155-8. (38) Boutelle, M. G.; Stanford, C.; Fillenz, M.; Albery, W. J.; Bartlett, P. N. Neurosci. Lett. 1986, 72, 283-8. (39) Martens, N.; Hindle, A.; Hall, E. A. H. Biosens. Bioelectron. 1995, 10, 393403.

EXPERIMENTAL SECTION Pt cylinders (PtC, 125-µm diameter, 1-mm length) were fabricated from Teflon-coated Pt wire. GluOx (EC 1.4.3.11, 200 units mL-1, Yamasa Corp.) was deposited onto the metal surface by dip-evaporation and immobilized by amperometric electropolymerization (+700 mV vs SCE) in 300 mM o-phenylenediamine, containing 5 mg mL-1 bovine serum albumin in phosphatebuffered saline (PBS, pH 7.4),40 as described previously to form PtC/GluOx/PPD-BSA biosensors.32 Pt disks (PtD) were fabricated by cutting the Teflon-coated wire transversely to produce 125µm-diameter disks, and PtD/GluOx/PPD-BSA biosensors fabricated in the same way as for PtC. After rinsing and a settling period at 700 mV in fresh PBS, calibrations were carried out to determine the sensitivity of the biosensors to Glu. A self-calibrating commercial membrane-covered amperometric oxygen sensor (∼1-cm diameter) was used to quantify solution oxygen concentration as described recently.33 The model used was a CellOx 325 connected to an Oxi 340A meter (Wissenschaftlich-Technische Werksta¨tten GmbH from Carl Stuart Ltd., Dublin, Ireland), incorporating a temperature probe for automatic compensation. Reliable quantification of O2 using this device required constant stirring of the solution at a rate of ∼3 Hz. The sensor range was 0.0-199.9% O2 (100% corresponding to air saturation) with a resolution of 0.1%. This percentage was converted to an estimated concentration of O2 by taking 200 µM to correspond to 100%.41,42 All experiments were performed in a standard three-electrode glass electrochemical cell containing 20 mL PBS at room temperature. A saturated calomel electrode (SCE) was used as the reference electrode, and a large stainless steel needle served as the auxiliary electrode. The applied potential for amperometric polymerizations and calibrations was +700 mV versus SCE. In oxygen dependence studies, to avoid contamination of the PBS by oxygen, the electrochemical cell was contained within an Atmosbag (Sigma),33 a two-hand 0.003-in.-gauge polyethylene bag that was sealed and filled with N2 during experiments, inflating to a volume of 280 L. After adding an aliquot of Glu, air was allowed into the system slowly by opening the bag slightly. Oxygen sensor data and biosensor data were recorded simultaneously through the transition from N2 saturation to air saturation. (40) Craig, J. D.; O’Neill, R. D. Analyst 2003, 128, 905-11. (41) Bourdillon, C.; Thomas, V.; Thomas, D. Enzyme Microb. Technol. 1982, 4, 175-80. (42) Zhang, Y. N.; Wilson, G. S. Anal. Chim. Acta 1993, 281, 513-20.

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Nonlinear regression analysis of the oxygen dependence of the Glu signal was carried out using a software package (Prism, GraphPad Software, Inc., San Diego, CA) and a Michaelis-Menten type equation (eq 4), whereas linear regression was performed on the Glu concentration dependence of the biosensor signal following conversion to current density. Data are presented as mean ( SEM, with n the number of biosensors. RESULTS AND DISCUSSION The concentration of Glu in bioanalytical applications rarely exceeds 100 µM. For example, values reported under physiological conditions are normally below 10 µM for cerebrospinal fluid43,44 and brain ECF,45-47 although 100 µM levels have been detected in the ECF following brain trauma.48 Calibrations were therefore performed in the range 0-150 µM Glu in quiescent air-saturated PBS to compare the Glu sensitivity of the two different biosensor designs. For the previously reported cylinder configuration,32,34,35 the PtC/GluOx/PPD-BSA biosensors characterized here showed linear responses to Glu in this concentration range: 8.7 ( 1.0 nA cm-2 µM-1, R2 ) 0.961, n ) 8. This level of Glu sensitivity was similar to that of a sensor used in a previous unsuccessful attempt to monitor brain ECF Glu.35 A new design was therefore adopted with the aim of increasing enzyme loading. Drops of GluOx solution were deposited onto the disk end of Teflon-coated Pt wire by dipping the wire vertically into the enzyme solution, removing it, and allowing to dry. These PtD/GluOx/PPD-BSA electrodes (1.23 × 10-4 cm2) were 32 times smaller than the corresponding cylinder sensors and showed significantly increased linear sensitivity to Glu: 32 ( 3 nA cm-2 µM-1, R2 ) 0.993, n ) 13. Thus, both the geometric area and the linear sensitivity of these PtDbased biosensors were similar to the more complicated carbon fiber cylinder designs involving catalytic oxidation of H2O2 using redox polymers and horseradish peroxidase.13 The oxygen dependence of the cylinder and disk biosensors was studied over the same Glu concentration range 0-150 µM. Figure 1 shows the effect of changing the concentration of oxygen from submicromolar levels to 50 µM on the biosensor signal recorded for 50 µM Glu. Here we quantified the oxygen dependence as KM(O2) defined in eq 4. The oxygen dependence was significantly greater for the cylinder-based electrode, KM(O2) ) 12 ( 1 µM (n ) 8) compared with 3 ( 1 µM (n ) 13) for PtD/ GluOx/PPD-BSA, p < 0.001. As shown for PtC/GOx/PPD-BSA glucose biosensors recently,33 the oxygen dependence of these first-generation devices is expected to increase with increased turnover of the enzyme associated with higher substrate concentrations. The KM(O2) values determined up to 150 µM Glu for PtD/GluOx/PPD-BSA and PtC/GluOx/PPD-BSA sensors are shown in Figure 2. There was a linear increase in KM(O2) for both sensor types, with the cylinder design displaying a significantly higher slope (p < 0.001). Using the slopes shown in Figure 2, and KM(O2) values for other biosensors of the same design at a variety of Glu concentra(43) Castillo, J.; Davalos, A.; Lema, M.; Serena, J.; Noya, M. Cerebrovasc. Dis. 1997, 7, 245-50. (44) Ince, E.; Karagoel, U.; Deda, G. Acta Paediatr. 1997, 86, 1333-6. (45) Segovia, G.; Porras, A.; Mora, F. Neurochem. Res. 1997, 22, 1491-7. (46) Lada, M. W.; Kennedy, R. T. Anal. Chem. 1996, 68, 2790-7. (47) Miele, M.; Berners, M.; Boutelle, M. G.; Kusakabe, H.; Fillenz, M. Brain Res. 1996, 707, 131-3. (48) Davalos, Antoni; Shuaib, Ashfaq; Wahlgren, Nils Gunnar J. Stroke Cerebrovasc. Dis. 2000, 9, 2-8.

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Figure 1. Examples of unfiltered data recorded amperometrically (+700 mV vs SCE) at 10 Hz with a PtD/GluOx/PPD-BSA (top) and PtC/GluOx/PPD-BSA (bottom) biosensor in 50 µM Glu expressed as a percentage of the maximum current (Imax, eq 4) and plotted against oxygen concentration recorded simultaneously using a CellOx sensor. The curves in each case represent the nonlinear regression analysis for eq 4. Regression parameters obtained for these examples: KM(O2) ) 2.3 ( 0.1 µM (arrow, R2 ) 0.976) for PtD/GluOx/ PPD-BSA; KM(O2) ) 10.8 ( 0.2 µM (R2 ) 0.973) for PtC/GluOx/ PPD-BSA. KM(O2) values for a range of Glu concentrations are plotted in Figure 2.

Figure 2. Mean ( SEM and linear regression analysis for KM(O2) values determined using the analysis shown in Figure 1 for 20, 50, 100, and 150 µM Glu. Good fits were obtained for both the PtC/GluOx/ PPD-BSA (open circles, n ) 4, R2 ) 0.990) and PtD/GluOx/PPDBSA (filled circles, n ) 8, R2 ) 0.993) designs, allowing extrapolation to lower Glu concentrations. Inset: closeup of the region 0-50 µM Glu for the cylinder-based biosensors (mean ( SD, n ) 4) confirming linearity for experimentally determined KM(O2) down to Glu concentrations as low as 5 µM.

tions, Figure 3 was constructed for a larger pool of electrodes. Thus, although the increase in KM(O2) expected with higher Glu concentrations was observed (Figure 2) for each design, a remarkable observation was that the biosensor design with the higher current density (PtD/GluOx/PPD-BSA) showed the lower oxygen dependence. This may be due, in part, to more efficient diffusion of oxygen to the small disk electrode (three-dimensional spherical diffusion) compared with two-dimensional cylindrical diffusion to the PtC/GluOx/PPD-BSA sensor. For example, there was no significant correlation between the Glu linear sensitivity and the corresponding KM(O2) slope for the cylinder-based biosensor: R2 ) 0.06, p > 0.55, n ) 8. This contrasts with a correlation observed previously for a glucose biosensor.42

Figure 3. Mean ( SEM of the slope for Glu calibrations carried out in the range 0-150 µM Glu (Glu sensitivity parameter, X-axis) plotted against the mean ( SEM of the slopes for KM(O2) data (e.g., Figure 2; O2 sensitivity parameter, Y-axis with units of µM oxygen per µM Glu) for GluOx/PPD-BSA biosensors based on cylinder and disk designs (schematic insets). The difference between the Glu sensitivity was significant for PtD vs PtC (p < 0.001), as was the difference between the O2 sensitivity (p < 0.001).

Preliminary analysis of full Michaelis-Menten calibration curves for Glu in a fixed concentration of O2 (air-saturated buffer) provides a consistent explanation of these results. The apparent enzyme kinetic constants, Vmax and KM(Glu), were significantly different for cylinder and disk biosensor designs. A 20 times higher value of Vmax was observed for disk biosensors, indicative of significantly increased active enzyme density compared with cylinders. In addition, the KM(Glu) values were also higher for the disk configuration, indicating an increased average diffusion barrier for Glu, consistent with high, possibly “stacked”, enzyme coverage observed for other oxidases on Pt.49 Thus, the turnover rate of each GluOx molecule in the linear Glu response region is less on PtD/GluOx/PPD-BSA biosensors, and this lowers O2 demand on the molecular scale. The lower values for KM(O2) demonstrated here for these same disk electrodes (Figure 3) show that the mass transport of the smaller neutral oxygen molecule is not adversely affected by the excess enzyme and, in conjunction with the more efficient hemispherical diffusion, leads to a lower oxygen dependence compared with the cylinder configuration. These preliminary data, and other results, will be explored in more detail in a full paper. Finally, as an alternative, and more intuitive, quantification of the level of oxygen dependence of these biosensors, we defined the concentration of oxygen at which 90% of the air-saturated signal (100%) is observed, [O2]90%. Using the slope of the KM(O2) data shown in Figure 3, a KM(O2) value can be determined for any concentration of Glu in the range 0-150 µM. Equation 4 (49) De Benedetto, G. E.; Malitesta, C.; Zambonin, C. G. J. Chem. Soc., Faraday Trans. 1994, 90, 1495-9. (50) Murr, R.; Berger, S.; Schuerer, L.; Peter, K.; Baethmann, A. Pflugers Arch. 1994, 426, 348-50. (51) Nair, P. K.; Buerk, D. G.; Halsey, J. H, Jr. Stroke 1987, 18, 616-22.

Figure 4. Theoretical curves generated using eq 4 and KM(O2) values calculated for 10 µM Glu at PtD/GluOx/PPD-BSA (0.65 ( 0.11 µM) and PtC/GluOx/PPD-BSA (2.35 ( 0.16 µM) biosensors from the data shown in Figure 3. This analysis indicates that 90% of the airsaturated 10 µM Glu signal can be maintained for PtD/GluOx/PPDBSA disk electrodes in media where oxygen concentrations fall to values as low as 5 µM; the corresponding [O2]90% for PtC/GluOx/PPDBSA cylinder sensors was 20 µM.

generates IGlu% as a function of [O2] for Imax ) 100%. This analysis is illustrated in Figure 4 for 10 µM Glu and shows that the PtD/ GluOx/PPD-BSA biosensor loses only 10% of its signal on changing the concentration of dissolved oxygen from 200 to 5 µM, whereas the same fraction of the current is lost for PtC/ GluOx/PPD-BSA electrodes by 20 µM. Since the average concentration of brain tissue oxygen has been estimated at ∼50 µM,50,51 we suggest that neither design would suffer significantly from oxygen fluctuations in brain ECF under all but extreme anaerobic conditions. CONCLUSIONS The data and analysis presented here enables the percentage oxygen interference for two types of first-generation Glu biosensor to be estimated as a function of both Glu and dissolved oxygen concentration. Of the two designs, the PtD/GluOx/PPD-BSA electrode offers the higher current density and lower oxygen dependence. The suitability of the design for a given application depends on the concentration of Glu being monitored, as well as the range of fluctuations in pO2 relevant to that medium. ACKNOWLEDGMENT This work was funded in part by Science Foundation Ireland (04/BR/C0198). We thank Dr. Kusakabe of Yamasa Corp., Japan for a generous gift of glutamate oxidase, and Enterprise Ireland for a postgraduate scholarship (C.Mc.M.). Received for review December 28, 2004.

September

3,

2004.

Accepted

AC048686R

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