Polymer-Enzyme Composite Biosensor with HighGlutamate Sensitivity and Low OxygenDependence

Colm P. McMahon and Robert D. O’Neill*

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

Two first-generation glutamate (Glu) biosensors are de-scribed: 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 designsincorporated the enzyme Glu-oxidase in a polymer (poly-o-phenylenediamine) matrix deposited on the Pt surface.Surprisingly, the smaller disk biosensor displayed acombination of higher Glu current density and loweroxygen dependence compared with the cylinder design.An analysis to estimate the oxygen interference in the Glusignal showed that 90% of the disk biosensor current for10 µM Glu remains on changing the dissolved oxygenconcentration from 200 to 5 µM. These results indicatethat brain Glu monitoring in vivo using this design,combined with an enzyme-inactive sensor for differentialelimination of electroactive interference, can now beexplored without significant influence by fluctuating tissuepO2.

The development of devices for monitoring L-glutamate (Glu)on-line has become an vibrant research area due to the importantrole 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

the most widespread neurotransmitter in the mammalian CNS,14

plays a major role in a wide range of brain functions, and hasbeen implicated in a number of neurological disorders.15 Systemsfor monitoring Glu in brain extracellular fluid (ECF) havetherefore been a key goal in the analytical and neurobiologicalsciences in recent years.

There have been two main approaches to the detection andquantification of brain ECF Glu concentrations: those usingmicrodialysis perfusion followed by ex situ analysis16-19 and, lesscommonly, those involving direct detection of Glu in the ECFusing implanted amperometric biosensors.10,11,13 The relativeadvantages and drawbacks of these two approaches have beenreviewed,20,21 but clearly, the high spatial and temporal resolutionsprovided by electrochemical sensors is particularly appealing forstudies of the neurochemical correlates of behavior.22-26 However,although considerable effort has gone into minimizing contamina-tion of biosensor signals by endogenous electroactive speciespresent in brain ECF,20,27,28 the problem of interference byfluctuating levels of the cosubstate (molecular oxygen, reaction2) 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 me-dium,4,13 and dissolved oxygen levels above 40 µM have beenidentified as being adequate for oxygen-independent signals.10,29

* Author to whom correspondence should be addressed. Fax: +353-1-7162127.Tel: +353-1-7162314. E-mail: Robert.ONeill@UCD.ie.(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, 1581-

5.(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, 5093-

100.

(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, 263-

74.(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, 69-

127.(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.

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(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.

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1196 Analytical Chemistry, Vol. 77, No. 4, February 15, 2005 10.1021/ac048686r CCC: $30.25 © 2005 American Chemical SocietyPublished on Web 01/11/2005

The oxidative deamination of Glu, catalyzed by GluOx,30 canbe represented by the following steps:

The H2O2 produced in reaction 2 can be oxidized, usuallyamperometrically, either directly on the electrode surface atrelatively high applied potentials (reaction 3)31,32 or catalyticallyat lower potentials.5,13

The oxygen dependence of a “first-generation” biosensor designedfor monitoring brain glucose has been characterized in vitro andin vivo.33 Based on Pt modified with a polymer-protein compositelayer consisting of glucose oxidase (GOx), poly(o-phenylenedi-amine) (PPD), and an albumin (BSA), this Pt/GOx/PPD-BSAbiosensor was shown not to suffer from significant oxygeninterference for glucose concentrations relevant to brain ECF invivo. Biosensors for Glu of a similar design (Pt/GluOx/PPD-BSA)32 have shown promising responses in monitoring brain Gluin alert rats implanted with probes of moderate sensitivity (0.02µA cm-2 µM-1),34 whereas sensors of lower sensitivity (0.01 µAcm-2 µM-1) failed to detect Glu changes associated with mildbehavioral stimulation (10-s tail pinch).35 Moreover, that latterstudy and others36,37 have indicated that brain H2O2 may representa potential interference for first-generation biosensors targetinganalytes with low ECF concentrations, such as Glu. It is clear,therefore, that successful monitoring of brain Glu using ampero-metric biosensors in vivo should involve the use of coupled “blank”(enzyme inactive) sensors for differential measurements, a strat-egy already employed in a number of biosensor applications tominimize interference from electroactive species reacting directlyon the electrode surface.11,13,31,38 Such “difference” Glu signals may,however, be susceptible to interference by limitations in oxygenavailability. Even “second-generation” biosensors incorporating aredox mediator to replace dioxygen in reaction 2 may be proneto oxygen interference due to competition between the mediatorand solution oxygen for the FADH2 moiety.39 The work presentedhere, therefore, aims to quantify the oxygen dependence of a

GluOx-based first-generation biosensor in terms of an apparentMichaelis-Menten constant for oxygen, KM(O2), to determinewhether this design is suitable for Glu monitoring in media withlow dissolved oxygen levels (eq 4). Surprisingly, miniaturization

of Pt/GluOx/PPD-BSA electrodes led to increased Glu currentdensity coupled with a lower oxygen dependence that providesimproved performance for Glu monitoring in oxygen-challengedsystems.

EXPERIMENTAL SECTIONPt cylinders (PtC, 125-µm diameter, 1-mm length) were

fabricated from Teflon-coated Pt wire. GluOx (EC 1.4.3.11, 200units mL-1, Yamasa Corp.) was deposited onto the metal surfaceby dip-evaporation and immobilized by amperometric electro-polymerization (+700 mV vs SCE) in 300 mM o-phenylenediamine,containing 5 mg mL-1 bovine serum albumin in phosphate-buffered saline (PBS, pH 7.4),40 as described previously to formPtC/GluOx/PPD-BSA biosensors.32 Pt disks (PtD) were fabricatedby cutting the Teflon-coated wire transversely to produce 125-µm-diameter disks, and PtD/GluOx/PPD-BSA biosensors fabri-cated in the same way as for PtC. After rinsing and a settling periodat 700 mV in fresh PBS, calibrations were carried out to determinethe sensitivity of the biosensors to Glu.

A self-calibrating commercial membrane-covered amperometricoxygen sensor (∼1-cm diameter) was used to quantify solutionoxygen concentration as described recently.33 The model usedwas a CellOx 325 connected to an Oxi 340A meter (Wissen-schaftlich-Technische Werkstatten GmbH from Carl Stuart Ltd.,Dublin, Ireland), incorporating a temperature probe for automaticcompensation. Reliable quantification of O2 using this devicerequired constant stirring of the solution at a rate of ∼3 Hz. Thesensor range was 0.0-199.9% O2 (100% corresponding to airsaturation) with a resolution of 0.1%. This percentage wasconverted to an estimated concentration of O2 by taking 200 µMto correspond to 100%.41,42

All experiments were performed in a standard three-electrodeglass electrochemical cell containing 20 mL PBS at room tem-perature. A saturated calomel electrode (SCE) was used as thereference electrode, and a large stainless steel needle served asthe auxiliary electrode. The applied potential for amperometricpolymerizations and calibrations was +700 mV versus SCE. Inoxygen dependence studies, to avoid contamination of the PBSby oxygen, the electrochemical cell was contained within anAtmosbag (Sigma),33 a two-hand 0.003-in.-gauge polyethylene bagthat was sealed and filled with N2 during experiments, inflatingto a volume of 280 L. After adding an aliquot of Glu, air wasallowed into the system slowly by opening the bag slightly.Oxygen sensor data and biosensor data were recorded simulta-neously through the transition from N2 saturation to air saturation.

(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.; NationalUniversity 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, 393-

403.

(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.

L-glutamate + H2O + GluOx/FAD f

R-ketoglutarate + NH3 + GluOx/FADH2 (1)

GluOx/FADH2 + O2 f GluOx/FAD + H2O2 (2)

H2O2 f O2 + 2H+ + 2e (3)

IGlu )IGlu

max

1 + KM(O2)/[O2](4)

Analytical Chemistry, Vol. 77, No. 4, February 15, 2005 1197

Nonlinear regression analysis of the oxygen dependence ofthe Glu signal was carried out using a software package (Prism,GraphPad Software, Inc., San Diego, CA) and a Michaelis-Mententype equation (eq 4), whereas linear regression was performedon the Glu concentration dependence of the biosensor signalfollowing conversion to current density. Data are presented asmean ( SEM, with n the number of biosensors.

RESULTS AND DISCUSSIONThe concentration of Glu in bioanalytical applications rarely

exceeds 100 µM. For example, values reported under physiologicalconditions are normally below 10 µM for cerebrospinal fluid43,44

and brain ECF,45-47 although 100 µM levels have been detectedin the ECF following brain trauma.48 Calibrations were thereforeperformed in the range 0-150 µM Glu in quiescent air-saturatedPBS to compare the Glu sensitivity of the two different biosensordesigns. For the previously reported cylinder configuration,32,34,35

the PtC/GluOx/PPD-BSA biosensors characterized here showedlinear responses to Glu in this concentration range: 8.7 ( 1.0 nAcm-2 µM-1, R2 ) 0.961, n ) 8. This level of Glu sensitivity wassimilar to that of a sensor used in a previous unsuccessful attemptto monitor brain ECF Glu.35 A new design was therefore adoptedwith the aim of increasing enzyme loading. Drops of GluOxsolution were deposited onto the disk end of Teflon-coated Pt wireby dipping the wire vertically into the enzyme solution, removingit, and allowing to dry. These PtD/GluOx/PPD-BSA electrodes(1.23 × 10-4 cm2) were 32 times smaller than the correspondingcylinder sensors and showed significantly increased linear sen-sitivity 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 PtD-based biosensors were similar to the more complicated carbonfiber cylinder designs involving catalytic oxidation of H2O2 usingredox polymers and horseradish peroxidase.13

The oxygen dependence of the cylinder and disk biosensorswas studied over the same Glu concentration range 0-150 µM.Figure 1 shows the effect of changing the concentration of oxygenfrom submicromolar levels to 50 µM on the biosensor signalrecorded for 50 µM Glu. Here we quantified the oxygen depen-dence as KM(O2) defined in eq 4. The oxygen dependence wassignificantly 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-BSAglucose biosensors recently,33 the oxygen dependence of thesefirst-generation devices is expected to increase with increasedturnover of the enzyme associated with higher substrate concen-trations. The KM(O2) values determined up to 150 µM Glu forPtD/GluOx/PPD-BSA and PtC/GluOx/PPD-BSA sensors areshown in Figure 2. There was a linear increase in KM(O2) for bothsensor types, with the cylinder design displaying a significantlyhigher slope (p < 0.001).

Using the slopes shown in Figure 2, and KM(O2) values forother biosensors of the same design at a variety of Glu concentra-

tions, Figure 3 was constructed for a larger pool of electrodes.Thus, although the increase in KM(O2) expected with higher Gluconcentrations was observed (Figure 2) for each design, aremarkable observation was that the biosensor design with thehigher current density (PtD/GluOx/PPD-BSA) showed the loweroxygen dependence. This may be due, in part, to more efficientdiffusion of oxygen to the small disk electrode (three-dimensionalspherical diffusion) compared with two-dimensional cylindricaldiffusion to the PtC/GluOx/PPD-BSA sensor. For example, therewas no significant correlation between the Glu linear sensitivityand the corresponding KM(O2) slope for the cylinder-basedbiosensor: R2 ) 0.06, p > 0.55, n ) 8. This contrasts with acorrelation observed previously for a glucose biosensor.42

(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 Cere-

brovasc. Dis. 2000, 9, 2-8.

Figure 1. Examples of unfiltered data recorded amperometrically(+700 mV vs SCE) at 10 Hz with a PtD/GluOx/PPD-BSA (top) andPtC/GluOx/PPD-BSA (bottom) biosensor in 50 µM Glu expressedas a percentage of the maximum current (Imax, eq 4) and plottedagainst oxygen concentration recorded simultaneously using a CellOxsensor. The curves in each case represent the nonlinear regressionanalysis for eq 4. Regression parameters obtained for these ex-amples: 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 areplotted 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/PPD-BSA (filled circles, n ) 8, R2 ) 0.993) designs, allowing extrapolationto lower Glu concentrations. Inset: closeup of the region 0-50 µMGlu for the cylinder-based biosensors (mean ( SD, n ) 4) confirminglinearity for experimentally determined KM(O2) down to Glu concentra-tions as low as 5 µM.

1198 Analytical Chemistry, Vol. 77, No. 4, February 15, 2005

Preliminary analysis of full Michaelis-Menten calibrationcurves for Glu in a fixed concentration of O2 (air-saturated buffer)provides a consistent explanation of these results. The apparentenzyme kinetic constants, Vmax and KM(Glu), were significantlydifferent for cylinder and disk biosensor designs. A 20 timeshigher value of Vmax was observed for disk biosensors, indicativeof significantly increased active enzyme density compared withcylinders. In addition, the KM(Glu) values were also higher forthe disk configuration, indicating an increased average diffusionbarrier for Glu, consistent with high, possibly “stacked”, enzymecoverage observed for other oxidases on Pt.49 Thus, the turnoverrate of each GluOx molecule in the linear Glu response region isless 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) showthat the mass transport of the smaller neutral oxygen moleculeis not adversely affected by the excess enzyme and, in conjunctionwith the more efficient hemispherical diffusion, leads to a loweroxygen dependence compared with the cylinder configuration.These preliminary data, and other results, will be explored in moredetail in a full paper.

Finally, as an alternative, and more intuitive, quantification ofthe level of oxygen dependence of these biosensors, we definedthe concentration of oxygen at which 90% of the air-saturatedsignal (100%) is observed, [O2]90%. Using the slope of the KM(O2)data shown in Figure 3, a KM(O2) value can be determined forany concentration of Glu in the range 0-150 µM. Equation 4

generates IGlu% as a function of [O2] for Imax ) 100%. This analysisis illustrated in Figure 4 for 10 µM Glu and shows that the PtD/GluOx/PPD-BSA biosensor loses only 10% of its signal onchanging the concentration of dissolved oxygen from 200 to 5µM, whereas the same fraction of the current is lost for PtC/GluOx/PPD-BSA electrodes by 20 µM. Since the averageconcentration of brain tissue oxygen has been estimated at ∼50µM,50,51 we suggest that neither design would suffer significantlyfrom oxygen fluctuations in brain ECF under all but extremeanaerobic conditions.

CONCLUSIONSThe data and analysis presented here enables the percentage

oxygen interference for two types of first-generation Glu biosensorto be estimated as a function of both Glu and dissolved oxygenconcentration. Of the two designs, the PtD/GluOx/PPD-BSAelectrode offers the higher current density and lower oxygendependence. The suitability of the design for a given applicationdepends on the concentration of Glu being monitored, as well asthe range of fluctuations in pO2 relevant to that medium.

ACKNOWLEDGMENTThis work was funded in part by Science Foundation Ireland

(04/BR/C0198). We thank Dr. Kusakabe of Yamasa Corp., Japanfor a generous gift of glutamate oxidase, and Enterprise Irelandfor a postgraduate scholarship (C.Mc.M.).

Received for review September 3, 2004. AcceptedDecember 28, 2004.

AC048686R

(49) De Benedetto, G. E.; Malitesta, C.; Zambonin, C. G. J. Chem. Soc., FaradayTrans. 1994, 90, 1495-9.

(50) Murr, R.; Berger, S.; Schuerer, L.; Peter, K.; Baethmann, A. Pflugers Arch.1994, 426, 348-50.

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Figure 3. Mean ( SEM of the slope for Glu calibrations carriedout 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 oxygenper µM Glu) for GluOx/PPD-BSA biosensors based on cylinder anddisk designs (schematic insets). The difference between the Glusensitivity was significant for PtD vs PtC (p < 0.001), as was thedifference between the O2 sensitivity (p < 0.001).

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

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