`
`Analyst, November 1997, Vol. 122 (1419–1424)
`
`1419
`
`Biosensor for Neurotransmitter L-Glutamic Acid
`Designed for Efficient Use of L-Glutamate Oxidase and
`Effective Rejection of Interference
`
`Michael R. Ryan, John P. Lowry and Robert D. O’Neill*
`Department of Chemistry, University College Dublin, Belfield, Dublin 4, Ireland.
`E-mail: Robert.ONeill@UCD.ie
`
`An amperometric biosensor for l-glutamic acid (Glu) was
`constructed by the adsorption and dip coating of
`l-glutamate oxidase (GluOx, 200 U ml21 phosphate
`buffer, pH 7.4) onto 60-mm radius Teflon-coated Pt wire
`(1 mm exposed length). The enzyme was then trapped on
`the surface by electropolymerisation of
`o-phenylenediamine that also served to block electroactive
`interference. This procedure afforded electrodes with
`similar substrate sensitivity compared with the classical
`approach of immobilising enzyme from a solution of
`monomer, and represents an approximately 10 000-fold
`increase in the yield of biosensors from a batch of enzyme.
`A number of strategies were examined to enhance the
`sensitivity and selectivity of the Pt/PPD/GluOx sensors
`operating at 0.7 V versus SCE. Pre-coating the Pt with
`lipid and incorporation of the protein bovine serum
`albumin into the polymer matrix were found to improve
`the performance of the electrode. The sensors had a fast
`response time, high sensitivity to Glu, with an LOD of
`about 0.3 mmol l21, and possessed selectivity
`characteristics suggesting that monitoring Glu in
`biological tissues in vivo may be feasible.
`Keywords: Enzyme-modified electrode; polymer-modified
`electrode; poly(o-phenylenediamine); ascorbic acid
`interference; amperometry; brain glutamate
`
`l-Glutamate (Glu) is a ubiquitous excitatory amino acid
`neurotransmitter in the mammalian CNS, playing a major role
`in a wide variety of brain functions.123 Glu concentrations in
`brain extracellular fluid (ECF) in vivo have been estimated in
`the 10 mmol l21 region using a variety of microdialysis
`techniques and detection systems,428 and a number of valuable
`studies on brain Glu have been reported using these method-
`ologies.1,9212 The dialysis approach to monitoring brain
`chemistry has certain restrictions, however, such as limited time
`resolution and depletion of the ECF. As an alternative approach
`to detecting species in the ECF, implantable amperometric
`biosensors provide a continuous signal13 with significantly less
`depletion.14
`A number of sensor types have been developed for
`the measurement of Glu based on a variety of metabolic
`enzymes.15–20 Attention has focused mainly on the use of
`l-glutamate oxidase (GluOx) that has FAD as the redox centre
`and molecular oxygen as a co-substrate,21 producing hydrogen
`peroxide that can be detected amperometrically19,20,22–30 or
`spectroscopically.31,32 The oxidative deamination catalysed by
`GluOx21 can be represented by the following steps:
`l-Glutamate + H2O + GluOx/FAD ? a-ketoglutarate + NH3 +
`(1)
`GluOx/FADH2
`GluOx/FADH2 + O2 ? GluOx/FAD + H2O2
`(2)
`The H2O2 produced (reaction 2) can be electro-oxidised and this
`is generally carried out amperometrically at relatively high
`applied potentials (reaction 3).
`
`H2O2 ? O2 + 2H+ + 2e
`(3)
`An important problem in the use of enzyme-modified electrodes
`in biological media is interference by endogenous electroactive
`reducing agents, especially ascorbic acid (AA). This problem
`has been resolved, to a great extent, by the use of electro-
`synthesised polymers, such as poly(o-phenylenediamine)
`(PPD), that block access to the electrode surface of even
`relatively small organic molecules33–39 without affecting sensi-
`tivity to hydrogen peroxide,40 and a Pt/PPD/GluOx sensor has
`been reported.17,24
`The classical procedure for incorporating oxidase enzymes
`into a protecting polymer film has been to co-deposit the
`enzyme and polymer onto the electrode from a solution of the
`enzyme and monomer.33–39 This is convenient and effective but
`is very cost-inefficient for expensive enzymes such as GluOx.
`The aim of this work was to develop a more efficient procedure
`for producing Pt/ PPD/GluOx sensors with high sensitivity to
`Glu. Since the intended application for these biosensors is
`monitoring Glu in brain ECF, where the concentration of AA is
`about 500 mmol l21,41,42 special attention was paid to blocking
`interference by reducing agents.
`
`Materials and Methods
`Reagents and Solutions
`The enzyme l-glutamate oxidase21 (GluOx from Streptomyces
`sp. X-119-6, EC 1.4.3.11, 200 U ml21 in 20 mmol l21
`potassium phosphate buffer, pH 7.4) was obtained as a generous
`gift from Yamasa Corporation, Chiba, Japan, and stored at
`220 °C. The enzyme glucose oxidase (GOx) from Aspergillus
`niger (EC 1.1.3.4, Type VII-S) was from Sigma Chemical Co.
`(St. Louis, MO, USA). The lipid phosphatidylethanolamine
`(PEA, Type II-S) and bovine serum albumin (BSA, fraction V)
`were also obtained from Sigma. Stearic acid (STA), Nafion
`(NAF, 1100 EW, 5% solution in alcohol) and d-glucose were
`obtained from Aldrich (Milwaukee, WI, USA). All chemicals,
`including o-phenylenediamine (PD, Sigma), l-glutamic acid
`(Glu, Sigma), l-glutamine (Sigma) and l-ascorbic acid (AA,
`Aldrich), were used as supplied.
`Solutions of PD (20–400 mmol l21) were made up in 10 ml
`of a phosphate-buffered saline solution (PBS) with dissolution
`achieved by sonication at 25 °C for 25 min. Stock solutions of
`100 mmol l21 Glu and AA were prepared in doubly distilled
`water and 0.01 mol l21 HCl, respectively, and stored at 4 °C.
`All experiments were carried out in vitro in PBS (pH 7.4) that
`consisted of 0.15 mol l21 NaCl (Merck, Poole, UK), 0.04
`mol l21 NaH2PO4 (Merck) and 0.04 mol l21 NaOH (Merck).
`Solutions were kept refrigerated when not in use.
`
`Instrumentation and Software
`Experiments were microcomputer controlled with data acquisi-
`tion achieved using a Biodata Microlink interface, a low-noise,
`low-damping potentiostat (Biostat II, Electrochemical and
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`1420
`
`Analyst, November 1997, Vol. 122
`
`Medical Systems, Newbury, UK) and in-house software. The
`linear and non-linear regression analyses were performed using
`the graphical software package Prism (GraphPad Software, San
`Diego, CA, USA). All experiments were done in a 25 ml glass
`cell at 25 °C, using a standard three-electrode set-up with a
`saturated calomel electrode (SCE) as the reference and a silver
`wire in a glass sheath as the auxiliary electrode.
`
`Preparation of the Working Electrodes
`All the working electrodes were based on Pt cylinders (60 mm
`radius) prepared by cutting strips of Teflon-coated platinum
`wire, sliding the Teflon along the wire to expose about 1 mm of
`metal and sealing the Teflon rim with cyanoacrylate adhesive.
`The exposed metal was then dipped a number of times into 20 ml
`of a buffered solution of GluOx or GOx to deposit the enzyme.
`The number of dips varied (see below) but in all cases the first
`dip was left in the enzyme solution for 5 min to allow adsorption
`of the enzyme to occur.17,24,43,44 For all subsequent dips the
`electrode was immersed in the enzyme solution, removed
`immediately and allowed to dry for 5 min.
`The enzyme-coated wire was introduced into PBS containing
`the monomer (20–400 mmol l21 PD) and, in some cases, the
`non-enzyme protein BSA (5 mg ml21),45 and electropolymer-
`isation carried out immediately at 0.7 V versus SCE. The
`polymerisation time for this self-sealing process was 30 min,
`unless stated otherwise. In some cases the lipid PEA45,46 or fatty
`acid STA was used to coat the Pt before enzyme deposition.
`This procedure involved dipping the bare Pt wire a number of
`times into PEA or STA dissolved in chloroform (100 mg ml21),
`allowing the solvent to evaporate each time (1 min).
`Calibrations were performed amperometrically at 0.7 V
`versus SCE in quiescent air-saturated PBS for glucose in the
`range 0–10 mmol l21 and for Glu in the ranges 0–100 mmol l21
`(for LOD and linearity studies) and 0–10 mmol l21 (for Km and
`Vmax determinations). Calibrations for interfering substances
`were also carried out under the same conditions except N2-
`saturated buffer was used: ascorbate (AA, 0–1 mmol l21),
`dopamine (DA, 0–10 mmol l21), 3,4-dihydroxyphenylacetic
`acid (DOPAC, 0–100 mmol l21) and uric acid (UA, 0–100 mmol
`l21). Air-saturated solutions were used in the determination of
`l-glutamine interference (0–1 mmol l21).
`Since the resting level of AA in brain ECF has been
`estimated41,42 at 500 mmol l21 and because the AA response at
`PPD-modified electrodes is non-linear (decreasing with higher
`concentrations),40,45 500 mmol l21 AA responses were deter-
`mined by adding 500 mmol l21 AA to 500 mmol l21 AA in PBS,
`corresponding to a doubling of the baseline ECF concentration
`of AA. This approach to quantifying interference by AA is
`further justified by the finding that doubling the concentration
`of AA in the ECF in vivo had no detectable effect on the current
`recorded with a Pt/PPD/GOx sensor, even several days after
`implantation.39
`
`Data Analysis
`Calibration plots for Glu were generated by plotting averaged
`steady-state currents versus substrate concentration and fitting
`the data using non-linear regression to obtain the apparent
`Michaelis-Menten constants Vmax (nA) and Km (mmol l21).
`Linear regression was used for the 0–100 mmol l21 data to
`determine sensitivity in the linear response region and correla-
`tion coefficients.
`The selectivity coefficient of each electrode type for Glu vs.
`AA (SAA) was calculated for individual sensors using eqn. (4),47
`and then averaged.
`
`=
`
`S
`AA
`
`I
`
`Glu
`I
`
`I
`
`- · 100%
`
`AA
`
`Glu
`
`(4)
`
`The ıIGluı and ıIAAı used here were the absolute steady-state
`currents for 10 mmol l21 Glu and 500 mmol l21 AA,
`respectively. The range of interest for SAA is 0–100%: 0%
`corresponds to IGlu and IAA being equal; negative values reflect
`interference current greater than the signal for the target
`substrate; and 100% corresponds to no AA signal for an
`increase in concentration of 500 mmol l21.
`All data are reported as mean ± s with n being the number of
`electrodes. Unpaired, two-tailed t-tests were used to compare
`responses for different electrode designs. Values of p < 0.05
`were taken to indicate statistically significant differences
`between groups of electrodes.
`
`Results and Discussion
`Pt/PPD/GOx Electrodes
`Previous studies on the immobilisation of enzymes into the non-
`conducting form of the polymer PPD have involved either
`electropolymerisation from a solution of the enzyme plus
`monomer33–39 or from a monomer solution using electrodes
`with pre-adsorbed enzyme.17,24 The former approach uses large
`amounts of enzyme, typically 5 mg ml21, whereas the latter
`leads to sensors with relatively poor sensitivity for substrate.17
`To develop a sensor for Glu, using the expensive enzyme
`GluOx, we investigated here the use of ‘dip coating’ to deposit
`enzyme on the electrode surface prior to electropolymerisation
`in solution of monomer, PD.
`To compare the effectiveness of the dip coating approach to
`the standard co-deposition of enzyme and polymer from
`solution, we used GOx as a model enzyme. Because our sensor
`development programme is motivated by applications in
`mammalian brain, we have used concentrations appropriate to
`this environment in many of the experiments.
`The average current density for 500 mmol l21 glucose,
`recorded amperometrically at 0.7 V versus SCE with Pt/PPD/
`GOx sensors produced by polymerisation in a 5-ml solution
`containing 5 mg ml21 GOx and 300 mmol l21 PD, was 1.8 ±
`0.9 mA cm22 (n = 13); this combination of enzyme and
`monomer has been shown previously to be optimal for these
`conditions.40 The currents for 500 mmol l21 glucose recorded
`under the same conditions with sensors produced by different
`numbers of dip coatings using a 200 U ml21 GOx solution (to
`mimic the GluOx solution supplied) followed by polymer-
`isation in 300 mmol l21 PD were: 1 dip, 0.7 ± 0.1 nA (n = 2);
`2 dips, 3.8 ± 2.3 nA (n = 3); 5 dips, 5.1 ± 2.0 nA (n = 3); 10
`dips, 10.9 ± 4.8 nA (n = 3); and 20 dips, 3.1 ± 1.6 nA (n = 2).
`There was a maximum in the response for 10 dips that
`corresponds to a current density of 2.8 ± 1.3 mA cm22 (n =
`3).
`Thus, the response of sensors produced by the dip coating
`method was at least as good as the classical co-deposition
`procedure. In addition, the reproducibility of the sensors’
`sensitivity to Glu (as measured by the coefficient of variation)
`was the same for the two methods of production.
`
`Pt/PPD/GluOx Electrodes
`Having established that GOx could be successfully immobilised
`by dip coating followed by polymerisation, the procedure was
`applied to GluOx. The amperometric responses at 0.7 V versus
`SCE for 10 mmol l21 Glu, recorded with sensors produced by
`different numbers of dip coatings using a 200 U ml21 GluOx
`solution followed by polymerisation in 300 mmol l21 PD, were:
`1 dip, 0.62 ± 0.02 nA (n = 2); 2 dips, 0.59 ± 0.14 nA (n = 2);
`5 dips, 0.78 ± 0.24 (n = 5); and 10 dips, 0.66 ± 0.35 (n = 5).
`
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`Surprisingly, and in contrast to GOx, there was no distinct peak
`in the current versus number of dips. Five dips was chosen as the
`standard for further experiments as this gave the largest (albeit
`not statistically different) response; this sensor is represented as
`Pt/PPD/GluOx. Approximately 60 sensors were made from
`each 20 ml aliquot of enzyme, representing an approximately
`10 000-fold increase in the efficiency of GluOx immobilisation
`compared with the co-deposition method.
`The sensitivity of the sensor corresponds to a current density
`of 0.20 ± 0.07 mA cm22 (n = 5) for 10 mmol l21 Glu and
`compares very favourably with other biosensors for Glu.16,17,20
`These basic sensors exhibited Michaelis–Menten kinetics with
`a Vmax of 6.2 ± 0.9 nA and a Km of 0.25 ± 0.10 mmol l21, n =
`4, the latter value being the same as that observed for GluOx in
`solution.21
`Considering the sensitivity of Pt to AA,40 bare cylinders of
`the dimensions used here would be expected to give about 400
`nA for 500 mmol l21 AA. The actual response to this
`concentration of AA determined using Pt/PPD/GluOx sensors
`was only 0.8 ± 0.3 nA (n = 5), indicating that the PPD film is
`blocking access to the Pt surface quite efficiently. This barrier,
`however, is not sufficient for neurochemical applications since
`the selectivity coefficient for 10 mmol l21 Glu versus 500
`mmol l21 AA, SAA (see eqn. 4), was 211 ± 40% (n = 5), i.e.,
`the current for AA was similar to that for Glu at these
`concentrations.
`
`Pt/PEA/PPD/GluOx Electrodes
`The improved selectivity afforded by undercoating the enzyme
`layer with large molecules, such as PEA,45,46 is due to the fact
`that different sites are involved in the oxidation of interference
`molecules (Pt surface) and the substrate (enzyme) (see Fig. 1).
`Thus, PEA efficiently blocks access to the Pt surface for AA,
`say, but does not hinder access of Glu to the enzyme (reaction
`1). Furthermore, the sensitivity of Pt to the small H2O2 molecule
`(reaction 3) is not affected by macromolecules on the metal
`surface.40
`Pre-coating Pt disks with the lipid PEA by drop coating from
`chloroform before electropolymerisation has been shown
`previously to enhance the interference blocking properties of
`biosensors.45,46 Since drop coating also led to a slower response
`time,45 the dip coating method was investigated here as an
`alternative means of depositing PEA onto bare Pt cylinders
`before immobilisation of the GluOx. The effect of 0–10 dips in
`a PEA solution (100 mg ml21 in chloroform) on the response of
`the sensor to 10 mmol l21 Glu and 500 mmol l21 AA was
`determined.
`The presence of lipid had no significant effect on the Glu
`response. Increasing the amount of PEA on the surface,
`however, decreased the AA response and had a maximum effect
`for 5 dips (0.33 ± 0.03 nA, n = 3, p < 0.05, compared with no
`PEA, 0.78 ± 0.29, n = 5). The observation that 10 dips was less
`effective than 5 dips may be caused by inhibition of PPD
`formation at this higher surface coverage of PEA. Although the
`characteristics of the Pt/PEA/PPD/GluOx sensor were im-
`
`Analyst, November 1997, Vol. 122
`
`1421
`
`proved by the PEA (SAA = 43 ± 10%, n = 3), the selectivity of
`the electrode was still not considered adequate for neu-
`rochemical analysis in vivo.
`
`Pt/PEA/PPD/BSA/GluOx Electrodes
`The ability of non-conducting PPD films formed at neutral pH
`(about 10 nm thick34,48) to block interference appears to be
`enhanced by the incorporation of protein (enzyme and non-
`enzyme) into the polymer matrix;40,45 both electrochemical and
`electron microscopy data suggest that PPD films are more
`compact when formed by electropolymerisation in monomer
`solution containing protein.40 Although enzyme is present on
`the Pt for the sensors described here so far, protein was not
`present in the polymerisation solution. Electropolymerisation in
`solutions of PD and BSA (5 mg ml21, a concentration that has
`been shown to be optimal for our conditions45) was therefore
`carried out in attempts to improve the selectivity further.
`The data in Table 1 show that there was indeed a significant
`(p < 0.01) improvement in SAA (83 ± 9%, n = 9) when BSA
`was incorporated into the PPD sensor. Surprisingly, this
`improvement was due to both an increase in the Glu current and
`a decrease in the AA response. Thus, the co-deposition of
`protein with the polymer may protect some GluOx molecules
`from inactivation by the polymerisation process in a similar way
`to that in which BSA protects enzymes during cross-linking
`with glutaraldehyde.49
`A schematic illustration of a Pt/PEA/PPD/BSA/GluOx
`electrode, based on both structural and electrochemical data, is
`shown in Fig. 1. The thickness of the non-conducting PPD film
`formed under neutral conditions (about 10 nm34,48) is similar to
`the diameter of oxidases such as GOx39 (about 9 nm, 180 kD)
`and GluOx (about 140 kD21), whereas the size of the smaller
`BSA molecule (about 70 kD) is not critical. One might expect,
`therefore, that the binding site of a population of GluOx
`molecules would not be hindered by the polymer.33 This view is
`supported by the reported rapid response times (e.g., 1 s34 or
`< 10 s39,40,45) for glucose at PPD/GOx electrodes and for Glu at
`PPD/GluOx sensors (see Fig. 2, below). Although PPD
`efficiently blocks access to the metal for molecules the size of
`AA, Glu, glucose, etc., the small H2O2 molecules produced
`enzymatically (reactions 1 and 2) can diffuse to the Pt surface to
`be oxidised40 (reaction 3). The PEA underlying the enzyme
`layer provides additional blocking of the Pt surface for
`interferences, such as AA, but does not hinder access of Glu to
`the enzyme (reaction 1).
`
`Other Modifications of the Sensor
`Stearic (octadecanoic) acid (STA) has been used in the past in
`attempts to block interference by AA in neurotransmitter
`detection using carbon electrodes.50,51 The rationale is that the
`presence of carboxylate anions on the surface (at pH 7.4) should
`reject AA anions by electrostatic repulsion. We therefore
`replaced the zwitterionic PEA with STA (5 dips in 100 mg ml21
`chloroform) in the dip coating of the Pt prior to enzyme
`
`Table 1 Average ± s Glu and AA currents calculated from individual calibrations in PBS (pH 7.4) at + 0.7 V versus SCE, the corresponding selectivity
`coefficients (SAA, see eqn. 4) ± s and LOD ± s. Number of dip coatings of each modifier was: phosphatidylethanolamine (PEA, 100 mg ml21 in chloroform:
`5), glutamate oxidase (GluOx, 200 U ml21 phosphate buffer: 5), stearic acid (STA, 100 mg ml21 in chloroform: 5) and Nafion (NAF, 1% alcohol solution:
`1)
`
`Electrode design
`Pt/PPD/GluOx, n = 5
`Pt/PEA/PPD/GluOx, n = 3
`Pt/PEA/PPD/BSA/GluOx, n = 9
`Pt/STA/PPD/BSA/GluOx, n = 6
`Pt/NAF/PPD/BSA/GluOx, n = 3
`
`10 mmol l21 Glu/nA
`0.78 ± 0.24
`0.61 ± 0.14
`1.14 ± 0.38
`1.29 ± 0.34
`0.43 ± 0.46
`
`500 mmol l21 AA/nA
`0.78 ± 0.29
`0.33 ± 0.03
`0.14 ± 0.12
`0.36 ± 0.17
`7 ± 11
`
`SAA (%)
`211 ± 40
`43 ± 10
`83 ± 9
`71 ± 10
`< 2100
`
`LOD/mmol l21
`0.35 ± 0.33
`0.23 ± 0.33
`0.27 ± 0.21
`0.15 ± 0.09
`1.5 ± 1.3
`
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`1422
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`Analyst, November 1997, Vol. 122
`
`deposition and subsequent polymerisation. The data in Table 1
`show that the replacement of blocking agent had the opposite
`effect to that expected, decreasing the selectivity coefficient (p
`< 0.05) due to a larger AA response (p < 0.02). It appears
`therefore that the hydrophobic properties of these molecules are
`equally, if not more, important than electrostatic factors for
`blocking interference.
`The perfluorinated polysulfonic acid Nafion has been used
`even more widely than STA to block AA interference in
`
`Teflon
`
`PEA
`
`Pt
`
`Glu
`
`GluOx
`
`H2O2
`
`PPD
`
`BSA
`
`AA
`
`Pt/PEA/PPD/BSA/GluOx
`
`125 mm
`Fig. 1 Schematic illustration of a Pt/PEA/PPD/BSA/GluOx sensor
`fabricated by: dip coating the lipid phosphatidylethanolamine (PEA) from
`a chloroform solution onto bare Pt; adsorption and dip coating of
`l-glutamate oxidase (GluOx) from an aqueous buffer onto the PEA layer;
`and electrosynthesis of the non-conducting form of polyphenylenediamine
`(PPD) in a PBS solution (pH 7.4) containing monomer and bovine serum
`albumin (BSA). See text for the rationale of the scheme. The GluOx-
`catalysed oxidative deamination of l-glutamate (Glu) yielding H2O2 is
`given in the text by reactions 1 and 2. The small H2O2 molecules can diffuse
`easily to the Pt surface to be electro-oxidised (reaction 3) with a
`characteristic fast response time (see Fig. 2). Access to the metal by larger
`electroactive species, such as ascorbic acid (AA), is severely restricted by
`the PPD, PEA and proteins, with a correspondingly small and slow
`response (see Fig. 2, inset).
`
`Fig. 2 Example of amperometric calibration data for glutamate (Glu,
`0–10 mmol l21) recorded at 0.7 V versus SCE with a Pt/PEA/PPD/BSA/
`GluOx electrode (see Fig. 1), also showing the lack of sensor response to
`5 injections of +200 mmol l21 glutamine. The inset shows an example of a
`500 mmol l21 AA response (see Table 1 for data).
`
`neurotransmitter detection.52 However, the replacement of PEA
`in the sensor using 1 dip of a 1% solution of Nafion in alcohol
`had a catastrophic effect on SAA, decreasing the Glu signal and
`increasing the AA current (see Table 1). Nafion may therefore
`inhibit both the deposition of GluOx on the electrode and the
`formation of PPD.
`As final modifications of the sensor we investigated the effect
`of monomer concentration and polymerisation time. At least
`four sensors were made for each monomer concentration: 20,
`100, 200, 300 and 400 mmol l21 PD. The value of SAA rose
`steadily from -360 ± 250% (n = 4) at 20 mmol l21 PD, -50 ±
`50% (n = 4) at 100 mmol l21, 15 ± 30% (n = 4) at 200 mmol
`l21 to a peak value of 83 ± 9% (n = 9) at 300 mmol l21 PD, and
`declined again to 66 ± 27% (n = 4) at 400 mmol l21, a
`concentration close to saturation. The SAA value was also
`sensitive to polymerisation time in 300 mmol l21 PD solutions,
`mainly due to a decrease in AA response for longer times: 12 ±
`16% (1 min, n = 3); 210 ± 100% (5 min, n = 3); 86 ± 9% (15
`min, n = 2); and 83 ± 9% (30 min, n = 9). Thus, although the
`polymerisation current associated with the formation of the self-
`sealing, non-conducting PPD (about 10 nm thick34,48) falls
`rapidly to low steady-state values, it appears that 15 min are
`needed to minimise any small pores in the polymer matrix40 (see
`Fig. 1).
`
`Other Characteristics of the Pt/PEA/PPD/BSA/GluOx
`Sensor
`The sensor type with the best average selectivity was the Pt/
`PEA/PPD/BSA/GluOx electrode produced by electropolymer-
`isation in 300 mmol l21 PD for 15–30 min: SAA = 84 ± 9% (n
`= 11) for 10 mmol l21 Glu and 500 mmol l21 AA. For use in a
`given application, however, electrodes can be chosen that have
`the best characteristics. For example, half of the total sample of
`11 electrodes gave SAA = 92 ± 3% (n = 5). This sensor design
`(see Fig. 1) may therefore be suitable for neurochemical studies
`and therefore further characterisation was carried out in vitro.
`The response time to Glu was of the order of the mixing time
`in the cell (about 10 s, see Fig. 2), i.e., similar to Pt/PPD/GOx
`electrodes33–39 but much faster than sensors incorporating PEA
`by the drop coating technique.45 The sensitivity was high in the
`linear range (0–100 mmol l21 Glu, 3.8 ± 1.3 nA cm22 mmol21
`l, r2 = 0.99 ± 0.01, n = 4) with a low LOD (2 3 s of baseline,
`see Table 1). Glutamine, a possible source of interference with
`an ECF level in the region of 100 mmol l21,53,54 had no effect
`on the sensor’s response up to 1 mmol l21 (see Fig. 2). These
`properties compare favourably with those of other GluOx-based
`sensors in the literature.16,17,20
`Fig. 2 (inset) shows a typical response of a Pt/PEA/PPD/
`BSA/ GluOx electrode to 500 mmol l21 AA. The average size of
`the response was small (0.14 ± 0.12 nA; see Table 1),
`representing an approximately 2500-fold decrease in sensitivity
`compared with bare Pt. Taken with the slow response time
`(about 2 min), the data indicate that the PEA and PPD films
`offer a significant barrier to AA diffusion to the Pt surface (see
`Fig. 1).
`AA has been shown to interfere with biosensors by another
`mechanism, i.e., a homogeneous reaction with hydrogen
`peroxide that decreases the sensors’ response to substrate.39
`Normally this reaction is too slow to be a problem,55,56 but it can
`be catalysed by heavy metal ions57 or peroxidases.58 Thus, trace
`metal impurities in the buffer can lead to negative interference
`by AA in vitro, but the effect can be blocked by the
`incorporation of EDTA in the buffer.39 Since AA has been
`shown to decrease the response to Glu of a GluOx-based
`assay,32 Glu responses were recorded in the presence and
`absence of AA (500 mmol l21) plus EDTA (1 mmol l21); there
`was no difference in Glu sensitivity under the two conditions
`(data not shown). This mode of interference by AA is also
`
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`Table 2 Comparison of the sensitivity of bare Pt and Pt/PEA/PPD/BSA/
`GluOx electrodes to some electroactive substances (other than AA) found
`in brain ECF. The ECF levels are estimated baseline concentrations.52 The
`responses at ECF levels for Pt/PEA/PPD/BSA/GluOx sensors are estimated
`from the calibration data
`
`Sensitivity
`bare Pt/
`Substance nA mmol21 l
`DA
`1.0 ± 0.1
`(n = 4)
`0.5 ± 0.1
`(n = 4)
`0.5 ± 0.1
`(n = 4)
`
`DOPAC
`
`UA
`
`Sensitivity
`Pt/PEA/PPD/
`BSA/GluOx/
`pA mmol21 l
`16 ± 1
`(n = 3)
`3.3 ± 0.3
`(n = 3)
`3.7 ± 0.5
`(n = 4)
`
`ECF level/
`mmol l21
`0.05
`
`Response at
`ECF levels/
`pA
`1
`
`10
`
`5
`
`35
`
`20
`
`absent in brain tissue, presumably owing to the absence of
`suitable catalysts.39
`Table 2 shows the response to a variety of other interfering
`substances found in brain ECF. Modification of the Pt with the
`PEA/PPD/BSA film (Fig. 1) reduced its sensitivity to DA,
`DOPAC and UA about 100 fold, and the responses were linear
`in the low concentration ranges tested. At the levels of these
`species found in the ECF, the corresponding current (see Table
`2) would be negligible compared with the 10 mmol l21 Glu
`signal ( >1 nA, see Table 1). Interference by UA is complicated
`by the finding that its concentration in brain ECF depends on the
`size of the electrode59 but for sensors of the size used here, UA
`concentrations would be < 5 mmol l21. Thus, although the
`presence of AA (and not other endogenous species, such as
`DA17) is the most significant limitation for the application of
`biosensors in brain tissue, the selectivity of the Pt/PEA/PPD/
`BSA/GluOx electrode developed here suggests suitability for
`neurochemical applications in vivo. A more complicated
`combination of Nafion, ascorbate oxidase, cellulose acetate,
`glutaraldehyde, BSA and GluOx has been used in one study to
`produce a sensor with an equivalent SAA of about 99%;16
`however, there has been a paucity of reports on applications
`using this sensor in the years since its first appearance.
`
`Conclusions
`The relatively simple fabrication of a sensor for Glu, based on
`the efficient use of GluOx, has been described. The sensor has
`high sensitivity for substrate and sufficient selectivity to provide
`a basis for neurochemical applications in vivo. Detailed
`characterisation remains to be carried out in brain tissue in vivo,
`however, before the sensor can be used to monitor Glu
`unambiguously in the ECF.
`
`We thank Dr. H. Kusakabe (Yamasa Shoyu Co., Japan) for the
`generous gift of l-glutamate oxidase. We are grateful to
`University College Dublin for financial support. JPL is a Marie
`Curie Fellow (Contract No. ERB FMB ICT 961319).
`
`References
`1 Fillenz, M., Behav. Brain Res., 1995, 71, 51.
`2 Obrenovitch, T. P., and Urenjak, J., Prog. Neurobiol., 1997, 51, 39.
`3 Conn, P. J., and Pin, J. P., Annu. Rev. Pharmacol. Toxicol., 1997, 37,
`205.
`4 Hazell, A. S., Butterworth, R. F., and Hakim, A. M., J. Neurochem.,
`1993, 61, 1155.
`5 O’Shea, T. J., Weber, P. L., Bammel, B. P., Lunte, C. E., Lunte, S. M.,
`and Smyth, M. R., J. Chromatogr., 1992, 608, 189.
`6 Choi, K. T., Illievich, U. M., Zornow, M. H., Scheller, M. S., and
`Strnat, M. A. P., Brain Res., 1994, 642, 104.
`
`Analyst, November 1997, Vol. 122
`
`1423
`
`7 Miele, M., Berners, M., Boutelle, M. G., Kusakabe, H., and Fillenz,
`M., Brain Res., 1996, 707, 131.
`8 Lada, M. W., and Kennedy, R. T., Anal. Chem., 1996, 68, 2790.
`9 Herrera-Marschitz, M., You, Z. B., Goiny, M., Meana, J. J., Silveira,
`R., Godukhin, O. V., Chen, Y., Espinoza, S., Pettersson, E., Loidl, C.
`F., Lubec, G., Andersson, K., Nylander, I., Terenius, L., and
`Ungerstedt, U., J. Neurochem., 1996, 66, 1726.
`10 Bogdanov, M. B., Tjurmina, O. A., and Wurtman, R. J., Brain Res.,
`1996, 736, 76.
`11 Miele, M., Boutelle, M. G., and Fillenz, M., J. Physiol. (London),
`1996, 497, 745.
`12 Meeusen, R., Smolders, I., Saare, S., De Meirleir, K., Keizer, H.,
`Serneels, M., Ebinger, G., and Michotte, Y., Acta Physiol. Scand.,
`1997, 159, 335.
`13 Lowry, J. P., and Fillenz, M., J. Physiol. (London), 1997, 498,
`497.
`14 Garguilo, M. G., and Michael, A. C., Anal. Chem., 1994, 66,
`2621.
`15 Albery, W. J., Boutelle, M. G., and Galley, P. T., J. Chem. Soc.
`Chem. Commun., 1992, 900.
`16 Hu, Y., Mitchell, K. M., Albahadily, F. N., Michaelis, E. K., and
`Wilson, G. S., Brain Res., 1994, 659, 117.
`17 Cooper, J. M., Foreman, P. L., Glidle, A., Ling, T. W., and Pritchard,
`D. J., J. Electroanal. Chem., 1995, 388, 143.
`18 Montagne, M., Durliat, H., and Comtat, M., Anal. Chim. Acta, 1993,
`278, 25.
`19 Mulchandani, A., and Wang, C. L., Electroanalysis, 1996, 8, 414.
`20 Cosnier, S., Innocent, C., Allien, L., Poitry, S., and Tsacopoulos, M.,
`Anal. Chem., 1997, 69, 968.
`21 Kusakabe, H., Midorikawa, Y., Fujishima, T., Kuninaka, A., and
`Yoshino, H., Agric. Biol. Chem., 1983, 47, 1323.
`22 Matsumoto, K., Sakoda, K., and Osajima, Y., Anal. Chim. Acta,
`1992, 261, 155.
`23 Boutelle, M. G., Fellows, L. K., and Cook, C., Anal. Chem., 1992, 64,
`1790.
`24 Cooper, J. M., and Pritchard, D. J., J. Mater. Sci., 1994, 5, 111.
`25 White, S. F., Turner, A. P. F., Bilitewski, U., Schmid, R. D., and
`Bradley, J., Anal. Chim. Acta, 1994, 295, 243.
`26 Walker, M. C., Galley, P. T., Errington, M. L., Shorvon, S. D., and
`Jefferys, J. G. R., J. Neurochem., 1995, 65, 725.
`27 Zilkha, E., Obrenovitch, T. P., Koshy, A., Kusakabe, H., and
`Bennetto, H. P., J. Neurosci. Methods, 1995, 60, 1.
`28 Yoshida, S., Kanno, H., and Watanabe, T., Anal. Sci., 1995, 11,
`251.
`29 Li, Q. S., Zhang, S. L., and Yu, J. T., Appl. Biochem. Biotechnol.,
`1996, 59, 53.
`30 Yao, T., Suzuki, S., Nishino, H., and Nakahara, T., Electroanalysis,
`1996, 7, 1114.
`31 Blankenstein, G., Preuschoff, F., Spohn, U., Mohr, K. H., and Kula,
`M. R., Anal. Chim. Acta, 1993, 271, 231.
`32 Stalikas, C. D., Karayannis, M. I., and Tzouwara-Karayanni, S. M.,
`Analyst, 1993, 118, 723.
`33 Sasso, S. V., Pierce, R. J., Walla, R., and Yacynych, A. M., Anal.
`Chem., 1990, 62, 1111.
`34 Malitesta, C., Palmisano, F., Torsi, L., and Zambonin, P. G., Anal.
`Chem., 1990, 62, 2735.
`35 Almeida, N. F., Wingard, L. B., Jr. and Malmros, M. K., Ann. NY
`Acad. Sci., 1990, 613, 448.
`36 Sohn, T. W., Stoecker, P. W., Carp, W., and Yacynych, A. M.,
`Electroanalysis, 1991, 3, 763.
`37 Reynolds, E. R., and Yacynych, A. M., Electroanalysis, 1993, 5,
`405.
`38 Bartlett, P. N., and Birkin, P. R., Anal. Chem., 1994, 66, 1552.
`39 Lowry, J. P., McAteer, K., El Atrash, S. S., Duff, A., and O’Neill, R.
`D., Anal. Chem., 1994, 66, 1754.
`40 Lowry, J. P., and O’Neill, R. D., Electroanalysis, 1994, 6, 369.
`41 Amatore, C., Kelly, R. S., Kristensen, E. W., Kuhr, W. G., and
`Wightman, R. M., J. Electroanal. Chem., 1986, 213, 31