Fabrication, Characterization, and Application of Boron-Doped

Department of Chemistry, Faculty of Science and Technology, Keio University, 3-14-1 Hiyoshi, Yokohama 223-8522, Japan,. Chemistry Department, Faculty ...
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Anal. Chem. 2007, 79, 8608-8615

Fabrication, Characterization, and Application of Boron-Doped Diamond Microelectrodes for in Vivo Dopamine Detection Akane Suzuki,† Tribidasari A. Ivandini,†,‡ Kenji Yoshimi,§ Akira Fujishima,| Genko Oyama,§ Taizo Nakazato,⊥,# Nobutaka Hattori,§ Shigeru Kitazawa,⊥,# and Yasuaki Einaga*,†

Department of Chemistry, Faculty of Science and Technology, Keio University, 3-14-1 Hiyoshi, Yokohama 223-8522, Japan, Chemistry Department, Faculty of Mathematics and Science, University of Indonesia, Kampus Baru UI Depok, Jakarta 16-424, Indonesia, Department of Neurology, Medical School, Juntendo University, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan, Kanagawa Academy of Science and Technology, KSP 3-2-1 Sakado, Kawasaki 213-0012, Japan, Department of Neurophysiology, School of Medicine, Juntendo University, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan, and CREST, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi 305-8575, Japan

Highly boron-doped diamond (BDD) was deposited on chemically etched micrometer-sized tungsten wires using microwave plasma assisted chemical vapor deposition (MPCVD), and these were used to fabricate BDD microelectrodes. BDD microelectrodes with very small diameter (about 5 µm) and 250 µm in length could be made successfully. In addition to the unique properties of BDD electrodes, such as a very low background current, high stability, and selective oxidation of dopamine (DA) in the presence of ascorbic acid (AA), other superior properties of the microelectrodes, including a constant current response, an increase in the mass transport, and the ability for use in high resistance media were also shown. An application study was conducted for in vivo detection of DA in mouse brain, where the BDD microelectrode was inserted into the corpus striatum of the mouse brain. A clear signal current response following medial forebrain bundle (MFB) stimulation could be obtained with high sensitivity. Excellent stability was achieved, indicating that the BDD microelectrodes are very promising for future in vivo electroanalysis. Microelectrodes have attracted great interest in recent years due to the advantages bestowed by their micrometer-sized dimensions. In addition to several unique properties, such as nonlinear diffusion, increased rate of mass transport, and reduced capacitance allowing a fast response, the very small size of microelectrodes have provided a major breakthrough in electrochemistry, because they have greatly extended the quality of the analysis and the range of experiments that can be done, for example, fast scan measurements and analysis in low conducting * To whom correspondence should be addressed. E-mail: chem.keio.ac.jp. † Keio University. ‡ University of Indonesia. § Juntendo University. | Kanagawa Academy of Science and Technology. ⊥ Department of Neurophysiology, Juntendo University. # CREST.

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media.1-3 In particular, the small size of the microelectrodes makes it possible to utilize them for in vivo detection, such as monitoring the brain via a neutrotransmitter, which is usually performed in a very small sample volume.4 Carbon fiber (CF) electrodes have already been established for some in vivo tests since they have good biocompatibility, are relatively cheap, and are easy to obtain.5-8 Moreover, CF is thin enough to penetrate biological tissue with the minimum disruption.4 However, CF has disadvantages, such as its brittleness and high adsorption behavior. On the other hand, the superior properties of highly borondoped diamond (BDD) electrodes compared to conventional carbon electrodes have already been reported, due to their superb electrochemical properties, such as their wide potential window, very small charging current, chemical inertness, and mechanical durability.9-12 These properties have enabled the establishment of a new field of electrochemical analysis, especially the detection of trace amounts of environment-related or biorelated substances.9,13-16 BDD also has good biocompatibility.9,12 (1) Bond, A. M. Analyst 1994, 119, R1. (2) Wiedemann, D. J.; Kawagoe K. T.; Kennedy, R.T.; Ciolkowski, E. L.; Wightman, R. M. Anal. Chem. 1991, 63, 2965. (3) Bond, A. M.; Fleischmann, M.; Robinson, J. J. Electroanal. Chem. 1984, 168, 299. (4) Cahill, P. S.; Walker, Q. D.; Finnegan, J. M.; Mickelson, G. E.; Travis, E. R.; Wightman, R. M. Anal. Chem. 1996, 68, 3180. (5) Paras, C. D.; Kennedy, R.T. Electroanal. 1997, 9, 203. (6) Kissinger, P. T.; Hart, J. B.; Adams, R. N. Brain Res. 1973, 55, 209. (7) Ponchon, J.-L.; Cespuglio, R.; Gonon, F.; Jouvet, M.; Pujol, J.-F. Anal. Chem. 1979, 51, 1483. (8) Jackson, B. P.; Dietz, S. M.; Wightman, R. M. Anal. Chem. 1995, 67, 1115. (9) Diamond Electrochemistry; Fujishima, A., Einaga, Y., Rao, T. N., Tryk, D. A., Eds.; Elsevier-BKC: Tokyo, Japan, 2005. (10) Yano, T.; Tryk, D. A.; Hashimoto, K.; Fujishima, A. J. Electrochem. Soc. 1998, 145, 1870. (11) Koppang, M. D.; Witek, M.; Blau, J.; Swain, G. M. Anal. Chem. 1999, 71, 1188. (12) Hartl, A.; Schmich, E.; Garrido, J. A.; Hernando, J.; Catharino, S. C. R.; Walter, S.; Feulner, P.; Kromka, A.; Steinmuller, D.; Stutzmann, M. Nat. Mater. 2004, 3, 736. (13) Suzuki, A.; Ivandini, T. A.; Kamiya, A.; Nomura, S.; Yamanuki, M.; Matsumoto, K.; Fujishima, A.; Einaga Y. Sens. Actuators, B 2007, 120, 500. (14) Ochiai, T.; Arihara, K.; Terashima, C.; Fujishima, A. Chem. Lett. 2006, 35, 1018. 10.1021/ac071519h CCC: $37.00

© 2007 American Chemical Society Published on Web 10/06/2007

Therefore, BDD meets the requirements for in vivo analysis. The fabrication and characterization of BDD microelectrodes have been reported previously, and the uniqueness of BDD microelectrodes for electrochemical analysis has been shown.17-25 However, to the best of our knowledge, until now, very few reports about the in vivo application of BDD microelectrodes in biological tissue have been published. The reason is mainly due to the difficulties in making BDD microelectrodes with very small tips that are less invasive of tissue. The diameter of reported BDD microelectrodes (10-30 µm)17-25 is still too big for applying them to in vivo detection. A diameter of ∼10 µm with a length of 25-500 µm is generally required for minimal tissue damage.26 In the present work, a very small BDD microelectrode was fabricated for application in in vivo detection. The tip of the BDD wire was prepared to be as small as that of a CF electrode, which has already been established for applications in in vivo detection.26 The BDD was deposited on tungsten wires. Prior to BDD deposition, the tungsten wire was conically shaped by electrochemical etching to achieve a very small tip diameter. With the use of the BDD coated tungsten wires, microelectrodes with very small tip diameters (∼5 µm) can be fabricated. Characterization was also performed, and the basic electrochemical behavior was compared with conventional CF electrodes. The electrodes were also investigated for the selective oxidation of dopamine. Dopamine (DA), 3,4-dihydroxyphenylethylamine, is known as an important neurotransmitter present in the central nervous system as an antecedent of adrenaline and noradrenaline which are used to control emotions or hormone balance.27 Normal levels of DA in the brain allow the usual freedom of movement, whereas excess DA in the brain often creates pleasurable, rewarding feelings and sometimes even euphoria. One of the most well-known and important effects of DA deficiency is Parkinson’s disease.28 The disease is characterized by degeneration and loss of midbrain substantia nigra neurons that produce the neurotransmitter DA, resulting in tremor at rest, inability to initiate or complete movements, muscle rigidity, postural instability, and lack of facial expression.29 As an example of an actual application of (15) Watanabe, T.; Ivandini, T. A.; Makide, Y.; Fujishima, A.; Einaga, Y. Anal. Chem. 2006, 78, 7857. (16) Ivandini, T. A.; Rao, T. N.; Fujishima, A.; Einaga, Y. Talanta 2007, 71, 648. (17) Sarada, B. V.; Rao, T. N.; Tryk, D. A.; Fujishima, A. J. Electrochem. Soc. 1999, 146, 1469. (18) Olivia, H.; Sarada, B. V.; Shin, D.; Rao, T. N.; Fujishima, A. Analyst 2002, 127, 1572. (19) Olivia, H.; Sarada, B. V.; Honda, K.; Fujishima, A. Electrochim. Acta 2004, 48, 2069. (20) Shin, D. C.; Sarada, B. V.; Tryk, D. A.; Fujishima, A. Anal. Chem. 2003, 75, 530. (21) Muna, G. W.; Quaiserova-Mocko, V.; Swain, G. M. Electroanalysis 2005, 17, 1160. (22) Park, J.; Quaiserova-Mocko, V.; Peckova, K.; Galligan, J. J.; Fink, G. D.; Swain G. M. Diamond Relat. Mater. 2006, 15, 761. (23) Park, J.; Galligan, J. J.; Fink, G. D.; Swain, G. M. Anal. Chem. 2006, 78, 6756. (24) Park, J.; Show, Y.; Quaiserova-Mocko, V.; Galligan, J. J.; Fink, G. D.; Swain, G. M. J. Electroanal. Chem. 2005, 583, 56. (25) Xie, S.; Shafer, G.; Wilson, C. G.; Martin, H. B. Diamond Relat. Mater. 2006, 15, 225. (26) Robinson, D. L.; Venton, B. J.; Heien, M. L. A. V.; Wightman, R. M. Clin. Chem. 2003, 49, 1763. (27) Zhao, Y.; Zhang, Y.; Yuan, Z. Analyst 2001, 126, 358. (28) Mo, J. W.; Ogorevc, B. Anal. Chem. 2001, 73, 1196. (29) Balcioglu, A.; Zhang, K.; Tarazi, F. I. Neuroscience 2003, 119, 1045.

Scheme 1. Schematic Drawing of a BDD Microelectrode

BDD microelectrodes, in vivo analysis of DA in mouse brain was successfully performed. Stimulation of the medial forebrain bundle (MFB) was conducted to control the release of DA. The MFB is a large collection of ascending and descending fibers that pass through the lateral region of the hypothalamus. Among those fibers, the MFB contains axons of dopaminergic neurons,30 where the stimulating electrode was set up. Release of DA was evoked in the corpus striatum of an anesthetized mouse, where the BDD microelectrode was inserted. A clear signal current response following electrical stimulation of dopaminergic neurons was monitored with high sensitivity and stability, indicating that the BDD microelectrodes are promising for future in vivo electrochemical analysis of biochemical compounds. EXPERIMENTAL SECTION Chemicals and Materials. Tungsten wire was obtained from Nilaco (30 or 50 µm i.d., 99.95% pure). Dopamine hydrochloride, L-ascorbic acid, and all other chemicals purchased from Wako Pure Chemical Industries were used without any further purification. Double distilled ultrapure water (>18 MΩ) was used for all solutions. Conductive silver paste (Dotite, Fujikura Kasei Co., Ltd.) and coated metal wire were used for making electrical connections to the microelectrodes. Resin (Cemedine 1516, Cemedine Co., Ltd.) and glass capillaries (purchased from Hirschmann Laborgerate GmbH & Co.) were utilized for insulating and strengthening the electrodes. Carbon fibers, supplied by Toho Tenax Co. Ltd, were used for comparison. Before use, the CF electrodes were pretreated by activation pulses. Activation pulsing was conducted by successively switching the potential from +2.5 to -2.5 V (vs Ag/AgCl) and from +2.1 to -2.1 V (vs Ag/AgCl) for a total of 100 cycles for about 15 min. The surface of the CF was refreshed by this treatment. Substrate Preparation and BDD Deposition. Tungsten wire was used as the substrate for BDD deposition. The wire was electrochemically etched in an aqueous solution of 2 M NaOH at +3.0 V (vs Ag/AgCl) for 20 s. During this process, tungsten wire was gradually lifted up from the etching solution. Finally the tip of the wire was conically shaped to leave a tip with a diameter of ∼3 µm. The sharpened wire was then immersed into HF solution (46.0%) for a few minutes to remove the tungsten oxide layer from the surface, and this was followed by a seeding process in an ultrasonic bath containing a 2-propanol suspension of diamond particles (0-500 nm particles, Kemet Co.) for 1 h. BDD thin film was deposited on the prepared tungsten wire using a microwave (30) Pillolla, G.; Melis, M.; Perra, S.; Muntoni, A. L.; Gessa, G. L.; Pistis, M. Psychopharmacology 2007, 191, 843.

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Scheme 2. Photo Image and Schematic Drawing of the in Vivo Mouse Brain Experimental Setup

plasma assisted chemical vapor deposition (MPCVD) system (ASTeX Corp.) with a plasma power of 2500 W. The deposition time was fixed at 3 h. A mixture of acetone and methanol in the ratio of 9:1 (v/v) was used as the carbon source and B2O3 at a concentration of 104 ppm (B/C) was dissolved in the mixture for the boron source. The surface morphology and crystalline structure of the BDD thin film was characterized using scanning electron microscopy (SEM, JEOL JSM 5400) and Raman spectroscopy (Renishaw System 2000). Microelectrode Preparation. The tungsten wire coated with the BDD thin film was connected to the coated metal wire using silver paste and then dried. This BDD wire then inserted through a prepulled glass capillary (using capillary puller, Narishige, Tokyo Japan) followed by resin infusion into the capillary for insulation. Resin was soaked up by capillary action. After drying overnight, the BDD microelectrode was completely fabricated. The details are illustrated in Scheme 1. CF electrodes were prepared by the same procedure as Scheme 1. The electrode length can be easily controlled by cutting the tip such that it had the same length as the BDD microelectrode. Electrochemical Measurements. The electrochemical measurements conducted in vitro were cyclic voltammetry (CV) and chronoamperometry (CA) using a potensiostat (Hokuto Denko, HZ-100). All the experiments were carried out at room temperature with a single compartment cell using a saturated Ag/AgCl electrode as the reference and a Pt wire as the counter electrode. A Faraday cage (Hokuto Denko, HS-101) was used to reduce external electromagnetic waves that might interfere with the small current responses. Before use, ultrasonication in 2-propanol followed by distilled water for 10 min each was adequate to clean the BDD macroelectrodes, whereas a pretreatment activation pulse was required for the CF electrodes. Activation pulses with the potential switching successively from +2.5 to -2.5 V (vs Ag/ AgCl) and from +2.1 to -2.1 V (vs Ag/AgCl) for a total of 100 cycles was employed to refresh the surface of the CF. This treatment was also required to recover the carbon electrodes when the reactivity had decreased. Before each measurement, an activation pulse was applied to CF at a potential from +1.285 V to -1.285 V (vs Ag/AgCl).31 Electrochemical measurements in a living organism were carried out in the brains of mice. Since the striatum of mammalian brain is rich in dopaminergic terminals, (31) Nakazato, T.; Akiyama, A. J. Neurosci. Methods 1999, 89, 105.

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it is a suitable model to evaluate whether the oxidation reactions of biologically released DA in vivo can be detected using BDD microelectrodes. Young adult C57BL/6J mice (male, 4 months old) were anesthetized first with pentobarbital (50 mg/kg, ip) and placed in a stereotaxic frame (Narishige, Tokyo Japan) according to the atlas of Paxinos and Franklin for surgical procedures. The working microelectrode was implanted in the neostriatum (0.5 mm rostral, 2.0 mm lateral to bregma, and 3.2 mm below the cortical surface) and and a pair of stainless bipolar stimulating electrodes (the stainless needle was insulated except for 0.3 mm at the tip) was implanted in the medial forebrain bundle (MFB, 2.0 mm caudal, 1.1 mm lateral to bregma, and approximately 5.0 mm below the cortical surface). The exact placement of the stimulating electrode in the dorsoventral coordinate was adjusted for maximal electrochemical response in the striatum. After surgery, the animals were maintained with isoflurane inhalation (1%, 30% O2, 70% N2O) and their rectal temperature was maintained at 36.0 ((1.0)°C during the electrochemical measurement. The in vivo experimental setup with the mouse brain is shown in Scheme 2. A Ag/AgCl wire and a stainless steel screw were placed on the cortical surface as reference and counter electrodes, respectively. Transient DA release into the corpus striatum was induced by MFB electrical stimulation (2 s, 50 Hz, biphasic 200 µA constant-current pulses with duration of 2 ms each, generated by a SEN-7203 stimulator and given through a photoisolator, SS201J, Nihon Kohden, Tokyo, Japan). In order to inhibit the DA uptake process into synapses, nomifensine was dissolved in saline solution and subcutaneously applied to the mouse with a dose of 7 mg/kg. Experimental protocols were approved by the Ethics Review Committee for Animal Experimentation of Juntendo University School of Medicine. The electrochemical measurements were conducted by differential pulse voltammetry (DPV) as mentioned below. RESULTS AND DISCUSSION A SEM image of the fabricated BDD microelectrode shows that the tip diameter is distinctly small (about 5 µm) with the polycrystalline diamond grain size being approximately ∼2 µm (Figure 1a). The tip size was almost the same as the conventional CF electrode used in this work (Figure 1b). On the basis of the experimental trial for in vivo measurement, the average tip length was made to be 250 µm (Figure 1c). Use of the same length of CF for in vivo monitoring of dopamine was also reported by

Figure 1. SEM image of (a) BDD wire and (b) CF electrode. (c) Shows a comparison of the optical microscope image of the BDD microelectrode (left side) and a conventional CF microelectrode (right side). (d) Raman spectra of the BDD wire.

Figure 2. Comparison of the CVs for 0.1 M Na2SO4 in the presence (a) and absence (b) of 1 mM each Fe(CN)62-/Fe(CN)63- with a CF electrode (- - -) and a BDD microelectrode (s) with a scan rate of 50 mV/s.

Gonon’s group.32 The Raman spectrum of the BDD microelectrode shows one clear peak at 1333 cm-1 for sp3 carbon bonds indicating that the BDD thin film has a fine quality with the absence of the peak at ∼1600 cm-1 generally attributed to nondiamond carbon impurities (Figure 1d). The shoulder before the sp3 carbon peak is attributed to the high boron doping concentration. The SEM image and Raman spectrum in this report are comparable with common BDD films on silicon wafers (Supporting Information, Figure S1). The results indicate that by optimizing the deposition conditions, similar BDD quality, in terms of the morphology and crystalline structure, can be deposited on different types and sizes of substrate. Prior to the in vivo analysis studies, the electrochemical behavior of the microelectrodes was investigated. Figure 2 shows a comparison of the cyclic voltammograms (CVs) of Fe(CN)62-/ Fe(CN)63- with CF and BDD microelectrodes in 0.1 M Na2SO4. A higher current is shown for the BDD microelectrode indicating a larger electroactive area than that of CF (Figure 2a). In spite of this, a lower background current and wider potential is found for the BDD microelectrode (Figure 2b). Table 1 shows a comparison of the signal, background, and signal to background ratio (S/B) (32) Dugast, C.; Suaud-Chagny, M. F.; Gonon, F. Neuroscience 1994, 62, 647.

of both CF and BDD microelectrodes extracted from the CVs in Figure 2. The BDD electrode has the advantage of a higher S/B ratio (more than 4 times), which gives rise to the very low detection limit. An important characteristic of microelectrodes, which is very useful for in vivo detection, is the ability to use them in high resistance media without the need to add any supporting electrolyte.1 This characteristic can be obtained because of the small current response, which is attributed to the limited electroactive area of microelectrodes. The IR drop problem encountered in the absence of added electrolyte is commonly minimized due to the small current range of the steady state measurement. Figure 3 shows the voltammetric response for the oxidation of Fe(CN)62-/Fe(CN)63- in double-distilled ultrapure water and in 0.1 M Na2SO4 (behaving as a supporting electrolyte) at a BDD microelectrode. The supporting electrolyte is generally added for charged species to suppress the migration current. If the supporting electrolyte is not sufficient, the electrochemical reaction is difficult to initiate. In the case of the BDD microelectrode, deceleration was not observed, as comparison with the ferricferrous redox reactions at the BDD microelectrodes show that the difference between the peaks in the presence or absence of Analytical Chemistry, Vol. 79, No. 22, November 15, 2007

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Table 1. Comparison of the Signal, Background, and Signal to Background (S/B) Ratio for Oxidation of 1 mM Fe(CN)62-/Fe(CN)63- in 0.1 M Na2SO4 at a CF Electrode (Extracted at a Potential of 0.6 V vs Ag/AgCl) and a BDD Microelectrode (Extracted at a Potential of 0.8 V vs Ag/AgCl)

CF electrode BDD microelectrode

S/nA

B/nA

S/B

67.6 108

0.70 0.25

97 428

Figure 3. CVs of Fe(CN)62-/Fe(CN)63- in 0.1 M Na2SO4 (- - -) and in double distilled ultrapure water (s) with BDD microelectrodes with a scan rate of 50 mV/s.

an additional supporting electrolyte is very small. In contrast, greater peak separation was obtained in the CVs of BDD macroelectrodes measured in ultrapure water suggesting that the BDD microelectrode can enlarge the surroundings for the electrochemical measurements (Supporting Information, Figure S2). Further, the effect of surface termination was also investigated since it is well-known that BDD electrodes are strongly influenced by the termination. As-deposited BDD (ad-BDD) electrodes are initially hydrogen-terminated as they are deposited in a hydrogen plasma CVD chamber.10 It has been reported that the surface charge of BDD electrodes can be converted to be relatively negative by the formation of surface carbon-oxygen functionalities by an anodic treatment,33 an oxygen plasma treatment,34 boiling in strong acid,35 or long-time exposure to air.36 Our group has reported the effect of surface termination of BDD electrodes on the oxidation of some compounds,37-40 including the selective detection of DA and ascorbic acid (AA).37 In living organisms, AA is present in concentrations of 10-100 times higher than DA. AA was found to have almost the same oxidation potential as DA (∼+0.6 V vs Ag/AgCl) in pH 2 buffer at ad-BDD, but the peaks were separated at an anodically oxidized BDD (aoBDD) surface due to the electrostatic interaction between AA and (33) Notsu, H.; Yagi, I.; Tatsuma, T.; Tryk, D. A.; Fujisima, A. Electrochem. SolidState Lett. 1999, 2, 522. (34) Yagi, I.; Notsu, H.; Kondo, T.; Tryk, D. A.; Fujishima, A. J. Electroanal. Chem. 1999, 472, 173. (35) Hayashi, K.; Yamanaka, S.; Watanabe, H.; Sekiguchi, T.; Okushi, H.; Kajimura, K. J. Appl. Phys. 1997, 81, 744. (36) Martin, H. B.; Argoitia, A.; Landau, U.; Andersin, A. B.; Angus, J. C. J. Electrochem. Soc. 1996, 143, L133. (37) Popa, E.; Notsu, H.; Miwa, T.; Tryk, D. A.; Fujishima, A. Electrochem. SolidState Lett. 1999, 2, 49. (38) Popa, E.; Kubota, Y.; Tryk, D. A.; Fujishima, A. Anal. Chem. 2000, 72, 1724. (39) Ivandini, T. A.; Sarada, B. V.; Rao, T. N.; Fujishima, A. Analyst 2003, 128, 924. (40) Ivandini, T. A.; Rao, T. N.; Fujishima, A.; Einaga, Y. Anal. Chem. 2006, 78, 3467.

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Figure 4. DA measurements in double distilled ultrapure water: (a) CVs for a mixture of 0.1 mM DA and 1 mM AA at ad-BDD (- - -) and ao-BDD (s) with a scan rate of 50 mV/s, (b) linear sweep voltammograms of various concentrations of DA (20-10 µM) in mixture solutions with 1 mM AA with a scan rate of 50 mV/s, and (c) chronoamperograms of various concentrations of DA (0.05-20 µM) in a mixture with 1 mM AA measured at an applied potential of 0.8 V (vs Ag/AgCl). The insets in parts b and c show linear calibration curves for current vs concentration.

Figure 5. Correlation diagrams for the signal current response vs applied potential for the oxidation of 1 µM DA (4) and 100 µM AA (+) in 0.1 M PBS measured by the DPV method. The inset shows a magnified view of the DA plot. DPV settings: frequency 50 Hz, potential step 200 mV, pulse amplitude 150 mV, starting potential 0.025 V vs Ag/AgCl.

the negative charge on the electrode surface.37,38 This behavior was also obtained for BDD microelectrodes, as shown in the comparison of the voltammetric responses for a mixture solution

Figure 6. Fast scan cyclic voltammograms of brain liquid without any stimulation at (a) an ao-BDD microelectrode and (b) CF electrode. Scan rate was 500 mV/s.

of DA and AA at ad- and ao-BDD microelectrodes (Figure 4a). Moreover, because of the constant current response, which is a typical characteristic of microelectrodes, the separation was clearer than that at the BDD macroelectrode (Supporting Information, Figure S3). The constant current response of the BDD microelectrode enabled us to analyze DA oxidation quantitatively with high precision. Good linearity (r2 ) 0.99) of the DA concentration in the range from 20 to 100 µM (n ) 6) in mixture solutions with 1 mM AA in ultrapure water could be obtained by the I-V method (Figure 4b). Furthermore, lower concentrations investigated by the chronoamperometric method (applied potential was ∼+0.8 V vs Ag/AgCl) show that the calibration was continuously linear (r2 ) 0.99) for the concentration range of 0.5 nM-100 µM with an experimental detection limit of 50 nM (Figure 4c) even without additional supporting electrolyte media. Selectivity of ao-BDD microelectrodes for DA oxidation in the presence of AA was also demonstrated by the different potential dependence between DA and AA in differential pulse voltammetry (DPV). Solutions of 1 µM DA and 100 mM AA, each in 0.1 M phosphate buffer solution (pH 7), were measured. Plots of the signal currents versus the applied potential are shown in Figure 5. The potential dependence of DA has a maximum peak at a potential of ∼0.8 V (vs Ag/AgCl), whereas that of AA showed a tendency to rise with increasing applied potential. These results are in good agreement with the I-V behavior shown in Figure 4a which shows the optimum oxidation potential of DA at ∼0.8 V (vs Ag/AgCl). The maximum point in the AA measurements was not reached up to a potential of 1.3 V (vs Ag/AgCl) but it should be found at a more positive potential (>1.4 V vs Ag/AgCl). The shifting of the peak to a much higher potential can be explained as the result of electrostatic repulsion of AA, which is negatively charged, by the negative potential of the aoBDD microelectrode surface. This behavior suggests that an unknown target (DA or AA) can be confirmed by observation of the potential dependence at ao-BDD microelectrodes. In the case of CF, this behavior could not be seen, as maxima at the same potential, ∼0.2 V (vs Ag/AgCl), were observed in the investigation of the potential dependence of DA and AA (data are not shown). This potential is even lower than the oxidation potential of DA and AA at ad-BDD (∼0.6 V vs Ag/AgCl in Figure 4a) because adsorption of DA and AA easily occurs at the CF electrode surface and accelerates the oxidation reactions.41,42 Because of their sp3 compact structures, BDD electrodes are well-known to have high stability for adsorption, including phys(41) Benoit-Marand, M.; Borelli, E.; Gonon, F. J. Neurosci. 2001, 21, 9134. (42) Terashima, C.; Rao, T. N.; Sarada, B. V.; Tryk, D. A.; Fujishima, A. Anal. Chem. 2002, 74, 895.

isorption and chemisorption.10,11 The behavior strongly affects the stability of the current response produced by an electrochemical reaction. As per the above details, the original termination of BDD, i.e., hydrogen termination, can be easily oxidized to become oxygen terminated. Once the BDD surface is oxidized, it is not easy to recover the hydrogen-termination.39,40 The stability of oxygen-terminated BDD electrodes has already been reported.43,44 Moreover, anodic oxidation is reported as an effective method for recovering the BDD surface, if the electrode loses sensitivity.44 Therefore, it can be said that oxygen-terminated BDD is more stable than hydrogen-terminated BDD. In the case of oxygenterminated BDD microelectrodes, the stability was demonstrated by 10 measurements of a mixture of 1 mM dopamine and 100 µM ascorbic acid with an RSD of 0.6% (data not shown). The above results indicated that BDD microelectrodes are superior to BDD macroelectrodes and to CF electrodes from the point of view of stability, selectivity, sensitivity, and availability for high resistance media. In order to evaluate the ability of BDD microelectrodes for in vivo detection, MFB stimulation was employed. Stimulation of dopaminergic neurons in the MFB caused the selective release of DA into the corpus striatum, where the BDD microelectrode was applied. Therefore, monitoring the changes in DA concentration can be performed. It is expected that the BDD microelectrodes developed in this work will have sufficient capability for in vivo analysis, because it is reported that the DA concentration after MFB stimulation is estimated to be up to several micromoles in the extracellular fluid.45 The stimulating electrode was carefully inserted into the brain cortex following the direction of the dopaminergic neurons. Electrical stimulation at a depth of 0.25 mm was applied. The position was then optimized by gradually increasing the depth in increments of 0.25 mm. The position of the electrode was fixed when the current response had reached the maximum value. In this report, a depth of 4.875 mm was fixed. Fast scan CVs of the background for in vivo measurements are shown for BDD microelectrodes (Figure 6a) and for a CF electrode (Figure 6b). In this potential range, no remarkable electroactive substance was recorded with the BDD microelectrode, and the background current was almost 1/10th lower than that of the CF electrode. The signal current response following (43) Rao, T. N.; Loo, B. H.; Sarada, B. V.; Terashima, C.; Fujishima, A. Anal. Chem. 2002, 74, 1578. (44) Allred, C. D.; McCreery, R. L. Anal. Chem. 1992, 64, 444. (45) 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.

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Figure 7. DPV monitoring of current response following MFB stimulation (50 Hz, 100 pulses for 2 s) measured at an applied potential of 0.9 V (vs Ag/AgCl) with (a) an ao-BDD microelectrode and (b) a CF electrode. The inset in Figure 7a shows the dependence of the signal current on the applied potential. DPV setting: frequency 50 Hz, potential step 100 mV, pulse amplitude 150 mV, starting potential 0.65 V vs Ag/AgCl. Table 2. Analytical Performance of in Vivo Dopamine Detection Following MFB Stimulation (50 Hz, 100 Pulses for 2 s) for 7 Measurements at a CF Electrode and a BDD Microelectrode Measured by the DPV Method with an Applied Potential of 0.9 V (vs Ag/AgCl)a S/nA B/nA N/nA S - B CF electrode 32.92 BDD microelectrode 4.88

30.93 4.56

0.2 0.02

1.99 0.33

S/N

RSD/% n)7

9.95 16.50

1.2 0.9

a

DPV setting: frequency 50 Hz, potential step 100 mV, pulse amplitude 150 mV, starting potential 0.65 V (vs Ag/AgCl).

MFB stimulation was monitored by the DPV method. The response for applied potentials between 0.8 and 1.1 V vs Ag/AgCl was monitored. Figure 7 shows that good responsivity and stability could be obtained for a BDD microelectrode. The average signal current response following MFB stimulation was 0.33 nA with very small noise (∼0.02 nA). An RSD of 0.88% was observed for 7 measurements using the BDD microelectrode. The potential dependence for the signal current (inset of Figure 7a) was found to have a maximum point at ∼0.9 V vs Ag/AgCl confirming that of the DA behavior shown in Figure 5. The potential shift to the higher value was probably caused by the absence of electrolyte in brain liquid, which leads to the deceleration of electron transfer in the dopamine oxidation reaction. Simultaneous measurements at CF electrodes show an average signal current response, noise, and RSD of 1.99 nA, 0.2 nA, and 1.2%, respectively. Comparison of in vivo measurements of dopamine at a BDD microelectrode and a CF electrode are summarized in Table 2. The results suggest the superiority of BDD microelectrodes over CF electrodes in terms of S/N ratio and stability. If the BDD microelectrodes need to be regenerated, an oxidation treatment in 0.1 M PBS at +2.0 V (vs Ag/AgCl) for 20 min is sufficient to recover the BDD microelectrode surface. To give further experimental corroboration for the fact that DA oxidation was monitored in vivo, the effect of nomifensine administration was observed at the BDD microelectrode. Nomifensine (1,2,3,4-tetrahydro-2-methyl-4-phenylisoquinolin-8amine) is a kind of medicine, which is known to inhibit the DA uptake process. Nomifensine increases the amount of synaptic dopamine available to receptors by blocking the dopamine’s re8614 Analytical Chemistry, Vol. 79, No. 22, November 15, 2007

Figure 8. DPV monitoring of current response following MFB stimulation before and after the administration of nomifensine (dose of 7 mg/kg) measured at an applied potential of 1.0 V (vs Ag/AgCl). The time at which nomifensine was administered was adjusted to 0 min. The MFB stimulation conditions are the same as in Figure 7. DPV setting: frequency 50 Hz, potential step 200 mV, pulse amplitude 150 mV, starting potential 0.5 V (vs Ag/AgCl).

uptake transporter.45-49 Normally, the DA concentration in the extracellular fluid is very low because the rate of DA uptake into the synapses and that of DA diffusion are quite fast.48 By nomifensine administration, the DA uptake rate can be inhibited, increasing the amount of DA in the extracellular fluid. Figure 8 shows the effect of nomifensine by using the DPV method. Before administration of nomifensine, small, but stable signal responses were observed following MFB stimulation. At the time of 0 min, nomifensine (dose: 7 mg/kg) was administered subcutaneously, and soon after, the signal current response rose remarkably. Therefore, there is no doubt that the DA was monitored precisely. CONCLUSIONS BDD microelectrodes with very small tips (5 µm diameter and 250 µm long) were fabricated by depositing BDD on etched tungsten wires. The superiority of BDD microelectrodes over conventional CF electrodes, in terms of a larger electroactive area with lower background current, higher sensitivity, and selectivity for DA oxidation in double distilled water, was demonstrated. (46) Vidal, L.; Alfonso, M.; Faro, L. F.; Campos, F.; Cervantes, R.; Duran, R. Toxicology 2007, 236, 42. (47) Hansard, M. J.; Smith, L. A.; Jackson, M. J.; Cheetham, S. C.; Jenner, P. J. Pharmacol. Exp. Ther. 2002, 303, 952 (48) Wightman, R. M.; Amatore, C.; Engstrom, R. C.; Hale, P. D.; Kristensen, E. W.; Kuhr, W. G.; May, L. J. Neuroscience 1988, 25, 513. (49) Robinson, D. L.; Venton, B. J.; Heien, M. L. A. V.; Wightman, R. M. Clin. Chem. 2003, 10, 1763.

Moreover, the different behavior of the potential dependence between DA and AA measured by different pulse voltammetry methods suggests a new method for selective detection of DA in the presence of AA. As an example of an application, in vivo detection of DA in a mouse brain was also performed. High sensitivity and stability of the peak currents were found following MBF stimulation. Selective in vivo detection of DA was confirmed by the use of nomifensine to inhibit the DA uptake process. The promising results found with BDD microelectrodes show them to have the ability for in vivo detection of DA, although further

investigation is required to enable measurement of the concentration. SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review July 18, 2007. Accepted August 25, 2007. AC071519H

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