Online Electrochemical Measurements of Ca2+ and Mg2+ in Rat Brain

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Anal. Chem. 2010, 82, 9885–9891

Online Electrochemical Measurements of Ca2+ and Mg2+ in Rat Brain Based on Divalent Cation Enhancement toward Electrocatalytic NADH Oxidation Zipin Zhang, Lingzhi Zhao, Yuqing Lin, Ping Yu, and Lanqun Mao* Beijing National Laboratory for Molecular Sciences, Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry, the Chinese Academy of Sciences (CAS), Beijing 100190, China This study describes a novel electrochemical approach to effective online monitoring of electroinactive Ca2+ and Mg2+ in the rat brain based on the current enhancement of divalent cations toward electrocatalytic oxidation of NADH. Cyclic voltammetry for NADH oxidation at the electrodes modified with the polymerized film of toluidine blue O (TBO) reveals that the current of such an electrocatalytic oxidation process is remarkably enhanced by divalent cations such as Ca2+ and Mg2+. The current enhancement is thus used to constitute an electrochemical method for the measurements of Ca2+ and Mg2+ in a continuous-flow system with the polyTBO-modified electrode as the detector. Upon being integrated with in vivo microdialysis, the electrochemical method is successfully applied in investigating on cerebral Ca2+ and Mg2+ of living animals in two aspects: (1) online simultaneous measurements of the basal levels of Ca2+ and Mg2+ in the brain of the freely moving rats by using ethyleneglcolbis(2-aminoethylether) tetraacetic acid (EGTA) as the selective masking agent for Ca2+ to differentiate the net current responses selectively for Ca2+ and Mg2+; and (2) online continuous monitoring of the cerebral Mg2+ following the global ischemia by using Ca2+-masking agent (i.e., EGTA) to completely eliminate the interference from Ca2+. Compared with the existing methods for the measurements of cerebral Ca2+ and Mg2+, the method demonstrated here is advantageous in terms of its simplicity both in instrumentation and in the experimental procedures and near real-time nature, and is thus highly anticipated to find wide applications in understanding of chemical events involved in some physiological and pathological processes. Simple and effective measurements of Ca2+ and Mg2+ in the brain of living animals are of great physiological and pathological importance due to their wide involvement in many physiological events. For example, Ca2+ is an important signal transduction element and is required for many functions in the central nervous systems including gene expression, neu* To whom correspondence should be addressed. Fax: +86-10-62559373. E-mail: [email protected]. 10.1021/ac102605n  2010 American Chemical Society Published on Web 11/08/2010

rotransmitter release, neurite outgrowth regulation, synaptogenesis, and synaptic transmission.1 Meanwhile, Mg2+ is an important mediator and regulator of Ca2+ signaling, and plays a classical role in defining the properties of adenosine triphosphate.2 Moreover, it was demonstrated that excessive Ca2+ in the extracellular fluid influx also plays a primary role in the development of several brain pathologies including those at ischemic/hypoxic conditions.3 While many methods have previously been reported for in vitro measurements of Ca2+ and Mg2+,4 simple but effective measurements of both species in the cerebral systems remain a longstanding challenge. Although atomic absorption spectroscopy (AAS) and inductively coupled plasma-mass spectrometry (ICPMS) have previously been employed for both discontinuous and continuous measurements of Ca2+ and Mg2+ in the rat brain,5 these methods were limited by an expensive instrumentation, or a poor time resolution caused by the sample collection for AAS measurements, or complex desalting procedures for ICPMS measurements. Compared with those methods, electrochemical methods are advantageous with respect to their relatively cheap instrumentation and simple procedures. More importantly, the smart design of electrode/ interface substantially endows this kind of methods with a high (1) For reviews,(a) Clapham, D. E. Cell 1995, 80, 259. (b) Mattson, M. P. Aging Cell 2007, 6, 337. (2) (a) For a review Saris, N.-E. L.; Mervaala, E.; Karppanen, H.; Khawaja, J. A.; Lewenstam, A. Clin. Chim. Acta 2000, 294, 1. (b) Tu ¨ rkyilmaz, C.; Tu ¨rkyilmaz, Z.; Atalaya, Y.; So¨ylemezogluc, F.; Celasun, B. Brain Res. 2002, 955, 133. (c) Killilea, D. W.; Ames, B. N. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 5768. (d) Slutsky, I.; Abumaria, N.; Wu, L.-J.; Huang, C.; Zhang, L.; Li, B.; Zhao, X.; Govindarajan, A.; Zhao, M.-G.; Zhuo, M.; Tonegawa, S.; Liu, G. Neuron 2010, 65, 165. (3) For reviews, (a) Lee, J.-M.; Zipfel, G. J.; Choi, D. W. Nature 1999, 399, A7. (b) Kristia´n, T. Cell Calcium 2004, 36, 221. (4) (a) Lee, S.; Lee, H. G.; Kang, S. H. Anal. Chem. 2009, 81, 538. (b) Deng, B.; Zhu, P.; Wang, Y.; Feng, J.; Li, X.; Xu, X.; Lu, H.; Xu, Q. Anal. Chem. 2008, 80, 5721. (c) Komatsu, H.; Miki, T.; Citterio, D.; Kubota, T.; Shindo, Y.; Kitamura, Y.; Oka, K.; Suzuki, K. J. Am. Chem. Soc. 2005, 127, 10798. (d) Farruggia, G.; Iotti, S.; Prodi, L.; Montalti, M.; Zaccheroni, N.; Savage, P. B.; Trapani, V.; Sale, P.; Wolf, L. I. J. Am. Chem. Soc. 2006, 128, 344. (e) Ishida, M.; Naruta, Y.; Tani, F. Angew. Chem., Int. Ed. 2010, 49, 91. (5) (a) Chung, Y. T.; Ling, Y. C.; Yang, C. S.; Sun, Y. C.; Lee, P. L.; Lin, C. Y.; Hong, C. C.; Yang, M. H. Anal. Chem. 2007, 79, 8900. (b) Yang, D.-Y.; Lee, J.-B.; Lin, M.-C.; Huang, Y.-L.; Liu, H.-W.; Liang, Y.-J.; Cheng, F.-C. J. Am. Colloid. Nutr. 2004, 23, 552S. (c) Lin, M.-C.; Huang, Y. L.; Liu, H.-W.; Yang, D.-Y.; Lee, C.-P.; Yang, L.-L.; Cheng, F. C. J. Am. Colloid. Nutr. 2004, 23, 561S.

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selectivity and improved stability.6 These properties potentially enable them to be readily integrated with in vivo sampling techniques,7 for a typical example, in vivo microdialysis that still remains very popular for monitoring brain chemistry,8 to generate online analytical systems.9 The online analytical systems developed by integrating in vivo microdialysis with selective electrochemical detection essentially avoid sample collection and separation and, consequently, have been demonstrated to be very effective for continuous monitoring of physiologically important species in the brain of living animals with an improved time resolution.10 While the excellent properties of electrochemical methods have substantially made them particularly attractive for effectively monitoring brain chemistry, the poor electroactivity of Ca2+ and Mg2+ unfortunately renders great difficulties in applying such kind of methods to monitor both species in the cerebral systems. In this study, we wish to report a simple but very effective electrochemical method for online monitoring of electrochemically inactive Ca2+ and Mg2+ in the rat brain. The rationale for the electrochemical detection of Ca2+ and Mg2+ is essentially based on the enhancement of both kinds of divalent cations toward the electrocatalytic oxidation of β-nicotinamide adenine dinucleotide (NADH) with organic redox dyes as the electrocatalyst.11 Although the divalent cation enhancement toward electrocatalytic NADH oxidation was previously reported,12 the utilization of this fundamental electrochemical phenomenon to establish a simple electrochemical method for effective moni(6) (a) Morita, K.; Yamaguchi, A.; Teramae, N. J. Electroanal. Chem. 2004, 563, 249. (b) Zhang, X.; Fakler, A.; Spichiger, U. E. Electroanalysis 1998, 10, 114. (c) Li, X.; Liu, Y.; Zhu, A.; Luo, Y.; Deng, Z.; Tian, Y. Anal. Chem. 2010, 82, 6512. (d) Lu, Y.; Li, X.; Zhang, L.; Yu, P.; Su, L.; Mao, L. Anal. Chem. 2008, 80, 1883. (e) Yan, J.; Zhou, Y.; Yu, P.; Su, L.; Mao, L.; Zhang, Q.; Zhu, D. Chem. Commun. 2008, 4330. (7) (a) Gaddum, J. H. J. Physiol. (Oxford, U.K.) 1958, 155, 2P. (b) Hamsher, A. E.; Xu, H.; Guy, Y.; Sandberg, M.; Weber, S. G. Anal. Chem. 2010, 82, 6370. (c) Xu, H.; Guy, Y.; Hamsher, A.; Shi, G.; Sandberg, M.; Weber, S. G. Anal. Chem. 2010, 82, 6377. (d) Kennedy, R. T.; Thompson, J. E.; Vickroy, T. W. J. Neurosci. Methods 2002, 114, 39. (e) Wang, M.; Roman, G. T.; Schultz, K.; Jennings, C.; Kennedy, R. T. Anal. Chem. 2008, 80, 5607. (8) (a) Ungerstedt, U.; Hallstrom, A. Life Sci. 1987, 41, 861. (b) For a review Watson, C. J.; Venton, B. J.; Kennedy, R. T. Anal. Chem. 2006, 78, 1391. (c) Wang, M.; Slaney, T.; Mabrouk, O.; Kennedy, R. T. J. Neurosci. Methods 2010, 190, 39. (d) Wang, M.; Roman, G. T.; Perry, M. L.; Kennedy, R. T. Anal. Chem. 2009, 81, 9072. (e) Robinson, T. E.; Justice Jr, J. B. Microdialysis In The Neurosciences; Elsevier Science Publishers BV, New York, 1991. (f) Nandi, P.; Lunte, S. M. Anal. Chim. Acta 2009, 651, 1. (g) Korf, J.; Huinink, K. D.; Posthuma-Tumpie, G. A. Trends Biotechnol. 2010, 28, 150. (9) (a) Niwa, O.; Torimitsu, K.; Morita, M.; Osborne, P.; Yamamoto, K. Anal. Chem. 1996, 68, 1865. (b) Pravda, M.; Bogaert, L.; Sarre, S.; Ebinger, G.; Kauffmann, J.-M.; Michotte, Y. Anal. Chem. 1997, 69, 2354. (c) Niwa, O.; Kurita, R.; Horiuchi, T.; Torimitsu, K. Anal. Chem. 1998, 70, 89. (d) Hayashi, K.; Kurita, R.; Horiuchi, T.; Niwa, O. Electroanalysis 2002, 14, 333. (e) Kurita, R.; Yabumoto, N.; Niwa, O. Biosens. Bioelectron. 2006, 21, 1649. (f) Jones, D. A.; Parkin, M. C.; Langemann, H.; Landolt, H.; Hopwood, S. E.; Strong, A. J.; Boutelle, M. G. J. Electroanal. Chem. 2002, 538, 243. (g) Hayashi, K.; Kurita, R.; Horiuchi, T.; Niwa, O. Biosens. Bioelectron. 2003, 18, 1249. (h) Lin, Y.; Zhang, Z.; Zhao, L.; Wang, X.; Yu, P.; Su, L.; Mao, L. Biosens. Bioelectron. 2010, 25, 1350. (10) (a) Pravda, M.; Marvin, C. A.; Sarre, S.; Michotte, Y.; Kauffmann, J.-M. Anal. Chem. 1996, 68, 2447. (b) Osborne, P.; Niwa, O.; Kato, T.; Yamamoto, K. J. Neurosci. Methods. 1997, 77, 143. (c) Niwa, O.; Horiuchi, T.; Kurita, R.; Torimitsu, K. Anal. Chem. 1998, 70, 1126. (d) Zhang, M.; Liu, K.; Gong, K.; Su, L.; Chen, Y.; Mao, L. Anal. Chem. 2005, 77, 6234. (e) Lin, Y.; Liu, K.; Yu, P.; Xiang, L.; Li, X.; Mao, L. Anal. Chem. 2007, 79, 9577. (f) Liu, K.; Lin, Y.; Yu, P.; Mao, L. Brain Res. 2009, 1253, 161. (g) Liu, K.; Lin, Y.; Xiang, L.; Yu, P.; Su, L.; Mao, L. Neurochem. Int. 2008, 52, 1247. (h) Lin, Y.; Zhu, N.; Yu, P.; Su, L.; Mao, L. Anal. Chem. 2009, 81, 2067.

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toring of cerebral Ca2+ and Mg2+ has not been reported so far. This study offers a novel, simple but effective electrochemical approach to online measurements of Ca2+ and Mg2+ in the rat brain of living animals. The method is envisaged to find wide applications in understanding of chemical events involved in some physiological and pathological processes. EXPERIMENTAL SECTION Reagents and Solutions. Sodium ascorbate (AA), dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), uric acid (UA), 5-hydroxytryptamine (5-HT), β-nicotinamide adenine dinucleotide (NADH) were all purchased from Sigma and used as supplied. CaCl2, MgCl2, Cu(NO3)2, CoCl2, ZnCl2, CdCl2, Mn(CH3COO)2, Ni(NO3)2, ethylenediamine-tetraacetic acid (EDTA), ethyleneglcol-bis(2-aminoethylether) tetraacetic acid (EGTA), and toluidine blue O (TBO) were purchased from Beijing Chemical Co. (Beijing, China). Tris-HCl buffer (pH 7.0) was used as supported electrolyte for offline voltammetric experiments. Ca2+- and Mg2+-free artificial cerebrospinal fluid (aCSF) was used as the perfusion solution for in vivo microdialysis and was prepared by mixing NaCl (128 mM), KCl (2.4 mM), KH2PO4 (0.5 mM), NaHCO3 (27.5 mM), Na2SO4 (0.5 mM) into Milli-Q water. Other chemicals were of at least analytical grade reagents and were used as received. All aqueous solutions were prepared with Milli-Q water. Electrodes and Thin-Layer Flow Cells. Glassy carbon (GC) electrodes used for offline voltammetric experiments (3 mm, diameter) and for online measurements in a thin-layer radial electrochemical flow cell were both purchased from Bioanalytical Systems Inc. (BAS, West Lafayette, IN). The thin-layer radial electrochemical flow cell consists of a thin-layer radial flow block with a 50 µm gasket, GC electrode (6 mm, diameter) as working electrode, stainless steel as auxiliary electrode, and Ag/AgCl electrode (3 M NaCl) as reference electrode. Both kinds of GC electrodes were polished first with emery paper and then with aqueous slurries of fine alumina powder (0.3 and 0.05 µm) on a polishing cloth, and finally rinsed with Milli-Q water under an ultrasonic bath for 10 min. Before surface modification with polymerized film of toluidine blue O (polyTBO), GC electrodes were electrochemically activated at +1.8 V in 0.10 M phosphate buffer (pH 7.0) for 30 s. PolyTBO-modified GC electrodes were prepared by cycling the electrodes in 0.10 M phosphate buffer (pH 7.0) containing 100 µM TBO within a potential range from -0.6 to +1.0 V at a scan rate of 50 mV/s for 20 cycles. After that, the electrodes were cycled in 0.10 M phosphate buffer (pH 7.0) containing no TBO within a potential range from -0.6 to +0.4 V at a scan rate of 50 mV/s until a stable voltammogram was obtained. For online electrochemical measurements, the polyTBOmodified GC electrode was fixed into the thin-layer radial electrochemical flow cell to serve as the detector for continuous measurements in a continuous-flow system. Experimental Setup. All electrochemical experiments were performed with a computer-controlled electrochemical analyzer (CHI 1030, CHI, Austin). Cyclic voltammetry was carried out in a conventional electrochemical cell with the polyTBO-modified GC electrode as working electrode, a platinum wire as counter electrode, and a Ag/AgCl electrode (KCl-saturated) as reference electrode. The integration of the thin-layer radial electrochemical flow cell with in vivo microdialysis to form online

Scheme 1. Schematic Illustration of The Online Electrochemical Method for (A) Simultaneous Measurements of Ca2+ and Mg2+ and (B) Continuous Monitoring of Mg2+ in The Rat Brain by Efficiently Integrating Electrochemical Detection with In Vivo Microdialysis

electroanalytical systems for simultaneous measurements of Ca2+ and Mg2+ (A) and for continuous monitoring of Mg2+ (B) in brain dialysate of rats was schematically shown in Scheme 1. For both kinds of measurements, brain dialysates continuously sampled from the cortex of living rats and standard solutions of Ca2+ and Mg2+ were delivered from gas-impermeable syringes (BAS) and pumped through tetrafluoroethylene hexafluoropropene (FEP) tubing by Pump 1 (CMA 100, CMA Microdialysis AB, Stockholm, Sweden) with aCSF as the perfusion solution at 1 µL/min. For simultaneous measurements of Ca2+ and Mg2+ in the rat brain, Tris-HCl buffer containing only NADH or NADH + Ca2+-chelating agent (i.e., EGTA) was perfused from Pump 2 with two syringes (i.e., Syringes 1 and 2) and was online mixed with the brain dialysates perfused from the rat brain from Pump 1 (Scheme 1A). For continuous monitoring of Mg2+ in the rat brain following global ischemia, Tris-HCl buffer containing NADH and EGTA was perfused from Pump 2 and was online mixed with the brain dialysates continuously perfused from the rat brain from Pump 1 (Scheme 1B). The polyTBO-modified electrode was polarized at 0.0 V both for simultaneous measurements of Ca2+ and Mg2+ and for continuous monitoring of Mg2+ in the brain microdialysate. Animal Surgery and Online Electrochemical Measurements. Surgeries for in vivo microdialysis were performed as reported previously.10f Briefly, adult male Sprague-Dawley rats (190-300 g) obtained from Health Science Center, Peking University, were housed on a 12:12 h light-dark schedule with food and water ad libitum. The animals were anaesthetized with pentobarbital (345 mg/kg, i.p.) and positioned onto a stereotaxic

frame. The microdialysis guide cannulas (BAS) were implanted in the cortex (AP ) 0 mm, L ) 5.2 mm from bregma; V ) 1 mm from dura) using standard stereotaxic procedures.13 The guide cannula was kept in place with three skull screws and dental cement. Stainless steel dummy blockers were inserted into the guide cannula and fixed until the insertion of the microdialysis probe (CMA, dialysis length, 4 mm; diameter, 0.24 mm). Throughout the surgery, the body temperature of the animals was maintained at 37 °C with a heating pad. Immediately after the surgery, the rats were placed into a warm incubator individually until they recovered from the anesthesia. The rats were allowed to recover for at least 24 h before the surgeries of global cerebral ischemia and in vivo microdialysis sampling. Surgeries for the global cerebral ischemia were performed as reported previously.10f In short, through a midline cervical incision, both common carotid arteries were exposed and isolated from surrounding connective tissue, with special care not to damage the vagus or the sympathetic nerves running close by. Global ischemia was induced (11) (a) Katz, E.; Lo ¨tzbeyer, T.; Schlereth, D. D.; Schuhmann, W.; Schmidt, H.L. J. Electroanal. Chem. 1994, 373, 189. (b) Mano, N.; Kuhn, A. Electrochem. Commun. 1999, 1, 497. (c) Mano, N.; Kuhn, A. J. Electroanal. Chem. 2001, 498, 58. (d) Cai, C.-X.; Xue, K.-H. Microchem. J. 2000, 64, 131. (12) (a) Milczarek, G. Langmuir 2009, 25, 10345. (b) Raj, C. R.; Behera, S. Langmuir 2007, 23, 1600. (c) Pariente, F.; Tobalina, F.; Moreno, G.; Herna´ndez, L.; Lorenzo, E.; Abruo`a, H. D. Anal. Chem. 1997, 69, 4065. (d) Munteanu, F. D.; Mano, N.; Kuhn, A.; Gorton, L. J. Electroanal. Chem. 2004, 564, 167. (e) Kubota, L. T.; Gorton, L. J. Solid State Electrochem. 1999, 3, 370. (13) Paxinos, G., Watson, C. The Rat Brain in Stereotaxic 542 Coordinates; Academic Press: San Diego, 1997.

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by occluding both carotid arteries with nontraumatic arterial clips for 50 min. Throughout the operation, supplements of pentobarbital (100 mg/kg) were given as required and the body temperature of the animals was maintained at 37 °C using a heating pad. Prior to the online electrochemical measurements, the microdialysis probes (CMA, dialysis length, 4 mm; diameter, 0.24 mm) were implanted into rat cortex and were perfused with aCSF solution at 1 µL/min for at least 90 min for equilibration. Online continuous monitoring of Mg2+ in the microdialysates sampled from the cortex of the rats was carried out concomitantly with the surgeries of global cerebral ischemia. After the microdialysis probe was implanted in the rat brain and was equilibrated through perfusing aCSF for at least 90 min, the surgeries of the cerebral ischemia were operated and the dialysate was continuously sampled with Pump 1 (Scheme 1B), After being online mixed with Tris-HCl buffer (pH 7.0) containing 1 mM NADH and 2 mM EGTA perfused from Pump 2, the dialysates were finally monitored in the electrochemical flow cell with the polyTBOmodified GC electrode as the detector. After the experiments, the animals were sacrificed with an overdose of pentobarbital. RESULTS AND DISCUSSION Amperometric Response of Ca2+ and Mg2+. To demonstrate the strategy for electrochemical detection of Ca2+ and Mg2+ through divalent cation enhancement toward electrocatalytic NADH oxidation, we compared cyclic voltammetry for the NADH oxidation at the polyTBO-modified GC electrode before and after addition of Ca2+ or Mg2+ into 0.10 M Tris-HCl buffer (pH 7.0). As depicted in Figure 1A, the oxidation of NADH occurs at 0.0 V at the polyTBO-modified GC electrode (black curve) under the conditions employed here. This potential remains much lower than that at bare GC electrode (i.e., +0.60 V under the same conditions), suggesting the good electrocatalytic activity of the polymerized TBO film toward the oxidation of NADH. The oxidation peak current was remarkably enhanced with the addition of Ca2+ or Mg2+ into the buffer (blue and red curves), which was elucidated by the formation of a ternary complex among TBO, divalent cations and NADH, as proposed by Katz, et al.11a and Mano, et al.14 The enhancement was subsequently exploited to constitute an electrochemical detector for effective measurements of electrochemically inactive Ca2+ and Mg2+ in the rat brain through efficiently integrating in vivo microdialysis (Scheme 1A and B). As displayed in Figure 1 B, the separated perfusion of Ca2+ and Mg2+ standards from Pump 1 and perfusion of 1 mM NADH in Tris-HCl buffer from Pump 2 (Scheme 1A) clearly produces current responses for each species, revealing the system with the polyTBO-modified electrode as the online detector was very responsive toward electroinactive Ca2+ and Mg2+ in a continuousflow system. In addition, the online system was relatively stable and reproducible for continuous and repeated measurements of Ca2+ and Mg2+ in a continuous-flow system (Supporting Information (SI) Figure S1). The current responses recorded for both Ca2+ and Mg2+ in the continuous-flow system remained unchanged after continuously running the measurements for

at least 2 h, and the relative standard deviations for the parallel measurements of Ca2+ (1.5 mM) and Mg2+ (1.5 mM), both for seven times, were 1.7% and 3.0%, respectively. More importantly, while the enhancement toward electrocatalytic oxidation of NADH could also be accomplished with other kinds of divalent cations, such as Cu2+, Mn2+, and Co2+, the perfusion of a mixture of other kinds of divalent cations at their physiological levels (i.e., Zn2+ (5 µM), Cu2+ (5 µM), Cd2+ (2 µM), Mn2+ (2 µM), Co2+ (2 µM), and Ni2+ (2 µM)) did not produce a recordable current response, as displayed in Figure 2, revealing that these kinds of divalent cations did not interfere with the measurements of Ca2+ and Mg2+, due to the high levels of Ca2+ and Mg2+ in the extracellular fluid of the rat brain (3-4 orders of magnitude higher than those of other kinds of divalent cations).5a Moreover, the perfusion of electroactive species endogenously existing in the rat brain, such as uric acid (UA), ascorbic acid (AA),10f,15 and electroactive neurotrans-

(14) (a) Mano, N.; Kuhn, A.; Menu, S.; Dufoure, E. J. Phys. Chem. Chem. Phys. 2003, 5, 2082. (b) Toh, C.-S.; Bartlett, P. N.; Mano, N.; Aussenac, F.; Kuhn, A.; Dufourc, E. J. Phys. Chem. Chem. Phys. 2003, 5, 588.

(15) (a) Roy, P. R.; Saha, M. S.; Okajima, T.; Park, S.-G.; Fujishima, A.; Ohsaka, T. Electroanalysis 2004, 16, 1777. (b) Raj, C. R.; Ohsaka, T. Electroanalysis 2002, 14, 679.

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Figure 1. (A) Typical CVs obtained at the poly(toluidine blue O)modified GC electrode in 0.10 M Tris-HCl buffer (pH 7.0) containing 2 mM NADH in the absence (black curve) and presence of 20 mM Ca2+ (blue curve) or 20 mM Mg2+ (red curve). Dashed black curve represents CV obtained at the poly(toluidine blue O)-modified GC electrode in 0.10 M Tris-HCl buffer (pH 7.0) containing no NADH. Scan rate, 20 mV/s. (B) Current-time responses obtained with the online electroanalytical system with the polyTBO-modified GC electrode as detector toward Ca2+ (1.5 mM) and Mg2+ (1.5 mM). The standard solutions of Ca2+ and Mg2+ (as indicated in the figure) in aCSF were perfused from Pump 1 and online mixed with 1 mM NADH solution in 0.10 M Tris-HCl buffer (pH 7.0) perfused from Pump 2. The perfusion rates for both pumps were 1 µL/min. The electrode was polarized at 0 V.

Figure 2. Current-time responses obtained with the system toward uric acid (UA, 10 µM), ascorbic acid (AA, 10 µM), dopamine (DA, 10 µM), 3,4-dihydroxyphenylacetic acid (DOPAC, 10 µM), 5-hydroxytryptamine (5-HT, 10 µM), a mixture of divalent cations (M2+, 5 µM Cu2+, 2 µM Mn2+, 2 µM Cd2+, 5 µM Zn2+, 2 µM Co2+, and 2 µM Ni2+), Ca2+ (1.5 mM), and Mg2+ (1.5 mM), as indicated in the figure. The standard solutions (as indicated in the figure) in a CSF were perfused from Pump 1 and online mixed with 1 mM NADH solution in 0.10 M Tris-HCl buffer (pH 7.0) perfused from Pump 2. Other conditions were the same as those in Figure 1B.

mitters did not produce current response either (Figure 2), suggesting that the measurements of Ca2+ and Mg2+ is also free from the interference from these species with the system developed in this study. Collectively, the utilization of the fundamental phenomenon on the divalent cation enhancement toward the electrocatalytic oxidation of NADH essentially constitutes an effective analytical scheme for reliable and durable measurements of electrochemically inactive Ca2+ and Mg2+ in a continuous-flow system. Such a feature substantially forms a strong basis both for simultaneous measurements of Ca2+ and Mg2+ and for continuous monitoring of Mg2+ in the rat brain with a technically simple electrochemical method, as described below. Toward Online Simultaneous Measurements of Cerebral 2+ Ca and Mg2+. While the results demonstrated above strongly suggest that the system integrating a polyTBO-based electrochemical detector with in vivo microdialysis is very attractive for simultaneous measurements of Ca2+ and Mg2+ in rat brain, the differentiation of the net current response for each species from the total response for a mixture containing both Ca2+ and Mg2+ remains critical for the transition of divalent cation enhancement toward catalytic NADH oxidation from a fundamental surface electrocatalysis to a novel analytical scheme for both species. This is the case because the current enhancement by Ca2+ and Mg2+ was actually based on the same mechanism, and as a result, the current enhancement by each species was eventually overlapped, as depicted in Figure 1A. To sort out and thus achieve the net current response for each kind of Ca2+ and Mg2+ from the total current for both Ca2+ and Mg2+, we tried to use various kinds of masking reagents to selectively chelate Ca2+, and it did not affect the current response of Mg2+. Among all masking reagents studied including calcichrome (eriochrome blue black), oxalate 8-hydroxyquinoline and tartaric acid, EGTA was found to predominantly chelate with Ca2+, due to the large difference in the stability of the complexes formed between Ca2+ and Mg2+ with EGTA (logKCa/logKMg ≈ 106) in a neutral or weakly acidic

Figure 3. (A) Typical current-time responses obtained with the system toward Ca2+ (1.5 mM) and Mg2+ (1.5 mM) under the same conditions as those in Figure 1 B, with an exception that 2 mM EGTA was mixed into 1 mM NADH in 0.10 M Tris-HCl buffer (pH 7.0), as indicated under the blue line in the figure. (B) Typical current-time responses obtained with the system toward the mixture of Ca2+ (1 mM) + Mg2+ (1 mM) or pure Ca2+ (1 mM), as indicated in the figure. The mixture and pure Ca2+ prepared in a CSF were perfused from Pump 1. A 1 mM pure NADH solution in Tris-HCl buffer (pH 7.0) or its mixture with 2 mM EGTA was perfused from Pump 2 (indicated under the blue lines). Perfusion rate for both pumps was 1 µL/min. Other conditions were same as Figure 1B.

solution.16 As a consequence, EGTA was used as the masking agent and was perfused with NADH from Pump 2 (Scheme 1 A) to selectively coordinate with Ca2+ and thus suppress its activity toward the current enhancement for NADH oxidation. By doing so, the simultaneous measurements of Ca2+ and Mg2+ in the rat brain could thus be performed by first measuring the total current response for both Ca2+ and Mg2+ with pure NADH solution in the Tris-HCl buffer as the perfusion solution for Pump 2, and then measuring the current response specific for Mg2+ with NADH solution in the Tris-HCl buffer containing EGTA masking reagent as the perfusion solution for Pump 2. The net current response for Ca2+ was thus able to calculate by subtracting the current obtained for both Ca2+ and Mg2+ with the current selectively recorded for Mg2+. As displayed in Figure 3 A, the addition of EGTA masking reagent in the NADH solution pumped from Pump 2 totally suppressed the current response of Ca2+, while the response of Mg2+ was still clearly maintained. On the other hand, we have run control experiments to clarify the possible effect of the presence of EGTA on the current response of Mg2+ at the polyTBO-modified electrode, as depicted in Figure 3B. With pure NADH solution as the perfusion solution for Pump 2, the online system produces current responses of 186 nA and 117 nA for a mixture of Ca2+ (1 mM) and Mg2+ (1 mM) and pure Ca2+ solution (1 mM), respectively. Meanwhile, with a mixture consisting of NADH and EGTA in Tris-HCl buffer as the perfusion solution for Pump 2, the online system produces a current response of 65 nA for Mg2+ (1 mM). The sum of the current responses obtained for each kind of Ca2+ and Mg2+ was almost close to the current response (16) Marhol, M.; Cheng, K. L. Anal. Chem. 1970, 42, 652.

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Figure 4. (A) Typical current-time responses obtained for the dialysate continuously sampled for the brain cortex of living rats with the online system. The dialysate and aCSF were perfused from Pump 1. A 1 mM NADH solution in Tris-HCl buffer containing 2 mM EGTA or EDTA (indicated in the figure) at the same concentration was perfused from Pump 2 (Scheme 1A). (B) Typical current-time responses obtained with the system for the brain microdialysate continuously sampled from the cortex of the rat brain. The brain dialysate was continuously sampled from Pump 1 and online mixed with pure NADH solution (1 mM) in Tris-HCl buffer (pH 7.0) or a mixture of NADH (1 mM) + EGTA (2 mM), perfused from Pump 2 (indicated under the lines). Other conditions were the same as those in Figure 1B.

achieved for the mixture of both species. This, on one hand, suggests that the presence of EGTA in the system did not greatly interfere with the selective measurement of Mg2+, while efficiently masked the current response of Ca2+, on the other hand, validates the method demonstrated here for sorting out the net current responses for Ca2+ and Mg2+. Under the conditions employed here, well-defined current responses were recorded for Ca2+ and Mg2+ (SI Figure S2). The current responses were linear with the concentrations of Ca2+ and Mg2+ with the same dynamic linear range from 0.1 to 2 mM for both species. The linear regression for Ca2+ and Mg2+ were I (nA) ) 24.19 + 102.9CCa2+ (mM), (γ ) 0.9955) and I (nA) ) 9.34 + 51.45CMg2+ (mM) (γ ) 0.9985), respectively. The dynamic linear ranges achieved with the online analytical system well cover the physiological levels of both species, further validating application of the system developed in this study based on the divalent cation enhancement toward electrocatalytic oxidation of NADH for simultaneous measurements of Ca2+ and Mg2+ in the rat brain. Considering the relatively complicated chemical environments involved in the cerebral systems, it remains very essential to investigate the selectivity of the method with the dialysate in vivo continuously sampled from the brain cortex of freely moving rats. To accomplish such a purpose, we separately added EGTA and EDTA into the mixture of NADH to selectively chelate Ca2+ and universally chelate Ca2+ and Mg2+, respectively. As displayed in Figure 4A, with the presence of EGTA into the NADH solution 9890

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and the resulting mixture being perfused from Pump 2, the switch of Pump 1 from pure aCSF into the brain dialysate clearly produced a current signal with the system, which was later decreased to the background level when Pump 1 was switched back to pure aCSF. This current response was ascribed to the current response for Mg2+ in the cortex in the brain, as described above. Contrarily, when EDTA was added into the NADH solution and the resulting mixture was perfused from Pump 2, the switch of Pump 1 from pure aCSF again into the brain dialysate did not produce an observable current response (Figure 4A). This was not surprising since EDTA can universally coordinate with almost all kinds of divalent cations and thus its presence into the perfusion solution was anticipated to suppress the current enhancement of divalent cations toward the electrocatalytic NADH oxidation. More importantly, a close look at the current responses in Figure 4 A reveals that the currents obtained for the brain dialysate with EDTA as the chelating agent was essentially the same as the background current obtained with the mixture of NADH and EGTA perfused from Pump 2 and with pure aCSF perfused from Pump 1 (Scheme 1 A). This result substantially demonstrates that no other chemical species endogenously existing in the cerebral systems were electrochemically oxidized or reduced at the polyTBO-modified electrode under the present conditions. This demonstration again ensures that the method developed in this study is very selective against the species endogenously existing in the rat brain. Figure 4B displays the current-time responses recorded with the online analytical system for the dialysate continuously sampled from the brain cortex of living rats. As described above, the current response obtained with pure NADH perfused from Pump 2 was used as the signal readout for both Ca2+ and Mg2+, while the response obtained a mixture of NADH and EGTA perfused from Pump 2 as the signal readout for Mg2+ in the rat cortex. The current response for Ca2+ was calculated as the difference between both. In this manner, the basal levels of Ca2+ and Mg2+ in the dialysate from the rat cortex were determined to be 285 ± 106.4 µM (n ) 3) and 240.3 ± 92.7 µM (n ) 3), respectively, which were almost consistent with the reported values.5a To further validate the simultaneous measurements of the basal levels of Ca2+ and Mg2+ in the rat brain, we have also determined the concentrations of both species with a traditional method, ICP-AES (SI Table S1). The basal levels of Ca2+ and Mg2+ in the rat cortex were determined to be 267.7 ± 106.2 µM (n ) 3) and 230.3 ± 124.3 µM (n ) 3), respectively. These values were quite close to those determined with the online system developed in this study, validating the application of our online system for effective measurements of both kinds of physiologically important metal cations in the cerebral systems. Toward Online Continuous Monitoring of Cortex Mg2+ Following Global Ischemia. In addition to the demonstration above, the use of masking agent EGTA to differentiate the net current responses selectively for each kind of Ca2+ and Mg2+, enabling the simultaneous measurements of the basal levels of both species in the rat brain, can also be used for continuous selective monitoring of Mg2+ in the rat brain following global ischemia. Moreover, in addition to its selectivity against the electroactive species including other kinds of divalent cations endogenously existing in the cerebral systems, the method

differences in the animals and in the brain regions used in our study from those in the previous reports. The observed decrease was understood by the competition of Mg2+ with Ca2+ for influx through the N-methyl-D-aspartate receptor gate, which was trigged to open by brain ischemia, as reported previously.3a,17

Figure 5. Typical current-time responses obtained for the cortex dialysate continuously sampled from the rats under normal and global ischemia conditions, as indicated in the figure. Other conditions were the same as those in Figure 1B.

developed here possesses a high selectivity against Ca2+ upon the addition of Ca2+-masking agent EGTA into the system (Scheme 1 B), as shown in SI Figure S3. Moreover, the online analytical system also bears a high tolerance against the changes in the chemical environments co-occurred following global ischemia including the fluctuation in levels of extracellular pH and O2, ascorbic acid, and NADH (SI Figure S4). This property, together with excellent analytical properties of this method, substantially validates its application for durable and reliable monitoring of Mg2+ in the rat brain following global ischemia, as described below. Figure 5 displays a typical current-time response recorded for the brain dialysate continuously sampled from the cortex of the rats following global ischemia. The level of Mg2+ in the cortex microdialysate was decreased by 26.3 ± 2.8% (n ) 3), following global brain ischemia induced by occluding the bilateral common carotid arteries of the rats. This value was lower than those reported in the literature,5c,17 presumably because of the (17) Lee, M.-S.; Wu, Y.-S.; Yang, D.-Y.; Lee, J.-B.; Cheng, F.-C. Clin. Chim. Acta 2002, 318, 121.

CONCLUSIONS By utilizing the fundamental electrochemical phenomena of divalent cation enhanced electrocatalytic NADH oxidation, we have developed a novel, simple but effective electrochemical platform both to simultaneous measurements of the basal levels of Ca2+ and Mg2+ and to continuous monitoring of Mg2+ change in the cerebral systems. It is very remarkable that, compared with the existing methods for the measurements described in this study, the method demonstrated here is advantageous in terms of its simplicity both in instrumentation and in the experimental procedures and near real-time nature, and is thus highly anticipated to find wide applications in understanding chemical events involved in physiological and pathological processes. ACKNOWLEDGMENT We thank the financial support from by NSFC (Grant Nos. 20975104, 20935005, 20625515, 90813032 for L.M. and 20905071 for Y.L.), National Basic Research Program of China (2007CB935603 and 2010CB933502), and Chinese Academy of Sciences (KJCX2YW-W25 and KJCX2-YW-H11). SUPPORTING INFORMATION AVAILABLE Figures S1-S4 and Table S1. This material is available free of charge via the Internet at http://pubs.acs.org.

Received for review September 4, 2010. Accepted October 26, 2010. AC102605N

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