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Physiologically Relevant Online Electrochemical Method for Continuous and Simultaneous Monitoring of Striatum Glucose and Lactate Following Global ...
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Anal. Chem. 2009, 81, 2067–2074

Physiologically Relevant Online Electrochemical Method for Continuous and Simultaneous Monitoring of Striatum Glucose and Lactate Following Global Cerebral Ischemia/Reperfusion Yuqing Lin, Ningning Zhu, Ping Yu, Lei Su, and Lanqun Mao* Beijing National Laboratory for Molecular Sciences, Institute of Chemistry, The Chinese Academy of Sciences (CAS), Beijing 100190, China This study demonstrates a new electroanalytical method with a high physiological relevance for simultaneous online monitoring of glucose and lactate in the striatum of the rat brain following global cerebral ischemia/reperfusion. The online analytical method is based on the efficient integration of in vivo microdialysis sampling with an online selective electrochemical detection with the electrochemical biosensors with dehydrogenases, i.e., glucose and lactate dehydrogenases, as recognition elements. The dehydrogenase-based electrochemical biosensors are developed onto the dual split-disk plastic carbon film (SPCF) electrodes with methylene green (MG) adsorbed onto single-walled carbon nanotubes (SWNTs) as the electrocatalyst for the oxidation of dihydronicotiamide adenine dinucleotide (NADH) at a low potential of 0.0 V (vs Ag/AgCl). Artificial cerebrospinal fluid (aCSF) containing NAD+ is externally perfused from a second pump and online mixed with the brain microdialysates to minimize the variation of pH that occurred following the cerebral ischemia/reperfusion and to supply NAD+ cofactor and O2 for the enzymatic reactions of dehydrogenases and ascorbate oxidase, respectively. As a result, the developed online electroanalytical method exhibits a high selectivity against the electrochemically active species endogenously existing in the cerebral systems and a high tolerance against the variation of pH and O2 following cerebral ischemia/reperfusion. This property, along with the good linearity and a high stability toward glucose and lactate as well as little cross-talk between two biosensors, substantially makes this method possible for the continuous, simultaneous, and online monitoring of glucose and lactate in the rat brain following global cerebral ischemia/reperfusion. This study establishes a new and effective platform for the investigation of the energy metabolism in physiological and pathological processes. As one kind of fatal diseases, ischemic stroke has drawn considerable attention over the last several decades because it can cause death or disability and thus result in a huge cost of * Corresponding author. Fax: +86-10-62559373. E-mail: [email protected]. 10.1021/ac801946s CCC: $40.75  2009 American Chemical Society Published on Web 02/19/2009

public health burden.1 Increasing evidence has demonstrated that understanding of the neurochemical processes, such as energy failure, anoxic depolarization, glutamate excitotoxicity, peri-infarct depolarization, and oxidative stress in the early stage of cerebral ischemia, is of great physiological and pathological importance because these neurochemical processes in the acute ischemic period trigger the pathologic damages to brain injury and eventually to neuron death.2 Thus, development of effective methods to probe these neurochemical processes could offer a straightforward basis for the early therapeutic intervention, which is of great importance in neuroprotective therapeutics for the ischemic injury. Motivated by understanding the neurochemical processes in acute ischemic period, we have recently been interested in developing new electrochemical methods to continuously monitor chemical species involved in the neurochemical processes with a real-time feature.3 For example, by taking advantage of the excellent electrocatalytic activity of carbon nanotubes toward the electrochemical oxidation of ascorbic acid (AA)4 and of the efficient integration of in vivo microdialysis and selective electrochemical detection, we have recently developed a new electrochemical method for continuous online measurements of AA in rat brain.3b The avoidance of sample collection and separation, good analytical properties, and near real-time nature as well as the less technical demand of this method substantially enabled its application for the comparative studies on the changes in the extracellular AA following different kinds of cerebral ischemia/ (1) For reviews (a) White, B. C.; Sullivan, J. M.; DeGracia, D. J.; O’Neil, B. J.; Neumar, R. W.; Grossman, L. I.; Rafols, J. A.; Krause, G. S. J. Neurol. Sci. 2000, 179, 1. (b) Rossi, D. J.; Brady, J. D.; Mohr, C. Nat. Neurosci. 2007, 10, 1377. (c) Gorelick, P. B. Stroke 2002, 33, 862. (d) Bronner, L. L.; Kanter, D. S.; Manson, J. E. N. Engl. J. Med. 1995, 333, 1392. (2) (a) Kaplan, B.; Brint, S.; Tanabe, J.; Jacewicz, M.; Wang, X.-J.; Pulsinelli, W. Stroke 1991, 22, 1032. (b) Lee, J.-M.; Grabb, M. C.; Zipfel, G. J.; Choi, D. W. J. Clin. Invest. 2000, 106, 723. (c) Dirnagl, U.; Iadecola, C.; Moskowitz, M. A. Trends Neurosci. 1999, 22, 391. (d) Faden, A. I. Clin. Neuropharmacol. 1987, 10, 193. (e) Wauquier, A.; Ashton, D.; Clincke, G. H. C. Ann. N.Y. Acad. Sci. 1988, 522, 478. (3) (a) Mao, L.; Osborne, P.; Yamamoto, K.; Kato, T. Anal. Chem. 2002, 74, 3684. (b) Zhang, M.; Liu, K.; Gong, K.; Su, L.; Chen, Y.; Mao, L. Anal. Chem. 2005, 77, 6234. (c) Lin, Y.; Liu, K.; Yu, P.; Xiang, L.; Li, X.; Mao, L. Anal. Chem. 2007, 79, 9577. (d) Zhang, M.; Mao, L. Front. Biosci. 2005, 10, 345. (4) (a) Zhang, M.; Gong, K.; Zhang, H.; Mao, L. Biosens. Bioelectron. 2005, 20, 1270. (b) Gong, K.; Zhang, M.; Yan, Y.; Su, L.; Mao, L.; Xiong, S.; Chen, Y. Anal. Chem. 2004, 76, 6500. (c) Wang, J.; Musameh, M.; Lin, Y. J. Am. Chem. Soc. 2003, 125, 2408.

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reperfusion.5 As an attempt that is concomitantly made with our further studies on profiling of AA levels in the acute ischemic period, we have been searching for an electroanalytical approach to understanding of the energy metabolism following the cerebral ischemia/reperfusion. This pursuit is motivated by the great importance of the energy metabolism in the cerebral ischemia and the fact that the change in the levels of glucose and lactate in the cerebral systems could offer information on cerebral energy metabolism with implication in the diagnosis of cerebral ischemia.1a,b,6 Although the pressing need of diabetes diagnosis has greatly activated intensive interests in the optical7 and electrochemical biosensing of glucose,8 the complexity of the cerebral environments, which becomes even more complicated following the cerebral ischemia/reperfusion, substantially enables the effective monitoring of glucose and lactate under pathological conditions a challenge for most kinds of the electrochemical biosensors reported so far.9 For instance, the cerebral ischemia/reperfusion normally occurs with a large fluctuation in the brain anoxia, resulting in the change in the O2 level in the brain.10 Such a change substantially renders difficulties in applying oxidasebased electrochemical biosensors for the monitoring of glucose and lactate following the cerebral ischemia/reperfusion because the current responses of this kind of biosensors based on the detection of O2 consumption, H2O2 production, or mediator regeneration are all O2-dependent.11 Although the current responses at the so-called third-generation electrochemical biosensors based on the direct electron transfer of the oxidases (5) Liu, K.; Lin, Y.; Xiang, L.; Yu, P.; Su, L.; Mao, L. Neurochem. Int. 2008, 52, 1247. (6) (a) Hillered, L.; Vespa, P. M.; Hovda, D. A. J. Neurotrauma 2005, 22, 3. (b) Shimojo, N.; Fujino, K.; Kitahashi, S.; Nakao, M.; Naka, K.; Okuda, K. Clin. Chem. 1991, 37, 1978. (c) Whitea, B. C.; Sullivana, J. M.; DeGraciaa, D. J.; O’Neila, B. J.; Neumar, R. W.; Pryce, J. D.; Gant, P. W.; Saul, K. J. Clin. Chem. 1970, 16, 562. (7) (a) Suri, J. T.; Cordes, D. B.; Cappuccio, F. E.; Wessling, R. A.; Singaram, B. Angew. Chem., Int. Ed. 2003, 42, 5857. (b) Nakayama, D.; Takeoka, Y.; Watanabe, M.; Kataoka, K. Angew. Chem., Int. Ed. 2003, 42, 4197. (8) (a) Maidan, R.; Heller, A. J. Am. Chem. Soc. 1991, 113, 9004. (b) Raitman, O. A.; Katz, E.; Bu ¨ ckmann, A. F.; Willner, I. J. Am. Chem. Soc. 2002, 124, 6487. (c) Tam, K. T.; Zhou, J.; Pita, M.; Ornatska, M.; Minko, S.; Katz, E. J. Am. Chem. Soc. 2008, 130, 10888. (d) Anicet, N.; Anne, A.; Moiroux, J.; Savea´nt, J. J. Am. Chem. Soc. 1998, 120, 7115. (e) Wang, J.; Lu, F. J. Am. Chem. Soc. 1998, 120, 1048. (f) Zayats, M.; Katz, E.; Willner, I. J. Am. Chem. Soc. 2002, 124, 2120. (g) Osborne, P.; Niwa, O.; Yamamoto, K. Anal. Chem. 1998, 70, 1701. (h) Chaubey, A.; Malhotra, B. D. Biosens. Bioelectron. 2002, 17, 441, and references therein. (9) (a) Szamocki, R.; Velichko, A.; Holzapfel, C.; Mu ¨ cklich, F.; Ravaine, S.; Garrigue, P.; Sojic, N.; Hempelmann, R.; Kuhn, A. Anal. Chem. 2007, 79, 533. (b) Ito, T.; Kunimatsu, M.; Kaneko, S.; Ohya, S.; Suzuki, K. Anal. Chem. 2007, 79, 1725. (c) Pereira, C. M.; Oliveira, J. M.; Silva, R. M.; Silva, F. Anal. Chem. 2004, 76, 5547. (d) Hrapovic, S.; Liu, Y.; Male, K. B.; Luong, J. H. T. Anal. Chem. 2004, 76, 1083. (e) Shin, J.; Marxer, S. M.; Schoenfisch, M. H. Anal. Chem. 2004, 76, 4543. (f) Muguruma, H.; Kase, Y.; Uehara, H. Anal. Chem. 2005, 77, 6557. (g) Yemini, M.; Reches, M.; Gazit, E.; Rishpon, J. Anal. Chem. 2005, 77, 5115. (h) Corvis, Y.; Walcarius, A.; Rink, R.; Mrabet, N. T; Rogalska, E. Anal. Chem. 2005, 77, 1622. (i) Zhang, M.; Mullens, C.; Gorski, W. Anal. Chem. 2007, 79, 2446. (j) Rowinski, P.; Rowinska, M.; Heller, A. Anal. Chem. 2008, 80, 1746. (k) Wang, J.; Chen, L.; Jiang, M.; Lu, F. Anal. Chem. 1999, 71, 5009. (10) (a) Persson, L.; Hillered, L. J. Neurosurg. 1992, 76, 72. (b) Back, T.; Hoehn, M.; Mies, G.; Bush, E.; Schmitz, B.; Kohno, K.; Hossmann, K. A. Ann. Neurol. 2000, 47, 485. (c) Sweeney, M. I. Neurosci. Rev. 1997, 21, 207. (d) Liu, S.; Shi, H.; Liu, W.; Furuichi, T.; Timmins, G. S.; Liu, K. J. Cereb. Blood Flow Metab. 2004, 24, 343. (11) (a) Shram, N. F.; Netchiporouk, L. I.; Martelet, C.; Jaffrezic-Renault, N.; Bonnet, C.; Cespuglio, R. Anal. Chem. 1998, 70, 2618. (b) Eklund, S.; Taylor, D.; Kozlov, E.; Prokop, A.; Cliffel, D. Anal. Chem. 2004, 76, 519.

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are theoretically O2-independent, these kinds of biosensors are still far from practical applications.12 On the other hand, the cerebral ischemia/reperfusion generally occurs with response to the acidity in the extracellular fluid.13 For instance, the extracellular pH in the core region of ischemia/reperfusion was decreased from 7.4 to 6.8.14 This change could make it difficult for applying the dehydrogenase-based electrochemical biosensors for the measurements of glucose and lactate following the cerebral ischemia/reperfusion because the electrocatalytic processes for the electrochemical oxidation of NADH cofactor involved in such a kind of biosensor are generally pHdependent.15 This study demonstrates a physiologically relevant online electrochemical method for continuously and simultaneously monitoring glucose and lactate with a high selectivity against the electrochemically active species endogenously existing in the cerebral systems and with a high tolerance against the variation of extracellular O2 and pH that occur following cerebral ischemia/reperfusion. The method is actually based on the utilization of dehydrogenases, rather than oxidases, as the recognition elements since the dehydrogenase-based catalytic chemical oxidation of glucose and lactate is O2-independent (Scheme 1). Moreover, an external solution containing NAD+ cofactor is pumped from a second pump and online mixed with the brain microdialysates to minimize the pH variation induced by the cerebral ischemia/reperfusion and to supply NAD+ cofactor and O2 needed for the enzymatic reactions with dehydrogenases and ascorbate oxidase, respectively. This study could offer a new electrochemical method with a high physiological relevance for continuous and simultaneous online monitoring of glucose and lactate in the rat brain following the cerebral ischemia/reperfusion. EXPERIMENTAL SECTION Reagents and Solutions. Sodium ascorbate (AA), dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), uric acid (UA), 5-hydroxytryptamine (5-HT), D(+)-glucose, nicotinamide adenine dinucleotide (NAD+), dihydronicotiamide adenine dinucleotide (NADH), glucose oxidase (GOx, EC 1.1.3.4, from Aspergillus niger), ascorbate oxidase (AOx, Cucurbita species, EC 1.10.3.3), glucose dehydrogenase (GDH, EC 1.1.1.47, from Pseudomonas sp.) and L-lactate dehydrogenase (LDH, EC 1.1.1.27, type II, from rabbit muscle) were all purchased from Sigma and used as supplied. Bovine serum albumin (BSA) was obtained from Proliant. L(+)-Lactate acid (90%) was bought from ACROS Organics. Methylene green (MG) was purchased from Beijing (12) (a) Khan, G. F.; Ohwa, M.; Wernet, W. Anal. Chem. 1996, 68, 2939. (b) Palmisano, F.; Zambonin, P. G. Anal. Chem. 2002, 74, 5913. (13) (a) Schurr, A. Int. J. Mol. Med. 2002, 10, 131. (b) Siesjo ¨, B. K. Brain Energy Metabolism; John Wiley & Sons, Ltd.: New York, 1978. (c) Sako, K.; Kobatake, K.; Yamamoto, Y. L.; Diksic, M. Stroke 1985, 16, 828. (d) Kobatake, K.; Sako, K.; Izawa, M.; Yamamoto, Y. L.; Hakim, A. M. Stroke 1984, 15, 540. (e) Astrup, J.; Symon, L.; Bronston, N. M.; Lassen, N. A. Stroke 1977, 8, 51. (14) (a) Jabre, A.; Bao, Y.; Spatz, E. L. Surg. Neurol. 2000, 54, 55. (b) Nakai, H.; Yamamoto, Y. L.; Diksic, M.; Worsley, K. J.; Takara, E. Stroke 1988, 19, 764. (c) Lipton, P. Physiol. Rev. 1999, 79, 1431. (15) (a) Gorton, L.; Domı´nguez, E. Rev. Mol. Biotechnol. 2002, 82, 371. (b) Karyakin, A. A.; Karyakina, E. E.; Schuhmann, W.; Schmidt, H.-L. Electroanalysis 1999, 11, 553. (c) Munteanu, F. D.; Kubota, L. T.; Gorton, L. J. Electroanal. Chem. 2001, 509, 2.

Scheme 1. Schematic Diagram of a Physiologically Relevant Online Electroanalytical System for Continuous and Simultaneous Monitoring of Striatum Glucose and Lactate Following Global Cerebral Ischemia/Reperfusion

Chemical Company (Beijing, China). Single-walled carbon nanotubes (SWNTs,