Microfluidic Chip-Based Online Electrochemical Detecting System

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Microfluidic Chip-Based Online Electrochemical Detecting System for Continuous and Simultaneous Monitoring of Ascorbate and Mg2+ in Rat Brain Xia Gao,† Ping Yu,‡ Yuexiang Wang,‡ Takeo Ohsaka,Δ Jianshan Ye,*,† and Lanqun Mao*,‡ †

College of Chemistry and Chemical Engineering, South China University of Technology, Guangzhou 510640, China 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 Δ Department of Electronic Chemistry, Interdisciplinary Graduate School of Science and Engineering, Tokyo Institute of Technology, 4259, Nagatsuta, Midori-ku, Yokohama 226-8502, Japan ‡

ABSTRACT: This study demonstrates a microfluidic chipbased online electrochemical detecting system for in vivo continuous and simultaneous monitoring of ascorbate and Mg2+ in rat brain. In this system, a microfluidic chip is used as the detector for both species. To fabricate the detector, a single-channel microfluidic chip is developed into an electrochemical flow cell by incorporating the chip with an indium− tin oxide (ITO) electrode as working electrode, an Ag/AgCl wire as reference electrode, and a stainless steel tube as counter electrode. Selective detection of ascorbate and Mg2+ is achieved by drop-coating single-walled carbon nanotubes (SWNTs) and polymerizing toluidine blue O (polyTBO) film onto the ITO electrode, respectively. Moreover, the alignment of SWNT-modified and polyTBO-modified electrodes and the solution introduction pattern are carefully designed to avoid any cross talk between two electrodes. With the microfluidic chip-based electrochemical flow cell as the detector, an online electrochemical detecting system is successfully established by directly integrating the microfluidic chip-based electrochemical flow cell with in vivo microdialysis. The microfluidic system exhibits sensing properties with a linear relationship from 5 to 100 μM for ascorbate and from 100 to 2000 μM for Mg2+. Moreover, this system demonstrates a high selectivity and stability and good reproducibility for simultaneous measurements of ascorbate and Mg2+ in a continuous-flow system. These excellent properties substantially render this system great potential for continuous and simultaneous online monitoring of ascorbate and Mg2+ in rat brain.

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Mg2+ in cerebral systems are important to understand the corelationship between these two species, ascorbate and Mg2+, in physiological and pathological processes. By integrating in vivo microdialysis with selective electrochemical detectors, we and others have successfully developed various online electrochemical detecting systems for continuous monitoring of one or two kinds of physiologically important species such as ascorbate,6 glucose/lactate,7 dopamine,8 and Ca2+/Mg2+,9 in cerebral systems. In these online electrochemical detecting systems, the electrochemical flow-through detector is composed of a commercially available flow cell integrated with a single or dual electrode that could be designed as the selective detectors for continuously monitoring one or two kinds of neurochemicals. Unfortunately, the uniform and the unchangeable electrode structure and alignment of the

ontinuous and simultaneous measurements of ascorbate and Mg2+ in the brain of living animals are of great physiological and pathological importance because both species in extracellular fluid are widely involved in some physiological events.1 For instance, as an electron donor, ascorbate serves as one of the most important small-molecular-weight antioxidants and free-radical scavengers and, as such, is normally neuroprotective.2 Moreover, ascorbate serves as a neuromodulator and is involved in neurotransmission processes.1e On the other hand, Mg2+ plays a critical role in defining the properties of adenosine-triphosphatases (ATPases) and serves as a cofactor in more than 300 enzymatic reactions.3 Moreover, Mg2+ and ascorbate are related to each other via glutamate release, a neurotransmission process that plays important roles in many physiological and pathological processes.4 To be specific, Mg2+ serves as a calcium channel blocker which is critical for glutamate release.5 On the other hand, ascorbate and glutamate affect each other through heteroexchange. Hence Mg2+ and ascorbate are two major factors that regulate glutamate release. Consequently, simultaneous measurements of ascorbate and © 2013 American Chemical Society

Received: June 10, 2013 Accepted: July 9, 2013 Published: July 9, 2013 7599

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commercially available flow cells offers limitations to these existing online analytical systems such as the capability for simultaneous monitoring of more than two kinds of neurochemicals and the tolerance against the cross talk for two components electrochemical analysis because of the fixed electrode alignment. To overcome this problem, we construct a new kind of electrochemical flow cells with a specially designed solution introduction strategy and an effective electrode alignment. In this study, we demonstrate a facile yet effective electrochemical platform for continuous and simultaneous online monitoring of ascorbate and Mg2+ in rat brain based on microfluidic chip technique. Over the past decades, the development of microfluidic systems for analyzing biological samples has dramatically increased.10 As a result, microfluidic systems have been used in various applications and specifically attractive in neurochemical detection because of their unique advantages such as versatility, easy fabrication and modification, negligible reagent/sample consumption and little waste generation, and, the most important, the capability to couple with various analytical systems.11 Additionally, compared to the commercially available flow cells, the capability to integrate multichannels into a microfluidic chip potentially makes the microfluidic techniques particularly attractive for high-throughput analysis for multicomponents, while the structural designability and flexibility including the design of solution introduction, electrode alignment, and microchannels substantially enable the microfluidic techniques to be particularly attractive for neurochemical analysis with less cross talk. The microfluidic chip-based online electrochemical detecting system demonstrated in this study is established by effectively integrating poly(dimethylsiloxane) (PDMS)-based microfluidic chip detector with in vivo microdialysis, as shown in Scheme 1 A. The microfluidic chip consists of a plate of PDMS containing a single channel and an indium−tin oxide (ITO) glass substrate for constructing the two working electrodes for the measurements of ascorbate and Mg2+, respectively (Scheme 1 B). The detection mechanisms of this chip-based online electrochemical platform are virtually based on the specific electrooxidation of ascorbate on carbon nanotubes and the divalent cation enhancement toward electrocatalytic NADH oxidation with polymerized film of toluidine blue O as the electrocatalyst, as demonstrated in our early study.9 Compared with the commercially available thin-layer flow cells, the microfluidic chip-based electrochemical flow cell demonstrated in this study is more flexible in structural design and electrode alignment with great potentiality for selective multicomponent analysis without cross talk. This study essentially paves a new approach to in vivo multiple neurochemical monitoring in a technically simple and experimentally designable fashion.

Scheme 1. (A) Schematic Illustration of the Microfluidic Chip-Based Online Detecting System for Continuous and Simultaneous Monitoring of Ascorbate and Mg2+ in Rat Brain and (B) Structure of the Microfluidic Chip-Based Electrochemical Detectora

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The microfluidic chip-based detector consists of two independent substrates: upper wafer, PDMS chip with a channel as a continuousflow microfluidic cell, 40 mm in length, 1 mm in width, and 500 μm micrometer in depth; lower wafer, SWNT-modified and polyTBOmodified electrodes as dual working electrodes. Ag/AgCl wire and stainless steel tube (also as outlet) were separately embedded into the PDMS chip and used as reference electrode (RE) and counter electrode (CE), respectively, for the electrochemical detection.

dissolving NaCl (128 mM), KCl (2.4 mM), CaCl2 (1.1 mM), KH2PO4 (0.5 mM), NaHCO3 (27.5 mM), and 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. Single-walled carbon nanotubes (SWNTs, < 2 nm in diameter and 0.5−50 μm in length) were purchased from Shenzhen Nanotech Port Co., Ltd. (Shenzhen, China). Prior to use, SWNTs were purified by refluxing the as-received SWNTs in 2.6 M nitric acid for 5 h, followed by centrifugation, resuspension, and filtration and air-drying to evaporate the solvent. The purified SWNTs were further heated under vacuum at 500 °C for 2 h. Fabrication of Microfluidic Chip-Based Electrochemical Flow Cells. As typically shown in Scheme 1 B, the microfluidic chip-based electrochemical flow cell developed in this study consists of a poly(dimethylsiloxane) (PDMS, Dow Corning, USA) cover plate containing a single channel and an indium−tin oxide (ITO) glass substrate for constructing SWNT-modified and polyTBO-modified electrodes as dual electrochemical detectors for ascorbate and Mg2+, respectively. The PDMS chip used in our experiments was fabricated via soft lithography.12 Briefly, a 4 cm × 1 mm straight-line pattern was designed and printed by a commercial printer. After lithography, a pattern (in the form of SU-8 photoresist) was made on a 4-in. silicon wafer to ensure a uniform height of ca. 500 μm and then was transferred to PDMS by replica molding. Finally, the PDMS replica was cut into slabs carrying the appropriate pattern and drilled go-through holes for placing an

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EXPERIMENTAL SECTION Reagents and Solutions. Sodium ascorbate, dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), uric acid (UA), and β-nicotinamide adenine dinucleotide (NADH) were all purchased from Sigma and used as supplied. MgCl2, CaCl2, Cu(NO3)2, CoCl2, ZnCl2, CdCl2, Mn(CH3COO)2, Ni(NO3)2, ethylene glycol bis(2-aminoethyl) 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 the electrolyte for offline voltammetric experiments. Mg2+-free artificial cerebrospinal fluid (aCSF) was used as a perfusion solution for in vivo microdialysis and was prepared by 7600

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clips. The wound was sutured immediately after the ischemia procedure was completed. Throughout the operation, supplements of chloral hydrate (100 mg/kg) were given as needed, and the body temperature of the animals was maintained at 37 °C by a heating pad. All electrochemical experiments were performed with a computer-controlled electrochemical analyzer (CHI 1030, CHI, Austin). For the online measurements, Mg2+-free aCSF was pumped through tetrafluoroethylene hexafluoropropene (FEP) tubing by Pump 1 (CMA 100, CMA Microdialysis AB, Stockholm, Sweden) into rat striatum at the perfusion rate of 1 μL/min (Scheme 1 B). Outlet brain dialysate was continuously delivered into inlet 1 from gas-impermeable syringes (BAS). On the upstream SWNT-modified electrode, ascorbate was detected. To eliminate the interference from Ca2+, Tris-HCl buffer (pH 7.0) containing NADH + Ca2+chelating agent (i.e., EGTA) was continuously perfused by Pump 2 into inlet 2 (inlet 2 was positioned in between the two electrodes on the substrate) and was online mixed with the outflow solution from the SWNT-modified electrode. Mg2+ was detected on the downstream polyTBO-modified electrode. For electrode calibration, the standard solutions of ascorbate and Mg2+ mixtures were delivered into inlet 1 instead of brain dialysate. The SWNT-modified and polyTBO-modified electrodes were polarized at 0.03 and 0.0 V for the measurements of ascorbate and Mg2+, respectively.

inlet tubing, a reference electrode, and a stainless steel tube outlet. Here, a microsized Ag/AgCl electrode, which was prepared by polarizing Ag wire (diameter, 0.2 mm) at 0.60 V in 0.10 M hydrochloride acid for 30 min, was used as reference electrode. A stainless steel tube with an inner diameter of 0.3 mm was used as the solution outlet and served as counter electrode in the microfluidic chip-based electrochemical flow cell as well. An ITO-coated glass plate was used as the substrate for preparing dual electrodes for simultaneous and selective detection of ascorbate and Mg2+. The ITO-coated glass plate was cleaned by sequentially sonicating the plate in acetone, 10% KOH, and distilled water, each for 10 min. After that, the ITO glass was partly covered with vaseline film with a thickness of ca. 2 mm, which was shaped into two separated, but serially lined electrodes as displayed in Scheme 1 B. The vaseline filmcovered ITO glass was then subjected to an etching process by immersing the glass into 5 M hydrochloride acid for 2 h. After that, the vaseline film was removed from the ITO surface, and the resulting ITO plate was sequentially rinsed in acetone and distilled water. For selective electrochemical detection of Mg2+, the downstream electrode was modified with polymerized film of toluidine blue O (polyTBO) under the procedures reported previously.9 Briefly, the polyTBO-modified electrode was prepared by cycling the electrode 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. The electrode was then 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. During the electropolymerization process, the part of exposed ITO employed as the upstream for detection of ascorbate was masked with tape to avoid the occurrence of electropolymerization of TBO at this part. After electropolymerization, the tape was removed, and the upstream electrode was modified with heat-treated SWNTs for selective detection of ascorbate. For this purpose, 2 mg/mL of the heattreated SWNTs was dispersed into ethanol, and 8 μL of the resulting dispersion was drop-coated onto the electrode. The electrode was then air-dried to evaporate the solvent to form SWNT-modified electrode. To form the microchip-based electrochemical flow cell for continuous electrochemical measurements, the PDMS chip with a microfluidic channel was tightly combined with the patterned ITO glass substrate under the external pressure (Scheme 1 B). Online Simultaneous Measurements of Ascorbate and Mg2+ in Brain Microdialysates. Experiments for in vivo microdialysis and global cerebral ischemia were performed based on the methods reported previously.6,7a−c Briefly, adult male Sprague−Dawley rats (250−300 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 anesthetized with chloral hydrate (345 mg/kg, i.p.) and positioned onto a stereotaxic frame. The microdialysis guide cannulas (BAS) were implanted in the striatum (AP = 0 mm, L = 4 mm from bregma; V = 4 mm from dura) using standard stereotaxic procedures. The rats were allowed to recover for at least 24 h before the surgeries of global two-vessel occlusion (2VO) cerebral ischemia and in vivo microdialysis sampling. Through a midline cervical incision, both common carotid arteries (CCA) were exposed and isolated from surrounding connective tissue. Global ischemia was induced by first occluding both carotid arteries with nontraumatic arterial

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RESULTS AND DISCUSSION By taking advantages of excellent properties of carbon nanotubes, which largely facilitate the electrochemical oxidation of ascorbate, we have previously developed a new mechanism for selective electrochemical detection of ascorbate.6a Such a mechanism was further exploited for continuous online monitoring of ascorbate in rat brain on a commercially available thin-layer radial electrochemical flow cell with singlewalled carbon nanotubes (SWNTs)-modified glassy carbon electrode as the selective electrochemical detector. Recently, we have also developed a new mechanism for electrochemical detection of electrochemically inactive Ca2+ and Mg2+ based on the current enhancement of divalent cations toward electrocatalytic oxidation of NADH with polymerized film of toluidine blue O (polyTBO) as the electrocatalyst.9 We further explored this mechanism for simultaneous online monitoring of both Ca2+ and Mg2+ in rat brain on a commercially available thinlayer radial electrochemical flow cell. In spite of these advancements, the utilization of these new mechanisms for simultaneous measurements of ascorbate and Mg2+ in rat brain on commercially available electrochemical flow cells remains challenging because NADH required for Mg2+ detection is readily oxidized on both SWNT-modified electrode13 (used for ascorbate detection) and polyTBO-modified electrode (used for Mg2+ detection) through redox-mediated surface electrocatalysis with PolyTBO as the electrocatalyst.9 Moreover, in addition to NADH oxidation on the SWNT-modified electrode, oxidation of high concentration (e.g., 100 μM) ascorbate is also observable on the polyTBO-modified electrode.14 These cross talk reactions on the two electrodes substantially invalidate the simultaneous measurements of both species with commercially available electrochemical flow cells. In this study, we use microfluidic techniques to circumvent this problem by taking advantage of the designable and flexible structure of the microfluidic chip-based flow cells. For continuous monitoring of ascorbate and Mg2+, a PDMS-based 7601

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NADH onto upstream SWNT-modified electrode (Figure 2 A). Figure 2 B shows the current responses obtained on the two

microfluidic chip-based detector was integrated with in vivo microdialysis to form an online electrochemical detecting platform. As displayed in Figure 1 A, the microfluidic chip-

Figure 2. (A) Schematic illustration of the microfluidic chip-based electrochemical detectors with two inlets (i.e., inlet 1) and 0.10 M Tris-HCl buffer (pH 7.0) containing NADH (2 mM) and EGTA (2 mM) (i.e., inlet 2). (B) Current−time responses obtained on the SWNT-modified (black line) and polyTBO-modified (red line) electrodes before and after adding NADH (2 mM) into 0.10 M Tris-HCl buffer (pH 7.0) containing EGTA (2 mM) perfused into inlet 2. The perfusion rates for both pumps were 1 μL/min. Other conditions were the same as those in Figure 1.

Figure 1. (A) Schematic illustration of the microfluidic chip-based electrochemical detectors with one inlet (i.e., inlet 1) for introducing 0.10 M Tris-HCl buffer (pH 7.0) containing NADH (2 mM) and EGTA (2 mM). (B) Current−time responses obtained on the SWNTmodified (black line) and polyTBO-modified (red line) electrodes before and after adding NADH (2 mM) into 0.10 M Tris-HCl buffer (pH 7.0) containing EGTA (2 mM). The perfusion rate for the single pump was 1 μL/min. The SWNT-modified and polyTBO-modified electrodes were polarized at 0.03 and 0 V, respectively.

working electrodes when NADH solution was introduced into inlet 2. No obvious current response was observed on the upstream SWNT-modified electrode (black line), while a welldefined current response was observed on the downstream polyTBO-modified electrode (red line). Since the NADH concentration did not change, the Mg2+ signal was measured as the current enhancement induced by the solution introduction into inlet 1. These results demonstrate that this new design essentially enables an easy optimization for solution introduction onto the microfluidic chip-based flow cell, which efficiently avoided the cross talk of NADH oxidation on the SWNTmodified electrode. Moreover, such cell design also well eliminates the cross talk of ascorbate oxidation on the downstream polyTBO-modified electrode, as depicted in Figure 3. The introduction of ascorbate standard from inlet 1 onto the microchip essentially produces a well-defined current response (i.e., 240 nA) on the SWNT-modified electrode (black line), and almost no current response was observed on the polyTBO-modified electrode (red line), suggesting that no ascorbate oxidation occurs on the polyTBO-modified electrode when the SWNT-modified electrode was on. At the same time, the simultaneous introduction of Mg2+ standard from inlet 1 and NADH from inlet 2 clearly produces a current response (i.e., 100 nA) on the polyTBO-modified electrode (red line), while no current response was observed on the SWNT-modified electrode (black line). Again, this result demonstrates that there is no cross talk of NADH oxidation on the SWNT-modified electrode.

based detector consists of a plate of PDMS containing a single channel and an ITO glass substrate, of which one part was coated with SWNTs for ascorbate detection and the other part was coated with polyTBO for Mg2+ detection. At a first try, we made three holes in the microchannel respectively for implantation of inlet 1, microsized Ag/AgCl reference electrode, and stainless steel outlet (also as counter electrode). We perfused 0.10 M Tris-HCl buffer (pH 7.0) containing EGTA (2 mM) and NADH (2 mM) into the chip through inlet 1 from a single pump. As mentioned above, NADH was needed for Mg2+ detection based on Mg2+-induced enhancement of the current response of catalytic oxidation of NADH with the polymerized film of TBO as the electrocatalyst. EGTA was used here as a masking agent to selectively chelate Ca2+ and to eliminate its interference toward Mg2+, as demonstrated in our earlier study. As depicted in Figure 1 B, the introduction of 0.10 M Tris-HCl buffer (pH 7.0) containing EGTA (2 mM) and NADH (2 mM) into the microchip produces current responses on both SWNT-modified (black line) and polyTBO-modified (red line) electrodes, suggesting NADH was oxidized electrochemically on both electrodes, generating a cross talk in the detection. This result unfortunately invalidates this type of microfluidic chip-based flow cell as a detector for simultaneous measurements of ascorbate and Mg2+. To overcome this problem, we added another inlet (i.e., inlet 2) onto the chip which was positioned in between the two electrodes (upstream SWNT-modified electrode and downstream polyTBO-modified electrode) to avoid introducing 7602

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Figure 3. Current−time responses obtained on the microfluidic chipbased online electrochemical detecting system with SWNT-modified (black line) and polyTBO-modified (red curve) electrodes as detectors for ascorbate (0.1 mM) and Mg2+ (2 mM). The standard solutions of ascorbate and Mg2+ (as indicated in the figure) in Mg2+-free aCSF were perfused into inlet 1 and then online mixed with 2 mM NADH solution in 0.10 M Tris-HCl buffer (pH 7.0) containing 2 mM EGTA perfused into inlet 2. Other conditions were the same as those in Figure 2.

Figure 4. Current−time responses obtained on the microfluidic chipbased online detecting system toward dopamine (DA, 1 μM), 3,4dihydroxyphenylacetic acid (DOPAC, 1 μM), uric acid (UA, 5 μM), Ca2+ (1 mM), a mixture of divalent cations (M2+, 5 μM Cu2+, 2 μM Mn2+, 2 μM Cd2+, 2 μM Zn2+, 2 μM Co2+, and 2 μM Ni2+), and a mixture of ascorbate (AA) and Mg2+ (50 μM and 2 mM), as indicated in the figure. The standard solutions prepared in Mg2+-free aCSF were perfused into inlet 1 and online mixed with 2 mM NADH solution in 0.10 M Tris-HCl buffer (pH 7.0) containing 2 mM EGTA perfused into inlet 2. Other conditions were the same as those in Figure 2.

More remarkably, we found that the electrode alignment in the microchip-based flow cell with the SWNT-modified electrode in the upstream and the polyTBO-modified electrode in the downstream well eliminates the cross talk of ascorbate oxidation on the downstream polyTBO-modified electrode, as depicted in Figure 3. When the upstream SWNT-modified electrode was turned on, being polarized at 0.03 V for the oxidation of ascorbate (yellow part, black line), no current response was observed on the downstream polyTBO-modified electrode (yellow part, red line). However, when the upstream SWNT-modified electrode was not polarized for ascorbate oxidation (i.e., the SWNT-modified electrode was turned off) (green part, black line), a current response was clearly observed on the downstream polyTBO-modified electrode (green part, red line). This result suggests that ascorbate oxidation could happen on the polyTBO-modified electrode but did not in the typical detection mode, because part of the ascorbate was oxidized at the turned-on SWNT-modified electrode before their arriving at the polyTBO-modified electrode and, as a result, the lowered concentration of ascorbate did not produce a current response at the downstream polyTBO-modified electrode. This result demonstrates that the alignment of the SWNT-modified electrode in the upstream of the microfluidic channel remarkably eliminates the cross talk of ascorbate oxidation on the polyTBO-modified electrode, further validating this method for continuous and simultaneous measurements of ascorbate and Mg2+. In addition to the no cross talk between the two electrodes, the microfluidic chip-based online detecting system exhibits a high selectivity for the simultaneous measurements of ascorbate and Mg2+. For example, when the SWNT-modified and polyTBO-modified electrodes were polarized at 0.03 and 0.0 V, respectively, the introduction of electroactive species endogenously existing in rat brain at their physiological levels did not yield any obvious current responses on either of the electrodes, as displayed in Figure 4, suggesting that the measurement of ascorbate and Mg2+ is free of interference from these species on our system. Moreover, while the

enhancement toward electrocatalytic oxidation of NADH on the polyTBO-modified electrode could also be achieved by other divalent cations, such as Ca2+, Cu2+, Mn2+, and Co2+, the perfusion of Ca2+ at its physiological level which was almost the same as Mg2+ in the extracellular fluid of rat brain9,15 did not produce any observable current response (data not shown) until the concentration was increased to 1 mM, as displayed in Figure 4, revealing that the Ca2+-masking agent EGTA successfully eliminated the interference from Ca2+. Meanwhile, a mixture of interfering 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 any obvious current response on either of the electrodes, demonstrating that these divalent cations did not interfere with the measurement of Mg2+. These results strongly suggest a high selectivity for Mg2+ detection by our system and further well validates the microfluidic chip-based online detecting system demonstrated in this study for continuous and simultaneous measurements of ascorbate and Mg2+ in rat brain. Figure 5 displays current responses of ascorbate and Mg2+ mixtures recorded on our online detecting systems. Our system produces well-defined current responses toward different concentrations of ascorbate and Mg2+, as typically shown in Figure 5 A. The dynamic linear ranges were from 5 to 100 μM (I (nA) = 2.90Cascorbate (μM) − 2.51, γ = 0.9999) for ascorbate and from 100 to 2000 μM (I (nA) = 37.8CMg2+ (μM) + 7.93, γ = 0.9987) for Mg2+. The reproducibility (Figure 5 B) and stability (Figure 5 C) of the online detecting system were studied with standard solutions of ascorbate and Mg2+ as well. The relative standard deviation (RSD) of the repeated measurements of ascorbate (50 μM) and Mg2+ (1 mM) standards with the same detecting system was 2.3% (n = 5) and 4.8% (n = 5), respectively. In addition, the system developed here was very stable for continuous measurements of ascorbate (50 μM) and Mg2+ (2 mM), as displayed in Figure 5 C; the current responses recorded for ascorbate and Mg2+ remain almost unchanged after continuously running the measurements for at least 2 h. Moreover, the online system was also 7603

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Figure 6. Online measurements of ascorbate and Mg2+ in the microdialysates from the striatum of ischemia-induced rats on the microfluidic chip-based online detecting system. The microdialysates or Mg2+-free aCSF was perfused into inlet 1. Tris-HCl buffer containing 2 mM NADH and 2 mM EGTA was perfused into inlet 2. Other conditions were the same as those in Figure 2.

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CONCLUSIONS In summary, by integrating microfluidic chip with in vivo microdialysis, we have successfully developed a facile yet effective online electrochemical detecting system for continuous and simultaneous monitoring of ascorbate and Mg2+ in rat brain. The designable and flexible cell structures of the microfluidic chip enable the simultaneous measurements of ascorbate and Mg2+ to be successfully achieved without cross talk but with technical simplicity and experimental reliability. The microfluidic chip-based online electrochemical system is very responsive, highly selective, stable, and reproducible and is thus reliable and durable for the continuous and simultaneous measurements of ascorbate and Mg2+ in cerebral systems. This study paves a new avenue to in vivo multiple-neurochemical monitoring in a technically simple and experimentally designable fashion, which is envisaged to find interesting applications in physiological and pathological applications.

Figure 5. (A) Current responses of standard ascorbate and Mg2+ with different concentrations. (B) Repeated measurements of standard ascorbate (AA, 50 μM) and Mg2+ (1 mM). (C) Continuous measurement of standard ascorbate (50 μM) and Mg2+ (2 mM). Other conditions were the same as those in Figure 2.

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AUTHOR INFORMATION

Corresponding Author

*Fax: +86-10-62559373. E-mail: [email protected] (L.M.), [email protected] (J.Y.).

stable for discontinuous measurements; we did not observe any obvious change of the current responses of ascorbate (50 μM) and Mg2+ (2 mM) for at least 5 days with continuous measurement for at least 4 h each day (data not shown). These results demonstrate that our microfluidic chip-based online detecting system possesses a high selectivity, good stability, and reproducibility for continuous and simultaneous measurements of ascorbate and Mg2+. Figure 6 displays current responses of the microdialysates continuously sampled from rat striatum following global ischemia. The basal levels of ascorbate (black line) and Mg2+ (red line and the dashed blue average line) in the striatum microdialysates before ischemia were determined to be 21.9 μM and 139.4 μM (blue part) according to electrode calibration, which were consistent with reported values.6a,9,15,16 When the animals were subjected to global ischemia induced by occluding the bilateral common carotid arteries, the basal levels of ascorbate and Mg2+ in the striatum microdialysate was decreased by about 20.9%, and 31.3%, respectively (green part). These values were smaller than those reported in the literature,6,17 presumably because of the differences of animals and brain regions as well as the length of global ischemia in our study compared to previous reports.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research is financially supported by the NSF of China (Grants 20975104, 20935005, 21127901, 21210007, and 91213305 for L.M. and 91132708 for P.Y.), the National Basic Research Program of China (973 programs, 2010CB33502, 2013CB933704), and The Chinese Academy of Sciences (KJCX2-YW-W25 and Y2010015).

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dx.doi.org/10.1021/ac401727d | Anal. Chem. 2013, 85, 7599−7605