Dual Electrochemical Microsensor for Real-Time Simultaneous

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Dual Electrochemical Microsensor for Real-Time Simultaneous Monitoring of Nitric Oxide and Potassium Ion Changes in a Rat Brain during Spontaneous Neocortical Epileptic Seizure Jungmi Moon, Yejin Ha, Misun Kim, Jeongeun Sim, Youngmi Lee, and Minah Suh Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b02396 • Publication Date (Web): 18 Aug 2016 Downloaded from http://pubs.acs.org on August 19, 2016

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Analytical Chemistry

Dual Electrochemical Microsensor for Real-Time Simultaneous Monitoring of Nitric Oxide and Potassium Ion Changes in a Rat Brain during Spontaneous Neocortical Epileptic Seizure Jungmi Moon,†,⊗ Yejin Ha,†,⊗ Misun Kim,† Jeongeun Sim,‡ Youngmi Lee,†,* Minah Suh‡,§,∥,* †Department of Chemistry and Nano Science, Ewha Womans University, Seoul, 03760, Republic of Korea ‡Center for Neuroscience Imaging Research (CNIR), Institute for Basic Science (IBS), Suwon, 16419, Republic of Korea §Department of Biomedical Engineering, Sungkyunkwan University, Suwon, 16419, Republic of Korea ∥Samsung Advanced Institute of Health Science and Technology (SAIHST), Sungkyunkwan University, Suwon, 16419, Republic of Korea ABSTRACT: In this work, we developed a dual amperometric/potentiometric microsensor for sensing nitric oxide (NO) and potassium ion (K+). The dual NO/K+ sensor was prepared based on a dual recessed electrode possessing Pt (diameter, 50 µm) and Ag (diameter, 76.2 µm) microdisks. The Pt disk surface (WE1) was modified with electroplatinization and the following coating with fluorinated xerogel; and the Ag disk surface (WE2) was oxidized to AgCl on which K+ ion selective membrane was loaded subsequent to the silanization. WE1 and WE2 of a dual microsensor were used for amperometric sensing of NO (106 ± 28 pA µM−1, n = 10, at +0.85 V applied vs. Ag/AgCl) and for potentiometric sensing of K+ (51.6 ± 1.9 mV pK−1, n = 10), respectively, with high sensitivity. In addition, the sensor showed good selectivity over common biological interferents, sufficiently fast response time and relevant stability (within 6 h in-vivo experiment). The sensor had a small dimension (end plane diameter, 428 ± 97 µm, n = 20) and needle-like sharp geometry which allowed the sensor to be inserted in biological tissues. Taking advantage of this insertability, the sensor was applied for the simultaneous monitoring of NO and K+ changes in living rat brain cortex at a depth of 1.19 ± 0.039 mm and near the spontaneous epileptic seizure focus. The seizures were induced with 4-aminopyridine injection onto the rat brain cortex. NO and K+ levels were dynamically changed in clear correlation with the electrophysiological recording of seizures. This indicates that the dual NO/K+ sensor’s measurements well reflect membrane potential changes of neurons and associated cellular components of neurovascular coupling. The newly developed NO/K+ dual microsensor showed the feasibility of real-time fast monitoring of dynamic changes of closely linked NO and K+ in vivo.

Nitric oxide (NO) is known to act as an important signaling molecule in a variety of biological/physiological events, such as vasorelaxation, neurotransmission, immune response, etc.1,2 NO is endogenously synthesized from L-arginine via the activity of a family of NO synthase (NOS) isozymes triggered by calcium ion (Ca2+) influx.3 The NO-mediating vasodilation pathway is known to be as follows. Once generated in the cells containing NOS, NO diffuses to smooth muscle cells and produces guanosine 3’,5’-cylic monophosphate (cGMP) by activating soluble guanylyl cyclase (sGC) and the intracellular accumulation of cGMP is followed. The increased cGMP activates potassium ion (K+) channels, causing K+ efflux and hyperpolarization of the cell membrane. The following closure of voltage-gated Ca2+ channels decreases intracellular Ca2+ levels in smooth muscle cells, mediating vasodilation.4,5 In addition to the regulation of vascular tone, this NO/cGMP/K+ channel pathway has been reported to be involved in other biological processes such as forebrain cholinergic neuron excitability on which learning and memory are critically dependent,6 and the action of antinociceptive drugs,7 etc.

K+ has been also considered as a fundamental factor in blood pressure regulation.8 There are some reports that saltsensitive hypertension may be attenuated by K+ supplementation by increasing NO production in Dahl rats9,10 and rabbits.11 Therefore, detailed analysis of NO and K+ in living biological systems is required to understand their functions. For the measurement of NO, spectroscopic (e.g., chemilunimescence,12,13 fluorescence14-16) and electrochemical methods17 have been mainly used. In particular, the latter one has a better applicability for in vivo and in vitro measurements18-23 due to the capability of real-time analysis and easy sensor miniaturization. Clinical analysis of K+ level is carried out generally using ion-selective electrode (ISE), atomic absorption spectroscopy, and flame photometry.24 ISE is advantageous in some aspects such as the wide linear dynamic range, simple operation, easy miniaturization, and fast analysis.25 As described above, NO and K+ are intimately connected in complex biological networks with probably very rapid temporal latency. However, there has been no report regarding simultaneous monitoring of NO and K+, to our best knowledge. Herein, we demonstrate the fabrication and char-

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acterization of a dual electrochemical microsensor intended for simultaneous detection of NO and K+. Integration of two different transduction schemes (amperometry and potentiometry) in a single sensor body makes it plausible to measure NO and K+ concentrations simultaneously in real time. The sensor has two electrodes: one electrode generates current proportional to NO concentration via the direct oxidation of NO; and the other electrode is an all-solid-state K+ ISE, developing junction potential change responding to K+ activity at the membrane/electronic conductor interface. Some trials to develop amperometric/potentiometric sensors were previously demonstrated for organophosphorus neurotoxins26 and for pH/glucose.27 We have also reported a similar amperometric/potentiometric dual microsensor for NO and Ca2+ analysis.28 The newly developed NO/K+ sensor has the tapered needlelike geometry, becoming insertable into biological tissues. Thus, this sensor is applied to measure the NO and K+ levels in a deep layer of a living rat brain tissue of which acute epileptic seizure is induced with 4-aminopyridine injection. This simultaneous monitoring of fast and dynamic changes of NO and K+ during neuronal hyperexcitation may allow better understanding of the physiological linkage between these two species. ■ EXPERIMENTAL SECTION Chemicals and materials. Theta type glass capillaries (diameter, 1.5 mm) were from World Precision Instrumentation Inc. (Sarasota, FL). Pt microwire (diameter, 50 µm) and Ag microwire (diameter, 76.2 µm) were purchased from Good Fellow (Hustingdon, England) and A. M System (Carlsborg, WA), respectively. Platinizing solution (3% H2PtCl6) was from YSI Incorporated (Yellow Spring, Ohio). (Heptadecafluoro-1,1,2,2,-tetrahydrodecyl)trimethoxysilane (17FTMS) was a product of Gelest (Tullytown, PA). Poly(vinyl chloride) carboxylated (c-PVC), polyurethane (PU), bis(2-ethylhexyl sebacate) (DOS), methyltrimethoxysilane (MTMOS), valinomycin, tetrahydrofuran (THF), tris(hydroxymethyl)aminomethane (Tris), 4-aminopyridine (4-AP), 3-aminopropyltriethoxysilane (APTES), chlorotrimethylsilane (TMCS), Trizma® base, HCl, potassium nitrate (KNO3), sodium nitrate (NaNO3), sodium cyanide (NaCN), sodium nitrite (NaNO2), sodium hydroxide (NaOH), 4-acetamidophenol (AP), Lascorbic acid (AA), uric acid (UA), dopamine (DA), ethanol and toluene were obtained from Sigma-Aldrich (St. Louis, MO). Phosphate buffered saline (PBS) was from Fisher Scientific (Rochester, NY). Ar and NO gases were obtained from Dong-A Gas Co. (Korea). All aqueous solutions were prepared using purified water (> 18 MΩ·cm) using reagent grade chemicals without additional purification. Preparation and characterization of a NO/K+ dual microsensor. First, a Pt/Ag dual microdisk electrode was fabricated via the method previously reported.28 Briefly, Pt (diameter, 50 µm) and Ag (diameter, 76.2 µm) microwires were sealed separately in a pulled theta-type glass capillary with thermal fusion under vacuum. Then, the end of the thermally sealed glass capillary was vertically burnished using a sand paper and 3-, 1-, 0.05-µm diamond papers in order. Pt and Ag microdisks embedded in a single working electrode (WE)

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body were electrochemically etched by being applied with 3 V amplitude ac voltage (60 Hz) for 1 s vs. Pt wire counter electrode in an aqueous solution containing 6 M NaCN and 0.1 M NaOH. Each bare Pt or Ag microdisk within the alcove of the glasssealed dual WE was modified independently. The resulting recessed Pt disk surface (WE1) was platinized with chronoamperometry (at −0.1 V vs. Ag/AgCl for 8 s) carried out in a platinizing solution (3% H2PtCl6). APTES (1 vol% in ethanol) was applied on the platinized Pt electrode surface using a fluid dispenser (1.0 psi, 0.0001s) followed by being dried overnight. Additionally, fluorinated-xerogel cocktail solution was loaded on the electrode surface and dried overnight to make a NO selective electrode (WE1). The xerogel cocktail was prepared by mixing 727.3 µL of ethanol, 18 µL of MTMOS, 14.5 µL of 17FTMS, 10 µL of 0.5 M HCl and 160 µL of water.29 The recessed Ag microdisk was chloridated to Ag/AgCl by being applied with a drop of 1 M FeCl3 solution for 1 h and then rinsed meticulously with deionized water. Then, TMCS (10 vol% in toluene) was loaded on the Ag/AgCl microdisk, followed by being dried for 12 h. Then, K+ ion selective polymer membrane cocktail was additionally applied onto the Ag/AgCl electrode using a dispenser (4 to 5 psi, 0.0200 s) and then dried for 8 h to produce a K+ ISE (WE2). K+ ion selective membrane cocktail consisted of 46.0 mg of c-PVC, 5.1 mg of PU, 2 mg of valinomycin, and 132.16 µL of DOS in 1.8 mL of THF. For each electrode modification, the silanizing solutions and ion selective membrane cocktail were loaded only on the desired single WE carefully using UltimusTM dispenser (NORDSON EFD, Ohio) under optical microscope, avoiding the other WE from being contaminated. The prepared dual sensor diagram is shown in Figure 1. The sensor overall end plane diameter was ca. 300 – 700 µm (n = 20). Two refillable miniature Ag/AgCl electrodes (eDAQ, electrode diameter = 2 mm) were independently used as the reference/counter electrodes for WE1 operated in amperometric mode and for WE2 operated in potentiometric mode. As prepared dual sensors were immersed in a PBS solution (pH 7.4) while WE1 was pre-polarized at +0.85 V (vs. Ag/AgCl) for 3 to 5 h before use. WE1 and WE2 of a dual microsensor were calibrated individually: The current response of WE1 to NO concentration increase (0 to 3.13 µM) was measured in a deoxygenated PBS solution (pH 7.4); and the potential change of WE2 with K+ concentration increase (10−5 to 10−1 M) was monitored in a 0.1 M Tris-buffer solution (pH 7.4). The tested concentration ranges were selected to cover both physiological NO levels30-33 and extracellular K+ levels.34-36 To change the concentration of NO or K+ in a test solution, a certain amount of the corresponding standard solutions was added successively. Potentiostat (CHI1000A, CH Instruments Inc., TX) was used for amperometry, linear sweep voltammetry (LSV), and cyclic voltammetry (CV) techniques. High impedance input 16-channel analog-to-digital converter (KOSENTECH, Korea) was used for all potentiometric measurements. Animal Preparation and in-vivo NO and K+ Measurements in a Rat Brain. Male rats (Sprague-Dawley, 250 – 400 g, n = 5) were under anesthesia with 2% isoflurane gas (Hana

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Figure 1. A schematic illustration for the preparation steps of an insertable NO/K+ dual microsensor. The dual sensor has two working electrodes, WE1 and WE2. The electrode dimension is as follows: a = 50 µm, b = 120 – 280 µm, c = 76.2 µm, and electrode overall end plane diameter (d) = 300 – 700 µm (n = 20).

Pharm., Korea) during the surgery and subsequent experiments. Animal’s skin over a head was cut and the skull, dura, and pia mater were removed in order. All animal treatments were approved by the Institutional Animal Care and Use Committee of Sungkyunkwan University. Experimental procedure for simultaneous NO and K+ measurement in a rat brain under acute seizure is similar to the previous method used for concurrent NO and CO measurements.37 An local field potential (LFP) electrode, Ag wire immersed in 0.9% saline filling a pulled glass capillary, was connected to CED Power 1401/Spike 2 system (Cambridge Electronic Design, UK). A pulled glass capillary containing 15 mM 4-AP (Sigma, MO) solution was connected to a nanoinjector. WE1 and WE2 of the prepared NO/K+ needle-like sharp microsensor were connected to CHI1000A multipotentiostat (CH Instruments) and high impedance input 16-channel analog-to-digital converter (KOSENTECH), respectively. The NO/K+ sensor, LFP electrode and glass capillary were positioned on a brain surface forming a triangle and then inserted into the brain tissue down to 1.19 ± 0.039-mm depth using micromanipulators (WPI) while maintaining an angle of 45° with respect to the brain surface. This made that three ends

Figure 2. Schematic experimental setup for simultaneous LFP, NO, and K+ measurements in rat brain cortical layer under epileptic seizure induced by 4-AP injection. The approximate brain cortical layers (1 to 6) were indicated with dotted lines.

could be located right next to one another (within a distance < 1 mm). Once the amperometric/potentiometric signals of a NO/K+ microsensor reached the stable levels, 15 mM 4-AP (70 µL) was injected with the nanoinjector to induce acute seizure with continuous measurements of the signals The WE1 current and WE2 potential recorded simultaneously were converted to NO and K+ concentrations using before and after calibration curves. The experimental set-up is shown in Figure 2. ■ RESULTS AND DISCUSSION Characterization of a dual NO/K+ microsensor. WE1 and WE2 of as prepared dual microsensor were characterized in amperometric and potentiometric modes, respectively. Current of WE1 (i.e., fluorinated-xerogel coated platinized Pt electrode) and potential of WE2 (i.e., K+ ISE) were monitored simultaneously while either NO or K+ concentrations were elevated. The applied potential to WE1 operating amperometrically was +0.85 V (vs. Ag/AgCl), which was optimized previously.28 Responding to successive increase of NO concentration (0 to 3.13 µM) in deoxygenated PBS (pH 7.4) solution, WE1 current increased stepwise in proportion to NO concentration but WE2 potential did not change (Figures 3A and 3B). In contrast, a stable sustained current was observed at WE1 while WE2 potential increased in response to K+ concentration elevation (10−5 M to 10−1 M) in 0.1 M Tris-buffer solution as presented in Figures 3C and 3D. This confirmed that WE1 and WE2 did not have noticeable crosstalk between their signals and independently sensed NO and K+, respectively. For the calibration of WE2, Tris-buffer without containing K+ was used, instead of PBS. As previously reported,28 the presilanization of Ag/AgCl surface of WE2 with TMCS before the K+ ion selective membrane loading resulted in better adhesion of the ion selective membrane to the electrode surface, and therefore provided the improved potential stability of WE2. Both current and potential signals of the dual sensor responded in linear proportion to NO and K+ concentrations: The sensitivity of WE1 towards NO was 106 ± 28 pA µM−1 (n = 10) and potential of WE2 showed near-Nernstian behavior for K+ (51.6 ± 1.9 mV pK−1, n = 10). The sensor response times (t90%, time to reach 90% of the steady state value) were estimated by analyzing typical sensor dynamic response

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curves like Figure 3; which were 3.7 ± 0.7 s (n = 10) for NO at WE1 and 2.3 ± 0.9 s (n = 10) for K+ at WE2. To obtain the dynamic response curve, a certain volume of NO or K+ standard solution was injected into a test solution being stirred magnetically. Thus, an additional time is required to attain the intended constant concentration after the injection of the standard solution. The actual sensor response time in a prehomogenized media is anticipated reasonably to be shorter than the measured t90%. In fact, the response time of WE1 is 34 times faster than that of the previously reported Clark-type amperometric NO sensors based on PTFE gas permeable membrane.32,33,38,39 The use of fluorinated xerogel membrane for the gas selectivity instead of diffusion resistive PTFE gaspermeable membrane allowed accelerated sensor response. Thus, fast dynamic biological processes would be studied more accurately in more realistic time resolution with current NO/K+ dual microsensor exhibiting fast response time. Detection limits of the sensors were determined as 9.55 nM NO at WE1 and 10−6 M K+ at WE2 (S/N = 3). Furthermore, excellent selectivity of the dual sensor was demonstrated for the targeted NO and K+. In fact, the current measured at WE1 was not altered noticeably with the additions of common oxidizable biological interfering species at/above their physiological levels (50 µM nitrite, 50 µM AP, 50 µM AA, 10 µM DA, 50 µM UA, 10 µM H2O2 and 2 µM H2S in a stirred PBS solution) as shown in Figure S1 (in Supporting Information). This verifies that hydrophobic fluorinat-

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ed-xerogel membrane provides a good sensor selectivity to NO over relatively less hydrophobic interferents. As previously reported,29 low resistance of a hydrated fluorous xerogel film possibly allows a conductive connection across the film, and therefore enables WE1 operate amperometrically. In addition, selectivity coefficient of WE2 to K+ over Na+ (log , ) was investigated experimentally through separate solution method (SSM). As seen in Figure S2, potential of WE2 slightly increased as the Na+ concentration increased higher than 10−2 M. The selectivity coefficient of ISE is required to be below −2.0 for the use in biological samples.40 The determined log , value of WE2 was −3.4, meeting the requirement for the biological applications. Simultaneous Measurement of NO and K+ in Rat Cortex under Epileptic Seizure. As prepared NO/K+ dual microsensor was diminutive particularly in the sensor end dimension and shaped like a sharp needle, and therefore the sensor was capable to be inserted into biological tissues. The end plane diameter was determined to be 428 ± 97 µm (n = 20). To demonstrate the sensor’s feasibility for real-time simultaneous measurement of NO and K+ in living tissues, chemically induced acute seizures of a rat cortex were monitored with the sensor. 4-AP, a K+ channel blocker, was focally injected into the cortical layer at a depth of 1.19 ± 0.039 mm, layer 5, using a nanoinjector that elicited acute focal seizures. 4-AP blocks voltage-gated K+ channels selectively and pro-

Figure 3. Typical dynamic response curves of (A) WE1 and (B) WE2 to NO concentration change (0 to 3.13 µM); and those of (C) WE1 and (D) WE2 to K+ concentration change (10−5 to 10−1 M). The applied potential to WE1 was +0.85 V vs. Ag/AgCl. (A and B) Changes in WE1 current and WE2 potential were simultaneously recorded with the successive additions of a NO standard solution to a deaerated PBS (pH 7.4) solution. (C and D) Changes in WE1 current and WE2 potential were simultaneously measured in 0.1 M Trisbuffer (pH 7.4) with the successive injections of a standard KNO3 solution. Insets show the corresponding calibration curves. (A) WE1 sensitivity to NO is 106 ± 28 pA µM−1, and (D) WE2 sensitivity to K+ is 51.6 ± 1.9 mV pK−1.

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motes synaptic transmission by retarding action potential repolarization that mimics epileptic seizure. It has been reported that brain cortical layer 5 shows the most prominent neural activity response to 4-AP.41 The sensor and local field potential (LFP) electrode were inserted through the brain cortex and positioned in close proximity to the 4-AP injection site as shown in Figure 2. Figure 4 is a representative result of simultaneous NO and K+ monitoring in a cortical tissue under 4-AP seizure. Based on the LFP signals, a seizure duration could be analyzed into three stages: initiation, propagation, and termination.42 At the initiation stage, both NO and K+ levels rose but the rising patterns were different one from the other. NO increased rapidly and then decreased rather gradually, generating the peaked signal change. On the other hand, K+ increase was slower but continued throughout the whole initiation period. There was a brief initial dip in K+ responses before rising stage. During the propagation stage, both NO and K+ sustained the quite stable levels. The decrease of NO started from the end part of this propagation stage. For the termination stage, NO decreased quickly down to its basal level right after the onset of termination. In contrast, the following onset of the K+ decrease was coincident with the seizure activity cessation along with the recovery of NO basal level. K+ responses returned to its baseline 30~40 s after the onset of termination. The monitored NO change during the acute seizure was well matched with the one observed with a NO/CO dual microsensor in our previous

Figure 4. Representative signal changes of LFP, NO and K+ levels in response to epileptic seizures which were measured in rat cortical layer. Three stages of seizure activity (initiation, propagation, and termination) were sectionalized with vertical dashed lines.

work.30 Increase in K+ level induced by 4-AP has been reported for juvenile rat hippocampal slice (CA3 subfield) in vitro.43 In a quick summary of this event, both NO and K+ responded rapidly to the onset of hyper-synchronous depolarization of neuronal membrane, but the release of vasomodulator NO started to decrease well before the onset of seizure activity termination. This suggests that NO signaling pathway is tightly correlated with neuronal signaling pathway more than meets the eye. Also, slow decaying of K+ releases suggests that ionic concentration changes may be slower than gas neurotransmitter diffusion at synapses. The absolute NO/K+ concentration increases (∆CNO and ∆CK+) during epileptic seizure events were calculated according to the following equation (eq 1). ΔCNO(or K+) = CNO(or K+),max − CNO(or K+),base

(1)

where CNO,max is the greatest NO concentration measured at the summit observed in the initiation period; CK+,max is the greatest K+ concentration measured as a nearly steady-state value in the propagation period; and CNO(or K+),base is the NO or K+ concentration averaged for 30 s before seizure starts. CNO(or K+),max values were obtained via averaging the data of 98 - 100% of the maximum value.

Figure 5. (A) The observed concentration changes of NO (red squares) and the corresponding concentration changes of extracellular K+ (blue circles) for all the animals examined (n = 5). The data points were presented intentionally in the order of K+ concentration changes from the greatest to the smallest in order to show the close relationship between the changed amounts of NO and extracellular K+ concentrations. (B) The observed time points of the maximum NO concentration changes (tNO,max, red bars) and the corresponding maximum K+ concentration changes (tK+,max, blue bars) for all the animals examined (n = 5).

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The measured ∆CNO and ∆CK+ values for five different rats were presented in Figure 5A. For all the animals studied, both NO and K+ levels were increased (i.e., both ∆CNO and ∆CK+ > 0) in response to epileptic seizure activity without exception. The mean ∆CNO and ∆CK+ values obtained from five rats exhibited the quite wide ranges: 0.015 – 0.31 µM for ∆CNO and 0.52 – 7.1 mM for ∆CK+. These large ∆CNO(or K+) ranges are due to high inter-individual variation. In fact, the standard deviations of the data obtained for single animal were relatively small as shown in Figure 5A. The measured ∆CNO at subµM level and ∆CK+ at a few mM level at rat cortex with 4-AP injection are similar to the previous reports.37,43 Interestingly, a tendency was observed that an animal with a relatively large ∆CK+ also exhibited a rather large ∆CNO, and vice versa (Figure 5A). This indicates a tight coupling between the changes of NO and K+ levels during seizure events. As seen in Figure 5B, a correlation was observed between the times to reach maximum values of NO (tNO,max) and K+ (tK+,max) for all the animals examined (n = 5). NO signal started increasing right after the onset of a seizure activity and reached the peak earlier. Then, the K+ attained the steady-state level later. tNO,max and tK+,max were compared with paired t-test and the p-value was under 0.05, indicating the significant difference between tNO,max and tK+,max. Preceding increase in NO level followed by a rather gradual increase in K+ level is supposed seemingly to be that the measured ∆CNO and ∆CK+ are related with cerebral vascular smooth muscle: The enhanced NO production in response to seizure mediates hyperpolarization (directly or via cGMP) causing K+ efflux from the smooth muscle cells.44 Subsequent further study is, however, required to clarify the interactive mechanism between NO and K+ at brain under epileptic seizure events. The sensor stability was assessed through comparing calibration curves recorded before and after the 6-h in-vivo experiments (Figure S3). The WE1 and WE2 maintained within 76 % and 91% of the initial sensitivities for NO and K+, respectively, supporting the reasonable stability. Changes of the sensor response times after in-vivo experiments (∆t90%) were 0.78 ± 0.55 s and 0.63 ± 0.49 s for WE1 and WE2, respectively (n = 5). Conclusively, the developed NO/K+ dual microsensor proved a good feasibility for the simultaneous real-time measurements of NO and K+ in vivo. ■ CONCLUSIONS A dual microsensor was fabricated aiming at simultaneous measurements of intimately linked NO and K+. The sensor composed of amperometric NO microelectrode (WE1, platinized Pt coated with fluorinated xerogel) and potentiometric all-solid-state K+ ISE (WE2, silanized Ag/AgCl loaded with K+ ion selective membrane). WE1 and WE2 showed the current and potential changes in proportion to NO and K+ concentrations, respectively, without crosstalk between WE1 and WE2 sensing signals. WE1 had adequate selectivity to NO over common oxidizable interferents (e.g., nitrite, AP, AA, DA, UA, H2O2, and H2S) while WE2 showed an appropriate selectivity coefficient (log , = −3.4). Due to the miniaturized dimension and tapered sharp geometry, the sensor could be inserted within a living rat’s cortical deep-layer and

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was utilized to monitor fast changes of NO and K+ levels in relation to seizure activity induced by 4-AP injection. The sensor sensitivities (106 ± 28 pA µM−1 for NO at +0.85 V applied vs. Ag/AgCl, n = 10; and 51.6 ± 1.9 mV pK−1 for K+, n = 10) and response times were sufficient to measure the NO and K+ dynamic changes. NO and K+ levels changed in different patterns while well correlated to three stages of seizure recognized with electrophysiological signals. Shorter tNO,max followed by tK+,max was also observed. Current work suggests the potential applicability of this dual sensor for many basic research areas in which the concurrent real-time measurements of NO and K+ are needed.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Amperometric responses of WE1 to typical interfering species (Figures S1); potentiometric responses of WE2 to K+ and Na+ ions (Figure S2); and dynamic response and calibration curves obtained pre- and post-in-vivo experiments (Figure S3).

AUTHOR INFORMATION Corresponding Authors * E-mail: [email protected]. Fax: +82-2-3277-2384. (Y. Lee) * E-mail: [email protected]. (M. Suh)

Author Contributions ⊗Authors

contributed equally to this work.

ACKNOWLEDGMENT This work was financially supported by the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning (2014R1A2A2A05003769) and by IBSR015-D1.

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Dual Electrochemical Microsensor for Real-Time Simultaneous

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