Reaction-Based Turn-on Electrochemiluminescent Sensor with a

Jan 9, 2015 - Hydrogen sulfide (H2S) has been drawing increasing attention because it plays an important role in the nervous system and has been deeme...
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Reaction-Based Turn-on Electrochemiluminescent Sensor with a Ruthenium(II) Complex for Selective Detection of Extracellular Hydrogen Sulfide in Rat Brain Xiaoxiao Yue,† Ziyu Zhu,† Meining Zhang,*,† and Zhiqiang Ye*,‡ †

Department of Chemistry, Renmin University of China, Beijing 100872, China State Key Laboratory of Fine Chemicals, School of Chemistry, Dalian University of Technology, Dalian 116024, China

‡

ABSTRACT: Hydrogen sulfide (H2S) has been drawing increasing attention because it plays an important role in the nervous system and has been deemed as a third endogenous gas signal molecule besides nitric oxide (NO) and carbon monoxide (CO). In this study, using a ruthenium complex, [Ru(bpy)2(bpyDPA)Cu]4+ (where bpy = 2,2′-bipyridine and bpy-DPA = 4-methyl-4′-[N,N-bis(2picolyl)aminomethylene]-2,2′-bipyridine) as recognition unit, we report a new reaction-based turn-on electrochemiluminescent (ECL) sensor to selectively detect extracellular H2S in rat brain, coupled with in vivo microdialysis for dialysate sampling. To prepare the sensor for sensing endogenous H2S, [Ru(bpy)2(bpyDPA)]2+ is first designed and synthesized, showing high ECL efficiency with tri-npropylamine (TPA) as a coreactant and quenching after reaction with Cu2+ (forming [Ru(bpy)2(bpy-DPA)Cu]4+). Then a Nafion membrane is coated on the surface of glassy carbon (GC) electrode and [Ru(bpy)2(bpy-DPA)Cu]4+ is confined onto the Nafion membrane through ion exchange. The resulting [Ru(bpy)2(bpy-DPA)Cu]4+/Nafion/GC sensor exhibits a low ECL signal. The [Ru(bpy)2(bpy-DPA)Cu]4+/Nafion/GC sensor demonstrates enhanced ECL signal after reacting with volatile H2S due to the high-affinity binding between sulfur and Cu2+, returning to [Ru(bpy)2(bpy-DPA)]2+/Nafion/GC. The changes of ECL signal at the sensor depend linearly on the concentration of Na2S in the range from 0.5 to 10 μM, with a detection limit of 0.25 μM. Moreover, the sensor demonstrates high selectivity, free from interference especially by other nonvolatile thiol-containing species, such as cysteine and glutathione. The basal dialysate level of H2S in the microdialysate from the cortex of adult male Sprague-Dawley rats is determined to be 2.3 ± 0.9 μM (n = 4). This method is reliable and is envisaged to help understand the regulation of H2S in physiological and pathological events.

H

with the coexistence of analogues including thiol-containing species (e.g., cysteine) in the cerebral system. In comparison with traditional photoinduced luminescence detectors, the instrumentation for electrochemiluminescence (ECL) detection is substantially less complicated and less expensive because the excitation laser and optical filters are eliminated.9 Moreover, ECL probes, such as Ru(bpy)32+, can be confined onto the surface of electrode to construct a ECL sensor.10 ECL sensors possess the advantages of both electrochemical and florescent sensors, such as high sensitivity, ease of control, adaptability for in situ/on-spot analysis and low background. Thus, ECL sensors have been extensively applied in a number of bioanalytical areas, including medical diagnosis (e.g., immunoassays and DNA probes) and disease prevention detection.10,11 For instance, Ru(bpy)32+ (where bpy = 2,2′bipyridine) has been widely used as an ECL sensor probe for biological molecules, in which the excited state of Ru(bpy)32+ is generated with a coreactant, for example, tri-n-propylamine (TPA).10,11a However, ECL sensors for small molecules, for

ydrogen sulfide (H2S), with an unpleasant rotten egg smell, is well-known as a toxic gas. Recently, increasing interest has been drawn to the determination of H2S because it has been recognized as an endogenous gas signal molecule,1 in addition to nitric oxide (NO) and carbon monoxide (CO).2,3 It plays a very important role in the central nervous system and cardiovascular and other biological systems under both physiological and pathological conditions.1,4 In central nervous system (CNS), H2S is produced through the enzymes cystathionine γ-lyase (CSE), cystathionine β-synthase (CBS), and 3-mercaptopyruvate sulfurtransferase (3-MST).5 Endogenous H2S has exerted multiple functions in both maintaining health and treating diseases. It modulates neurotransmission, acts as a neuroprotectant via its antioxidant, anti-inflammatory, and antiapoptotic effects, and shows potential therapeutic value in several CNS diseases including Alzheimer’s disease, Parkinson’s disease, ischemic stroke, and traumatic brain injury.1c,d,5a,6 Although some methods, such as electrochemistry, high-performance liquid chromatography, colorimetric methods, and fluorescence spectroscopy, have been reported for in vitro H2S detection,7,8 the high complexity of the cerebral system substantially makes direct selective detection of cerebral H2S a long-standing challenge, especially © XXXX American Chemical Society

Received: October 16, 2014 Accepted: December 23, 2014

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DOI: 10.1021/ac503875j Anal. Chem. XXXX, XXX, XXX−XXX

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H2S with ECL sensor, which offers an alternative platform for monitoring brain chemistry.

example H2S, that do not participate in the redox reaction generating the excited state remain largely underexplored.12,11c In this study, we develop a highly selective reaction-based turn-on ECL sensor by incorporating a new ruthenium(II) complex [Ru(bpy)2(bpy-DPA)Cu]4+ (where bpy-DPA = 4methyl-4′-[N,N-bis(2-picolyl)aminomethylene]-2,2′-bipyridine) into Nafion membrane coated onto glassy carbon (GC) electrode, for sensing cerebral H2S, coupled with in vivo microdialysis for dialysate sampling, shown in Scheme 1.

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EXPERIMENTAL SECTION Reagents and Materials. Tris(2,2′-bipyridyl)ruthenium(II) dichloride hexahydrate [Ru(bpy)3Cl2·6H2O, minimum 98%] and tri-n-propylamine (TPA, 98%), were purchased from Alfa Aesar. [Ru(bpy)2(bpy-DPA)](PF6)2 (where bpy = 2,2′-bipyridine and bpy-DPA = 4-methyl-4′-[N,N-bis(2picolyl)aminomethylene]-2,2′-bipyridine) and[Ru(bpy)2(bpyDPA)Cu](PF6)4 were synthesized as we previously reported.14b Nafion (5 wt %) was purchased from Dupont. L-Cysteine (Cys), glutathione (GSH), and homocysteine (Hcy) were purchased from Sigma. All other chemicals were of at least analytical reagent grade and were used without further purification. The pH of the phosphate buffer (pH = 7.4) containing TPA was adjusted with concentrated NaOH or phosphoric acid. Artificial cerebrospinal fluid (aCSF) was prepared by dissolving NaCl (126 mM), KCl (2.4 mM), KH2PO4 (0.5 mM), MgCl2 (0.85 mM), NaHCO3 (27.5 mM), Na2SO4 (0.5 mM), and CaCl2 (1.1 mM) into water, and the pH of the solution was adjusted to pH 7.4. All solutions were prepared with deionized water (Milli-Q, Millipore). Preparation of [Ru(bpy) 2 (bpy-DPA)Cu] 4+ /Nafion/ Glassy Carbon Sensor. Before modification, the glassy carbon (GC) electrodes (3 mm in diameter) were polished successively with 0.1 and 0.05 μm alumina powders, rinsed thoroughly with deionized water, and then sonicated in deionized water for 3 min. Afterward, a 0.8 μL aliquot of 0.5 wt % Nafion solution was drop-coated on GC electrode, and the solvent was evaporated at room temperature to give a Nafion/GC electrode. The Nafion/GC electrode was immersed in 0.05 μM [Ru(bpy)2(bpy-DPA)Cu]4+ solution for 30 min. After being taken out and rinsed with deionized water, the electrodes (denoted as [Ru(bpy)2(bpy-DPA)Cu]4+/Nafion/ GC electrodes) were used for ECL measurement and H2S sensing. Electrochemical and Electrochemiluminescent Measurements. All electrochemical and ECL experiments were conducted in a standard three-electrode cell by use of a computer-controlled CHI 760D electrochemical analyzer (CH Instruments, Shanghai Chenhua Instrument Corp.). [Ru(bpy)2(bpy-DPA)Cu]4+/Nafion/GC electrodes were used as working electrodes. A Pt spiral wire and Ag/AgCl (filled with saturated KCl) were used as the counter and reference electrodes, respectively. ECL intensities were measured through the bottom of the electrochemical cell with a BPCL ultra-weak luminescence analyzer (Jian Xin Li Tuo Science and Technology Co., Ltd., Beijing). The photomultiplier tube (PMT) used in the BPCL ultra-weak luminescence analyzer was biased at −1100 V. All electrochemical experiments were performed at room temperature. Luminescence spectra were recorded on a Hitachi F-4600 luminescence spectrometer (Hitachi Co.). ECL spectra were recorded on a luminescence spectrometer without excitation light with indium tin oxide (ITO) electrode as working electrode. The applied potential for ECL spectra was 1.4 V. ECL signal was measured in 5.0 mL of 0.1 M phosphate buffer (pH = 7.4) containing 1.0 mM TPA. The increment of ECL peak height ΔI (ΔI = I − I0) was recorded for its linear relationship with H2S, where I0 and I represent the ECL peak heights of [Ru(bpy)2(bpy-DPA)Cu]4+/Nafion/GC electrode before and after reaction with H2S, respectively. One droplet of

Scheme 1. Turn-on Electrochemiluminescent Sensor with a Ruthenium(II) Complex for Sensing Extracellular H2S in Rat Brain, Coupled with In Vivo Microdialysis

Chemoselective reaction is one general strategy for monitoring small molecules in living biological specimens.13 Accordingly, a few fluorescent or chemiluminescent turn-on probes have been designed for sensing H2S, based on the specific H2S-induced reaction.8,13b−e,14,15a In the H2S-induced reaction, H2S is typically used as the nucleophile to attack activated electrophiles or precipitate metals salts (such as Cu2+), or as a reductant to reduce azides on masked fluorophores.13b−e,14,15a Although these probes have been elegantly applied to detect cellular H2S or blood H2S, they still face a challenge in selectivity and photostability for sensing cerebral H2S. On the basis of these design considerations, we designed and synthesized a new Ru(II)−bipyridine complex containing a di(2-picolyl)amine (DPA) moiety, [Ru(bpy)2(bpy-DPA)]2+, showing high ECL efficiency with TPA as a coreactant. The ECL of [Ru(bpy)2(bpy-DPA)]2+/TPA was strongly quenched after reaction with Cu2+ to form a heterobimetallic Ru(II)− Cu(II) complex, [Ru(bpy)2(bpy-DPA)Cu]4+, through an excited-state electron-transfer or energy-transfer mechanism as fluorescence.16 Then [Ru(bpy)2(bpy-DPA)Cu]4+ recovered and emitted enhanced ECL in the presence of sulfide, forming CuS and [Ru(bpy)2(bpy-DPA)]2+ due to the high affinity between sulfide and Cu 2+ . 14 H 2 S can volatilize from physiological sample (e.g., cerebral microdialysate), and [Ru(bpy)2(bpy-DPA)Cu]4+ was incorporated into Nafion membrane coated onto the surface of GC electrode through ion exchange (Scheme 1). Hence, [Ru(bpy)2(bpy-DPA)Cu]4+ can selectively respond to H2S without interference, especially from nonvolatile thiol-containing species. As far as we know, this is the first report on the direct selective sensing of cerebral B

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Analytical Chemistry 10 μL phosphate buffer (pH = 7.4) was coated on the [Ru(bpy)2(bpy-DPA)Cu]4+/Nafion/GC electrode. The droplet can be compared to a reservoir to dissolve H2S volatized from the Na2S solution. Subsequently, the modified electrode was hung over different concentrations of Na2S solution (30 μL) for 1 h (Scheme 1). In this experiment, Na2S acted as a H2S donor because its purity(>98%) highly exceeds that of commercially available NaHS. The linearity was measured according to the relationship between ΔI and the concentration of Na2S. In Vivo Microdialysis. Animal surgery and in vivo microdialysis were carried out with the procedures reported in our earlier works.17 Briefly, adult male Sprague-Dawley (SD) rats (250−300 g) purchased from Health Science Center, Peking University, were housed on a 12 h/12 h light/dark schedule with food and water ad libitum. The microdialysis guide cannula (BAS/MD-2250, BAS) was implanted into the cortex via standard stereotaxic surgical procedures; the body temperature of the animals was maintained at 37 °C with a heating pad. After the rats were allowed to recover for at least 24 h, a microdialysis probe was implanted into the rat cortex to perfuse aCSF solution at 1.0 μL/min. After continuously perfusing the probe for at least 90 min for equilibration, the microdialysate was collected in a sealed tube with ice bath for ECL analysis. The [Ru(bpy)2(bpy-DPA)Cu]4+/Nafion/GC electrode was hung over the microdialysate sampled from cortex (30 μL) for 1 h. Afterward, the ECL signal (I) was measured in 0.1 M phosphate buffer (pH = 7.4) containing 1.0 mM TPA, and the ECL peak intensity change ΔI (ΔI = I − I0) was calculated.

the same characteristic metal-to-ligand charge transfer (MLCT)-based luminescence.18 Luminescence quantum yield of [Ru(bpy)2(bpy-DPA)]2+ was calculated to be 94%, with [Ru(bpy)3]Cl2 as a standard. As expected, [Ru(bpy)2(bpyDPA)Cu]4+ shows low luminescence through an electrontransfer or energy-transfer mechanism (Figure 1, blue curve). These properties substantially form a strong basis for ECL sensing of H2S. [Ru(bpy)3]2+ has been a widely used ECL-active molecule because of its high ECL efficiency, good solubility in both aqueous and nonaqueous solutions, and ability to undergo reversible one-electron-transfer redox processes.9 A number of [Ru(bpy)3]2+ derivatives and other ruthenium(II) N,Nchelating complexes have been designed and their ECL behaviors have been studied.12,18 These compounds generally displayed one-electron oxidation and reduction waves in their cyclic voltammograms and demonstrated strong ECL signal with TPA as coreactant.9,18 The redox property of [Ru(bpy)3]2+ enables it to be widely immobilized onto electrode surfaces, fabricating a regenerable ECL sensor for various analysis.10,11a Among them, the confinement of Ru(bpy)32+ in Nafion films is a powerful approach to construct an ECL sensor.19 Such an ECL sensor can be used to determine oxalate, amines, nicotinamide adenine dinucleotide (NADH), and peptides.10e,20 In the present study, [Ru(bpy)2(bpy-DPA)]2+ was incorporated into Nafion film coated on the surface of GC electrode to fabricate one sensor. A typical cyclic voltammogram of [Ru(bpy)2(bpy-DPA)]2+/Nafion/GC electrode is shown in Figure 2A (black curve). One pair of well-defined and peak-shaped redox waves were obtained at ca. +1.08 V (vs Ag/AgCl with saturated KCl), which was ascribed to the electron transfer process of [Ru(bpy)2(bpy-DPA)]2+ at this electrode. The redox potential is almost the same as that observed for Ru(bpy)32+/Nafion/GC electrode (Figure 2A, red curve), and this redox peak current is diffusion-controlled (data not shown). Figure 2B shows typical ECL emission obtained at [Ru(bpy)2(bpy-DPA)]2+/Nafion/GC electrode (black curve) and Ru(bpy)32+/Nafion/GC electrode (red curve) in 0.1 M phosphate buffer containing 1.0 mM TPA. The ECL curve depicted one typical ECL peak, pointing to the formation of an electronically excited state of [Ru(bpy)2(bpy-DPA)]2+ via the coreactant mechanism. The ECL spectrum of [Ru(bpy)2(bpyDPA)]2+ in 0.1 M phosphate buffer containing 1.0 mM TPA is almost the same as that of Ru(bpy)32+ (Figure 2C). Thus, the ECL mechanism was assumed to be similar to that of Ru(bpy)32+/TPA,9,21 summarized in typical reactions in eq 1.

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RESULTS AND DISCUSSION Spectral, Electrochemical, and Electrochemiluminescent Properties of [Ru(bpy)2(bpy-DPA)]2+. Figure 1

TPA − e → TPA• + → TPA• + H+ [Ru(bpy)2 (bpy‐DPA)]2 + + TPA• → [Ru(bpy)2 (bpy‐DPA)]+ + product

Figure 1. Excitation and emission spectra (λex = 456 nm, λem = 612 nm) of 10 μM [Ru(bpy)2(bpy-DPA)]2+ (red curve), Ru(bpy)32+ (black curve), and [Ru(bpy)2(bpy-DPA)Cu]4+ (blue curve) in 0.1 M phosphate buffer (pH = 7.4).

[Ru(bpy)2 (bpy‐DPA)]2 + − e → [Ru(bpy)2 (bpy‐DPA)]3 + [Ru(bpy)2 (bpy‐DPA)]3 + + [Ru(bpy)2 (bpy‐DPA)]+ → [Ru(bpy)2 (bpy‐DPA)]2 +* [Ru(bpy)2 (bpy‐DPA)]2 +* → [Ru(bpy)2 (bpy‐DPA)]2 + + hν

illustrates the excitation and emission spectra of [Ru(bpy)2(bpy-DPA)]2+ (red curve), [Ru(bpy)3]2+ (black curve), and [Ru(bpy)2(bpy-DPA)Cu]4+ (blue curve) in 0.1 M phosphate buffer (pH = 7.4). Figure 1 shows that the maximum excitation and emission peaks of [Ru(bpy)2(bpyDPA)]2+ are 456 and 612 nm, respectively. These features are similar to the properties of well-studied Ru(bpy)32+, indicating

(1)

The ECL emission efficiency was defined as the number of photons generated from an electrochemical event, described in eq 2.22 In eq 2, I is the integrated emission intensity integrated C

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ECL efficacy of [Ru(bpy)2(bpy-DPA)]2+ are perhaps due to the substitution group of DPA. t′

ΦECL =

∫0 I dt Q a,c

(2)

Electrochemiluminescent Behavior of [Ru(bpy)2(bpyDPA)Cu]4+/Nafion/Glassy Carbon Electrode toward Sensing H2S. H2S is a weak acid and a highly volatile gas. In aqueous solution containing H2S, the equilibrium in eq 5 exists, with pKa1 = 7.04 (6.75 at 37 °C) and pKa2 = 11.96.5a In extracellular fluid, H2S exists in the approximate ratio of 20% H2S, 80% HS−, and trace S2− at 37 °C and pH 7.4.5a In the present study, Na2S acts as a H2S donor in phosphate buffer (pH = 7.4), with the equilibrium in eq 3 existing. H2S volatilizes easily in the sealed system and drives the equilibrium to the left until reaching balance. One drop of phosphate buffer (ca. 10 μL, pH = 7.4) was maintained on the surface of [Ru(bpy)2(bpy-DPA)Cu]4+/Nafion/GC electrode. This droplet can be compared to a reservoir to dissolve the free H2S gas (equilibria in eqs 4 and 5). Dissociated S2− can react with [Ru(bpy)2(bpy-DPA)Cu]4+, forming CuS and [Ru(bpy)2(bpyDPA)]2+ (equilibrium in eq 6), because of large value of pKsp(CuS) (35.02), as reported previously.14 Therefore, the DPA-Cu(II) group in the complex has two functions in fluorescence.14b It acts as both transducer and recognizer of function. The greater ECL properties of [Ru(bpy)2(bpyDPA)]2+ than that of [Ru(bpy)2(bpy-DPA)Cu]4+ before reaction with sulfide anions makes the developed biosensor produce an enhanced signal, which can prevent false positive signals resulted from signal-off methods. This reaction-induced ECL restoration, combined with the system (shown in Scheme 1) of selectively recognizing volatilized H2S, would be a good strategy for specific measurement of free H2S gas. S2 − ↔ HS− ↔ H 2S(aq)

(3)

H 2S(aq) ↔ H 2S(g) ↔ H 2S(aq)

(4)

H 2S(aq) ↔ HS− ↔ S2 −

(5)

S2 − + [Ru(bpy)2 (bpy‐DPA)Cu]4 + ↔ CuS + [Ru(bpy)2 (bpy‐DPA)]2 +

(6)

Figure 3 shows the ECL response obtained at [Ru(bpy)2(bpy-DPA)Cu]4+/Nafion/GC electrode before (black curve) and after (blue curve) reaction with H2S in 0.1 M phosphate buffer containing 1.0 mM TPA. The [Ru(bpy)2(bpyDPA)Cu]4+/Nafion/GC electrode shows a very low ECL response (black curve). In contrast, after the [Ru(bpy)2(bpyDPA)Cu]4+/Nafion/GC electrode captured H2S gas, volatilized from 20 μM Na2S solution, a high ECL intensity was observed. It is far beyond the ECL response of [Ru(bpy)2(bpyDPA)Cu]4+/Nafion/GC electrode and near the response of [Ru(bpy)2(bpy-DPA)]2+/Nafion/GC electrode (red curve), indicating high recovery efficiency. These properties substantially form a strong basis for signal-on ECL sensing of H2S. Linearity, Selectivity, and Reproducibility. To determine the sensitivity of our H2S detection method, we employed the [Ru(bpy)2(bpy-DPA)Cu]4+/Nation/GC electrode to react with different amounts of H2S using Na2S as donor. The typical response curve (Figure 4) shows that the ECL response was enhanced with increasing amounts of Na2S. The ECL

Figure 2. (A) Cyclic voltammograms (CVs) obtained at [Ru(bpy)2(bpy-DPA)]2+/Nafion/GC electrode (black curve) and Ru(bpy)32+/Nafion/GC electrode (red curve) in 0.1 M phosphate buffer. Scan rate 50 mV·s−1. (B) ECL response of [Ru(bpy)2(bpy-DPA)]2+/ Nafion/GC electrode (black curve) and Ru(bpy)32+/Nafion/GC electrode (red curve) in 0.1 M phosphate buffer containing 1.0 mM TPA. Scan rate 50 mV·s−1. (C) ECL spectra of [Ru(bpy)2(bpyDPA)]2+ (black curve) and Ru(bpy)32+(red curve) in 0.1 M phosphate buffer containing 1.0 mM TPA.

over a period of time t′, and Q is equal to the total anodic (Qa) charge over the same time period. ECL property of [Ru(bpy)2(bpy-DPA)]2+/TPA was further studied and the relative ECL efficacy of [Ru(bpy)2(bpy-DPA)]2+ was calculated to be 0.46, giving an ECL efficacy of Ru(bpy)32+ of 1. Comparing to Ru(bpy)32+, the larger ΔEp obtained at [Ru(bpy)2(bpy-DPA)]2+/Nafion/GC electrode and a low D

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Figure 5. ECL response obtained at [Ru(bpy)2(bpy-DPA)Cu]4+/ Nafion/GC sensors toward 50 μM GSH, 50 μM Cys, 50 μM Hcy, 300 mM HCO3−, and 8 μM Na2S.

Figure 3. ECL responses obtained at [Ru(bpy)2(bpy-DPA)Cu]4+/ Nafion/GC electrode (black curve), [Ru(bpy)2(bpy-DPA)]2+/Nafion/ GC electrode (red curve), and [Ru(bpy)2(bpy-DPA)Cu]4+/Nafion/ GC electrode after reaction with H2S (blue curve) with 20 μM Na2S as H2S donor in 0.1 M phosphate buffer (pH = 7.4) containing 1.0 mM TPA. Scan rate 50 mV·s−1.

successfully avoided the nonvolatile species interfering with H2S and made high selectivity possible. The unique ECL properties of [Ru(bpy)2(bpy-DPA)Cu]4+/Nation/GC sensors, together with their demonstrated selectivity, reproducibility, and linearity, make these sensors particularly attractive for sensing H2S in complicated biological samples, for example, rat brain. Electrochemiluminescent Sensing of H2S in Rat Brain. To demonstrate the application of the developed [Ru(bpy)2(bpy-DPA)Cu]4+/Nation/GC sensor for selectively sensing H2S in the rat brain, the sensor was hung over 30 μL of undiluted brain microdialysate for 1 h (as shown in Scheme 1). The ECL intensity before (I0) and after (I) incubation over microdialysate was measured and the results are exhibited in Table 1. The average H2S concentration in the cortex of rat Table 1. Measurement of H2S Concentrations in Rat Brain

Figure 4. ECL response obtained at [Ru(bpy)2(bpy-DPA)Cu]4+/ Nation/GC electrode in 0.1 M phosphate solution (pH = 7.4) containing 1.0 mM TPA with increasing Na2S donor concentrations (0, 0.5, 2, 4, 6, 8, and 10 μM from bottom to top). Potential range 0.6−1.35 V; scan rate 50 mV·s−1. (Inset) Linear relationship between (I − I0) and Na2S donor concentration.

sample

concn (μM)

1 2 3 4

1.5 2.7 3.4 1.5

added

found (μM)

4 μM Cu2+

0.09

2 μM Na2S

3.4

brain was 2.3 ± 0.9 μM (n = 4). In order to verify that the ECL response we obtained was due to H 2 S in the brain microdialysate, 2 μM Na2S was added into brain microdialysate, and the recovery was 95%. A control experiment was also conducted. Cu2+ (4 μM) was added into microdialysate to combine all sulfur. The sensor was hung over this solution. A very small ECL change (ΔI) was obtained, comparing to the large changes (ΔI) of microdialysate. These results suggested that the signal change we obtained resulted from H2S in the microdialysate. It shall be noted that other sulfur-containing species existing in the rat brain, such as cysteine, could not volatilize from microdialysate nor be changed to H2S by enzymes (CBS or CSE in the brain) that are cut off by the microprobe. On the other hand, brain tissue contains metal ions, for example, Cu2+, Zn2+, and Ca2+, with concentrations ranging from several to hundreds of nanomoles per liter (nanomolar), varying with animal models, brain regions, and experimental conditions, as reported previously.23 In chemistry, some metal ions could form precipitates with free H2S. But this may not be the case under physiological conditions. At this moment, we could not provide the possible reasons underlying

enhancement (ΔI) was linearly related to Na2S concentration over the range from 0.5 to 10 μM [ΔI = 1182.5CNa2S (μM) + 1391.7, R2 = 0.9976]. The detection limit was 0.25 μM (S/N = 3). The detection limit is comparable to those obtained with fluorescent approaches based on reaction-based strategy.8,13b,e,14c The relative standard deviation is 2.75% (n = 6). The volatility of H2S and the developed [Ru(bpy)2(bpyDPA)Cu]4+ /Nation/GC electrode make it possible to selectively detect H2S in the cerebral system without the interference of coexisting species, even other sulfur-containing species. As shown in Figure 5, the [Ru(bpy)2(bpy-DPA)Cu]4+/ Nation/GC sensor did not give any observable response upon reaction with 50 μM sulfur-containing species GSH, Cys, and Hcy and 300 mM HCO3−. In contrast, the ECL response increased significantly after reaction with H2S (8 μM Na2S). Moreover, the way that the sensor hung over the sample E

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Article

Analytical Chemistry

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the high concentration of H2S existing in rat brain, which will be one of the interesting areas of our future studies. However, the concentration of H2S determined with our ECL method was almost consistent with the reported values,6e,15b,24 which demonstrates that the method developed in this study could be potentially used in understanding the molecular events involved in some physiological and pathological processes associated with H2S.

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CONCLUSIONS In summary, by designing the [Ru(bpy)2(bpy-DPA)Cu]4+ ECL complex, we demonstrate a reaction-based signal-on [Ru(bpy)2(bpy-DPA)Cu]4+/Nafion/GC sensor to selectively recognize H2S. In this way, we succeed in reliably monitoring extracellular H2S in rat brain coupled with microdialysate. As far as we know, this is the first report of ECL sensing of H2S in the brain. The sensor has advantages in selectivity, easy portability, and instrumental demands and thus can be very attractive for reliable monitoring H2S in rat brain, which will help to explore the regulation and function of H2S. This demonstration also proves that the designed new derivative of Ru(bpy)23+ to recognize small molecules, especially without electroactive and luminescent properties, is a good candidate for monitoring neurochemicals. Synthesis of these complexes to construct selective ECL biosensors is underway in our group.

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AUTHOR INFORMATION

Corresponding Authors

*Fax +86 10 62516444; e-mail [email protected]. *Fax +86 10 62516444; e-mail [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We gratefully acknowledge financial support from the National Natural Science Foundation of China (Grants 21175151 and 21475149) and the youth elite project of Beijing Higher School. We also greatly appreciate Jie Hao and Qin Jiang for preparing microdialysate.

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DOI: 10.1021/ac503875j Anal. Chem. XXXX, XXX, XXX−XXX