Reduction of Ammineruthenium(III) by Sulfide Enables In Vivo

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Reduction of Ammineruthenium(III) by Sulfide Enables In Vivo Electrochemical Monitoring of Free Endogenous Hydrogen Sulfide Shujun Wang, Xiaomeng Liu, and Meining Zhang* Department of Chemistry, Renmin University of China, Beijing 100872, China ABSTRACT: The discovery of endogenous sulfide in mammalian brain opens up a door to understanding of the physiological function of hydrogen sulfide (H2S). The transformation of different forms of sulfide (i.e., S2−, HS−, H2S, bound sulfane sulfur, et al.) in various physiological conditions hurdles the direct detection of hydrogen sulfide in vivo. Here, we find that ammineruthenium(III) (Ru(NH3)63+) can catalyze the electrochemical oxidation of free sulfide including HS− and H2S in a neutral solution (pH 7.4). This property is used to constitute an electrochemical mechanism for selective detection of hydrogen sulfide. By coupling in vivo microdialysis with selective electrochemical detection, we successfully developed an integrated microchip-based online electrochemical system (OECS) for continuous monitoring of free endogenous hydrogen sulfide in the central nervous system (CNS). The microchip-based OECS is well responsive toward hydrogen sulfide with high stability, sensitivity and selectivity. Compared with the existing methods, the OECS does not require offline treatment of brain tissue or adjustment of the detection solutions into acidic or strong basic atmosphere. These priorities essentially enable the system to accurately and reliably track dynamics of hydrogen sulfide in the CNS.

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online electrochemistry combined with in vivo microdialysis, as it could record the dynamic change of neurochemistry in vivo with high temporal and spatial resolution.25−31 To be specific, hydrogen sulfide is electroactive (ES/H2S = −0.274 V vs NHE, pH 7.4) and could be electrochemically oxidized. Unfortunately, the electrochemical oxidation normally occurs at a high overpotential, which could result in great interference from other electroactive species in the CNS. Although hydrogen sulfide could be electrochemically oxidized at special metal electrodes such as Ni, V 2O 5 at a low potential, the electrochemical reaction generally occurs in acidic (pH < 2) or basic (pH > 12) media, which could lead to the release of hydrogen sulfide from bound sulfane sulfur and overestimation of free hydrogen sulfide in the real tissue.32,33 On the other hand, some potentiometric methods based on silver/silver sulfide ion selective electrode or on a suitable ionophore have also been reported. However, these methods normally monitor S2− concentration and require strong basic conditions for the measurements.34,35 Amperometric and polarographic sulfide sensors have been used for real-time analysis of hydrogen sulfide.36−40 However, these sensors normally required the use of a H2S-permeable membrane that is easily blocked for in vivo measurements. Therefore, an in vivo electrochemical method

he demonstration of endogenous sulfide in mammalian brain has prompted great interest in studying the physiological role of hydrogen sulfide in the central nervous system (CNS).1−4 Increasing researches have demonstrated that hydrogen sulfide is one of the important neuromodulators in the CNS and the third gasotransmitter joined with nitric oxide and carbon monoxide. It not only protects neurons and glia and induces hippocampal long-term potential, but also is related to CNS diseases, such as Alzheimer’s disease, Parkinson’s disease, ischemic stroke, and traumatic brain injury.5−8 Although there are some methods to determine hydrogen sulfide (i.e., sulfide), the transformation of different forms of sulfide (S2−, HS−, H2S, bound sulfane sulfur, et al.) and the chemical instability (easily chemically oxidized) of hydrogen sulfide under physiological conditions render a great challenge to accurately track free hydrogen sulfide in the brain under physiological condition (pH 7.4).9−20 As in the CNS, hydrogen sulfide primarily exists in forms of H2S, HS−, and a trace amount of S2− and stores in the form of bound sulfide. The stored sulfide will release when pH is changed to be more acid or alkaline, or under reducing condition.21,22 Moreover, hydrogen sulfide easily volatiles and is readily oxidized in oxygen atmosphere.23,24 Therefore, a reliable method for detecting free hydrogen sulfide with high temporal resolution under physiological conditions is highly needed for probing the function of hydrogen sulfide. Electrochemistry has shown great power for in vivo analysis including in vivo electrochemistry using microelectrode and © 2017 American Chemical Society

Received: January 7, 2017 Accepted: April 19, 2017 Published: April 19, 2017 5382

DOI: 10.1021/acs.analchem.7b00069 Anal. Chem. 2017, 89, 5382−5388

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Scheme 1. (A) Potentials of Typical Species Related to Hydrogen Sulfide; (B) Schematic of the Microchip-Based Online Electrochemical System for Measurement of Hydrogen Sulfide; (C) Relative Proportion of H2S, HS−, and S2− as a Function of pH

oxygen (i.e., the concentration attenuated by 10 and 30% after 30 and 60 min, respectively). All other chemicals were of at least analytical reagent grade. All solutions were prepared with deionized water (Milli-Q, Millipore). Apparatus and Measurements. The electrochemical measurement was conducted in a three-electrode cell using a computer-controlled CHI 660A electrochemical analyzer (Chenhua Instrument, Shanghai, China). ITO electrode was first cleaned with ethanol and distilled water, and the electrode area was controlled by acrylate adhesive, the area is 4 × 2 mm2. A Pt spiral wire and an Ag/AgCl (filled with saturated KCl) were used as counter and reference electrodes, respectively. The oxidation state of the sulfur element on ITO electrode before and after reaction with ruthenium complex was analyzed by X-ray photoelectron spectroscopy (XPS). XPS was conducted on an ESCALab220i-XL electron spectrometer from VG Scientific with 300 W Al Kα radiation. Fabrication of Microchip-Based Electrochemical Flow Cells. The device was composed of a polydimethylsiloxane (PDMS) chip with microchannel (1.8 cm in length, 170 μm in width and 150 μm in depth) and a piece of indium tin oxide (ITO) glass (4 cm long, 2.5 cm wide), as illustrated in Scheme 1B. The PDMS chip was fabricated by standard soft lithography.44 Briefly, a patterned mask designed by AutoCAD was printed by commercial printer, and then the pattern was transferred onto a silicon wafer through lithography. Afterward, a 10:1 mixture of PDMS and cross-linking agent was poured on a silicon template and baked to cure completely. The PDMS replica was cut into slabs, carrying the appropriate pattern and drilled go-through holes for placing inlet tubing, reference electrode and Pt tube outlet. The ITO glass plate was first sonicated in acetone, cleaned with ethanol and distilled water, and then etched in 5 M HCl for 2 h, leaving a small area (3 mm, 1.6 cm) covered by tapes as working electrode and connected with an electrochemical station. The PDMS chip and ITO glass plate were subjected to plasma clean for 3 min, assembled tightly, and baked for an hour. To connect the microchip-based electrochemical detection system with the external fluidics, tetrafluoroethylene hexafluoropropene (FEP) tubing was used. The fluidics were perfused through FEP tubing delivered from syringes, which were pumped by a microinjection Pump 11 Elite (Harvard

with high selectivity is urgently needed for tracking free hydrogen sulfide in the brain. Motivated by this need, we have been dedicated to developing an in vivo electrochemical method for selective monitoring free hydrogen sulfide in the brain. For example, by taking advantage of the specific H2S-induced chemical reaction (i.e., precipitate metal salts), we previously synthesized an electrochemiluminescent (ECL) probe and developed a reaction-based turn-on ECL sensor to selectively detect extracellular hydrogen sulfide in the microdialysate based on the volatile property of hydrogen sulfide.41 In the present work, we demonstrate an electrochemical method for in vivo monitoring of total free hydrogen sulfide in a neutral solution at a low potential based on the reduction property of hydrogen sulfide (Scheme 1A).42 The method developed here is essentially based on the coupling of the chemical reaction between Ru(NH3)63+ (oxidant) and hydrogen sulfide (reductant)43 with good electrochemical property of Ru(NH3)63+/2+. By doing so, the concentration of hydrogen sulfide is determined according to the reoxidation current of Ru(NH3)62+ at electrode. Based on this electrochemical mechanism and the combination with in vivo microdialysis, we develop a microchip-based online electrochemical system (OECS) for determination of hydrogen sulfide (Scheme 1B). The system is well responsive to hydrogen sulfide with high selectivity and sensitivity and, as such, could be used for the in vivo measurement of hydrogen sulfide in the CNS.

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EXPERIMENTAL SECTION Chemicals and Instruments. Hexaammineruthenium chloride (Ru(NH3)6Cl3), Na2S·9H2O, L-cysteine (Cys), glutathione (GSH), ascorbic acid (AA), and dopamine (DA) were purchased from Sigma-Aldrich. 3,4-Dihydroxyphenylacetic acid (DOPAC), uric acid (UA), and serotonin hydrochloride (5HT) were purchased from Alfa Aesar. Artificial cerebrospinal fluid (aCSF) was prepared by dissolving NaCl (126 mM), KCl (2.4 mM), KH 2 PO 4 (0.5 mM), MgCl 2 (0.85 mM), NaHCO3(27.5 mM), Na2SO4 (0.5 mM), and CaCl2 (1.1 mM) into water, and the solution was adjusted to pH 7.4. A stock solution of sulfide was prepared by dissolving Na2S·9H2O (as H2S donor) in aCSF and used immediately, for sulfide solution exposed in air for long time would be oxidized by 5383

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reversible redox probe with a potential of about −0.19 V versus Ag/AgCl (KCl-sat.), which falls between the potentials of S/ H2S and electroactive species such as DA. We speculated that both forms of sulfide (i.e., H2S and HS−) could be oxidized by Ru(NH3)63+ in the neutral solution, as the standard potential of Ru(NH3)63+/Ru(NH3)62+ is higher than those potentials of sulfide. Note that it has not been determined which form of H2S (H2S, HS−, or S2−, the mix of free inorganic sulfides) is active in the CNS, the term “hydrogen sulfide” has been used due to its neurotoxicity in the neurocommunity, which in fact refers to total free sulfide.9 Therefore, we made use of the ammineruthenium(III) complex as an electrocatalyst to catalyze the oxidation of hydrogen sulfide (i.e., total sulfide) at a potential that is lower than those for the oxidation of other kinds of electroactive species in the CNS. To demonstrate the strategy for the electrochemical detection of hydrogen sulfide, we studied the oxidation of hydrogen sulfide at ITO electrode in phosphate buffer (pH 7.4) in the absence and presence of 1 mM Ru(NH3)63+ when the scan rate was 10 mV s−1. As shown in Figure 1A (also in Figure

Apparatus). Ru(NH3)63+ and a certain concentration of sodium sulfide (Na2S·9H2O, as H2S donor) were injected into microchannel form inlet 1 and 2, respectively, both at a flow rate of 2 μL·min−1. One microsized Ag/AgCl wire were inserted in outlet as reference electrode and a hollow stainless steel as counter electrode (Scheme 1B). In Vivo Measurements of Sulfide in Brain Microdialysate of Guinea Pig. All animal procedures were approved by the Animal Care and Use Committee at National Center for Nanoscience and Technology of China. Surgeries for in vivo microdialysis were performed based on the procedures reported previously. Briefly, adult male guinea pigs (350−400 g) were housed on a 12:12 h light−dark schedule with food and water ad libitum. Guinea pigs were anesthetized with chloral hydrate (345 mg/kg, i.p.) and positioned onto a stereotaxic frame afterward. The microdialysis guide cannulas (BAS) were implanted in the hippocampus (AP = −6.2 mm, L = 6.8 mm from bregma, V = −5 mm from dura) using standard stereotaxic procedures. Afterward, the guinea pigs were allowed to recover for at least 24 h before in vivo microdialysis sampling and were fed food without AA. For online measurements, brain microdialysate was sampled from the guinea pig hippocampus through microdialysis probes CMA 12 (20 kDa, 4 mm), which were purchased from CMA Microdialysis AB (Stockholm, Sweden) at the perfusion rate 2 μL·min−1 and were delivered into a microchannel from inlet 1, while Ru(NH3)63+ was continuously perfused into the channel from inlet 2. After continuously perfusing microdialysate for at least 90 min, the amperometric response was recorded. For electrode calibration, the standard solutions of sulfide were delivered from inlet 1 instead of microdialysate. The electrode was polarized at 0.0 V for the determination of sulfide.

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RESULTS AND DISCUSSION Electrocatalytic Oxidation of Hydrogen Sulfide with Ammineruthenium(III) Catalyst. In an aqueous solution of sulfide at 20 °C, equilibrium exists H2SpKa1 HS−pKa2 S2− with ⇀ ⇀ 37 pKa1 = 6.88 and pKa2 = 14.15. As shown in Scheme 1C, in the phosphate buffer (pH 7.4), H2S and HS− coexist. When the solution pH values are lower than 6.0 and higher than 9.0, the majority form of sulfide is H2S and HS−, respectively. The standard redox potentials of H2S/S (shown in eq 1) and HS−/S (eq 2) are +0.144 V and −0.476 V (both vs NHE), respectively.45 Based on the Nernst equation, RT [H+]2 θ ES/H2S = E(S/H + nF ln [H S] and 2S)

Figure 1. Cyclic voltammograms (CVs) obtained at ITO electrode in different solutions: (A) 0.1 M pure phosphate buffer (pH 7.4) in the absence (black curve) and presence (red curve) of 3 mM Na2S, scan rate, 10 mV s−1; (B) 0.1 M phosphate buffer (pH 7.4) containing 1 mM Ru(NH3)63+ in the absence (black curve) and presence of Na2S with different concentrations of 3 mM (red curve), 6 mM (blue curve), and 9 mM (purple curve), scan rate, 10 mV s−1; (C, D) 0.1 M phosphate buffer (pH 7.4) containing 1 mM Ru(NH3)63+ in the absence (black curve) and presence (red curve) of Na2S (3 mM); 0.1 M phosphate buffer (pH 7.4) containing Na2S (3 mM) (blue curve), scan rate, 500 mV s−1 (C); 2 V s−1 (D).

2

θ − + ES/HS− = E(S/HS ) −

RT nF

ln

1 , [HS−][OH−]

where the amount of

H2S and HS could be inferred from Scheme 1C, the oxidation potentials of H2S/S and HS−/S at pH 7.4 were calculated as −0.473 V and −0.477 V (both vs Ag/AgCl, KCl-sat), respectively (Scheme 1A). H 2S → S + 2H+ + 2e−

E θ = +0.144 V vs NHE

HS− + OH− → S + 2e− + H 2O

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E θ = − 0.476 V vs NHE

1C,D, blue curve), at ITO electrode in the absence of Ru(NH3)63+ in solution, cyclic voltammogram (CV) for the oxidation of sulfide was ill-defined, with tailed current response at a potential as high as +0.60 V, suggesting such a process was rather sluggish. As shown in Figure 1B, Ru(NH 3 ) 6 3+ demonstrates a pair well-defined reversible redox wave at a formal potential of −0.19 V versus Ag/AgCl (KCl-sat.) at ITO

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On the basis of the potentials of the redox couples shown above, it occurs to us the coupling of a chemical reaction of hydrogen sulfide with an electrochemical one would form a new mechanism for electrocatalytic oxidation of hydrogen sulfide. Ru(NH3)63+/Ru(NH3)62+ is a typical electrochemical 5384

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the Figure 2B,D, the redox potential of Ru(NH3)63+/2+ is essentially pH-independent. These results demonstrate that the catalytic reaction between Ru(NH3)63+ and sulfide including H2S and HS− could occur at pH 7.4 phosphate buffer, shown in Figure 1B. To further verify the reaction between Ru(NH3)63+ and sulfide occurs in phosphate buffer (pH 7.4), we carried out Xray photoelectron spectroscopy (XPS) to characterize the electrochemical products produced at ITO electrode applied 0.0 V for 30 min in phosphate buffer containing 10 mM Na2S and 1 mM Ru(NH3)63+. Figure 3 (black curve) shows typical

electrode (black curve). The addition of sulfide into solution led to the increase in the oxidation current of Ru(NH3)62+ (ipa) and the decrease in the reduction current of Ru(NH3)63+ (ipc), suggesting that the catalytic oxidation of sulfide occurs with Ru(NH3)63+ as the electrocatalyst. Furthermore, with 3 mM Na2S added into the solution, when the scan rate was 10 mV s−1, the value of ipa/ipc was 1.59. This value was decreased to 1.08 (Figure 1C) and 0.99 (Figure 1D) when the scan rates were increased to 500 mV s−1 and 2 V s−1, respectively. This result presumably suggests that catalytic reaction is a redoxmediated process.46 Note that, the addition of sulfide in solution remarkably increases the initial current response starting from −0.3 V in the voltammogram (Figure 1B, red), which was due to the consumption of Ru(NH3)63+ caused by the proceeding reaction between Ru(NH3)63+ and sulfide. The reaction between Ru(NH3)63+ and hydrogen sulfide lays a good foundation for detecting hydrogen sulfide. As mentioned above, H2S and HS− coexist in the phosphate buffer with pH = 7.4, whereas, at pH 9, HS− is the major form and H2S is the major form at pH 6 (Scheme 1C). To clarify which form of sulfide is involved into the catalytic reaction, we investigated the catalytic reaction in the buffers with different pH values. As typically depicted in Figure 2, in the buffers with

Figure 3. XPS for S 2p of the product at ITO electrode. The electrode was first polarized at 0.0 V for 30 min in phosphate buffer containing 10 mM Na2S with (black curve) or without (blue curve) 1 mM Ru(NH3)63+, took out of solution, and rinsed with water.

XPS results of ITO electrode after electrochemical experiments. The spectra exhibit two peaks at about 163.46 and 164.7 eV, which correspond to 2p3 and 2p3/2 of S0, respectively. In contrast, there are no peaks of S0 in the spectra at ITO electrode in the same conditions with exception that no Ru(NH3)63+ was added into the buffers (Figure 3, blue curve). These results ensured our catalytic reaction. First, H2S or HS− is chemically oxidized by Ru(NH3)63+ to elemental sulfur (eq 3), then the produced Ru(NH3)62+ is electrochemically oxidized into Ru(NH3)63+ (eq 4), leading to the increase of the oxidation current of Ru(NH3)63+. 2Ru(NH3)36 + + H 2S or HS− → S + 2H+ + 2Ru(NH3)62 + (3)

2Ru(NH3)62 + ⇌ 2Ru(NH3)36 + + 2e− Figure 2. CVs obtained at ITO electrode in different solutions: (A, C) 0.1 M phosphate buffer (A, pH 6; C, pH 9) in the absence (black curve) and presence (red curve) of 3 mM Na2S, scan rate, 10 mV s−1; (B, D) 0.1 M phosphate buffer (B, pH 6; D, pH 9) containing 1 mM Ru(NH3)63+ in the absence (black curve) and presence of 3 mM Na2S (red curve), scan rate, 10 mV s−1.

(4)

Microfluidic Chip-Based Online Electrochemical System (OECS) for Detection of Hydrogen Sulfide. The electrochemical mechanism between Ru(NH3)63+ and H2S enables the electrochemical monitoring of hydrogen sulfide. To do this, we designed a microfluidic-based chip on which there are two inlets, one for infusion of Ru(NH3)63+, and the other for standard sulfide solution or microdialysate. The two inlets solution mixed and the current signals were recorded at the ITO electrode inlet in the mixed channel, as shown in Scheme 1B. Before online measurements of brain microdialysate, the performance of the microchip-based OECS was studied in vitro. As shown in Figure 4A, the system produces well-defined current responses toward different concentrations of hydrogen sulfide. Current response is linear with the concentration of hydrogen sulfide within a range from 0.5 to 10 μM (I (nA) =

pH values of 6.0 (A) and 9.0 (C), the oxidation of sulfide (i.e., 3 mM Na2S added into the buffers) at ITO electrode was illdefined, which was quite similar to that in the phosphate buffer with pH 7.4 (Figure 1A), showing that the oxidation of sulfide at both pH values were rather slow. In contrast, the addition of 3 mM Na2S into both buffers clearly increases the oxidation currents at a quite low potential of −0.15 V, while decreasing the reduction ones in the CVs (B, D). As could be seen from 5385

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Figure 4. (A) Amperometric response recorded with microchip-based OECS toward standard solutions containing different concentrations of hydrogen sulfide as indicated in the figure. Inset, plot of current response versus the concentration of sulfide. (B) Amperometric response recorded with microchip-based OECS toward (black curve) 20 μM GSH, 20 μM Cys, 5 μM AA, 10 μM DA, 20 μM DOPAC, 50 μM UA, 10 μM 5-HT, and 10 μM sulfide; (red curve) 2 μM sulfide. The perfusion rate, 2 μL min−1. Applied potential, 0 V vs Ag/AgCl (aCSF).

−1.1241 (nA) + 0.2814 CH2S (μM), r = 0.9871). The detection limit was 0.17 μM (S/N = 3). We studied the interference from other sulfur-containing compounds and the electroactive species in brain. As shown in Figure 4B (black curve), the perfusion of 20 μM Cys, 20 μM GSH, 5 μM AA, 10 μM DA, 20 μM DOPAC, 50 μM UA, or 10 μM 5-HT into the system did not produce obvious current responses as compared with 10 μM sulfide. These results demonstrate the good selectivity of the OECS. In addition, the stability of the system was also studied. As depicted in Figure 4B (red curve), the system was quite stable for continuously sensing of sulfide and the current responses (0.4 nA) remain almost unchanged for almost 1 h for 2 μM sulfide. In Vivo Monitoring of Free Hydrogen Sulfide in the Hippocampus of Guinea Pig. Microdialysis is a powerful sampling technique for small molecules, which can be performed in awake, freely moving animals with minimum invasion to organism, which enables continuous monitoring of analyte in the extracellular fluid of animal brain and other tissues for a long time.47−49 Microdialysis sampling coupled with microchip could achieve promoted temporal resolution (several seconds) and fewer samples (nL to pL).50−52 Many species, such as glucose, AA, and Ca2+, have been electrochemically detected by integrating microdialysis and microchip.53,54 Here, we combined a microchip with microdialysis sampling to electrochemically detect hydrogen sulfide in hippocampus of guinea pig, the result was shown in Figure 5A. We observed that, as solution of inlet 1 switched from pure aCSF to microdialysate (Scheme 1B), an obvious increase of current response was recorded. The basal level of hydrogen sulfide in the hippocampus was determined to be 3.10 ± 1.01 μM (n = 4). The basal concentration of hydrogen sulfide in the microdialysate was reported to range from 2.3 to 12.2 μM,41,55,56 and the basal concentration of hydrogen sulfide in the brain was from 28.9 to 45.6 μM.38,57,58 The differences in the basal level of hydrogen sulfide might be due to the differences in animal models, brain regions, and experimental conditions employed, as reported previously.59,60 Note that we used a guinea pig as the animal model in our study because the concentration of AA in the brain of guinea pig is lower than those in other animal models such as rat and mouse,61 and our system is also responsive to AA when AA concentration was higher than 10 μM. Nevertheless, we believe that our system may also be applicable to other animal models with higher

Figure 5. Typical amperometric responses recorded with microchipbased OECS for the brain microdialysate of free moving guinea pig (A) perfused with 1 mM Ru(NH3)63+ or 1 mM Ru(NH3)63+ containing 10 μM GSSG; (B) before and after intraperitoneal injection (labeled with red star) of 40.8 mg kg−1 Na2S·9H2O (black curve) and saline (blue curve). Perfusion rate, 2 μL min−1. Applied potential, 0 V vs Ag/AgCl (aCSF).

concentration of AA because the interference from AA could be readily eliminated with some techniques such as Nafion coating onto the electrode or preoxidization of AA in the upstream of microchip, as demonstrated previously.62−65 In order to verify that the current signal obtained was due to sulfide in brain microdialysate, 1 mM Ru(NH3)63+ solution containing 10 μM glutathione disulfide (GSSG) was delivered into microchannel, in which GSSG could consume hydrogen sulfide, as reported previously.45 As shown in Figure 5A, with the addition of GSSG, the current response of microdialysate decreased almost to the basal level (−0.69 ± 0.34 nA, n = 4), suggesting that the current responses obtained here were resulted from hydrogen sulfide in the microdialysate. These results again ensure that our method exhibits high selectivity toward hydrogen sulfide. Compared with the existing methods conducted in strongly acidic or basic solutions for monitoring of free hydrogen sulfide, our method bears the advantage in terms of the capability of the measurements of hydrogen sulfide in the physiological solution without perturbing the equilibria between free and bound sulfide.66−68 5386

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Intraperitoneal (i.p.) injection sulfide is an effective way to supplement hydrogen sulfide in the brain, which is reported to decrease injury of neurons caused by ischemia.69 Here, we injected sulfide to free moving guinea pig and monitored the change of sulfide concentration in hippocampus. Figure 5B (black line) recorded current responses before and after injection. After sulfide injection, the current increased apparently (n = 3). For comparison, we conducted intraperitoneal injection of the same amount of saline as control experiment to ensure that the current increase was due to the injection of sulfide. There was no obvious change (n = 3) in current response after i.p injection of saline (Figure 5B, blue line), further validating the method for the measurements of hydrogen sulfide.

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CONCLUSION In conclusion, we have demonstrated an electrocatalytic reaction between Ru(NH3)63+ and hydrogen sulfide and, based on this reaction mechanism, developed a microchipbased OECS for continuously monitoring of free hydrogen sulfide at physiological conditions. The system shows good performance for hydrogen sulfide analysis with high stability and selectivity. We utilized this system for detection of sulfide in living animal brain, showing that the system is capable of continuously monitoring of hydrogen sulfide in vivo. The present method opens a new opportunity and offers a simple and effective platform for investigations of hydrogen sulfide in physiological and pathological processes.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Meining Zhang: 0000-0002-7061-6025 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We gratefully acknowledge the financial support from National Natural Science Foundation of China (Grant Nos. 21475149 and 21522509). We also greatly appreciate Wenliang Ji and Tongfang Xiao for animal surgery.

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