Contents lists available at ScienceDirect

Sensors and Actuators B: Chemical

journal homepage: www.elsevier.com/locate/snb

Selective electrochemiluminescent detection of sulfide based on a dual-quenching cyclometalated Ir(III) complex

Hoon Jun Kim1, Taemin Kim1, Jong-In Hong*Department of Chemistry, Seoul National University, Seoul 08826, Republic of Korea

A R T I C L E I N F O

Keywords:Electrochemiluminescent (ECL) sensorDual-quenchingSulfideChemodosimeterIridium complex

A B S T R A C T

Hydrogen sulfide (H2S) is known to be an important mediator of diverse cellular functions in health and indisease. It is of great importance to develop a selective and efficient detection method for H2S. Herein, we reportan iridium-based electrochemiluminescent (ECL) sulfide probe (1) composed of the acrylate and dini-trobenzenesulfonyl (DNBS) units as both reactive and quenching groups. While control probes (2 and 3) havingeither acrylate or a DNBS group exhibited moderate selectivity for sulfide and other biothiols, the dual-quenching chemodosimetric ECL probe 1 installed with both acrylate and DNBS units enabled selective sensingof sulfide anions over various anions and biothiols with high turn-on ratio. The sensing mechanism of probe 1was elucidated using MALDI-TOF mass and 1H NMR analyses. Probe 1 was successfully applied to the detectionof H2S in human serum by the ECL method. This ECL-based detection method can be developed into a point-of-care testing method for selective sensing of sulfide.

1. Introduction

Hydrogen sulfide (H2S), which is best known for its rotten egg smell,is regarded as a toxic gas. However, H2S is also produced in our bodyendogenously from cysteine by the action of enzymes such as cy-stathionine β-synthase (CBS) [1], cystathionine γ-lyase (CSE) [2], and3-mercaptopyruvate sulfurtransferase (3MST) [3]. Endogenously pro-duced H2S in the human body plays an important role as a cell-signalingmolecule in diverse physiological processes such as neuromodulation inthe brain, smooth muscle relaxation in the vascular system, and mod-ulation of the blood pressure [4]. H2S is also involved in several ther-apeutic pathways, including inflammation, insulin release, angiogen-esis, and reduction of ischemia reperfusion injuries [5]. Changing levelsof endogenous H2S can be an indicator of various diseases, such asDown’s syndrome and Alzheimer’s disease [6]. Therefore, in recentyears there has been increasing interest in selective and accurate de-termination of H2S.

In addition to traditional H2S detection methods based on gaschromatography [7], sulfide precipitation [8], and colorimetric assays[9], fluorescence-based assays [10–12] have been actively studied inrecent years because of the high sensitivity and relatively facile ana-lysis. Most H2S fluorescent probes have been developed based on theunique characteristics of sulfide anions, including their reducing or

nucleophilic properties. When designing H2S probes, one strategy is tointroduce a Michael-acceptor-type α,β-unsaturated ester (acrylate) unit[11] or a dinitrophenyl ether group [12] as a nucleophilic attack site forthe sulfide anions. However, fluorescence-based probes have funda-mental limitations when applied to a portable point-of-care (POC)system because they require additional bulky equipment during theanalysis process.

Electrochemiluminescence (ECL) is a light emitting process in whichchemical species undergo electron transfer reactions at the electrodesurfaces to generate excited states that emit light. ECL-based chemo-sensors are powerful candidates for POC testing because of their simplesensing processes and easy handling [13]. Thus far, most ECL sensingsystems have been developed based on efficient ECL emissions thatoccur via an oxidative reduction process between the ruthenium tris(bipyridine) complex (Ru[bpy]32+), a well-known ECL luminophore,and tripropylamine (TPA) as a co-reactant. Several different types of Ru[bpy]32+-based biosensors and immunoassays have been developed[14], some of which are commercially available. However, efforts arestill being made to overcome the limitations of Ru(II) species such astheir narrow emission range and relatively low ECL efficiency.

Cyclometalated Ir(III) complexes have been studied as a substitutefor Ru(II) derivatives. Ir(III) complexes have several advantages com-pared to Ru(II) derivatives, including higher photoluminescence (PL)

https://doi.org/10.1016/j.snb.2020.127656Received 16 September 2019; Received in revised form 10 December 2019; Accepted 2 January 2020

⁎ Corresponding author.E-mail address: jihong@snu.ac.kr (J.-I. Hong).

1 These authors contributed equally to this work.

Sensors & Actuators: B. Chemical 307 (2020) 127656

Available online 03 January 20200925-4005/ © 2020 Published by Elsevier B.V.

T

efficiency, easy control of the wide emission range via modification ofeither main or ancillary ligands, and suitability for multiplexing de-tection [15]. In addition, it has been reported that several Ir(III) com-plexes show much stronger ECL than Ru[bpy]32+ under the sameconditions [16]. For these reasons, various different cyclometalatediridium-complex-based ECL sensors have been developed [17].

In this study, we designed a dual-quenching ECL chemodosimetricprobe 1 with two reaction sites that led to the recovery of the ECL signalin the presence of the sulfide anion (Scheme 1). Probe 1 possesses anα,β-unsaturated ester (acrylate) moiety which acts as a Michael ac-ceptor in the phenylisoquinoline (piq) main ligands and a 2,4-dini-trobenzenesulfonyl (DNBS) group in the 3-hydroxypicolinate ancillaryligand. The reaction between unsaturated ester of 1 and sulfide anionresults in a change in the electronic distribution of 1, which induces theenhancement of PL and ECL signals. The DNBS group is a well-knownstrong electron acceptor that can also operate as a highly efficientphoto-induced electron transfer (PET) quencher [18]. The reaction ofprobe 1 with sulfide or other biothiols such as cysteine (Cys) andhomocysteine (Hcy) resulted in the cleavage of the DNBS moiety vianucleophilic aromatic substitution to produce red emission in both PLand ECL. For comparison, two probes having either acrylate (2) orDNBS moiety (3) were designed. The detailed procedures for thesynthesis of the probes described in this study are given in the Sup-plementary material (SI).

2. Experimental section

2.1. Chemicals and materials

All commercial chemicals were obtained from Tokyo ChemicalIndustry (TCI), Sigma-Aldrich, Alfa Aesar and Acros Organics and wereused without additional purification. The probe stock solutions (1∼3)for all ECL and photophysical experiments were prepared from 2mMdimethyl sulfoxide (DMSO, spectrophotometric grade) solutions, di-luted with acetonitrile (CH3CN, spectrophotometric grade), and storedin a fridge for use. Analytes were dissolved in 10mM HEPES buffer (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, pH 7.4).

2.2. Syntheses of probes

Probes 1, 2 and 3 were prepared as shown in Scheme 2. Ligands 5and 7 as the main ligand were synthesized by the conventional Suzuki-Miyaura cross-coupling reaction and condensation reaction. Ir(III)

complex dimers were synthesized in refluxing 2-ethoxyethanol andH2O. Then auxiliary ligands were attached in the presence of base andthe final products were purified by flash column chromatography(SiO2). Probes 1, 2 and 3 were fully characterized by 1H NMR, 13CNMR, and HRMS (SI).

3. Results and discussion

3.1. Properties of probe 2

Initially, we examined the PL and ECL properties of probe 2, aprecursor of 1, in order to confirm the effect of unsaturated estergroups. Probe 2 showed a maximum PL intensity at 654 nm in H2O/DMSO (1:1 v/v, pH 7.4, 10mM HEPES buffer), which is red-shifted by41 nm with weaker emission compared to (piq)2Ir(pic) (pic: picolinate)(Fig. 1a). This red shift can be attributed to the electron-withdrawingcharacter of the substituted unsaturated ester on the phenyl ring of 2,which contributes to more stabilization of the LUMO than the HOMO incomparison with (piq)2Ir(pic) (Fig. S6). The unsaturated ester moiety of2 also caused the partial quenching of emission, resulting in a weakerPL emission (see Fig. S7 for details). However, the nucleophilic attack ofthe sulfide ion on the unsaturated ester group in probe 2 attenuated itsquenching effect. When Na2S was added as a sulfide donor to 2, therewas a 3.4-fold increase in the emission, which was blue-shifted by13 nm. Fig. 1a shows the changes in the phosphorescence intensity ofprobe 2 when varying concentrations of the sulfide donor were added(see also Fig. S8).

We also performed ECL experiments using probe 2 in H2O/CH3CNcontaining 10 % DMSO (1:1 v/v, pH 7.4, 10mM TPA, 10mM HEPES,and 0.1M tetrabutylammonium perchlorate (TBAP) as the supportingelectrolyte). The initial ECL intensity of 2 was observed at ∼1.4 Vduring cyclic voltammetry (CV) experiments. However, after the addi-tion of the sulfide ion, 2-S2– showed strong ECL enhancement, as ex-pected (Fig. 1b). The addition of 5mM of sulfide led to a 2.3-fold signalenhancement at the same potential and the ECL signal was maintainedfor more than 20-sweep CV cycles (Fig. S9). A linear relationship be-tween the sulfide concentration and ECL intensity was observed in theconcentration range of 0.5–4mM (Fig. 1c). The limit of detection (LOD)was estimated to be 550 nM. Selectivity tests revealed that only sulfideincreased the ECL intensity of 2 (Fig. 1d).

Density functional theory (DFT) calculations were performed inorder to identify the HOMO/LUMO electronic distributions of 2 and 2-S2– (Scheme 3). The HOMO of 2 is located on the phenyl group of the

Scheme 1. (a) Molecular structures of probes 1-3. (b) Proposed sensing mechanism of probe 1 for sulfide and biothiols.

H.J. Kim, et al. Sensors & Actuators: B. Chemical 307 (2020) 127656

2

main ligand but the LUMO is delocalized on the entire main ligand,including the acrylate moiety. This delocalized electronic distributioncan be attributed to the additional electron-withdrawing character ofunsaturated ester. The HOMO of 2-S2– is similar to that of 2, while theLUMO of 2-S2– is localized only on the isoquinoline moiety excludingacrylate, similar to the general C^N chelating cyclometalated Ir(III)complexes. These results indicate that an electron-withdrawing groupattached to the C-ring of the C^N main ligands of cyclometalated Ir(III)complexes stabilizes the LUMO more than the HOMO. In the PL andECL emission processes, the changes in the LUMO energy level andelectronic distribution on reaction with the sulfide anions affects thelight emission region and intensity because of the changes in theHOMO-LUMO energy gap.

3.2. Properties of probe 3

Probe 3, which has a DNBS group in the ancillary ligand, shows adynamic color change from colorless to yellow upon the addition ofsulfide (Fig. 2a). 3 displays intense absorptions at 280 and 340 nm,which correspond to the spin-allowed ligand-centered (1LC) transition.In addition, the broad absorption at 430–550 nm is the typical shape ofmetal-to-ligand charge transfer (1MLCT and 3MLCT) transitions. How-ever, an intense absorption band appeared at ∼450 nm upon addition

of sulfide to 3, which indicated cleavage of the DNBS moiety because ofthe reaction with sulfide.

Although probe 3 exhibited no phosphorescence when excited at500 nm, a large phosphorescent enhancement at 606 nm was observedwhen sulfide ions were added (Fig. 2b). Furthermore, probe 3 showedhigh sensitivity and fast response toward sulfide ions in comparison toprobe 2. The phosphorescence intensity of 3 increased gradually withincreasing concentrations of sulfide, and this enhancement of thephosphorescence intensity was found to have a linear relationship withthe concentration of sulfide in the range of 0–20 μM (Fig. S11). Thedetection limit measured by evaluating PL was determined to be1.3 μM. The reaction between 10 μM 3 and 20 μM sulfide in H2O/DMSO(1:1 v/v, pH 7.4, 10mM HEPES) was complete within 60min (Fig.S12).

Fig. 3a shows the ECL spectrum of 3 during the potential sweep at aPt electrode in the range of 0–1.5 V. A similar trend was observed in thePL study. Probe 3 did not show any ECL during the potential sweep inthe absence of sulfide ions. This indicates that the PET-drivenquenching mechanism of the DNBS group was still operational in theECL process with the additional heterogeneous electron transfer processfrom the working electrode during the oxidation scan. With increasingsulfide concentration, the ECL intensity gradually increased and be-came saturated when the concentration of sulfide reached 4 equiv.

Scheme 2. Synthetic scheme of (a) probes 1, 2 and (b) 3: (i) 1-chloroisoquinoline, Pd(PPh3)4, K2CO3, THF, H2O, reflux. (ii) triethyl phosphonoacetate, DBU, THF, rt,5 h. (iii) Iridium(III) chloride hydrate, 2-ethoxyethanol, H2O, reflux. (iv) 3-hydroxypicolinic acid, Na2CO3, 2-ethoxyethanol, reflux. (v) 2,4-dinitrobenzenesulfonylchloride, Et3N, CH2Cl2, rt.

Fig. 1. (a) Phosphorescent emission spectra of2 (10 μM) in the presence of 0−5mM of sul-fide in H2O/DMSO (1:1 v/v, pH 7.4, 10mMHEPES). (b) ECL intensity of 2 (10 μM) uponthe addition of sulfide (Na2S, 5mM) in H2O/CH3CN containing 10 % DMSO (1:1 v/v, pH7.4, 10mM TPA, 10mM HEPES, and 0.1MTBAP as the supporting electrolyte). (c)Isothermal binding curve obtained for ECL ti-tration upon the addition of sulfide in H2O/CH3CN containing 10 % DMSO (1:1 v/v, pH7.4, 10mM TPA, 10mM HEPES, and 0.1MTBAP as the supporting electrolyte). (d) ECLresponses of 2 (10 μM) in the presence of var-ious analytes (sodium salt, 5 mM each); (1)Probe 2 only (2) Br− (3) I− (4) NO3

− (5) SO3−

(6) N3− (7) CO3

2- (8) SO42- (9) CN− (10) Ser

(11) Val (12) Cys (13) Hcy (14) S2-.

H.J. Kim, et al. Sensors & Actuators: B. Chemical 307 (2020) 127656

3

(Fig. 3b). In particular, the ECL intensity showed a linear correlationfrom 0 to 2 equiv. of sulfide (Fig. 3c). The detection limit was calculatedto be 20 nM, which was much lower than the value determined byconsidering PL (1.3 μM).

The selectivity test was carried out by adding other anions, aminoacids or biothiols to the solution of 3. As shown in Fig. 3d, no ECLenhancement was observed upon addition of an excess amount of otheranalytes. However, the sulfide anion could not be discriminated fromother biothiols such as cysteine (Cys) and homocysteine (Hcy). There-fore, it was necessary to develop a probe that could distinguish betweensulfide and other biothiols. The PL selectivity tests also showed similarresults (Fig. S13).

DFT calculations were carried out in order to explain the turn-onmechanism of 3 upon the addition of sulfide (Scheme 4). The HOMO islocated on the phenyl moiety of the piq main ligand, but the LUMO islocalized at the DNBS group of the ancillary ligand. This separatedHOMO/LUMO distribution induces the electron transfer process fromthe Ir(III) complex to the DNBS group, which resulted in non-lumi-nescent excited states. The cleavage of the DNBS group on reaction withsulfide shifted the LUMO mainly to the isoquinoline moiety, whichresulted in red PL and ECL.

3.3. Properties of probe 1

Finally, we designed probe 1 by combining the main ligands of 2and the ancillary ligand of 3. This strategy is based on the followingrationale: (i) the DNBS group in the ancillary ligand almost completelyquenches the phosphorescence and ECL of iridium complexes and ex-hibits a primary selectivity toward sulfide and biothiols against otheranions; (ii) the unsaturated ester moiety in the main ligands contributes

to unique selectivity toward sulfide and further quenches the residualphosphorescence and ECL. Based on this principle, we developed themore advanced probe 1 for detection of sulfide ions, which displayed anexcellent turn-on ratio and good selectivity that overcomes the short-comings of 3.

Fig. 4a shows an ECL titration curve of 1 upon the addition of sul-fide. The ECL emission of 1 increased as a result of two reaction eventsin the presence of sulfide ions. First, the cleavage of the DNBS groupincreased the ECL intensity upon the addition of less than 0–2 equiv. ofsulfide, which was an almost similar behavior as that of probe 3. Next,the second reaction between the unsaturated ester group and sulfidetook place in the presence of over 50 equiv. of sulfide. This Michael-type reaction resulted in complete recovery of the ECL intensity, whichis identical to that of 2-S2– (Fig. 1b and 4b). In particular, the secondreaction occurred only with sulfide ions, which enabled probe 1 toovercome the selectivity problem of 3 against biothiols (as shown inFig. 3d).

The PL measured in H2O/DMSO solution (1:1 v/v, pH 7.4, 10mMHEPES) illustrates the two-step luminescence recovery pattern moreclearly. In the first step, ∼25 % of the phosphorescence intensity wasrecovered with the addition of small amount of sulfide ions (0–30 μM).Subsequently, further PL enhancements were observed with an addi-tional amount of sulfide (1–2mM) (Fig. S14).

The ECL selectivity of 1 toward sulfide and biothiols was also in-vestigated. The ECL intensity of 1 in the presence of biothiols (10mM)showed a relatively small enhancement in CH3CN/HEPES (1:1 v/v)containing 10 % DMSO, which was ∼30 % of the intensity seen with 1in the presence of an excess amount of sulfide (10mM) (Fig. 4c). Theseexperiments confirmed that the addition of biothiols eliminated theDNBS moiety from probe 1 and thus blocked the PET-quenching from

Scheme 3. Calculated HOMO/LUMO energy levels and electronic distributions of 2 and 2-S2−.

Fig. 2. (a) UV–vis absorption of 3 (10 μM) be-fore (black) and after (red) addition of sulfide(20 μM) in H2O/DMSO (1:1 v/v, pH 7.4,10mM HEPES). (b) Phosphorescence emissionspectra (λex =500 nm) of 3 before and afteraddition of sulfide (20 μM) in H2O/DMSO (1:1v/v, pH 7.4, 10mM HEPES). (For interpreta-tion of the references to colour in this figurelegend, the reader is referred to the web ver-sion of this article.).

H.J. Kim, et al. Sensors & Actuators: B. Chemical 307 (2020) 127656

4

DNBS. However, the quenching effect still remained because of theunreacted acrylate moieties in the main ligands. Competitive ECL as-says were also performed to evaluate the selectivity of probe 1 forsulfide by adding anions, amino acids, and biothiols. Other analytesexcept sulfide and biothiols showed no changes in the ECL intensity.However, the ECL intensity of probe 1 drastically increased whenadding 10mM sulfide to the mixtures, except iodide which undergoesoxidative chemisorption to form a close-packed layer of zerovalent io-dine at the platinum electrode surface (Fig. 4d) [19]. Because PLcompetition assay showed that there was no interference from iodide inthe detection of sulfide, this result suggests that probe 1 selectivelydetects sulfide by the PL method (Fig. S16).

To elucidate the sensing mechanism of probe 1, MALDI-TOF MS and1H NMR analyses were carried out after addition of sulfide to probe 1.The reaction between a small amount of sulfide (3 equiv.) and probe 1showed an MS peak (935.725 (m/z)) corresponding to probe 2, in-dicating that sulfide reacted only with the DNBS group of 1 (Fig. S17).MS data also confirmed the formation of the 1-S2− adduct (1003.138

(m/z)) after addition of excess amounts of sulfide (1000 equiv.) (Fig.S17). 1H NMR analysis revealed that 1 reacted with sulfide (3 equiv.) toproduce 2, resulting from removal of the DNBS group from 1 (Fig. S18).Furthermore, 1H NMR spectrum of 1 + S2- (3 equiv.) is the same as thatof 2. 1H NMR spectrum obtained after addition of excess amounts ofsulfide (1000 equiv.) showed disappearance of vinyl protons at5.95 ppm and 5.76 ppm in 1 and appearance of new peaks at 6.66 ppmand 5.95 ppm in 1-S2-. MALDI-TOF MS and 1H NMR analysis clearlyrevealed that the reaction between the two groups (DNBS, acrylate) ofprobe 1 and sulfide occurs sequentially rather than simultaneously.

DFT theoretical calculations supported these experimental results(Scheme 5). The HOMO of 1 is mainly localized on the Ir(III) center andthe phenyl ring of piq ligand, whereas the LUMO is localized on theDNBS, similar to probe 3. We can also confirm that the LUMO+3 of 1is very similar to the LUMO of 2, which is delocalized on entire piqmain ligand. These electronic distributions support the dual-quenchingmechanism of 1 by the unsaturated ester and DNBS groups. The reac-tion product of 1 with sulfide (1-S2–) is identical to 2-S2–, and the LUMO

Fig. 3. (a) ECL intensity of 3 (10 μM) upon theaddition of sulfide (20 μM) in H2O/CH3CN (1:1v/v, pH 7.4, 10mM TPA, 10mM HEPES, and0.1M TBAP as the supporting electrolyte)while the potential is swept at a Pt disk elec-trode (diameter: 2 mm) in the range 0–1.6 V(scan rate: 0.1 V/s). (b) Isothermal bindingcurve obtained for ECL titration upon the ad-dition of sulfide (0–100 μM). (c) Linear plot ofthe ECL intensity of 3 at 1.4 V upon the addi-tion of varying concentrations of sulfide. (d)ECL responses of 3 (10 μM) in the presence ofvarious analytes (sodium salt, 20 μM each); (1)Probe 3 only (2) Br− (3) I− (4) NO3

− (5) SO3−

(6) N3− (7) CO3

2- (8) SO42- (9) CN− (10) Ser

(11) Val (12) Cys (13) Hcy (14) S2-.

Scheme 4. Calculated HOMO/LUMO energy levels and electronic distributions of 3 and 3-S2−.

H.J. Kim, et al. Sensors & Actuators: B. Chemical 307 (2020) 127656

5

is mainly localized on the isoquinoline moiety, which is consistent withthe other well-known iridium complexes that show high luminescence.In other words, the electron-withdrawing DNBS group highly stabilizesthe LUMO level, and thus the DNBS group could act as an electronacceptor from the Ir complex to decrease the phosphorescence intensitythrough donor-excited PET (probes 1 and 3). The acrylate group has aweaker electron-withdrawing effect than DNBS, but this also stabilizesthe LUMO level and acts as a partial PET quencher when attached to theC-ring of the C^N main ligand (probe 2) of the cyclometalated Ir(III)complexes. Consequently, probe 1 with two types of quenching groupsis able to show a high turn-on signal in response to sulfide by blockingdual quenching effect (Fig. S19). On the other hand, the reaction pro-duct of 1 with other biothiols is identical to that formed with 2, whichpartially quenched the PL and ECL because of the electron-withdrawingacrylate unit in the piq main ligand.

To demonstrate the potential clinical application of probe 1, ECLexperiments were carried out in diluted (x10) human serum in 10mMHEPES buffer solution and CH3CN containing 10 % DMSO (1:1, v/v,10mM TPrA, and 0.1M TBAP as the supporting electrolyte) (Fig. 5).

The standard addition curve showed a good linear relationship(R2= 0.959) in the concentration range of 1–30 μM. The initial amountof sulfide in diluted serum was determined to be 2.09 μM. By multi-plying the dilution factor 10, the sulfide level in human serum wasobtained as 20.9 μM, which is within the serum sulfide levels(10.5–22.5 μM) for healthy people [20]. These results suggest thatprobe 1 could potentially be applied to ECL-based clinical diagnosis.

4. Conclusion

We developed an iridium complex-based chemodosimetric ECL

Fig. 4. (a) Isothermal binding curve of 1 (10μM) obtained for ECL titration upon the addi-tion of sulfide (0−10mM) in H2O/CH3CNcontaining 10 % DMSO (1:1 v/v, pH 7.4,10mM TPA, 10mM HEPES, and 0.1M TBAP asthe supporting electrolyte). (inset) Linear plotof the ECL intensity at 1.4 V upon the additionof varying concentrations of sulfide (0–15 μM).(b) ECL Intensity of 1 (10 μM) upon the addi-tion of sulfide (30 μM and 10mM each) whilethe potential is swept at a Pt disk electrode(diameter: 2 mm) in the range 0–1.6 V (scanrate: 0.1 V/s). (c) ECL intensity of 1 (10 μM) inthe presence of sulfide (3 equiv. and 1000equiv. each), cysteine (1000 equiv.), andhomocysteine (1000 equiv.). (d) CompetitiveECL assays performed by addition of sulfide(10 mM) to probe 1 (10 μM) in the presence ofvarious analytes (10mM, 1000 equiv.): (1)Probe 1 only (2) Br− (3) I− (4) NO3

− (5) SO3−

(6) N3− (7) CO3

2- (8) SO42- (9) CN− (10) Ser

(11) Val (12) Cys (13) Hcy (14) S2- (30 μM, 3equiv.) (15) S2-. (inset) Images of a Pt electrodein a solution of 1 in the presence of sulfide,cysteine, and homocysteine (10mM) each. ECLimages were taken during CV between 0.2 and1.6 V vs. Ag/AgCl at 0.1 V/s.

Scheme 5. Calculated HOMO/LUMO energy levels and electronic distributions of 1 and 1-S2−.

H.J. Kim, et al. Sensors & Actuators: B. Chemical 307 (2020) 127656

6

probe for sulfide detection. The ECL sulfide probe was equipped withtwo reactive groups, acrylate and DNBS units, which also showed aquenching effect. Sulfide selectively reacted with the acrylate Michaelacceptor group to revive the ECL emission. The DNBS group acting as aquenching unit was cleaved by sulfides and biothiols to enhance theECL. Control probe 2 having the acrylate unit showed moderate ECLselectivity for sulfide. Control probe 3 having the DNBS unit displayedgood ECL selectivity for sulfide and other biothiols. Combining the tworeaction/quenching sites allowed probe 1 to selectively detect sulfideover various anions and biothiols with high turn-on ratio. The sensingmechanism of probe 1 was clarified using MALDI-TOF mass spectro-metry and 1H NMR analyses. DFT calculations were performed to ra-tionalize the turn-on mechanism of the sensing process. Probe 1 wassuccessfully applied to the detection of H2S in human serum by the ECLmethod. It is expected that ECL analysis based on dual-quenchingprobes can be utilized for the development of POC diagnostic tools.

Declaration of Competing Interest

The authors declare that they have no known competing financialinterests or personal relationships that could have appeared to influ-ence the work reported in this paper.

Acknowledgements

This work was supported by the National Research Foundation(Grant No. 2018R1A2B2001293) funded by the MSIT.

Appendix A. Supplementary data

Supplementary material related to this article can be found, in theonline version, at doi:https://doi.org/10.1016/j.snb.2020.127656.

References

[1] S. Singh, D. Padovani, R.A. Leslie, T. Chiku, R. Banerjee, Relative contributions ofcystathionine β-synthase and γ-cystathionase to H2S biogenesis via alternativetrans-sulfuration reactions, J. Biol. Chem. 284 (2009) 22457–22466.

[2] T. Chiku, D. Padovani, W. Zhu, S. Singh, V. Vitvitsky, R. Banerjee, H2S biogenesis byhuman cystathionine gamma-lyase leads to the novel sulfur metabolites lanthionineand homolanthionine and is responsive to the grade of hyperhomocysteinemia, J.Biol. Chem. 284 (2009) 11601–11612.

[3] N. Shibuya, M. Tananka, M. Yoshida, Y. Ogasawara, T. Togawa, K. Ishii, H. Kimura,3-Mercaptopyruvate sulfurtransferase produces hydrogen sulfide and bound sulfane

sulfur in the brain, Antioxid. Redox. Signal 11 (2009) 703–714.[4] (a) G. Yang, L. Wu, B. Jiang, W. Yang, J. Qi, K. Cao, Q. Meng, A.K. Mustafa, W. Mu,

S. Zhang, S.H. Snyder, R. Wang, H2S as a physiologic vasorelaxant: hypertension inmice with deletion of cystathionine gamma-lyase, Science 322 (2008) 587–590;(b) R.A. Dombkowski, M.J. Russell, K.R. Olson, Hydrogen sulfide as an endogenousregulator of vascular smooth muscle tone in trout, Am. J. Physiol. Regul. Integr.Comp. Physiol. 286 (2004) R678–R685.

[5] (a) Y. Kaneko, Y. Kimura, H. Kimura, I. Niki, l-Cysteine inhibits insulin releasefrom the pancreatic β-cell, Diabetes 55 (2006) 1391–1397;(b) R.C.O. Zanardo, V. Brancaleone, E. Distrutti, S. Fiorucci, G. Cirino, J.L. Wallace,Hydrogen sulfide is an endogenous modulator of leukocyte-mediated inflammation,FASEB J. 20 (2006) 2118–2120.

[6] (a) K. Eto, T. Asda, K. Arima, T. Makifuchi, H. Kimura, Brain hydrogen sulfide isseverely decreased in Alzheimer’s disease, Biochem. Biophys. Res. Commun. 293(2002) 1485–1488;(b) P. Kamoun, M.-C. Belardinelli, A. Chabli, K. Lallouchi, B. Chadefaux-Vekemans,Endogenous hydrogen sulfide overproduction in down syndrome, Am. J. Med.Genet. A 116A (2003) 310–311;(c) W. Yang, G. Yang, X. Jia, L. Wu, R. Wang, Activation of KATP channels by H2S inrat insulin-secreting cells and the underlying mechanisms, J. Physiol. 569 (2005)519–531.

[7] U. Hannestad, S. Margheri, B. Sorbo, A sensitive gas chromatographic method fordetermination of protein-associated sulfur, Anal Biochem. 178 (1989) 394–398.

[8] T. Nagata, S. Kage, K. Kimura, K. Kudo, M. Noda, Sulfide concentrations in post-mortem mammalian tissues, J. Forensic. Sci. 35 (1990) 706–712.

[9] (a) W. Lei, P.K. Dasgupta, Determination of sulfide and mercaptans in causticscrubbing liquor, Anal. Chim. Acta 226 (1989) 165–170;(b) M.M. Hughes, M.N. Centelles, K.P. Moore, Making and working with hydrogensulfide: the chemistry and generation of hydrogen sulfide in vitro and its mea-surement in vivo: a review, Free Radic. Biol. Med. 47 (2009) 1346–1353;(c) D. Jimenez, R. Martinez-Manez, F. Sancenon, J.V. Ros-Lis, A. Benito, J. Soto, Anew chromo-chemodosimeter selective for sulfide anion, J. Am. Chem. Soc. 125(2003) 9000–9001.

[10] (a) A.R. Lippert, E.J. New, C.J. Chang, Reaction-based fluorescent probes for se-lective imaging of hydrogen sulfide in living cells, J. Am. Chem. Soc. 133 (2011)10078–10080;(b) H. Peng, Y. Cheng, C. Dai, A.L. King, B.L. Predmore, D.J. Lefer, B. Wang, Afluorescent chemoprobe for fast and quantatative detection of hydrogen sulfide inblood, Angew. Chem. Int. Ed. 50 (2011) 9672–9675;(c) F. Yu, P. Li, P. Song, B. Wang, J. Zhao, K. Han, An ICT-based strategy to acolorimetric and ratiometric fluorescence probe for hydrogen sulfide in living cells,Chem. Commun. 48 (2012) 2852–2854;(d) L.A. Montoya, M. Pluth, Selective turn-on fluorescent probes for imaging hy-drogen sulfide in living cells, Chem. Commun. 48 (2012) 4767–4769;(e) S. Chen, Z.-J. Chen, W. Ren, H.-W. Ai, Reaction-based genetically encodedfluorescent hydrogen sulfide sensors, J. Am. Chem. Soc. 134 (2012) 9589–9592.

[11] (a) Y. Qian, J. Karpus, O. Kabil, S.-Y. Zhang, H.-L. Zhu, R. Banerjee, J. Zhao, C. He,Selective fluorescent probes for live-cell monitoring of sulphide, Nat. Commun. 2(2011) 495;(b) C. Liu, J. Pan, S. Li, Y. Zhao, L.Y. Wu, C.E. Berkman, A.R. Whorton, M. Xian,Capture and visualization of hydrogen sulfide by a fluorescent probe, Angew. Chem.Int. Ed. 50 (2011) 10327–10329;(c) C. Liu, B. Peng, S. Li, C.-M. Park, A.R. Whorton, M. Xian, Reaction basedfluorescent probes for hydrogen sulfide, Org. Lett. 14 (2012) 2184–2187;(d) X. Li, S. Zhang, J. Cao, N. Xie, T. Liu, B. Yang, Q. He, Y. Hu, An ICT-basedfluorescent switch-on probe for hydrogen sulfide in living cells, Chem. Commun. 49(2013) 8656–8658;(e) L.L. Mittapelli, G.N. Nawale, S.P. Gholap, O.P. Varghese, K.R. Gore, A turn-onfluorescent GFP chromophore analog for highly selective and efficient detection ofH2S in aqueous and in living cells, Sens. Actuators, B 298 (2019) 126875.

[12] (a) X. Cao, W. Lin, K. Zheng, L. He, A near-infrared fluorescent turn-on probe forfluorescence imaging of hydrogen sulfide in living cells based on thiolysis of dini-trophenyl ether, Chem. Commun. 48 (2012) 10529–10531;(b) T. Liu, Z. Xu, D.R. Spring, J. Cui, A lysosome-targetable fluorescent probe forimaging hydrogen sulfide in living cells, Org. Lett. 15 (2013) 2310–2313;(d) Y. Liu, G. Feng, A visible light excitable colorimetric and fluorescent ESIPTprobe for rapid and selective detection of hydrogen sulfide, Org. Biomol. Chem. 12(2014) 438–445;(e) S.-Y. Kim, H.J. Kim, J.-I. Hong, Electrochemiluminescent chemodosimetricprobes for sulfide based on cyclometalated Ir(III) complexes, RSC Adv. 7 (2017)10865–10868.

[13] X. Yue, Z. Zhu, M. Zhang, Z. Ye, Microfluidic chip-based online electrochemicaldetecting system for continuous and simultaneous monitoring of ascorbate andMg2+ in rat brain, Anal. Chem. 87 (2015) 1839–1845.

[14] (a) W. Miao, A.J. Bard, Electrogenerated chemiluminescence. 77. DNA hybridiza-tion detection at high amplification with [Ru(bpy)3]2+-containing microspheres,Anal. Chem. 76 (2004) 5379–5386;(b) W. Zhan, A.J. Bard, Electrogenerated chemiluminescence. 83. Immunoassay ofhuman C-reactive protein by using Ru(bpy)32+-encapsulated liposomes as labels,Anal. Chem. 79 (2007) 459–463;(c) F. Deiss, C.N. Lafratta, M. Symer, T.M. Blicharz, N. Sojic, D.R. Walt, Multiplexedsandwich immunoassays using electrochemiluminescence imaging resolved at thesingle bead level, J. Am. Chem. Soc. 131 (2009) 6088–6089;(d) K. Muzyka, Current trends in the development of the electrochemiluminescentimmunosensors, Biosens. Bioelectron. 54 (2013) 393–407.

[15] (a) Z. Mao, M. Wang, J. Liu, L.-J. Liu, S.M.Y. Lee, C.H. Leung, D.L. Ma, A long

Fig. 5. Determination of sulfide in human serum using 1(10μM) by the standardaddition method. Condition: Human serum was diluted (x10) with H2O/CH3CNcontaining 10 % DMSO (1:1 v/v, pH 7.4, 10mM HEPES, and 0.1M TBAP as thesupporting electrolyte).

H.J. Kim, et al. Sensors & Actuators: B. Chemical 307 (2020) 127656

7

lifetime switch-on iridium(III) chemosensor for the visualization of cysteine in livezebrafish, Chem. Commun. 52 (2016) 4450–4453;(b) D. Han, M. Qian, H. Gao, B. Wang, H. Qi, C. Zhang, A “switch-on” photo-luminescent and electrochemiluminescent multisignal probe for hypochlorite via acyclometalated iridium complex, Anal. Chim. Acta 1074 (2019) 98–107;(c) J.B. Liu, C. Wu, F. Chen, C.H. Leung, D.L. Ma, A simple iridium(III) dimer as aswitch-on luminescent chemosensor for carbon disulfide detection in water sam-ples, Anal. Chim. Acta 1083 (2019) 166–171.

[16] (a) J.I. Kim, I.-S. Shin, H. Kim, J.-K. Lee, Efficient electrogenerated chemilumi-nescence from cyclometalated iridium(III) complexes, J. Am. Chem. Soc. 127(2005) 1614–1615;(b) K.N. Swanick, S. Ladouceur, E. Zysman-Colman, Z. Ding, Bright electro-chemiluminescence of iridium(III) complexes, Chem. Commun. 48 (2012)3179–3181;(c) B.D. Stringer, L.M. Quan, P.J. Barnard, D.J.D. Wilson, C.F. Hogan, Iridiumcomplexes of N-heterocyclic carbene ligands: investigation into the energetic re-quirements for efficient electrogenerated chemiluminescence, Organometallics 33(2014);(d) Y. Zhou, W. Li, L. Yu, Y. Liu, X. Wang, M. Zhou, Highly efficient electro-chemiluminescence from iridium(III) complexes with 2-phenylquinoline ligand,Dalton Trans. 44 (2015) 1858–1865.

[17] (a) I.-S. Shin, Y.-T. Kang, J.-K. Lee, H. Kim, T.H. Kim, J.S. Kim, Evaluation ofelectrogenerated chemiluminescence from a neutral Ir(III) complex for quantitativeanalysis in flowing streams, Analyst 136 (2011) 2151–2155;(b) H.J. Kim, K.-S. Lee, Y.-J. Jeon, I.-S. Shin, J.-I. Hong, Electrochemiluminescentchemodosimeter based on iridium(III) complex for point-of-care detection ofhomocysteine levels, Biosen. Bioelectron. 91 (2017) 497–503;(c) H. Rhee, T. Kim, J.-I. Hong, Ir(III) complex-based phosphorescence and elec-trochemiluminescence chemodosimetric probes for Hg(II) ions with high selectivityand sensitivity, Dalton Trans. 47 (2018) 3803–3810;(d) K.-R. Kim, H.J. Kim, J.-I. Hong, Electrogenerated chemiluminescent chemo-dosimeter based on a cyclometalated iridium(III) complex for sensitive detection ofthiophenol, Anal. Chem. 91 (2019) 1353–1359;(e) J. Park, T. Kim, H.J. Kim, J.-I. Hong, Iridium(III) complex-based electro-chemiluminescent probe for H2S, Dalton Trans. 48 (2019) 4565–4573;(f) T. Kim, J.-I. Hong, Photoluminescence and electrochemiluminescence dual-signaling sensors for selective detection of cysteine based on iridium(III) complexes,ACS Omega 7 (2019) 12616–12625;(g) C.K.P. Truong, T.D.D. Nguyen, I.-S. Shin, Electrochemiluminescent chemo-sensors for clinical applications: a review, BioChip J. 13 (2019) 203–216.

[18] (a) S. Ji, H. Guo, X. Yuan, X. Li, H. Ding, P. Gao, C. Zhao, W. Wu, W. Wu, J. Zhao, Ahighly selective OFF-ON red-emitting phosphorescent thiol probe with large stokes

shift and long luminescent lifetime, Org. Lett. 12 (2010) 2876–2879;(b) H. Guo, Y. Jing, X. Yuan, S. Ji, J. Zhao, X. Li, Y. Kan, Highly selective fluor-escent OFF–ON thiol probes based on dyads of BODIPY and potent intramolecularelectron sink 2,4-dinitrobenzenesulfonyl subunits, Org. Biomol. Chem. 9 (2011)3844–3853;(c) Y. Tang, H.-R. Yang, H.-B. Sun, S.-J. L, J.-X. Wang, Q. Zhao, X.-M. Liu, W.-J. Xu,S.-B. Li, W. Huang, Rational design of an “OFF–ON” phosphorescent chemodosi-meter based on an iridium(III) complex and its application for time-resolved lu-minescent detection and bioimaging of cysteine and homocysteine, Chem. Eur. J. 19(2013) 1311–1319.

[19] (a) Y. Zu, A.J. Bard, Electrogenerated chemiluminescence. 66. The role of directcoreactant oxidation in the ruthenium tris(2,2′)bipyridyl/tripropylamine systemand the effect of halide ions on the emission intensity, Anal. Chem 72 (2000)3223–3232;(b) M. Schmittel, S. Qinghai, A lab-on-a-molecule for anions in aqueous solution:using Kolbe electrolysis and radical methylation at iridium for sensing, Chem.Commun. 48 (2012) 2707–2709.

[20] X. Yang, J. Du, Y. Zhou, Rapid and point of care measurement of sulfide in humanserum with a light emitting diode-based photometer by marriage of gas separationwith paper enrichment, Sens. Actuators, B 232 (2016) 738–743.

Hoon Jun Kim received his B.S. (2008) in Chemistry at Seoul National University, andPh.D. degree (2017) in Organic Chemistry at Seoul National University. After post-doctoral studies with Prof. Jong-In Hong at Seoul National University, He joined LG Chemin 2018. His research interest focuses on design and synthesis of cyclometalated iridium(III) complexes as electrogenerated chemiluminescence (ECL) sensors for biologicallyimportant molecules and environmentally toxic chemicals.

Taemin Kim received his B.S. (2012) and M.S. (2015) degrees in Chemistry at SoongsilUniversity. He is currently undertaking PhD studies at the Department of Chemistry at theSeoul National University under the supervision of Prof. Jong-In Hong. His current re-search interest focuses on design and synthesis of electrogenerated chemiluminescence(ECL) sensors for point of care detection.

Jong-In Hong received his Ph.D. in Chemistry from Columbia University in 1990 withProf. W. Clark Still. After postdoctoral studies with Prof. Julius Rebek, Jr. at M.I.T.(1991–1992), he joined Seoul National University in 1993 as an Assistant Professor, andbecame a Professor in 2004. His research interests include electrogenerated chemilumi-nescence (ECL) sensors, molecular recognition and sensing of biologically significantcompounds and the development of organic electronic materials such organic light-emitting diodes and organic thermoelectrics.

H.J. Kim, et al. Sensors & Actuators: B. Chemical 307 (2020) 127656

8