Chromogenic and fluorogenic chemosensors for hydrogen sulfide: review of detection mechanisms since the year 2009

Abstract

The development of probes for the biologically important gas hydrogen sulfide (H₂S) has been an active area of research in recent years. This review summarizes recent work on the recognition mechanisms used by chromogenic and fluorogenic sensors and their applications in the detection of H₂S. Several types of recognition mechanisms have been reported, including cleavage of the alcoxyl (R–O) bond, cleavage of the S–O bond, reduction of the azide group, the reduction of nitro groups to amines, the replacement of a copper complex and double bond addition reactions. In all instances the reactions are accompanied by changes in color and/or emission. These recognition mechanisms are important and straightforward procedures for use in the design of highly selective colorimetric or fluorimetric probes for the detection of H₂S in living cells.

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Jianfang Li is studying for a master's degree at the Institute of Molecular Science at Shanxi University. She received her BSc in chemistry at Shanxi University in 2009.

Caixia Yin obtained her doctoral degree in chemistry at Shanxi University in 2005. She is now a professor in the Key Laboratory of Chemical Biology and Molecular Engineering of the Ministry of Education, Institute of Molecular Science at Shanxi University, specializing in inorganic chemistry. Her current research interests are molecular recognition and sensor chemistry.

Fangjun Huo obtained his doctoral degree in chemistry at Shanxi University in 2007. He is now an associate professor in the Research Institute of Applied Chemistry at Shanxi University, specializing in organic chemistry. His current research interests are sensors and supramolecular chemistry.

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1. Introduction

Hydrogen sulfide (H₂S) is a colorless gas with a pungent taste. Exposure to this flammable gas, which has a distinctive odor of rotten eggs, can trigger irritation of the eyes and respiratory tract.¹ H₂S is emitted from various industries, such as mining, smelting, sugar beet, the manufacture of carbon disulfide and organophosphate pesticides, as well as industries producing leather, sulfur dyes, paints and animal glue. H₂S is also emitted from places of organic decomposition such as swamps, sewers, septic tanks and sewage settling ponds. People who work in these locations for a long time are therefore at risk of poisoning by the gas.

Once inhaled, H₂S is rapidly absorbed by the lungs and this may lead to unconsciousness with attendant neurological sequelae, or even to death. The gas has also been associated with deaths from cardiovascular causes.² However, recent studies have challenged this traditional view of H₂S as a toxin and have shown that some animals can also produce H₂S in a controlled fashion,³ suggesting that this reactive sulfur species is important in maintaining normal physiology.⁴ H₂S may be produced by non-enzymatic processes, including release from sulfur stores and the metabolism of polysulfides.^5,6

A variety of disease phenotypes have been associated with inadequate levels of H₂S, including Alzheimer's disease,⁷ impaired cognitive ability in patients deficient in cystathionine beta synthase⁸ and hypertension in cystathionine γ lyase knockout mice.⁹ In addition, excessive H₂S production in vital organs may be responsible for the pathogenesis of other diseases such as diabetes.^10–12 These studies established H₂S as an essential physiological mediator and cellular signaling species,^13,14 but our understanding of H₂S chemistry and its far-ranging contributions to physiology and pathology is still in its infancy.

The multitude of physiological functions in which H₂S is involved requires the development of useful methods for its detection. To date, several detection methods have been developed for H₂S, including gas chromatography,^15,16 electrochemical analysis,^17,18 colorimetric and/or fluorescent methods^19–22 and metal nanoparticle sensors.²³ These methods are useful for monitoring hydrogen sulfide in environmental samples, such as air, water, sediments and sludge.²⁴ Among these detection methods, colorimetric and/or fluorescent probes have been developed rapidly as a result of their excellent properties and simplicity, especially turn-on fluorescent probes, which not only have high sensitivity, selectivity and ease of application,^25,26 but also have a high signal-to-noise ratio.^27,28 In addition, detection with these probes always involves fluorescent or ultraviolet emissions, which can be detected visually without instrumentation. Many fluorescent imaging probes^29–39 for H₂S detection have been developed. All these probes use one of the following mechanisms: (1) cleavage of the alcoxyl (R–O) bond; (2) cleavage of the S–O bond; (3) the reduction of azides; (4) the reduction of nitro groups to amines; (5) the replacement of copper complexes; or (6) a double bond addition reaction.

Pandey et al.² reviewed H₂S gas sensors based on the type of material and/or sensing principle. Lin and Chang⁵ reported fluorescent probes for imaging H₂S in biological systems. Yu et al.⁴⁰ reviewed the synthesis and design strategies of probes. Our review focuses on the recognition mechanism in the detection of H₂S.

2. Recognition mechanisms for H₂S

2.1. Cleavage of the alcoxyl (R–O) bond

A representative reaction involving the cleavage of the R–O bond is the thiolysis of dinitrophenyl ether, which is often used to protect tyrosine in the synthesis of peptides. The dinitrophenyl protective group is removed using thiols as the thionyl agents under basic conditions.^41,42 H₂S is a small gas molecule and has a pK_a value of about 6.9,⁴³ whereas typical free thiols in cells (e.g. glutathione and cysteine) have higher pK_a values of about 8.5.^44,45 Therefore, based on significant differences such as size and the pK_a value at physiological pH, the thiolysis of the dinitrophenyl ether reaction can be used to detect H₂S over biologically abundant glutathione and cysteine⁴⁶ (Scheme 1).

Scheme 1 Strategy for the design of fluorescent probes based on cleavage of the R–O bond.

Cao et al.⁴⁶ designed and synthesized a near-infrared (NIR) H₂S probe (1), a unique type of fluorescent turn-on probe for H₂S based on the chemistry of dinitrophenyl ether (Fig. 1). Free probe 1 was essentially non-fluorescent in 50 mM PBS buffer (pH 7.0) with 3 mM cetyltrimethylammonium bromide and 10% ethanol. However, on the addition of NaHS (note that NaHS or Na₂S is the source of H₂S), the fluorescence intensity of the probe increased significantly (an 18-fold fluorescence enhancement), giving a strong emission at 708 nm in the NIR region, which is favorable for fluorescent imaging studies.³⁶ The detection limit was 5 × 10⁻⁸ M (S/N = 3) and the probe was used for biological imaging in living cells.

Fig. 1 Reaction of probe 1 with NaHS.

Liu and Feng⁴⁷ reported a probe based on 3-hydroxyflavone excited state intramolecular proton transfer (ESIPT) (2), which was able to rapidly detect H₂S in both aqueous solution and in biological serum samples with high selectivity and sensitivity. Free probe 2 in pH 7.4 PBS buffer (20 μM) was light yellow and was itself not fluorescent; the addition of HS⁻ led to emission at 538 nm with a color change from light yellow to deep yellow. However, other anions, including the thiol group, did not cause any significant change in the emission of probe 2. The optical changes of probe 2 in the presence of HS⁻ suggested that HS⁻ specifically triggered the thiolysis of dinitrophenyl ether and simultaneously released 4′-dimethylamino-3-hydroxyflavone (Fig. 2), which is a known visible light excitable ESIPT dye with a high fluorescence quantum yield.

Fig. 2 Reaction of probe 2 with HS⁻. Color changes of probe 2 (20 μM) solution after the addition of NaHS (200 μM) (a) under room light and (b) under 365 nm light. Reprinted with permission from Org. Biomol. Chem., 2014, 12, 438–445. Copyright 2014, Royal Society of Chemistry.

Sayed et al.⁴⁸ reported the synthesis and sensing features of fluorescent probe 3 (Fig. 3) based on quinoline for the detection of H₂S in water and living cells. The addition of HS⁻ induced the hydrolysis of the ether with a subsequent 345-fold enhancement of emission. Other competitor ions, such as OH⁻, F⁻, Cl⁻, Br⁻, I⁻, N₃⁻, CN⁻, SCN⁻, AcO⁻, CO₃²⁻, PO₄³⁻, SO₄²⁻, SO₃²⁻, S₂O₃²⁻, H₂O₂, Cys, Me–Cys, Hcy, GSH and lipoic acid, had no obvious effect. The selectivity toward HS⁻ was ascribed to the HS⁻ induced hydrolysis of the 2,4-dinitrophenyl ether moiety, which yielded the highly fluorescent 8-hydroxyquinoline group.^48–50 Probe 3 is non-toxic and can selectively and sensitively detect the HS⁻ anion in HeLa cells. The good detection limit (60 nM) and excellent solubility in HEPES–DMSO (99 : 1 v/v) indicated that 3 was a useful probe for hydrogen sulfide.

Fig. 3 Structure of probe 3.

Two new colorimetric and fluorescent turn-on probes (4 and 5) were developed by Wei et al.⁵¹ that detected H₂S based on the thiolysis of the 7-nitrobenz-2-oxa-1,3-diazole (NBD) ether bond (Fig. 4 and 5), which was based on previous reports.^52,53 Both 4 and 5 showed no obvious fluorescence in PBS buffer (pH 7.4). The addition of H₂S resulted in a 1000-fold enhancement in fluorescence for 4 and a 77-fold enhancement in fluorescence for 5. The difference in fluorescence enhancement for the two probes is related to the molecular structure. The electron-withdrawing oxygen moiety in 4 enhances the reactivity of the electrophilic NBD ether and increases the stability of the phenol anions, which results in a three-fold faster H₂S reactivity for 4 than for 5 under physiological conditions, accompanied by a stronger fluorescence enhancement for 4. The fluorescent turn-on response can be observed visually under a 365 nm UV lamp. At the same time, the color of 4 changes from colorless to red at 490 nm, whereas 5 emits at an NIR wavelength of 662 nm after reaction with H₂S. The high selectivity to H₂S helps visualization by the naked eye.

Fig. 4 Reaction of probe 4 with H₂S.

Fig. 5 Reaction of probe 5 with H₂S.

2.2. Cleavage of the S–O bond

Some workers have reported that the fluorescence of the fluorophores can be efficiently quenched when electron-withdrawing groups such as dinitrobenzenesulfonyl are bonded with the fluorophores;^54–57 cleavage of these groups will release the fluorophores and recover the fluorescence. Using this general approach, several elegant H₂S probes have been developed based on the cleavage of the dinitrophenyl ether or dinitrobenzenesulfonyl group as a result of the nucleophilic properties of H₂S (Scheme 2).

Scheme 2 Summary of strategies for the design of fluorescent probes based on cleavage of the S–O bond.

Yang et al.⁵⁸ developed a probe (6) for the sensitive detection of the sulfide anion based on its nucleophilic properties in aqueous solution. In 25% v/v aqueous acetone, the probe itself had an absorption peak at about 453 nm and no obvious fluorescence in the emission spectrum. On mixing with the sulfide anion in 25% v/v aqueous acetone, both the fluorescence and absorbance of the reaction solution dramatically increased as a result of the efficient removal of the 2,4-dinitrobenzenesulfonyl group of 6 and the release of fluorescein (Fig. 6). Based on the obvious color change, the probe can be used for the visual detection of sulfide anions.

Fig. 6 Reaction of probe 6 with H₂S.

Fu et al.⁵⁹ designed a probe (7) for sensing HS⁻ that had a strongly red-emitting styryl-containing BODIPY and a 2,4-dinitrobenzenesulfonyl unit, known as an electron acceptor that is able to quench fluorescence by electron transfer.^60–62 The optical response of probe 7 to various species was investigated in acetone (10 μM). A red shift of about 10 nm of the absorption band can be seen in the UV-visible spectrum. On the addition of HS⁻, probe 7 was cleaved and dinitrobenzenesulfonyl was released, leading to significantly enhanced fluorescence (Fig. 7). Probe 7 can permeate cell membranes and could potentially detect HS⁻ in living cells.

Fig. 7 Reaction of probe 7 with HS⁻.

Zheng et al.⁵⁷ designed a fluorescent turn-on probe (8) to selectively detect H₂S based on the nucleophilic properties of S²⁻. The probe had obvious fluorescence in pH 7.4 HEPES-buffered water. In the presence of competitor ions (F⁻, Cl⁻, Br⁻, I⁻, CO₃²⁻, NO₃⁻, S₂O₃²⁻, SO₃²⁻, HSO₃⁻ and SO₄²⁻), only the addition of S²⁻ resulted in the appearance of a new peak at 518 nm in the emission spectra; this was enhanced up to 340-fold. The enhancement of the fluorescence was ascribed to the cleavage of the strong electron-withdrawing dinitrobenzenesulfonate ester group from probe 8 and the release of the fluorescein fluorophore (Fig. 8). The probe has low cytotoxicity and can permeate the cell membrane to monitor sulfide anions in live cells.

Fig. 8 Reaction of probe 8 with S²⁻.

2.3. Reduction of azides

On the basis of the ability of H₂S to reduce azides, it is possible to design and synthesize fluorescent probes for the biological messenger molecule H₂S based on an ESIPT fluorescence control mechanism and ICT-induced blue or red shifts in the emission spectrum^63–65 by controlling the electron-donating ability of different substituent groups (Scheme 3).

Scheme 3 Summary of strategies for the design of fluorescent probes based on the reduction of azides.

Yu et al.⁶⁵ reported a colorimetric and ratiometric fluorescent probe (9) with a selective response to H₂S. The probe was based on heptamethine cyanine with a chemically responsive azide unit (Cy–N₃). Fig. 9 shows the detection mechanism of the probe. When the azides were reduced to amines by H₂S, an NIR fluorescence maximum appeared at 750 nm (F = 0.12), accompanied by a color change from blue to green. Probe 9 had an excellent selectivity for H₂S among a series of anions and its detection limit was 0.08 μM. The probe could permeate cells and was able to sense different levels of intracellular H₂S.

Fig. 9 Reaction of probe 9 and H₂S.

Zheng et al.⁶⁶ reported PI–N₃ (Fig. 10) as a fluorescent chemosensor for H₂S based on a phenanthroimidazole dye. The free probe 10 showed a maximum absorption band at 358 nm and a relatively weak fluorescence (Φ = 0.030) in pH 7.4, 25 mM PBS buffer–ethanol (7 : 3 v/v) at ambient temperature. The enhancement in fluorescence intensity on the addition of NaHS to probe 10 was approximately 20-fold (Fig. 9). Unfortunately, the detection limit for probe 10 was 0.879 μM, which is inferior to other reported probes such as probe 9 (0.08 μM). To examine the specificity of probe 10 for H₂S, species such as N₃⁻, ClO⁻, Cl⁻, F⁻, H₂O₂, CO₃²⁻, HCO₃⁻, HPO₄²⁻, NO₂⁻, OAc⁻, S₂O₃²⁻, SO₄²⁻ and citrate were introduced; these species had almost no fluorescence response within 5 min. However, cellular thiols such as GSH and cysteine had a <8.0-fold enhancement in fluorescence, which may interfere with the detection of HS⁻. The sensing process was confirmed by both NMR spectrometry and mass spectrometry. This method was also suitable for monitoring changes in the levels of H₂S in living cells as a result of cell permeability.

Fig. 10 Structure and fluorescence titration of probe 10 with sulfide ions. Reprinted with permission from Org. Biomol. Chem., 2012, 10, 9683–9688, Copyright 2012, Royal Society of Chemistry.

Zhou et al.²⁴ reported a fluorescent probe (11) (Fig. 11) based on the NBD fluorophore combined with a azide group. The absorption spectrum showed that the addition of NaHS resulted in a red shift of the absorption peak from 396 to 468 nm, which produced a colorimetric change from pale yellow to deep yellow. The free probe 11 is weakly fluorescent as a result of the quenching effect of the azido group; with the addition of NaHS, the fluorescence intensity increased up to 16-fold. MCF-7 cells were incubated with 11 to image H₂S and the detection limit was 680 nM.

Fig. 11 Structure of probe 11.

The fluorescence probe 12 (Fig. 12), based on 8-aminopyrene-1,3,6-trisulfonate, was designed and used for the detection of H₂S by Hartman and Dcona.⁶⁷ The presence of the electron-rich azide dramatically quenches the fluorescence of 12. However, after the reduction of the azides to amines by H₂S, the fluorescence of the solution showed a seven-fold enhancement. The probe could detect H₂S in serum samples and HS⁻ was easily quantifiable by fluorescence when its concentration was between 2 and 100 μM.

Fig. 12 Structure of probe 12.

Two coumarin-based probes 13 and 14 (Fig. 13) were designed by Li et al.⁶⁸ The optical response of probes 13 and 14 to various species was investigated in 100 mM sodium phosphate buffer (pH 7.4, 0.05% DMF). The free probes had no or very weak fluorescence. Because of the stronger electron-donating ability of –NEt₂ than that of –OH, probe 14 showed a larger fluorescence enhancement after reduction by H₂S. The quantum yields significantly increased from (0.16 ± 0.013)% and (0.58 ± 0.02)% to (0.66 ± 0.015)% and (10.93 ± 0.15)%, respectively. Li et al.⁶⁸ also showed the selectivity of the two probes among species including cysteine, glutathione, thiophenol, 4-chlorophenyl thiophenol, 2-amino thiophenol, NaSCN and NaHSO₃. It was also confirmed that the recognition mechanism was the reduction of azides to amines controlled by the strong ICT effects. Both probes 13 and 14 could be used to determine H₂S in biological samples.

Fig. 13 Structure of probes 13 and 14.

A naphthalimide derivative as a fluorescent turn-on probe (15) for H₂S detection was reported by Montoya and Pluth⁶⁹ (Fig. 14). Probe 15 is weakly fluorescent in its unreacted state; on the addition of H₂S, 15 was efficiently converted to a fluorescent amine (Φ = 0.096 ± 0.001) with a turn-on fluorescent response. These changes were attributed to the reduction of azides to amines, which was confirmed by both NMR spectrometry and mass spectrometry. To demonstrate the selectivity of probe 15, other reactive sulfur, oxygen and nitrogen species (RSONS), including cysteine, glutathione, alpha-lipoic acid, NO, H₂O₂, SO₃²⁻ and S₂O₃²⁻ were examined. All these species showed no obvious change in their fluorescence spectra under the same conditions. Probe 15 was used to study the physiological roles of endogenous H₂S and a detection limit of 5–10 μM was obtained.

Fig. 14 Structure of probe 15.

Lippert et al.⁷⁰ designed and synthesized two azide-caged rhodamine analogues as fluorescent probes 16 and 17 (Fig. 15) for the sensitive detection of H₂S. The fluorescence properties of these two probes were tested in 20 mM HEPES-buffered solutions (pH 7.4). There was no absorption feature in the visible region as a result of the closed lactone conformation of the two probes. On the addition of HS⁻, both the probes showed new absorption bands in the visible region and there was a significant enhancement in the fluorescence intensities (Φ = 0.51 and 0.60). The change in fluorescence was due to the products of the reactions between 16 and 17 with HS⁻; the corresponding rhodamine dye structures were confirmed by ¹H-NMR and liquid chromatography-mass spectrometry analyses. These probes were able to sense different levels of H₂S in HEK293T cells using confocal microscopy.

Fig. 15 Reaction of probes 16 and 17 with H₂S.

Chen et al.⁷¹ synthesized two chemoprobes 18 and 19 (Fig. 16) for the detection of H₂S based on the coumarinyl moiety. Neither of these probes showed fluorescence when they were free. On the addition of 100 μM NaHS to a solution of 19, there was a strong enhancement in fluorescence at 445 nm, which reached 40–90-fold after 50 min. Probe 18, unlike 19, had a weak response to 100 μM H₂S and had a low selectivity over other species, which indicated that 18 was not a good probe for the detection of H₂S. The pH variation of 19 was evaluated and it was found that it increased up to 170-fold in pH 7.9 buffer after reaction with H₂S. Imaging of H₂S was achieved in the cardiac tissues of normal and atherosclerotic rats.

Fig. 16 Structure of probes 18 and 19.

The benzopyran derivative 20 (Fig. 17) reported by Sun et al.⁷² was used for the fluorescence turn-on detection of H₂S. On the addition of H₂S, a color change from yellow to orange red was observed as a result of a large red shift of 90 nm in the absorption band to 505 nm. At the same time, a strong fluorescence enhancement at 670 nm appeared (λ_ex = 520 nm), as a result of the reduction of the azido group of 20 to a fluorescent amino group. Probe 20 was successfully used to image H₂S in living mice. Although the probe had excellent selectivity for H₂S over a wide pH range of 2.5–10, the water solubility (PBS buffer/DMSO 1 : 1) could be improved for better imaging in living systems.

Fig. 17 Structure of probe 20.

Bailey and Pluth⁷³ reported two reaction-based chemiluminescent sulfide sensors, 21 and 22, with strong luminescence responses toward H₂S. Both 21 and 22 completely converted each azide to the corresponding amine (Fig. 18) after the addition of H₂S and the products were confirmed by ¹H-NMR spectrometry. The response of 21 and 22 to biologically relevant RSONS was also tested. Probes 21 and 22 showed a 128-fold and 45-fold enhancement, respectively, in the turn-on response to H₂S and high selectivity for H₂S over reactive oxygen and nitrogen species. However, 21 showed a poor selectivity over cysteine-derived reductants, whereas 22 has a high selectivity. The detection limits were 0.7 ± 0.3 and 4.6 ± 2.0 μM, respectively.

Fig. 18 Reaction of probes 21 and 22 with H₂S.

Jiang et al.⁷⁴ reported a turn-on fluorescent probe (23) (Fig. 19) for H₂S imaging in living cells based on an ESIPT mechanism. This probe consisted of a p-aminobenzyl moiety and 2-(20-hydroxyphenyl)-benzothiazole. When the azido group was reduced to an amino group, the p-aminobenzyl moiety could self-immolate to release the ESIPT chromophore 2-(20-hydroxyphenyl)-benzothiazole, with a corresponding remarkably ratiometric fluorescence signal. Control experiments in the presence of other reactive species showed that 23 exhibited good selectivity for H₂S. This probe was able to sense H₂S in HeLa cells using confocal fluorescence microscopy imaging. The detection limit was 2.4 μM under the test conditions.

Fig. 19 Upper panel: structure of probe 23. Lower panels: fluorescence microscopy results for imaging of H₂S with probe 23 (5 μM) in HeLa cells. (A) Fluorescent image of HeLa cells incubated with probe 23 for 60 min and (B) bright-field image of HeLa cells in (A). Fluorescent images (top row) and bright-field images (lower row) of HeLa cells incubated with probe 23 for 30 min followed by incubation with different concentrations of NaHS for another 30 min: (C and D) 20 μM; (E and F) 50 μM; (G and H) 100 μM. E_x = 330 nm and E_m = 450–600 nm. Reprinted with permission from Talanta, 2014, 12, 1122–126, 2014 Elsevier B.V. All rights reserved.

Chen et al.⁷⁵ reported a red emission fluorescent probe (24) (Fig. 20) for H₂S based on the reduction of azides with H₂S. Because of its long excitation and emission wavelengths, dicyanomethylenedihydrofuran was selected as the fluorophore. After incubation with H₂S, the azide of this probe was reduced to form a highly fluorescent amine-derived compound. The fluorescence enhancement at 619 nm was up to 55-fold. The sensing mechanism was confirmed by both ¹H-NMR and ¹³C-NMR spectrometry. Probe 24 had low toxicity toward HUVEC cells and could be used to detect H₂S in living cells. Despite the excellent selectivity, the time-dependent fluorescence response assay showed that the reaction was complete within 60 min, which is very long. In addition, the water solubility needs to be improved.

Fig. 20 Structure of probe 24.

A BODIPY azide-based colorimetric and fluorescence probe (25) (Fig. 21) for the selective and sensitive detection of H₂S was reported by Saha et al.⁷⁶ This probe was not fluorescent as a result of the quenching effect of the electron-rich azido group. The BODIPY azide could be reduced to a BODIPY amide by H₂S with a turn-on fluorescent response. The probe was used to detect H₂S in HeLa cells. The probe had high solubility in water and could measure H₂S in serum samples with a 28-fold increase in fluorescence. The detection limit was 265 nM. It was found that probe 25 could be used to determine fluctuating concentrations of H₂S in biological systems.

Fig. 21 Structure of probe 25.

Sun et al.⁷⁷ reported a polymeric fluorescent sensor (26) (Fig. 22) for the detection of H₂S in PBS–CH₃CN (1 : 1 v/v, pH 7.4). The intensity of fluorescence increased with increasing Na₂S concentration. Na₂S induced a three-fold increase in the fluorescence intensity of probe 26. Probe 26 also showed selectivity for H₂S among other biological anions. Confocal microscopy images of HeLa cells incubated with 26 indicated that the probe could be used for the detection of H₂S in vivo. The mechanism of the response of 26 to H₂S was the reduction of the sulfonyl azide to sulfonamide in the presence of Na₂S, as confirmed by IR and ¹H-NMR spectrometry.

Fig. 22 Structure of probe 26.

2.4. Replacement of copper complexes

Complexes of a dye and metal(s) have been designed for sensing anions; the coordinated metal(s) strongly interact with anions and the compounds formed can be detected.^78–82 Copper complexes can be used to sensitively detect thiols such as cysteine and glutathione over other amino acids in aqueous media.^83–85 The recognition mechanism is based on metal–anion affinity, which has also been used as another method of sensing anions.^86–90 Here we focus on the copper complex, in which copper is released when H₂S binds to the Cu²⁺ center (Scheme 4).

Scheme 4 Summary of strategies for the design of fluorescent probes based on replacement of copper complexes.

Boron dipyrromethene–Cu²⁺ (27) is a suitable probe for detecting H₂S in HEPES buffer solution (50 mM, pH 7.4, 5% DMSO).⁸⁵ After the addition of H₂S, the color of the solution of 27 turns from orange to pink. This indicates the capability of probe 27 to detect HS⁻ by the release of Cu²⁺ and the formation of compound 27₁ in aqueous solution (Fig. 23). The detection limit for HS⁻ was 1.67 × 10⁻⁷ M under the same experimental conditions. Probe 27 did not show any significant change in absorption spectra with the addition of excess amounts of other anions such as F⁻, Cl⁻, Br⁻, I⁻, SO₄²⁻, SO₃²⁻, NO₃⁻, HCO₃⁻, H₂PO₄⁻, N₃⁻, NO₂⁻, SCN⁻ and CN⁻, indicating the excellent selectivity of 27 over other competitive anions. Unfortunately, probe 27 has almost no fluorescence response to HS⁻, which hinders the application of the probe in vivo.

Fig. 23 Reaction of probe 27 with H₂S.

Zhang and Jin⁹¹ reported another copper complex of an azo dye (28) for the detection of H₂S based on a displacement method. The association constant between compound 28₁ and Cu²⁺ (K_ass = 1.82 × 10⁴) was much smaller than that of Cu²⁺ and S²⁻ (K_ass = 7.87 × 10³⁵), showing that Cu²⁺ was easily captured by S²⁻ and free compound 28₁ was released from the complex. When Cu²⁺ was grabbed by S²⁻ from 28, the color of the solution changed from pink to yellow. Probe 28 retained a sensing response to H₂S in the presence of most competing anions. The ¹H-NMR signal and the absorption spectrum indicated a displacement reaction mechanism (Fig. 24).

Fig. 24 Reaction of probe 28 and Na₂S.

A benzimidazole-based tripodal fluorescence derivative 29₁ has been shown to capture Cu²⁺ to form complex 29, which was used to detect H₂S.⁹² Probe 29 consists of a benzimidazole-based tripodal ligand which binds with Cu²⁺ ions to induce quenching of the fluorophore. The copper complex 29 releases Cu²⁺ after reaction with S²⁻, resulting in both the absorption and fluorescence spectra returning to the initial state. Copper complex 29 could be used to detect hydrogen sulfide and may be a retrievable fluorescence sensor for H₂S (Fig. 25).

Fig. 25 Reaction of probe 29 with S²⁻.

A hetero-bimetallic Ru(II)–Cu(II) complex 30 was used for the detection of H₂S.⁹³ Compound 30₁ is a highly luminescent Ru(II)–bipyridine complex, which has a strong fluorescence (λ_ex = 456 nm, λ_em = 612 nm). The fluorescence of 30₁ could be 99% quenched after reaction with 1.0 molar equiv. of Cu²⁺ ions through an electron-transfer or energy-transfer mechanism to form the Ru(II)–Cu(II) complex 30. Complex 30 has a very high sensitivity for the detection of sulfide in 100% aqueous solutions. On the addition of S²⁻, the fluorescence intensity of the system gradually increases to the initial value as a result of the formation of stable CuS species (Fig. 26). Probe 30 was used to detect H₂S in various wastewater samples.

Fig. 26 Reaction of probe 30 with S²⁻.

A new sulfide-selective chemo-signaling system based on a Cu²⁺ complex of a fluorescein derivative was reported by Choi et al.⁹⁴ Probe 31 had no fluorescence, which is attributed to the effective binding of Cu²⁺ in the three nitrogen coordination pocket of compound 31₁, which induces a strong affinity between Cu²⁺ and the dipicolylamine binding moiety. On the addition of S²⁻, the emission intensity of 31 at 517 nm increased quickly with increasing amounts of S²⁻ as a result of the release of free compound 31₁ (Fig. 27). The detection limit was 420 nM in 100% water solution. The combination of good photochemical properties, perfect water solubility, a good detection limit and excellent selectivity makes this probe an effective tool for the detection of sulfides.

Fig. 27 Upper panel: reaction of probe 31 and S²⁻. Lower panel: fluorescence titration (left) of compound 31₁ with sulfide ions and emission spectra (right) of probe 31 with different species. Reprinted with permission from Chem. Commun., 2009, 7390–7392, Copyright 2008, Royal Society of Chemistry.

Another copper complex fluorescence sensor 32 (Fig. 28) was reported by Sasakura et al.⁹⁵ for the selective detection of H₂S. Probe 32 is not fluorescent because the paramagnetic Cu²⁺ center has a significant quenching effect on fluorophores. Cu²⁺ is captured by S²⁻ from the aza-macro-cyclic ring when H₂S binds to the Cu²⁺ center, resulting in a 50-fold enhancement in fluorescence. Probe 32 has a fast response to H₂S within seconds and exhibited a high selectivity over other species in the detection of H₂S. Copper complex 32 was shown to be appropriate for the fluorescence imaging of cellular H₂S.

Fig. 28 Reaction of probe 32 with H₂S.

Probe 33 was designed by Hou et al.⁹⁶ as a probe for H₂S in PBS–CH₃CN (1 : 1 v/v) at pH 7.2. The addition of Cu²⁺ led to compound 33₁ displaying a visible pink to yellow color change and green fluorescence quenching. Density functional theory (DFT) and ESI-MS analysis proved the formation of the copper complex 33 in a 1 : 1 stoichiometry (Fig. 29). Probe 33 is highly selective for the detection of H₂S. When S²⁻ is added to a solution of 33, the fluorescence of the system returns as CuS is formed. Chemosensor 33 can image H₂S in HeLa cells.

Fig. 29 Reaction of probe 33 with S²⁻.

Hou⁹⁷ et al. reported a probe (34) based on fluorescein for the detection of hydrogen sulfide. On the addition of Cu(II), the fluorescence intensity of 34₁ showed a 28-fold quenching with a small blue shift of the absorption band. The formation of 34 in a 1 : 1 stoichiometry was confirmed by DFT calculations. It is remarkable that copper complex 34 has a high selectivity and specificity for the recognition of hydrogen sulfide. With increasing additions of H₂S to 34, the fluorescence intensity of the system gradually enhances (25–30-fold) until the initial states are recovered. This phenomenon suggests the release of Cu²⁺ from 34 and the formation of CuS (Fig. 30). Probe 34 was used to monitor H₂S in live-cell imaging.

Fig. 30 Upper panel: reaction of probe 34 with H₂S. Lower panel: confocal fluorescence images in HepG2 cells (Zeiss LSM 510META confocal microscope, 40× objective lens). (a) HepG2 cells without adding indicator; (b) fluorescence image of HepG2 cells incubated with 34₁ (10 μM); (c) dark-field and (d) bright-field images of cells supplemented with 10 μM 34₁ in growth media for 60 min at 37 °C and then incubated with 15 μM CuCl₂ for 30 min at 37 °C. Cells incubated with 34₁ and CuCl₂ and (e) 200 μM and (f) 400 μM NaHS for another 30 min. Reprinted with permission from Dalton Trans., 2012, 41, 5799–5804. Copyright 2012, Royal Society of Chemistry.

A highly selective fluorescent probe (35) for the detection of sulfide that could be regenerated was reported by Wu et al.⁹⁸ With the addition of Cu²⁺, the fluorescence of compound 35₁ was gradually quenched until a non-fluorescent stable copper complex 35 was formed at (I₀ − I)/I₀ × 100% = 98%. On the addition of S²⁻ to the solution of 35, compound 35₁ will be completely regenerated and the fluorescence (both the intensity and the maximum emission peak) of the system will return (Fig. 31). The non-fluorescent stable copper complex 35 is highly reactive to sulfide and has a high selectivity for S²⁻ in aqueous solution. The copper ions were captured by S²⁻, resulting in the regeneration of compound 35₁, as confirmed by ¹H-NMR, matrix-assisted laser desorption ionization time-of-flight mass spectrometry and single-crystal X-ray diffraction data.

Fig. 31 Reaction of probe 35 with S²⁻.

A displacement method for detecting H₂S in aqueous media based on copper complex 36 was reported by Wang et al.⁹⁹ The fluorescence of compound 36₁ can be efficiently quenched by paramagnetic Cu²⁺ as a result of complexation with a large red shift (80 nm) in the absorption spectrum between 360 and 440 nm, which indicates that compound 36₁ binds with Cu²⁺. The addition of sulfide induced a significant enhancement in fluorescence (20-fold) at 445 nm, ascribed to the release of the free compound 36₁ (Fig. 32). To investigate the selectivity of copper complex 35 to sulfide, representative species, including the anions Cl⁻, Br⁻, I⁻, AcO⁻, N₃⁻, CO₃²⁻, NO₂⁻, H₂O₂, ClO⁻, NO, S₂O₃²⁻, SO₃²⁻ and ascorbic acid, were introduced; these species did not generate the same response. Therefore copper complex 36 can be used as a highly selective probe for the detection of S²⁻. Complex 36 can also be used to detect sulfide in living cells as it can permeate the cell membrane and has a low toxicity.

Fig. 32 Reaction of probe 36 with S²⁻.

Tang et al.¹⁰⁰ reported a novel benzimidazole derivative that exhibited highly selective and successive recognition of Cu²⁺ and sulfide anions in 100% water solution. Compound 37₁ has a strong fluorescence emission (λ_ex = 338 nm, λ_em = 475 nm) in water at pH 6.0, whereas the addition of an appropriate amount of Cu²⁺ will cause complete fluorescence quenching [(I₀ − I)/I₀ × 100 = 99%]. The time-of-flight electrospray ionization high-resolution mass spectrum and IR spectrum indicate that the stoichiometric ratio in copper complex 37 is 1 : 1. It is worth noting that on the addition of 1.0 equiv. of S²⁻ to the solution of 37, the fluorescence of the system was completely regenerated at 475 nm in both intensity and shape as a result of the release of Cu²⁺ (Fig. 33). This probe has excellent selectivity toward S²⁻ in aqueous solution and is highly reactive to sulfide with a fast response (30 s). Probe 36 was used for the detection of S²⁻ in real water samples.

Fig. 33 Reaction of probe 37 with S²⁻.

A selective fluorescent sensor for S²⁻ based on a 2-methylquinoline derivative was designed by Gao et al.¹⁰¹ Compound 38₁ can bind with Cu²⁺ along with significant fluorescence quenching (Φ = 0.059) and a color change from colorless to yellow. These changes are attributed to the formation of copper complex 38, as confirmed by DFT calculations. In particular, the addition of S²⁻ leads to total regeneration of the fluorescence and a color change. This indicated that Cu²⁺ was released from 38, as confirmed by XRD measurements and electrospray ionization mass spectrometry analysis (Fig. 34). However, other anions and various forms of sulfate did not generate the same changes. Therefore 38 may be a retrievable and highly selective fluorescent sensor for detecting sulfide. The detection limit of this probe for S²⁻ was 9.49 × 10⁻⁷ M.

Fig. 34 Reaction of probe 38 with S²⁻.

Guo et al.¹⁰² reported a selective fluorescent chemosensor (39) based on a rhodamine B derivative. In the presence of Cu²⁺, the spirocyclic structure of compound 39₁ opens and binds with Cu²⁺ to form 39, resulting in a color change from colorless to clear pink and a seven-fold enhancement in fluorescence. Adding S²⁻ to a solution of 39 will totally regenerate compound 39₁ with fluorescence quenching and restoration of the color of the system (Fig. 35). This shows that Cu²⁺ released from complex 39 was captured by S²⁻ to produce CuS. Control experiments in the presence of other common amino acids revealed that 39 exhibited good selectivity for S²⁻. The detection limit of this probe for S²⁻ was 2.43 × 10⁻⁸ M. This probe allows visual detection as a result of the color change and turn-on fluorescence response. The disadvantage is that the water solubility could be better, although the detection limit is excellent.

Fig. 35 Reaction of probe 39 with S²⁻.

Chemosensor 40 was reported by Huang et al.¹⁰³ as a suitable probe for H₂S. Compound 40₁ showed a strong fluorescence (λ_ex = 360 nm) as a result of a disubstituted polyacetylene (P2)-bearing cyclen moiety on the side chains, whereas the addition of Cu²⁺ led to the complete quenching of the fluorescence of the compound as a result of the formation of copper complex 40 (Fig. 36). The quenched fluorescence could recover to more than 62% of the original intensity of free 40₁ after the addition of increasing concentrations of sulfide anions to a solution of 40. No fluorescence change was observed with the addition of other anions. These results indicated that 40 had a high selectivity to detecting the sulfide anion. Probe 40 was used for the detection of S²⁻ in real water samples and the detection limit was 2.0 × 10⁻⁷ M.

Fig. 36 Reaction of probe 40 with S²⁻.

Tang et al.¹⁰⁴ reported a simple quinoline-derived thiosemicarbazone as a colorimetric and fluorescent sensor for the relay recognition of Cu²⁺ and sulfide in aqueous solution. Compound 41₁ tightly binds to Cu²⁺ through a 1 : 1 interaction and a deprotonation process with a color change from colorless to yellow. The fluorescence of compound 41₁ is quenched after binding to Cu²⁺ due to the formation of copper complex 41 (Fig. 37). Subsequent to the addition of S²⁻ to the solution of 41, the fluorescence of the system is enhanced and restored to the initial emission properties of free 41₁ and the absorption spectrum is also restored. Experimental results with competing ions revealed that only S²⁻ leads to the regeneration of free compound 41₁. The detection limit of this probe for S²⁻ was 7.2 × 10⁻⁷ M.

Fig. 37 Reaction of probe 41 with S²⁻.

Qu et al.¹⁰⁵ reported a red fluorescent turn-on probe for hydrogen sulfide. Compound 42₁, containing a phenanthrene-fused dipyrromethene structure, had a fluorescence emission at 600 nm. After binding with Cu²⁺, a red shift and fluorescence quenching (75% quenching, Φ = 0.002) were observed as a result of the formation of copper complex 42 (Fig. 38). These changes were mainly associated with compound 42₁ to Cu²⁺ charge transfer transitions associated with the Cu²⁺ center. As expected, the Cu–42 solution had very weak fluorescence. On the addition of HS⁻ to the solution of 42, the fluorescence intensity of the mixture increased dramatically until the intensity and shape of the emission band were totally restored. The fluorescence intensity of 42 changed little after the addition of excess amounts of common inorganic anions and other biothiols. Compound 42 can permeate cells and responds to intracellular HS⁻ anions.

Fig. 38 Reaction of probe 42 with HS⁻.

Coumarin derivative 43₁ containing a di-2-picolylamine moiety was developed as a probe for Cu²⁺ and S²⁻ by Hou et al.¹⁰⁶ On the addition of Cu²⁺, the fluorescence of compound 43₁ at 500 nm was completely quenched as a result of the formation of 43 (Fig. 39). As a result of the low solubility constant (k_sp = 6.3 × 10⁻³⁶) of CuS, free 43₁ is released after the addition of S²⁻ to the solution of 43, resulting in a dramatic enhancement in fluorescence. However, almost no change was observed in the fluorescence intensity of 43 after the addition of excess competing anions. This probe was able to sense different levels of H₂S in HeLa cells using confocal microscopy imaging. The detection limit was 1.3 × 10⁻⁷ M.

Fig. 39 Upper panel: reaction of probe 43 with S²⁻. Lower panel: calculated energy-minimized structures of 43₁ (left) and 43 (right). Reprinted with permission from Chem. Commun., 2013, 49, 7510–7512. Copyright 2013, Royal Society of Chemistry.

Lou et al.¹⁰⁷ reported a displacement-based sensing method using Cu²⁺-based receptors for sulfide anion recognition. After binding with Cu²⁺, the strong fluorescence of compound 44₁ was completely quenched with a color change from yellow-green to almost colorless as a result of the formation of copper complex 44. The subsequent addition of S²⁻ to the solution of 44 fully restored the fluorescence of 44₁ as a result of the release of free 44₁ and the formation of CuS (Fig. 40). The fluorescent intensity recovered to more than 73% of the original intensity of 44₁. Probe 44 has a high selectivity for S²⁻ over other anions.

Fig. 40 Upper panel: reaction of probe 44 with S²⁻. Lower panel: fluorescence emission response profiles of 44₁ + Cu²⁺. Inset: fluorescence emission spectra of 44₁ (5 μM) in H₂O–CH₃OH (3 : 1 v/v, Britton–Robinson buffer, pH 7.1) after the addition of Cu²⁺ (2.7 × 10⁻⁶ mol L⁻¹) and turn-on by S²⁻ (9.0 × 10⁻⁶ mol L⁻¹, photo J). The concentrations of the different anions were 9.0 × 10⁻⁵ mol L⁻¹. Excitation wavelength (nm) = 451. Reprinted with permission from Analyst, 2011, 136, 684–687, Copyright 2011, Royal Society of Chemistry.

2.5. Reduction of nitro groups

The nitro group is considered to be a strong quencher of fluorophores and can be reduced by Na₂S to produce the corresponding amino group, which provides a way of designing and synthesizing new types of fluorescent probe for H₂S detection (Scheme 5).⁶⁷

Scheme 5 Summary of strategies for the design of fluorescent probes based on reduction of the nitro group.

Wu et al.¹⁰⁸ reported colorimetric and ratiometric fluorescent probes 45–47 based on an ICT strategy for the detection of H₂S. The reaction of probe 45 with H₂S in DMF triggered the chemo-selective reduction of the nitro group to the amine with a color change from red to green. However, on the addition of H₂S, the fluorescence intensity of the probe decreased at 602 nm and a concomitant increase at 482 nm (λ_ex = 397 nm) with a blue shift of 120 nm was observed. Probe 45 has an excellent selectivity compared with other species. The detection limit for H₂S was 2.5 μM. With one more double bond than probe 45, probe 46 showed similar ultraviolet and fluorescent properties. Probe 46 had excellent selectivity for H₂S over other RSONS and anions, but more equivalents of H₂S were needed to establish the reaction. Probe 47 could not react with H₂S and almost no fluorescence change was found (Fig. 41–43).

Fig. 41 Reaction of probe 45 with S²⁻.

Fig. 42 Reaction of probe 46 with S²⁻.

Fig. 43 Structure of probe 47.

Bae et al.¹⁰⁹ reported a phenylseleno-nitrobenzoxadiazole derivative (48) for the colorimetric signaling of hydrogen sulfide. The reduction of the nitro group of the nitrobenzoxadiazole framework to an amino group by H₂S resulted in a pronounced color change from yellow to pink. The mechanism of this reaction was the reduction of the nitro group to an amine (Fig. 44), which was confirmed by ¹H-NMR spectrometry. It was proved that probe 48 has excellent selectivity compared with other species. Applications to tap water and simulated wastewater have rarely been reported, but the detection limit of 48 in 50% aqueous DMSO solution was determined to be 2.1 × 10⁻⁶ M. If the water solubility and detection limit could be improved, this could be a useful probe with potential for cell imaging.

Fig. 44 Reaction of probe 48 with H₂S.

Wang et al.¹¹⁰ designed a turn-on NIR fluorescent probe (49) (Fig. 45) based on reduction of the nitro group for the intracellular detection of H₂S. Using a heptamethine cyanine dye with m-nitrophenol, the fluorescence of the cyanine platform was quenched by an electron-transfer process between the modulator and the fluorophore. After the nitro group had been reduced to an amino group by H₂S, probe 49 showed an increase in fluorescence intensity with a slight blue shift. This probe also showed remarkable turn-on fluorescence for H₂S in the presence of other biologically relevant species. Probe 49 was used to detect intracellular H₂S in RAW264.7 cells.

Fig. 45 Reaction of probe 49 with H₂S.

2.6. Double bond addition reaction

Nucleophilic HS⁻ adds quickly to an electron-poor C=C double bond (Scheme 6). Zhao et al.¹¹¹ reported a highly selective colorimetric probe (50) (Fig. 46) for the fast and quantitative detection of hydrogen sulfide. Probe 50 itself has an absorption band at 527 nm under simulated physiological conditions (20 mM PBS, pH 7.4). However, when Na₂S was gradually added to a solution of 50, the absorption intensity significantly decreased and the color of the solution turned from red to colorless. To investigate the selectivity, probe 50 was incubated with various biological species. The results showed that probe 50 has an excellent selectivity for H₂S over other analytes, including Cys and GSH.

Scheme 6 Summary of strategies for the design of fluorescent probes based on the double bond addition reaction.

Fig. 46 Reaction of probe 50 with H₂S.

Qian et al.¹¹² reported a sensitive and selective fluorescent probe (51) (Fig. 47) for H₂S detection based on a BODIPY–coumarin platform. Free 51 has a very weak fluorescence at 512 nm (λ_ex = 458 nm). After incubation with H₂S for 20 min, probe 51 displayed a significant enhancement in fluorescence at 512 nm (ε = 1.69 × 104 M⁻¹ cm⁻¹, Φ = 0.19, >26-fold), whereas other representative biological thiols and amino acids in PBS buffer induced negligible optical changes in 51 under similar conditions. The sensing mechanism was confirmed by high-resolution mass spectrometry. This probe was used to detect H₂S in fresh mouse blood plasma and for imaging H₂S in live cells. The fluorescence response is complete within 20 min, which needs to be improved to realize the fast, accurate and real-time detection of intracellular H₂S.

Fig. 47 Reaction of probe 51 with Na₂S.

Liu et al.¹¹³ reported a fluorescent probe (52) (Fig. 48) for the detection of H₂S based on a flavylium derivative. Free probe 52 has an NIR emission peak at 690 nm. The addition of H₂S to a solution of 52 leads to a decrease in fluorescence based on the selective nucleophilic attack of H₂S on the electrically positive benzopyrylium moiety. Probe 52 has excellent selectivity for H₂S over other analytes. This probe detected H₂S in HeLa cells and in human serum samples by preliminary confocal laser scanning microscopy and the detection limit was 0.14 μM (S/N = 3).

Fig. 48 Reaction of probe 52 with H₂S.

3. Conclusion

We have summarized recent reports on the recognition mechanisms for H₂S and their applications in the detection of H₂S. The reported examples can be classified according to their mechanism of reaction (Table 1), such as the cleavage of alcoxyl (R–O) bonds, the cleavage of S–O bonds, the reduction of azides, the reduction of nitro groups, the replacement of the copper complex and double bond addition reactions. The cleavage of the alcoxyl (R–O) bond occurs with probes containing dinitrophenyl ether. The cleavage of the S–O bond takes place mainly with probes containing electron-withdrawing groups, such as dinitrobenzenesulfonyl. The reduction of azides and nitro groups to amines is based on the reduction ability of hydrogen sulfide. The replacement of the copper complex is based on the higher stability of CuS than the copper complex. The double bond addition reaction is mainly a result of the fast H₂S nucleophilic addition to the double bond in nearly neutral medium. Many of the examples can be used for cell imaging as a result of cell permeability and low toxicity.^{24,46,59,66,68–72,75–77,95–97,99,105,106,110,113} The recognition process of many probes is accompanied by color changes that can be monitored visually.^{24,47,51,58,65,70,72,76,85,91,92,96,97,99,101,102,104,107–109,111,113} We hope that this review will help with the design of highly selective simple colorimetric or fluorimetric turn-on probes for the detection of H₂S in living cells.

Table 1 References for recognition mechanism for the detection of hydrogen sulfide

Type of reaction	Cell imaging	Chromogenic reaction	Fluorogenic reaction
Cleavage of alcoxyl (R–O) bond	46	47, 51	46–51
Cleavage of S–O bond	57	56	55–57
Reduction of azide	24, 64, 66–70, 73–75	24, 63, 68, 70, 74	24, 63–75
Replacement of copper complex	93–95, 97, 103, 104	83, 89, 90, 94, 95, 97, 99, 100, 102, 105	90–105
Reduction of nitro groups to amine	108	106, 107	106–108
Double bond addition reaction	111	109, 111	109–111

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Acknowledgements

This work was supported by the National Natural Science Foundation of China (nos 21102086, 21472118), the Shanxi Province Science Foundation for Youths (nos 2012021009-4 and 2013011011-1), the Shanxi Province Foundation for Returnee (no. 2012-007), the Taiyuan Technology Star Special (no. 12024703), the Program for the Top Young and Middle-aged Innovative Talents of Higher Learning Institutions of Shanxi (no. 2013802), the Talents Support Program of Shanxi Province (no. 2014401) and Chinese Academy of Sciences Key Laboratory of Analytical Chemistry for Living Biosystems Open Foundation (no. ACL201304).

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