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Detection of hydrogen sulphide based on a novel G-quadruplex selective fluorescent probe
Sensors & Actuators: B. Chemical 272 (2018) 308–313
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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Detection of hydrogen sulphide based on a novel G-quadruplex selective fluorescent probe
T
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Yinghui Hua,1, Xiang Lia,1, Zhe Zhanga, Guanfeng Chena, Haoqi Lianga, Dan Zhanga,b, , ⁎ Changlin Liua, a b
Key Laboratory of Pesticide and Chemical Biology, Ministry of Education and School of Chemistry, Central China Normal University, Wuhan 430079, PR China Institute of Public Health and Molecular Medicine Analysis, Central China Normal University, Wuhan 430079, PR China
A R T I C LE I N FO
A B S T R A C T
Keywords: H2S detection G-quadruplex probes 3-Hydroxychromone derivatives Fluorescent sensor Reduction
It is of great importance to detect hydrogen sulfide (H2S) simply and sensitively for its role in various physiological processes as well as its inherent toxicity. In this work, two 3-Hydroxychromone (3HC) derivatives were prepared from 3HC-N3 based on HS− mediated reduction of azides, which was found to recognize G-quadruplexes (G4) with strong fluorescence emission through strong π-π stacking interactions and hydrogen bonding. Moreover, the mixture of 3HC-N3 and G4 was used as the H2S sensor with a limit of detection (LOD) as low as 2.64 nM in aqueous solution, which was much lower than most of current approaches. Given the fine performances and striking properties, this G-quadruplex-selective fluorescent turn-on probe would achieve a promising application of H2S detection in low fluorescence interfering environment samples.
1. Introduction Hydrogen sulfide (H2S) is a highly toxic and flammable gas with rotten egg smell, which is mainly produced in sewage plants, oil and natural gas industries, and coal mines. Besides, H2S gas is also extensively used in many chemical industries, scientific research institutions and companies of heavy water production. H2S is also a broadspectrum poison, meaning that it can poison several different systems in the body through inhibition of cytochrome c oxidase, formation of reactive oxygen species (ROS) and direct toxic effects on the brain [1,2]. Amongst all pollutants in the environment, H2S is the major threat to human health even at very low concentrations [3]. Therefore, it is imperatively necessary to develop highly selective and sensitive detection methods of H2S. Variety of methods have been applied for H2S detection, such as colorimetry [4], inductively coupled plasma-atomic emission [5], electrochemical analysis [6], gas chromatography [7] and optical sensors [8]. Colorimetric assays of H2S mainly involves the reaction of sulfide with N, N-dimethyl-p-phenylenediamine sulfate to form methylene blue [9]. However, methylene blue only obeys Beer's Law at very low concentrations and the accuracy of colorimetric is very low [10]. Besides, inductively coupled plasma-atomic emission, electrochemical analysis and gas chromatography usually require expensive
and sophisticated instruments, complicated preprocessing and postmortem processing. Based on polyaniline nanowires-gold nanoparticles, electrochemical analysis of H2S can obtain extremely low detection limit (0.1ppb), but the preparation of this sensor are relatively complicated [11]. Besides, optical H2S sensors are mainly prepared using metal organic framework (MOF) fluorescent materials, fluorescent organic molecules, and fluorescent nanoclusters, which can offer high sensitivity and selectivity [12–15]. Furthermore, based on surface plasmon resonance (SPR) and lossy mode resonance (LMR) techniques, Gupta and coworkers prepared various fiber optic H2S with high sensitivity and long-term stability, but the experimental setup of these excellent performance sensors is relatively complex [16]. Complex synthetic steps or experimental setups [16,17], lower specificity [18], high working temperature [19], and dry testing conditions [19] are hindrances that need to be overcome in the development of H2S sensors. One famous DNA conformation widely employed in sensing is Gquadruplexes (G4), which are non-canonical four-stranded structures formed by at least four tracts of consecutive guanines [20]. The rich diversity in structural topologies of G4 has made them attractive and versatile signal-transducing elements for the development of label-free detection of important species in biology or environment [21,22]. Therefore, G4 has already been used to develop various sensors for
⁎ Corresponding authors at: Key Laboratory of Pesticide and Chemical Biology, Ministry of Education and School of Chemistry, Central China Normal University, Wuhan 430079, PR China. E-mail addresses: [email protected] (D. Zhang), [email protected] (C. Liu). 1 These authors contributed to this work equally.
https://doi.org/10.1016/j.snb.2018.05.177 Received 7 December 2017; Received in revised form 1 May 2018; Accepted 30 May 2018 Available online 31 May 2018 0925-4005/ © 2018 Elsevier B.V. All rights reserved.
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Scheme 1. Compound 3HC-N3, 3HC-NH2 and sensing of hydrogen sulfide based on G-quadruplex recognition.
in 50 mM Tris-buffer at 37 °C, pH 7.4. Fluorescence spectra were obtained on a Cary Eclipse fluorescence spectrophotometer (Varian, USA) using a 1 cm quartz cuvette with a total volume of 400 μL, unless otherwise stated, 5 nm slit widths and a photomultiplier tube power of 700 V were used. The LOD was defined as 3 × standard deviation of the blank, which was calculated according to standard methods in linearity H2S response range of 3HC-N3/ILPR [37]. Absorption spectra were recorded on a SPECORD 210 Ultra-visible spectrophotometer (Analytic Jena AG) using 1 cm path length quartz cuvette with a total volume of 200 μL.
protein [23], nucleic acids [24], metal ions [25] and small molecules [26]. 3-hydroxychromone (3HC) derivatives with excellent sensitivity to the polarity of microenvironment [27], which are important excitedstate intramolecular proton transfer (ESIPT) based molecules, have been used to investigate micelles [28], phosphor lipid vesicles [29], metals [30] and proteins [31]. According to our previous reports, hydrophobic microenvironment provided by low-polar solvent [32], high α-helical level proteins [32,33], and intracellular environment [33], could enhance the fluorescence intensity of ESIPT molecules. Recently, many works have also been done to use 3HC dyes in the studies of structures and interactions of nucleic acids [34]. For example, a 3HC fluorophore being conjugated to polycationic spermine was able to easily distinguish its binding to double-stranded DNA (dsDNA) from single-stranded DNA (ssDNA) by dramatic changes in the spectra [35]. Thus, application of environment-sensitive dyes undergoing ESIPT constitutes a new approach for H2S probing. In this work, we screened out a 3HC derivative 3HC-NH2, which was able to recognize G4 with obvious. Then we modified the −NH2 group of 3HC-NH2 to −N3 group and obtained 3HC-N3, and further applied 3HC-N3 to detect HS− in the presence of G4, as the −N3 group could be reduced to the −NH2 group through HS− [36]. The results displayed that the mixture of 3HC-N3 and G4 was capable of acting as the HS− sensor with a limit of detection (LOD) as low as 2.64 nM in aqueous solution, which was much lower than most of the previous methods. Through strong π-π stacking interactions and hydrogen bonding, G4 DNA could function as fluorescent activator for 3HC-NH2. Therefore, the studies here would provide a new perspective to develop the sensing application of the ESIPT-based 3HC derivatives, and then be helpful for future potential biology imaging.
3. Results and discussion 3.1. Design and synthesis of 3HC-N3 and 3HC-NH2 ESIPT-based 3HC derivatives have excellent sensitivity to the polarity of microenvironment [27], and the hydrophobic microenvironment could enhance the fluorescence intensity of certain 3-hydroxychromone derivatives. Because of the hollow structure inside G4 [38], it might enhance the fluorescence intensity of 3HC derivatives by the hydrophobic interactions similar to bovine serum albumin. However, so far as we know, few examples of the interaction between 3HC derivatives and G4 DNA are available in the literature. In our study, we synthesized two novel 3HC derivatives, 3HC-NH2 and 3HC-N3, which were able to transform into each other via the diazotization reaction and reductive reaction, respectively (Scheme 1 and S1). The fluorescence experiments revealed that the two 3HC dyes had a single and weak emission band in Tris-buffer (50 mM Tris-HCl, 50 mM K+, pH 7.4), however, when in the presence of most, but not all, G4-forming sequences, a dramatic increase can be observed in 3HC-NH2 emission intensity, whereas the presence of control dsDNA and ssDNA showed only moderate effect on emission. As H2S can reduce 3HC-N3 and yield 3HC-NH2 under mild conditions, herein, the mixture of 3HC-N3 and G4 was used as a fluorescence sensor for H2S. The synthesis of 3HC-N3 was achieved based on reported procedures for 3HC derivatives (Scheme S1) [39]. An oxidative cyclization between 2′-hydroxyacetophenone and 4-acetamidobenzaldehyde yield the flavonol unit (the intermediate 1), in which the amino group was protected. Afterward, 3HC-NH2 was obtained via deprotection using HCl. Then, 3HC-N3 was formed based on the diazotization reaction. The chemical structures of the two 3HC derivatives were fully characterized by NMR (1H and 13C) and HRMS. Besides, 3HC-NH2 was additionally characterized by elemental analysis as well as X-ray single crystal diffraction analysis (ESI, detailed crystallographic data were summarized in Table S1).
2. Material and methods 2.1. Materials and oligonucleotides Unless otherwise noted, materials were purchased from commercial suppliers without further purification. All the solvents were treated according to standard methods. The oligonucleotides were purchased from Invitrogen Technology (Shanghai, China), purified over polyacrylamide gel electrophoresis (PAGE), and then dissolved in deionized water, which was finally stored at −20 °C. Concentrations of oligonucleotides were determined by ultraviolet (UV) spectrometry using extinction coefficients according to the manufacturer’s instructions. All other reagents were purchased from commercial suppliers as analytical grade. Full synthesis details and characteristics of the novel compounds were shown in ESI.
3.2. Fluorescence properties of 3HC-NH2 in the presence of G4 DNA 2.2. General spectroscopic methods In order to demonstrate the selective fluorescent recognition of 3HC-NH2 toward the G4-forming oligonucleotides of ILPR (sequence was shown in Table S2), having parallel and antiparallel G4 forms coexisted under physiological conditions [40], ILPR was used to evaluate the interactions through absorption, fluorescence spectroscopy and CD
Deionized water was used to prepare all aqueous solutions. The compounds were dissolved in dimethyl sulfoxide (DMSO) to make 10 mM stock solutions, which were diluted to the required concentrations for measurement. All spectroscopic measurements were working 309
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Fig. 1. (a) Fluorescence spectra of 3HC-NH2 (5 μM) in titrations with ILPR (0.5, 1, 2, 3, 5, 7 μM). The arrow indicated the development of bands with increasing ILPR concentration. (b) Fluorescence intensity of 3HC-NH2 (1 μM) at λem (maximum emission wavelength) = 508 nm in the presence of various DNA structures in final concentrations of 20 μM. ss-DNA: G4TTA-C; ds-DNA: CT-DNA (base concentration); G4: TBA, NRAS, C-KIT, G4TTA, GUK1, c-MYC, AAVS1, PSMA4, AS1411 and ILPR. All spectra were acquired at 37 °C in 50 mM Tris-buffer (including 50 mM K+, pH 7.4). λex (maximum excitation wavelength) = 395 nm.
be mainly caused by the strong π-π stacking interactions with G4, and the weaker interactions of 3HC-NH2 with ds-DNA and anti-parallel Gquadruplex of TBA led to no fluorescence response. Overall, it was suggested that 3HC-NH2 could be sensitive to most G4 DNA (TBA, was the exception) leading to fluorescence response through strong intermolecular interactions with DNA, and G4 DNA might also be used as the fluorescent activator for 3-hydroxychromone derivatives.
melting in Tris-buffer. The data of UV–vis titration shown in Fig. S1 exhibited clear red-shift phenomenon (from 374 nm to 395 nm) and hypochromic effect upon titration of ILPR, which indicated the binding between 3HC-NH2 and the folded ILPR. Furthermore, when excited at 395 nm, the characteristic emission of 3HC-NH2 (5 μM) located at 508 nm increased obviously even in the presence of 0.1 equiv of ILPR, and then increased dramatically when the concentration of ILPR was increased to 1.4 equiv to 3HC-NH2, which illustrated the sensitive fluorescent response and sensing property of 3HC-NH2 to ILPR G4 (Fig. 1a). In addition, the G4-binding selectivity of 3HC-NH2 was determined by recording the fluorescence intensity of 3HC-NH2 (1 μM) at 508 nm in the presence of various DNA structures (Fig. 1b), such as ssDNA (G4TTA-C), CT-DNA and G4 DNA (TBA, NRAS, C-KIT, G4TTA, GUK1, c-MYC, AAVS1, PSMA4, AS1411 and ILPR, sequence were shown in Table S2). The data showed that 3HC-NH2 was able to recognize almost all of the G4 with obvious emission intensity (thrombinbinding aptamer, TBA, was the only exception), whereas the moderate effect on emission could be observed in the presence of control ssDNA and CT-DNA. Moreover, CD melting experiment was used to further evaluate the influence of 3HC-NH2 on the stability of the formed ILPR G4 (Fig. S2). As shown in Fig. S2, the melting temperature (Tm) of the natural ILPR G4 in Tris-buffer was 44.2 °C, and then increased to 47.0 °C, 46.9 °C and 48.1 °C when in the presence of 1, 3 or 7 equiv of 3HC-NH2, respectively, which indicated the moderate stabilization effect of 3HC-NH2 on ILPR G4. Considering the sensitive fluorescent response of 3HC-NH2 to G4 and the moderate stabilization effect of 3HC-NH2 on ILPR G4, molecular docking simulations were carried out using the AutoDock program based on the structural data of different DNA to demonstrate the interaction between 3HC-NH2 and G4. As shown in the docking between 3HC-NH2 and ds-DNA (PDB: 1FKY), only two intermolecular hydrogen bonds were formed (Fig. 2a), which indicated the weak interaction between ds-DNA (PDB: 1FKY) and 3HC-NH2. For anti-parallel quadruplex formed by TBA (PDB: 1HAO), there were three intermolecular hydrogen bonds and π-π stacking interaction (between phenyl in chromone group and thymine) existing between anti-parallel G-quadruplex of TBA and 3HC-NH2 (Fig. 2b). Due to lack of structural data of ILPR in the database, docking between 3HC-NH2 and parallel Gquadruplex of c-MYC (PDB: 2L7 V) was carried out to elucidate the mechanism of fluorescence response of 3HC-NH2 to G4. Surprisingly, strong π-π stacking interactions and one hydrogen bond existed between 3HC-NH2 and parallel G-quadruplex of c-MYC, and these π-π stacking interactions were mainly formatted by guanine in c-MYC (Fig. 2c). Therefore, the fluorescence response of 3HC-NH2 to G4 would
3.3. The application of 3HC-N3/ILPR for H2S detection H2S-mediated reduction of azides to amines has been applied to a series of H2S probes which display a range of fluorescent responses and sensitivities [36]. Because of strong and selective fluorescent response of 3HC-NH2 to G4 DNA, 3HC-N3/ILPR might work as a fluorescent sensor of H2S through H2S-mediated reduction of azides to amines under mild conditions. In order to confirm the reduction of 3HC-N3 by H2S, UV–vis absorption spectra of 3HC-N3 with excessive NaHS at various times (1, 10, 30, 60, 90, 100 min) was carried out in 50 mM Tris-buffer, pH 7.4. As shown in Fig. S3, when 10 equiv of NaHS was added to 3HC-N3 at various times, new UV absorbance peaks at about 355 nm were found, which indicated the generation of 3HC-NH2. Surprisingly, when ILPR was added into the mixture of fully reacted 3HCN3 and NaHS, the peak at about 355 nm shifted to 379 nm (Fig. S3), and this peak shift was corresponded to the UV–vis titration of 3HC-NH2 using ILPR (Fig. S1). All the results indicated the reduction of 3HC-N3 to 3HC-NH2 and the combination of the newly generated 3HC-NH2 with ILPR under mild conditions. At the same time, NaHS or ILPR alone could not increase the fluorescence of 3HC-N3, and only coexistence of NaHS and ILPR could significantly enhance the fluorescence of 3HC-N3 (Fig. 3), which also confirmed that 3HC-N3/ILPR could be used as the H2S sensor. Furthermore, we determined the excitation wavelength, content of G4, reaction time and temperature of the H2S sensor (3HC-N3/ILPR). Because of the red-shift of 3HC-NH2 by ILPR, we next determined the excitation wavelength of the H2S sensor using UV–vis titration of 3HCN3/NaHS with ILPR. After full reaction of 3HC-N3 and NaHS, various concentrations of ILPR were added into the mixture, and then an obvious red shift phenomenon was obtained (from 376 nm to 397 nm) (Fig. S4), which was similar to the UV–vis titration of 3HC-NH2 with ILPR (from 374 nm to 395 nm) (Fig. S1). So, 395 nm was used as excitation wavelength in the following fluorescence experiments. For the dramatical enhancement of fluorescence intensity of 3HC-NH2 by ILPR, the working concentration of G4 DNA in 3HC-N3/ILPR was determined by fluorescence titration of 3HC-N3/NaHS with ILPR. The fluorescence intensity of 3HC-N3/NaHS enhanced with increasing concentrations of 310
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Fig. 2. The interactions between 3HC-NH2 and ds-DNA (PDB: 1FKY), the parallel quadruplex motif (PDB: 2L7V) and the anti-parallel quadruplex formed by TBA (PDB: 1HAO) based on molecular docking simulation. DNA and 3HCNH2 were represented as cartoon sticks and spheres. C, gray; N, blue; H, white; O, red. Light blue dotted lines represented the hydrogen bonding between bases and 3HC-NH2, and yellow cylinders represented π∼π stacking interactions between bases and 3HCNH2. (a) Side view of ds-DNA with 3HC-NH2. (b) The interactions of 3HC-NH2 with ds-DNA in the region of groove binding. (c) Side view of parallel G-quadruplex of c-MYC with 3HCNH2. (d) The interaction of 3HC-NH2 with the top quartet of c-MYC as well as 5′-terminal bases. (e) Side view of anti-parallel G-quadruplex of TBA with 3HC-NH2. (f) The interaction of 3HC-NH2 with TBA G-quadruplex through groove binding. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
(0–20 μM), this sensor underwent a significant increase in fluorescence with maximum peak at 508 nm, and almost kept unchanged after about one equal NaHS was added (Fig. 4b), which might indirectly verify that the reaction ratio of this sensor with H2S would be 1:1. Plotting the relative fluorescence intensity F (F, the emission intensity of 3HC-N3 (4 μM)/ILPR (40 μM) at 508 nm in the presence and in the absence of NaHS, respectively) against the concentration of NaHS, a linear response of fluorescence intensity versus NaHS concentration could be observed over the range of 0∼0.1 μM with a correlation coefficient (R) of 0.99 (Fig. 4c), and LOD of this H2S sensor was calculated as 2.64 nM by standard method [37]. Furthermore, the selectivity of H2S-induced fluorescence response of 3HC-N3 (4 μM)/ILPR (40 μM) was determined by recording the emission spectra of this sensor at 508 nm after the treatment with a variety of anions or biological thiols, such as HS−, F−, Cl−, Br−, I−, NO3−, NO2−, AcO−, SCN−, CO32−, HCO3−, H2PO4−, SO42−, HSO4−, SO32−, S2O32−, S2O72−, cysteine (Cys), homocysteine (Hcy), GSH and NAC (10 equiv). As shown in Fig. 5, the presence of the tested sulphurcontaining anions, other anions, Hcy and GSH had no obvious fluorescence response, indicating the high selectivity of 3HC-N3/ILPR toward H2S (except for Cys and NAC). As to Cys and NAC, it could be explained by Cys and NAC might cause slight reduction of azides to amines. Therefore, this Cys and NAC-mediated reduction would not obviously affect the detection of H2S using 3HC-N3/ILPR in the presence of not too much Cys or NAC.
Fig. 3. Fluorescence spectra of 3 μM 3HC-N3 (black), upon addition of 15 μM NaHS (red), 90 μM ILPR (blue) and the mixture of the former two components (pink), respectively. Fluorescence scans were recorded after incubating at 37 °C for 15 min in 50 mM Tris-buffer (include 50 mM K+, pH 7.4). λex = 395 nm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
ILPR, but fluorescence intensity did not increase any more after ILPR concentration reached 10 equiv (Fig. S4). Therefore, 10 equiv of ILPR was used as working concentration of H2S sensor. Besides, real-time monitoring the fluorescence increase of 3HC-N3/ILPR with NaHS was carried out under 0 or 37 °C, aiming to determine response time and operating temperature of H2S sensor. The reaction of 3HC-N3 and NaHS at 37 °C could be faster with stronger fluorescence intensity compared with 0 °C, and a dramatic increase in fluorescence intensity at 37 °C was observed in the first 15 min, after which its fluorescence intensity increased very slowly (Fig. S5). So the reaction temperature and time of the H2S sensor were optimized to 37 °C and 15 min, respectively. After full optimization as above, 3HC-N3 (4 μM)/ILPR (40 μM) in 50 mM Tris-buffer (pH 7.4) was designed to be an H2S sensor. Besides, 395 nm, 37 °C and 15 min were used as the excitation wavelength, reaction temperature and reaction time of this sensor, respectively. Then, the H2S-induced fluorescence response of this sensor was investigated by fluorescence titration in 50 mM Tris-buffer (pH 7.4). As shown in Fig. 4a, the background fluorescence of 3HC-N3 (4 μM)/ILPR (40 μM) was weak when excited at 395 nm, upon titration with NaHS
4. Conclusions In this study, we reported the preparation and photophysical evaluation of a novel H2S sensor 3HC-N3/ILPR, in which 3HC-N3 would selectively response to H2S and ILPR worked as a fluorescent activator. Through strong π-π stacking interactions and hydrogen bonding between 3HC-NH2 and G4 DNA (except for anti-parallel G4 of TBA), ILPR remarkably enhanced the fluorescence of 3HC-NH2, and 3HC-NH2 could stabilize the structure of ILPR at the same time. The results of UV and fluorescence titrations indicated that 3HC-NH2 could be generated by the reductive reaction of 3HC-N3, and then strong fluorescence response could be obtained by the interactions between 3HC-NH2 and ILPR. Besides, 3HC-N3 (4 μM)/ILPR (40 μM) could function as a H2S sensor with low LOD as 2.64 nM in aqueous solution, which had high selectivity toward H2S. Overall, G-quadruplex-selective fluorescent turn-on probe 3HC-N3 with fine performances and striking properties 311
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Fig. 5. Fluorescence responses of 3HC-N3 (4 μM) and ILPR (40 μM) at 508 nm toward various analytes (final concentration: 40 μM). From left to right: None, HS−, F−, Cl−, Br−, I−, NO3−, NO2−, AcO−, SCN−, CO32−, HCO3−, H2PO4−, SO42−, HSO4−, SO32−, S2O32−, S2O72−, Cys, Hcy, GSH and NAC. Fluorescence scans were recorded after incubating at 37 °C for 15 min at in Tris-buffer (50 mM Tris-HCl, pH 7.4, with 50 mM K+). λex = 395 nm.
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Fig. 4. (a) Fluorescence spectra of 3HC-N3 (4 μM) and ILPR (40 μM) in titrations with NaHS (0, 0.01, 0.02, 0.05, 0.1, 1, 3, 6, 10, 15, 20 μM). The arrow indicates an increment in NaHS concentration from 0 to 20 μM. (b) Plot of the emission intensity at λem = 508 nm against the concentration of NaHS. (c) A linear response of emission intensity at λem = 508 nm versus NaHS concentration was observed over the range of 0–0.1 μM (R2 = 0.99). All full fluorescence scans were recorded after incubating at 37 °C for 15 min in Trisbuffer (50 mM Tris-HCl, pH 7.4, with 50 mM K+). λex = 395 nm.
would realize a promising application of H2S detection under mild aqueous environment through the combination of ILPR. Furthermore, G4 DNA might also be used as the fluorescent activator for ESIPT based 3-hydroxychromone derivatives.
Acknowledgments This work was financially supported by National Natural Science Foundation of China [21302059]; Self-determined research funds of 312
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Yinghui Hu is studying for MS degree at Central China Normal University (CCNU). Her research focuses on cancer cell imaging. Xiang Li is studying for PhD degree at CCNU. His research focuses on the interactions between proteins and DNA. Zhe Zhang is studying for PhD degree at CCNU. Her research focuses on polymer synthesis. Guanfeng Chen is a MS student graduated from CCNU. Her research focuses on G4 DNA. Haoqi Liang is studying for MS degree at CCNU. His research focuses on novel fluorescent probes. Dan Zhang is an associate professor of the College of Chemistry, CCNU. She received her PhD in 2011 from Wuhan University. She joined the faculty at CCNU in 2012. Her recent research focuses on molecular recognition, prostate cancer and G-quadruplex. Changlin Liu is a professor of the College of Chemistry, CCNU. He received her PhD in 1998 from Huazhong University of Science and Technology (HUST). He worked in HUST from 1986 to 2006. He joined the faculty at CCNU in 2006. His recent research focuses on the regulation of ROS and the interactions of proteins and DNA.
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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Detection of hydrogen sulphide based on a novel G-quadruplex selective fluorescent probe
T
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Yinghui Hua,1, Xiang Lia,1, Zhe Zhanga, Guanfeng Chena, Haoqi Lianga, Dan Zhanga,b, , ⁎ Changlin Liua, a b
Key Laboratory of Pesticide and Chemical Biology, Ministry of Education and School of Chemistry, Central China Normal University, Wuhan 430079, PR China Institute of Public Health and Molecular Medicine Analysis, Central China Normal University, Wuhan 430079, PR China
A R T I C LE I N FO
A B S T R A C T
Keywords: H2S detection G-quadruplex probes 3-Hydroxychromone derivatives Fluorescent sensor Reduction
It is of great importance to detect hydrogen sulfide (H2S) simply and sensitively for its role in various physiological processes as well as its inherent toxicity. In this work, two 3-Hydroxychromone (3HC) derivatives were prepared from 3HC-N3 based on HS− mediated reduction of azides, which was found to recognize G-quadruplexes (G4) with strong fluorescence emission through strong π-π stacking interactions and hydrogen bonding. Moreover, the mixture of 3HC-N3 and G4 was used as the H2S sensor with a limit of detection (LOD) as low as 2.64 nM in aqueous solution, which was much lower than most of current approaches. Given the fine performances and striking properties, this G-quadruplex-selective fluorescent turn-on probe would achieve a promising application of H2S detection in low fluorescence interfering environment samples.
1. Introduction Hydrogen sulfide (H2S) is a highly toxic and flammable gas with rotten egg smell, which is mainly produced in sewage plants, oil and natural gas industries, and coal mines. Besides, H2S gas is also extensively used in many chemical industries, scientific research institutions and companies of heavy water production. H2S is also a broadspectrum poison, meaning that it can poison several different systems in the body through inhibition of cytochrome c oxidase, formation of reactive oxygen species (ROS) and direct toxic effects on the brain [1,2]. Amongst all pollutants in the environment, H2S is the major threat to human health even at very low concentrations [3]. Therefore, it is imperatively necessary to develop highly selective and sensitive detection methods of H2S. Variety of methods have been applied for H2S detection, such as colorimetry [4], inductively coupled plasma-atomic emission [5], electrochemical analysis [6], gas chromatography [7] and optical sensors [8]. Colorimetric assays of H2S mainly involves the reaction of sulfide with N, N-dimethyl-p-phenylenediamine sulfate to form methylene blue [9]. However, methylene blue only obeys Beer's Law at very low concentrations and the accuracy of colorimetric is very low [10]. Besides, inductively coupled plasma-atomic emission, electrochemical analysis and gas chromatography usually require expensive
and sophisticated instruments, complicated preprocessing and postmortem processing. Based on polyaniline nanowires-gold nanoparticles, electrochemical analysis of H2S can obtain extremely low detection limit (0.1ppb), but the preparation of this sensor are relatively complicated [11]. Besides, optical H2S sensors are mainly prepared using metal organic framework (MOF) fluorescent materials, fluorescent organic molecules, and fluorescent nanoclusters, which can offer high sensitivity and selectivity [12–15]. Furthermore, based on surface plasmon resonance (SPR) and lossy mode resonance (LMR) techniques, Gupta and coworkers prepared various fiber optic H2S with high sensitivity and long-term stability, but the experimental setup of these excellent performance sensors is relatively complex [16]. Complex synthetic steps or experimental setups [16,17], lower specificity [18], high working temperature [19], and dry testing conditions [19] are hindrances that need to be overcome in the development of H2S sensors. One famous DNA conformation widely employed in sensing is Gquadruplexes (G4), which are non-canonical four-stranded structures formed by at least four tracts of consecutive guanines [20]. The rich diversity in structural topologies of G4 has made them attractive and versatile signal-transducing elements for the development of label-free detection of important species in biology or environment [21,22]. Therefore, G4 has already been used to develop various sensors for
⁎ Corresponding authors at: Key Laboratory of Pesticide and Chemical Biology, Ministry of Education and School of Chemistry, Central China Normal University, Wuhan 430079, PR China. E-mail addresses: [email protected] (D. Zhang), [email protected] (C. Liu). 1 These authors contributed to this work equally.
https://doi.org/10.1016/j.snb.2018.05.177 Received 7 December 2017; Received in revised form 1 May 2018; Accepted 30 May 2018 Available online 31 May 2018 0925-4005/ © 2018 Elsevier B.V. All rights reserved.
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Scheme 1. Compound 3HC-N3, 3HC-NH2 and sensing of hydrogen sulfide based on G-quadruplex recognition.
in 50 mM Tris-buffer at 37 °C, pH 7.4. Fluorescence spectra were obtained on a Cary Eclipse fluorescence spectrophotometer (Varian, USA) using a 1 cm quartz cuvette with a total volume of 400 μL, unless otherwise stated, 5 nm slit widths and a photomultiplier tube power of 700 V were used. The LOD was defined as 3 × standard deviation of the blank, which was calculated according to standard methods in linearity H2S response range of 3HC-N3/ILPR [37]. Absorption spectra were recorded on a SPECORD 210 Ultra-visible spectrophotometer (Analytic Jena AG) using 1 cm path length quartz cuvette with a total volume of 200 μL.
protein [23], nucleic acids [24], metal ions [25] and small molecules [26]. 3-hydroxychromone (3HC) derivatives with excellent sensitivity to the polarity of microenvironment [27], which are important excitedstate intramolecular proton transfer (ESIPT) based molecules, have been used to investigate micelles [28], phosphor lipid vesicles [29], metals [30] and proteins [31]. According to our previous reports, hydrophobic microenvironment provided by low-polar solvent [32], high α-helical level proteins [32,33], and intracellular environment [33], could enhance the fluorescence intensity of ESIPT molecules. Recently, many works have also been done to use 3HC dyes in the studies of structures and interactions of nucleic acids [34]. For example, a 3HC fluorophore being conjugated to polycationic spermine was able to easily distinguish its binding to double-stranded DNA (dsDNA) from single-stranded DNA (ssDNA) by dramatic changes in the spectra [35]. Thus, application of environment-sensitive dyes undergoing ESIPT constitutes a new approach for H2S probing. In this work, we screened out a 3HC derivative 3HC-NH2, which was able to recognize G4 with obvious. Then we modified the −NH2 group of 3HC-NH2 to −N3 group and obtained 3HC-N3, and further applied 3HC-N3 to detect HS− in the presence of G4, as the −N3 group could be reduced to the −NH2 group through HS− [36]. The results displayed that the mixture of 3HC-N3 and G4 was capable of acting as the HS− sensor with a limit of detection (LOD) as low as 2.64 nM in aqueous solution, which was much lower than most of the previous methods. Through strong π-π stacking interactions and hydrogen bonding, G4 DNA could function as fluorescent activator for 3HC-NH2. Therefore, the studies here would provide a new perspective to develop the sensing application of the ESIPT-based 3HC derivatives, and then be helpful for future potential biology imaging.
3. Results and discussion 3.1. Design and synthesis of 3HC-N3 and 3HC-NH2 ESIPT-based 3HC derivatives have excellent sensitivity to the polarity of microenvironment [27], and the hydrophobic microenvironment could enhance the fluorescence intensity of certain 3-hydroxychromone derivatives. Because of the hollow structure inside G4 [38], it might enhance the fluorescence intensity of 3HC derivatives by the hydrophobic interactions similar to bovine serum albumin. However, so far as we know, few examples of the interaction between 3HC derivatives and G4 DNA are available in the literature. In our study, we synthesized two novel 3HC derivatives, 3HC-NH2 and 3HC-N3, which were able to transform into each other via the diazotization reaction and reductive reaction, respectively (Scheme 1 and S1). The fluorescence experiments revealed that the two 3HC dyes had a single and weak emission band in Tris-buffer (50 mM Tris-HCl, 50 mM K+, pH 7.4), however, when in the presence of most, but not all, G4-forming sequences, a dramatic increase can be observed in 3HC-NH2 emission intensity, whereas the presence of control dsDNA and ssDNA showed only moderate effect on emission. As H2S can reduce 3HC-N3 and yield 3HC-NH2 under mild conditions, herein, the mixture of 3HC-N3 and G4 was used as a fluorescence sensor for H2S. The synthesis of 3HC-N3 was achieved based on reported procedures for 3HC derivatives (Scheme S1) [39]. An oxidative cyclization between 2′-hydroxyacetophenone and 4-acetamidobenzaldehyde yield the flavonol unit (the intermediate 1), in which the amino group was protected. Afterward, 3HC-NH2 was obtained via deprotection using HCl. Then, 3HC-N3 was formed based on the diazotization reaction. The chemical structures of the two 3HC derivatives were fully characterized by NMR (1H and 13C) and HRMS. Besides, 3HC-NH2 was additionally characterized by elemental analysis as well as X-ray single crystal diffraction analysis (ESI, detailed crystallographic data were summarized in Table S1).
2. Material and methods 2.1. Materials and oligonucleotides Unless otherwise noted, materials were purchased from commercial suppliers without further purification. All the solvents were treated according to standard methods. The oligonucleotides were purchased from Invitrogen Technology (Shanghai, China), purified over polyacrylamide gel electrophoresis (PAGE), and then dissolved in deionized water, which was finally stored at −20 °C. Concentrations of oligonucleotides were determined by ultraviolet (UV) spectrometry using extinction coefficients according to the manufacturer’s instructions. All other reagents were purchased from commercial suppliers as analytical grade. Full synthesis details and characteristics of the novel compounds were shown in ESI.
3.2. Fluorescence properties of 3HC-NH2 in the presence of G4 DNA 2.2. General spectroscopic methods In order to demonstrate the selective fluorescent recognition of 3HC-NH2 toward the G4-forming oligonucleotides of ILPR (sequence was shown in Table S2), having parallel and antiparallel G4 forms coexisted under physiological conditions [40], ILPR was used to evaluate the interactions through absorption, fluorescence spectroscopy and CD
Deionized water was used to prepare all aqueous solutions. The compounds were dissolved in dimethyl sulfoxide (DMSO) to make 10 mM stock solutions, which were diluted to the required concentrations for measurement. All spectroscopic measurements were working 309
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Fig. 1. (a) Fluorescence spectra of 3HC-NH2 (5 μM) in titrations with ILPR (0.5, 1, 2, 3, 5, 7 μM). The arrow indicated the development of bands with increasing ILPR concentration. (b) Fluorescence intensity of 3HC-NH2 (1 μM) at λem (maximum emission wavelength) = 508 nm in the presence of various DNA structures in final concentrations of 20 μM. ss-DNA: G4TTA-C; ds-DNA: CT-DNA (base concentration); G4: TBA, NRAS, C-KIT, G4TTA, GUK1, c-MYC, AAVS1, PSMA4, AS1411 and ILPR. All spectra were acquired at 37 °C in 50 mM Tris-buffer (including 50 mM K+, pH 7.4). λex (maximum excitation wavelength) = 395 nm.
be mainly caused by the strong π-π stacking interactions with G4, and the weaker interactions of 3HC-NH2 with ds-DNA and anti-parallel Gquadruplex of TBA led to no fluorescence response. Overall, it was suggested that 3HC-NH2 could be sensitive to most G4 DNA (TBA, was the exception) leading to fluorescence response through strong intermolecular interactions with DNA, and G4 DNA might also be used as the fluorescent activator for 3-hydroxychromone derivatives.
melting in Tris-buffer. The data of UV–vis titration shown in Fig. S1 exhibited clear red-shift phenomenon (from 374 nm to 395 nm) and hypochromic effect upon titration of ILPR, which indicated the binding between 3HC-NH2 and the folded ILPR. Furthermore, when excited at 395 nm, the characteristic emission of 3HC-NH2 (5 μM) located at 508 nm increased obviously even in the presence of 0.1 equiv of ILPR, and then increased dramatically when the concentration of ILPR was increased to 1.4 equiv to 3HC-NH2, which illustrated the sensitive fluorescent response and sensing property of 3HC-NH2 to ILPR G4 (Fig. 1a). In addition, the G4-binding selectivity of 3HC-NH2 was determined by recording the fluorescence intensity of 3HC-NH2 (1 μM) at 508 nm in the presence of various DNA structures (Fig. 1b), such as ssDNA (G4TTA-C), CT-DNA and G4 DNA (TBA, NRAS, C-KIT, G4TTA, GUK1, c-MYC, AAVS1, PSMA4, AS1411 and ILPR, sequence were shown in Table S2). The data showed that 3HC-NH2 was able to recognize almost all of the G4 with obvious emission intensity (thrombinbinding aptamer, TBA, was the only exception), whereas the moderate effect on emission could be observed in the presence of control ssDNA and CT-DNA. Moreover, CD melting experiment was used to further evaluate the influence of 3HC-NH2 on the stability of the formed ILPR G4 (Fig. S2). As shown in Fig. S2, the melting temperature (Tm) of the natural ILPR G4 in Tris-buffer was 44.2 °C, and then increased to 47.0 °C, 46.9 °C and 48.1 °C when in the presence of 1, 3 or 7 equiv of 3HC-NH2, respectively, which indicated the moderate stabilization effect of 3HC-NH2 on ILPR G4. Considering the sensitive fluorescent response of 3HC-NH2 to G4 and the moderate stabilization effect of 3HC-NH2 on ILPR G4, molecular docking simulations were carried out using the AutoDock program based on the structural data of different DNA to demonstrate the interaction between 3HC-NH2 and G4. As shown in the docking between 3HC-NH2 and ds-DNA (PDB: 1FKY), only two intermolecular hydrogen bonds were formed (Fig. 2a), which indicated the weak interaction between ds-DNA (PDB: 1FKY) and 3HC-NH2. For anti-parallel quadruplex formed by TBA (PDB: 1HAO), there were three intermolecular hydrogen bonds and π-π stacking interaction (between phenyl in chromone group and thymine) existing between anti-parallel G-quadruplex of TBA and 3HC-NH2 (Fig. 2b). Due to lack of structural data of ILPR in the database, docking between 3HC-NH2 and parallel Gquadruplex of c-MYC (PDB: 2L7 V) was carried out to elucidate the mechanism of fluorescence response of 3HC-NH2 to G4. Surprisingly, strong π-π stacking interactions and one hydrogen bond existed between 3HC-NH2 and parallel G-quadruplex of c-MYC, and these π-π stacking interactions were mainly formatted by guanine in c-MYC (Fig. 2c). Therefore, the fluorescence response of 3HC-NH2 to G4 would
3.3. The application of 3HC-N3/ILPR for H2S detection H2S-mediated reduction of azides to amines has been applied to a series of H2S probes which display a range of fluorescent responses and sensitivities [36]. Because of strong and selective fluorescent response of 3HC-NH2 to G4 DNA, 3HC-N3/ILPR might work as a fluorescent sensor of H2S through H2S-mediated reduction of azides to amines under mild conditions. In order to confirm the reduction of 3HC-N3 by H2S, UV–vis absorption spectra of 3HC-N3 with excessive NaHS at various times (1, 10, 30, 60, 90, 100 min) was carried out in 50 mM Tris-buffer, pH 7.4. As shown in Fig. S3, when 10 equiv of NaHS was added to 3HC-N3 at various times, new UV absorbance peaks at about 355 nm were found, which indicated the generation of 3HC-NH2. Surprisingly, when ILPR was added into the mixture of fully reacted 3HCN3 and NaHS, the peak at about 355 nm shifted to 379 nm (Fig. S3), and this peak shift was corresponded to the UV–vis titration of 3HC-NH2 using ILPR (Fig. S1). All the results indicated the reduction of 3HC-N3 to 3HC-NH2 and the combination of the newly generated 3HC-NH2 with ILPR under mild conditions. At the same time, NaHS or ILPR alone could not increase the fluorescence of 3HC-N3, and only coexistence of NaHS and ILPR could significantly enhance the fluorescence of 3HC-N3 (Fig. 3), which also confirmed that 3HC-N3/ILPR could be used as the H2S sensor. Furthermore, we determined the excitation wavelength, content of G4, reaction time and temperature of the H2S sensor (3HC-N3/ILPR). Because of the red-shift of 3HC-NH2 by ILPR, we next determined the excitation wavelength of the H2S sensor using UV–vis titration of 3HCN3/NaHS with ILPR. After full reaction of 3HC-N3 and NaHS, various concentrations of ILPR were added into the mixture, and then an obvious red shift phenomenon was obtained (from 376 nm to 397 nm) (Fig. S4), which was similar to the UV–vis titration of 3HC-NH2 with ILPR (from 374 nm to 395 nm) (Fig. S1). So, 395 nm was used as excitation wavelength in the following fluorescence experiments. For the dramatical enhancement of fluorescence intensity of 3HC-NH2 by ILPR, the working concentration of G4 DNA in 3HC-N3/ILPR was determined by fluorescence titration of 3HC-N3/NaHS with ILPR. The fluorescence intensity of 3HC-N3/NaHS enhanced with increasing concentrations of 310
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Fig. 2. The interactions between 3HC-NH2 and ds-DNA (PDB: 1FKY), the parallel quadruplex motif (PDB: 2L7V) and the anti-parallel quadruplex formed by TBA (PDB: 1HAO) based on molecular docking simulation. DNA and 3HCNH2 were represented as cartoon sticks and spheres. C, gray; N, blue; H, white; O, red. Light blue dotted lines represented the hydrogen bonding between bases and 3HC-NH2, and yellow cylinders represented π∼π stacking interactions between bases and 3HCNH2. (a) Side view of ds-DNA with 3HC-NH2. (b) The interactions of 3HC-NH2 with ds-DNA in the region of groove binding. (c) Side view of parallel G-quadruplex of c-MYC with 3HCNH2. (d) The interaction of 3HC-NH2 with the top quartet of c-MYC as well as 5′-terminal bases. (e) Side view of anti-parallel G-quadruplex of TBA with 3HC-NH2. (f) The interaction of 3HC-NH2 with TBA G-quadruplex through groove binding. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
(0–20 μM), this sensor underwent a significant increase in fluorescence with maximum peak at 508 nm, and almost kept unchanged after about one equal NaHS was added (Fig. 4b), which might indirectly verify that the reaction ratio of this sensor with H2S would be 1:1. Plotting the relative fluorescence intensity F (F, the emission intensity of 3HC-N3 (4 μM)/ILPR (40 μM) at 508 nm in the presence and in the absence of NaHS, respectively) against the concentration of NaHS, a linear response of fluorescence intensity versus NaHS concentration could be observed over the range of 0∼0.1 μM with a correlation coefficient (R) of 0.99 (Fig. 4c), and LOD of this H2S sensor was calculated as 2.64 nM by standard method [37]. Furthermore, the selectivity of H2S-induced fluorescence response of 3HC-N3 (4 μM)/ILPR (40 μM) was determined by recording the emission spectra of this sensor at 508 nm after the treatment with a variety of anions or biological thiols, such as HS−, F−, Cl−, Br−, I−, NO3−, NO2−, AcO−, SCN−, CO32−, HCO3−, H2PO4−, SO42−, HSO4−, SO32−, S2O32−, S2O72−, cysteine (Cys), homocysteine (Hcy), GSH and NAC (10 equiv). As shown in Fig. 5, the presence of the tested sulphurcontaining anions, other anions, Hcy and GSH had no obvious fluorescence response, indicating the high selectivity of 3HC-N3/ILPR toward H2S (except for Cys and NAC). As to Cys and NAC, it could be explained by Cys and NAC might cause slight reduction of azides to amines. Therefore, this Cys and NAC-mediated reduction would not obviously affect the detection of H2S using 3HC-N3/ILPR in the presence of not too much Cys or NAC.
Fig. 3. Fluorescence spectra of 3 μM 3HC-N3 (black), upon addition of 15 μM NaHS (red), 90 μM ILPR (blue) and the mixture of the former two components (pink), respectively. Fluorescence scans were recorded after incubating at 37 °C for 15 min in 50 mM Tris-buffer (include 50 mM K+, pH 7.4). λex = 395 nm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
ILPR, but fluorescence intensity did not increase any more after ILPR concentration reached 10 equiv (Fig. S4). Therefore, 10 equiv of ILPR was used as working concentration of H2S sensor. Besides, real-time monitoring the fluorescence increase of 3HC-N3/ILPR with NaHS was carried out under 0 or 37 °C, aiming to determine response time and operating temperature of H2S sensor. The reaction of 3HC-N3 and NaHS at 37 °C could be faster with stronger fluorescence intensity compared with 0 °C, and a dramatic increase in fluorescence intensity at 37 °C was observed in the first 15 min, after which its fluorescence intensity increased very slowly (Fig. S5). So the reaction temperature and time of the H2S sensor were optimized to 37 °C and 15 min, respectively. After full optimization as above, 3HC-N3 (4 μM)/ILPR (40 μM) in 50 mM Tris-buffer (pH 7.4) was designed to be an H2S sensor. Besides, 395 nm, 37 °C and 15 min were used as the excitation wavelength, reaction temperature and reaction time of this sensor, respectively. Then, the H2S-induced fluorescence response of this sensor was investigated by fluorescence titration in 50 mM Tris-buffer (pH 7.4). As shown in Fig. 4a, the background fluorescence of 3HC-N3 (4 μM)/ILPR (40 μM) was weak when excited at 395 nm, upon titration with NaHS
4. Conclusions In this study, we reported the preparation and photophysical evaluation of a novel H2S sensor 3HC-N3/ILPR, in which 3HC-N3 would selectively response to H2S and ILPR worked as a fluorescent activator. Through strong π-π stacking interactions and hydrogen bonding between 3HC-NH2 and G4 DNA (except for anti-parallel G4 of TBA), ILPR remarkably enhanced the fluorescence of 3HC-NH2, and 3HC-NH2 could stabilize the structure of ILPR at the same time. The results of UV and fluorescence titrations indicated that 3HC-NH2 could be generated by the reductive reaction of 3HC-N3, and then strong fluorescence response could be obtained by the interactions between 3HC-NH2 and ILPR. Besides, 3HC-N3 (4 μM)/ILPR (40 μM) could function as a H2S sensor with low LOD as 2.64 nM in aqueous solution, which had high selectivity toward H2S. Overall, G-quadruplex-selective fluorescent turn-on probe 3HC-N3 with fine performances and striking properties 311
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Fig. 5. Fluorescence responses of 3HC-N3 (4 μM) and ILPR (40 μM) at 508 nm toward various analytes (final concentration: 40 μM). From left to right: None, HS−, F−, Cl−, Br−, I−, NO3−, NO2−, AcO−, SCN−, CO32−, HCO3−, H2PO4−, SO42−, HSO4−, SO32−, S2O32−, S2O72−, Cys, Hcy, GSH and NAC. Fluorescence scans were recorded after incubating at 37 °C for 15 min at in Tris-buffer (50 mM Tris-HCl, pH 7.4, with 50 mM K+). λex = 395 nm.
CCNU from the colleges’ basic research and operation of MOE [CCNU16A02042, CCNU14A05004]; Natural Science Foundation of Hubei Province of China [2014CFB654]. The support from 111 Program (B17019) was also acknowledged. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.snb.2018.05.177. References [1] D.H. Truong, M.A. Eghbal, W. Hindmarsh, S.H. Roth, P.J. O'Brien, Molecular mechanisms of hydrogen sulfide toxicity, Drug Metab. Rev. 38 (4) (2006) 733–744. [2] T.H. Milby, R.C. Baselt, Hydrogen sulfide poisoning: clarification of some controversial issues, Am. J. Ind. Med. 35 (2) (1999) 192–195. [3] H.N. Saiyed, Hydrogen sulfide human health aspects, concise international chemical assessment document no. 53, Indian J. Med. Res. 123 (1) (2006) 96. [4] M.F. Choi, Fluorimetric optode membrane for sulfide detection, Analyst 123 (7) (1998) 1631–1634. [5] M. Colon, J.L. Todolí, M. Hidalgo, M. Iglesias, Development of novel and sensitive methods for the determination of sulfide in aqueous samples by hydrogen sulfide generation-inductively coupled plasma-atomic emission spectroscopy, Anal. Chim. Acta 609 (2) (2008) 160–168. [6] N.S. Lawrence, J. Davis, L. Jiang, T.G.J. Jones, S.N. Davies, R.G. Compton, The electrochemical analog of the methylene blue reaction: a novel amperometric approach to the detection of hydrogen sulphide, Electroanalysis 12 (18) (2000) 1453–1460. [7] M.G. Choi, S. Cha, H. Lee, H.L. Jeon, S.K. Chang, Sulfide-selective chemosignaling by a Cu2+ complex of dipicolylamine appended fluorescein, Chem. Commun. 47 (2009) 7390–7392. [8] R. Dalapati, S.N. Balaji, V. Trivedi, L. Khamaria, S. Biswas, A dinitro-functionalized Zr (IV)-based metal-organic framework as colorimetric and fluorogenic probe for highly selective detection of hydrogen sulphide, Sens. Actuators B Chem. 245 (2017) 1039–1049. [9] N.S. Lawrence, J. Davis, L. Jiang, Tim G.J. Jones, S.N. Davis, R.G. Compton, Selective determination of thiols: a novel electroanalytical approach, Analyst 125 (4) (2000) 661–663. [10] M.N. Hughes, M.N. Centelles, K.P. Moore, Making and working with hydrogen sulfide: the chemistry and generation of hydrogen sulfide in vitro and its measurement in vivo: a review, Free Radic. Biol. Med. 47 (10) (2009) 1346–1353. [11] M.D. Shirsat, M.A. Bangar, M.A. Deshusses, N.V. Myung, A. Mulchandani, Polyaniline nanowires-gold nanoparticles hybrid network based chemiresistive hydrogen sulfide sensor, Appl. Phys. Lett. 94 (2009) 083502–083504. [12] C.R. 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. 123 (44) (2011) 10511–10513. [13] 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. [14] A.R. Lippert, E.J. New, C.J. Chang, Reaction-based fluorescent probes for selective imaging of hydrogen sulfide in living cells, J. Am. Chem. Soc. 133 (26) (2011) 10078–10080. [15] R. Dalapati, S.N. Balaji, V. Trivedi, L. Khamaria, S. Biswas, A dinitro-functionalized Zr (IV)-based metal-organic framework as colorimetric and fluorogenic probe for
Fig. 4. (a) Fluorescence spectra of 3HC-N3 (4 μM) and ILPR (40 μM) in titrations with NaHS (0, 0.01, 0.02, 0.05, 0.1, 1, 3, 6, 10, 15, 20 μM). The arrow indicates an increment in NaHS concentration from 0 to 20 μM. (b) Plot of the emission intensity at λem = 508 nm against the concentration of NaHS. (c) A linear response of emission intensity at λem = 508 nm versus NaHS concentration was observed over the range of 0–0.1 μM (R2 = 0.99). All full fluorescence scans were recorded after incubating at 37 °C for 15 min in Trisbuffer (50 mM Tris-HCl, pH 7.4, with 50 mM K+). λex = 395 nm.
would realize a promising application of H2S detection under mild aqueous environment through the combination of ILPR. Furthermore, G4 DNA might also be used as the fluorescent activator for ESIPT based 3-hydroxychromone derivatives.
Acknowledgments This work was financially supported by National Natural Science Foundation of China [21302059]; Self-determined research funds of 312
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Yinghui Hu is studying for MS degree at Central China Normal University (CCNU). Her research focuses on cancer cell imaging. Xiang Li is studying for PhD degree at CCNU. His research focuses on the interactions between proteins and DNA. Zhe Zhang is studying for PhD degree at CCNU. Her research focuses on polymer synthesis. Guanfeng Chen is a MS student graduated from CCNU. Her research focuses on G4 DNA. Haoqi Liang is studying for MS degree at CCNU. His research focuses on novel fluorescent probes. Dan Zhang is an associate professor of the College of Chemistry, CCNU. She received her PhD in 2011 from Wuhan University. She joined the faculty at CCNU in 2012. Her recent research focuses on molecular recognition, prostate cancer and G-quadruplex. Changlin Liu is a professor of the College of Chemistry, CCNU. He received her PhD in 1998 from Huazhong University of Science and Technology (HUST). He worked in HUST from 1986 to 2006. He joined the faculty at CCNU in 2006. His recent research focuses on the regulation of ROS and the interactions of proteins and DNA.
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