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A Highly Sensitive and Selective Fluorescent Probe for Thiophenol Designed via a Twist-Blockage Strategy Qi Sun,§,† Shu-Hou Yang,§,† Lei Wu,† Wen-Chao Yang,*,† and Guang-Fu Yang*,†,‡ †

Key Laboratory of Pesticide & Chemical Biology of Ministry of Education, College of Chemistry, Central China Normal University, Wuhan 430079, P.R. China ‡ Collaborative Innovation Center of Chemical Science and Engineering, Tianjin 30071, P.R. China S Supporting Information *

ABSTRACT: A benzoquinolizine coumarin-based fluorescent probe was developed for detecting thiophenols, demonstrating the superior fluorescence properties caused by the decay of the twisting effect of N,N-diethylamino group of coumarin. It discriminated thiophenols from various analytes including aliphatic thiols with good selectivity and displayed ∼700-fold fluorescence intensity enhancement and a remarkable limit of detection (4.5 nM). The new probe also can be applied to quantitative determine the concentrations of thiophenol in water samples and living cells.

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mechanism or photoinduced electron transfer (PET) mechanism or through-bond energy transfer (TBET) mechanism. Among those currently available fluorescent thiophenol probes, some intrinsic defects, including weak fluorescence intensity (FI), dependence on organic cosolvents or high pH solutions, impede their application in environmental and biological samples. Therefore, it remains highly desirable to develop a fluorescent probe with improved thiophenol sensing properties Previously, we have designed a coumarin-based selective thiophenol probe 1 with good features of a large Stokes shift (145 nm), more than 280-fold FI enhancement, and good limit of detection (LOD) of 30 nM in aqueous solution.23 Probe 1 (Scheme 1) was designed by using N,N-diethylaminosubstituted coumarin as the fluorophore moiety and the 2,4dinitrobenzenesulfonic amide group both as the recognition unit and as a fluorescence quencher. N,N-diethylaminosubstituted coumarin derivatives are known to adopt a twisted intramolecular charge transfer (TICT) state, and restricting the twist of the N,N-diethylamino group can significantly affect the TICT effect.29,30 Thus, we expected the blockage of the twisting for the N,N-diethylamino group at the 7-position of the coumarin ring would increase the fluorescence of the fluorophore, thus leading to superior LOD and sensing performance in comparison with our last work.23 To validate our hypothesis and obtain new thiophenol probe with superior property, we designed a novel fluorescent probe 2 based on the concept of blocking the twisting effect of N,N-diethylamino group of the coumarin ring (Scheme 1, in blue). In design of

hiophenols, also named benzenethiols, are extensively used in organic synthesis for preparing various products in the agrochemical industry and the pharmaceutical industry. However, thiophenols are poisonous to aquatic bioorganisms and animals, possessing a median lethal concentration (LC50) of 0.01−0.4 mM in fish and a median lethal dose (LD50) of 46.2 mg/kg in mouse.1−3 Moreover, prolonged exposure to thiophenols in most common environmental samples such as water or soil is highly detrimental to human health, triggering many serious systemic injuries, including increased respiration, central nervous system damage, muscle weakness, vomiting, coma, and even death.3 Thus, an easy, rapid, and efficient method to quantitatively determine thiophenol levels is necessary. At present, the exploration of fluorescent probes for thiols is highly preferable to other methods, because fluorescent probes could directly and reliably indicate the target levels in environmental or biological samples or the redox changes for biological processes.4−15 However, thiophenol and biothiol share similar physical and chemical properties, and thus, it is extremely challenging to develop fluorescent probes that can differentiate them. Compared to the substantial progress in exploring fluorescent probes for biothiols, much less consideration has been given to discovering the highly selective thiophenol probes. Then it was found that the sulphonamide bond was prone to break for the nucleophilic attack of thiophenols (pKa 6.5) under physiological pH but not aliphatic thiols (pKa 8.5) at physiological pH, which makes the discrimination very much possible. To date, only a dozen studies have reported the discovery of thiophenol probes.16−28 Most of the fluorescent probes targeting thiophenols were designed on the basis of intramolecular charge transfer (ICT) © 2016 American Chemical Society

Received: October 23, 2015 Accepted: January 20, 2016 Published: January 20, 2016 2266

DOI: 10.1021/acs.analchem.5b04029 Anal. Chem. 2016, 88, 2266−2272

Article

Analytical Chemistry Scheme 1. Design Mechanism of Fluorescent Probe (2) for Thiophenol Detection

Scheme 2. Synthetic Route of Probe 2*

* Reagents and conditions: (a) n-BuOH, acetic acid, piperidine, reflux; (b) tin(II) chloride dehydrate, hydrochloric acid, r.t.; (c) 2,4dinitrobenzene-1-sulfonyl chloride, DCM, r.t.

this new probe, benzoquinolizine coumarin, a typical coumarin structure31,32 that exhibits better quantum yield compared to coumarin, was used as the fluorophore, while the 2,4dinitrobenzenesulfonic amide group was again introduced both as the recognition unit and as a fluorescence quencher for benzoquinolizine coumarin. As expected, besides the good practicality in living cells and water samples, this benzoquinolizine coumarin-based probe shows significantly improved properties, shown as the following: (1) a larger Stokes shift (155 nm) than probe 1 and most of the other reported thiophenol probes; (2) about 700-fold FI enhancement in aqueous solution; and (3) a remarkable LOD of 4.5 nM. It is noteworthy that the signaling mechanism in the designed probe should be majorly attributed to the PET according to our experimental observation and the comprehensive understanding of some relevant literature.33,34 In the meantime, the restriction of the twist of the N,N-diethylamino group significantly affected the fluorescence of the fluorophore and led to improved sensing properties.

(1.00 g, 4.6 mmol) and ethyl nitroacetate (673 mg, 5.06 mmol) were dissolved in 30 mL of n-butanol and refluxed with stirring overnight. Then the reaction mixture was cooled to room temperature and filtered. After being washed with petroleum ether and dried, M1 was produced as a purple solid (1.0 g, 77% yield) which was used for the subsequent synthesis without further purification. 1H NMR (400 MHz, CDCl3): δ 8.55 (s, 1H), 6.98 (s, 1H), 3.41 (q, J = 5.3 Hz, 4H), 2.76−2.86 (m, 4H), 1.99 (s, 4H). (10-amino-2,3,6,7-tetrahydro-1H,5H,11H-pyrano[2,3-f ] pyrido[3,2,1-ij] quinolin-11-one) (M2). In a 50 mL roundbottomed flask, concd HCl (20 mL), tin(II) chloride dehydrate (5.9 g, 26.25 mmol) were added in order. To this mixture, M1 (1 g, 3.5 mmol) was added at room temperature in small portions over a period of 30 min. After it was stirred overnight, the reaction mixture was cooled in an ice bath, followed by alkalization with sodium hydroxide solution (5 M). The resulting solution was extracted with diethyl ether. The organic layer was washed with water (50 mL), dried with anhydrous Na2SO4, and further concentrated under reduced pressure. The combined residue was subjected to silica gel chromatography to give M2 as a yellow solid (546 mg, 61% yield). mp: 123−124 °C. 1H NMR (400 MHz, DMSO- d6): δ 6.79 (s, 1H), 6.62 (s, 1H), 4.97 (s, 2H), 3.11−3.14 (m, 4H), 2.67−2.74 (m, 4H), 1.85−1.91 (m, 4H). HRMS calcd for [M+]: 256.1206. Found: 256.12113. (2,4-dinitro-N-(11-oxo-2,3,6,7-tetrahydro-1H,5H,11Hpyrano[2,3-f ]pyrido[3,2,1-ij]quinolin-10-yl)benzenesulfonamide) (Probe 2). M2 (256 mg, 1.0 mmol) was dissolved in dichloromethane, pyridine (158 mg, 2 mmol) was added, and the resulting solution was cooled in an ice bath. Then the solution of 2,4-dinitrobenzenesulfonyl chloride (452 mg, 2 mmol) in CH2Cl2 was added dropwise to the above solution. The reaction mixture was stirred for 6 h at room temperature, then diluted with H2O and extracted with CH2Cl2. The organic layer was dried over anhydrous Na2SO4 and then concentrated under reduced pressure. The combined residue was subjected to silica gel chromatography to generate 2 as black solid with yield of 86%. mp: 206−207 oC. 1H NMR (600 MHz, CDCl3): δ 8.76 (d, J = 1.8 Hz, 1H), 8.41 (d, J = 6 Hz, 1H), 8.13 (d, J = 8.4 Hz, 1H), 7.78 (d, J = 3.6 Hz, 2H), 6.94 (s, 1H), 3.25−3.30 (m, 4H), 2.77 (q, J = 8.0 Hz, 4H), 1.92−1.98 (m, 4H). 13C NMR (100 MHz, DMSO-d6): δ 164.32, 154.90,

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EXPERIMENTAL SECTION Materials and Chemicals. All the chemical and biological reagents were purchased from commercialized companies and prepared in stock with standard methods before usage. Silica gel (200−300 mesh, Qingdao Makall Group Co., Ltd. in Qingdao city of China) was used in silica gel column chromatography. Solvents employed in this study were dried in a regular method and redistilled. Both 1H NMR and 13C NMR spectra were measured and analyzed in trichloromethane-d or dimethyl sulfoxide-d6 by a Varian Mercury 600 MHz spectrometer. In NMR, the chemical shift (δ) is the corresponding resonances signal that was recorded in ppm with tetramethylsilane (TMS) as reference. The chemical shift mutiplicities were given in following format: singlet (s), doublet (d), triplet (t), multiplet (m), broad (br). High-resolution mass spectra (HRMS) were obtained with WATERS MALDI SYNAPT G2 HDMS (MA, USA). Melting points were derived from Buchi B-545 melting point instrument without further correction. In general, the designed probe 2 was synthesized as depicted in Scheme 2, and the experimental details are shown below the scheme. Synthesis of Intermediates and Probe 2. M1 and M2 Synthesized According to Reported Literature.35,36 (10nitro-2,3,6,7-tetrahydro-1H,5H,11H-pyrano[2,3-f ]pyrido[3,2,1-ij]quinolin-11-one) (M1). 9-Formyl-8-hydroxyjulolidine 2267


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Analytical Chemistry

Figure 1. (a) Fluorescence spectra of 3-amino-7-diethylamino-chromene-2-one in aqueous solution upon the addition of glycerin. (b) Timedependent fluorescence emission spectra (λex = 380 nm) for probe 2 (10 μM) and thiophenol (40 μM) in aqueous solution (0.01 M phosphate buffer, pH 7.4). Left inset: photographs of samples with and without thiophenol (40 μM) that were taken under hand-hold UV light (λem = 535 nm). Right inset: time profile of FI at the emission wavelength of 535 nm.

media containing 0.1% (V/V) DMSO was added into the cells that were seeded 1 day before, and the cells were incubated for 30 min at 37 °C. As control experiments, the cells were treated with PBS for 1 h or treated with probe 2 (20 μM) alone for 1 h. The cell samples were pretreated with thiophenol (50 μM) for 30 min at 37 °C, and then treated by the probe 2 (20 μM) in the culture media containing 0.1% (V/V) DMSO. After 30 min incubation, the treated cells and control cells were imaged by inverted fluorescence microscopy (Olympus IX71, Japan). MTT assay for probe 2 were performed three times, and the cytotoxic effect against HEK293 cells was evaluated accordingly. Determination of Thiophenol in Water Sample. As described previously,20 the capability of probe 2 for thiophenol detection in crude water samples (Changjiang River and East Lake in Wuhan city of China) was tested with standard addition method. Three concentrations of thiophenol (0.05, 0.5, 5 μM) were applied here, and the resultant recovery for each sample was calculated as the basic criteria.

154.65, 152.34, 150.94, 145.58, 142.95, 137.05, 131.84, 130.54, 124.59, 123.66, 118.07, 111.83, 110.16, 59.78, 54.22, 53.69, 31.72, 25.68, 24.65. HRMS calcd for [M + H]+: 487.0918. Found: 487.0914. Validation by Direct Chemical Reaction. A direct chemical reaction of thiophenol with probe 2 was carried out for mechanistic validation. In 5 mL of DMSO-PBS (1:1, V/V, pH 7.4) solution, Probe 2 (200 mg, 0.41 mmol) was added and well-dissolved, followed by the addition of thiophenol (45 mg, 0.41 mmol). The resultant mixture was stirred overnight at 30 °C. Then the reaction system was cooled to room temperature, washed with a certain amount of water, and further extracted by dichloromethane. The crude product was obtained from the evaporation under reduced pressure and further purified by flash column chromatography, providing compounds 3 (yield, 72%) and 4 (yield, 65%) as the products. The detailed 1H NMR spectra of compounds 3 and 4 were listed in Figure 2. Mechanism Study for the Fluorescence Sensing. The off−on fluorescence sensing mechanism of probe 2 upon recognition of thiophenol was validated by HRMS and LC-MS. Figure S1 shows the HRMS peak of probe 2, Figure S2 indicates the HRMS peak of M2, and Figure S3 monitored the reaction system of probe 2 and thiophenol. In Figure S3, we can find the HRMS peaks of compounds 3, 4, and probe 2. Determination of Quantum Yield. The quantum yields of fluorescence for probe 2 and the corresponding fluorophore were determined by the previously reported method,4,20 using rhodamine B as reference. The quantum yields for probe 2 and the corresponding fluorophore were measured with Abbe’s refractometer and then calculated with eq 1.

ϕs =

Fs·Ac ϕ Fc·A s c

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RESULTS AND DISCUSSION Probe Synthesis and Primary Analysis. As mentioned before, probe 1 showed very weak fluorescence and produced a strong fluorescence signal due to the generation of 3-amino-7diethylamino-chromene-2-one (fluorophore of probe 1) when reacting with thiophenols. N,N-diethylamino-substituted cou-

(1)

Determination of Fluorescence lifetime. The lifetime of fluorescence for the fluorophores of probe 1 and 2 were determined by fluorescence spectrophotometer (FP6500, Jasco), using rhodamine B as reference. The compounds were prepared in water at 100 μM concentration. The lifetime values were obtained by fitting the time-dependent decay data to a single exponential decay function using origin 8.0 software. Cell Imaging and Cytotoxicity Testing. The cellular imaging and the 3-(4,5-dimethylthiazol-2-yl)-3,5-diphenytetrazolium bromide (MTT) assay for the newly developed probe 2 were carried out according the well-established protocol in our previous study.20 Again, HEK293 cells were utilized as models for cellular studies. Then probe 2 (20 μM) dissolved the culture

Figure 2. Partial 1H NMR spectra of probe 2 (I) in CDCl3, compound 3 (II) in CDCl3, compound 4 (III) in CDCl3, and probe 2 + thiophenol (IV) in CDCl3. 2268


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Figure 3. Spectra of fluorescence emission of probe 2 (10 μM) as a function of the concentration of thiophenol (a) and p-methoxy-thiophenol (b) in aqueous solution (0.01 M phosphate buffer, pH 7.4) at the excitation wavelength of 380 nm. Insets: plots of the relative maximum FI (F/F0) vs the concentration of (a) thiophenol and (b) p-methoxy-thiophenol.

much higher than that of M2 (fluorophore of probe 2), indicating the involvement of the TICT effect. In addition, the fluorescence lifetime of fluorophores for probe 1 and 2 was determined to be 1.40 and 1.55 ns, respectively. The extension of the lifetime suggests that the blockage of N,N-diethylamino group lead to superior optical property of M2. Thus, we established an off−on fluorogenic sensing method against thiophenol with probe 2, which exhibited a larger Stokes shift (155 nm) and better quantum yield improvement (Φ = 0.30) in comparison with probe 1. Next, the mechanism of off−on fluorescence sensing for probe 2 upon the recognition of thiophenol was validated by the combination of direct chemical reaction and monitoring of the thiophenol sensing progression. The direct chemical reaction of designed probe with thiophenol in buffer solution (DMSO-PBS, V/V = 1:1, pH = 7.4), generated the compounds 3 and 4 as the products that were confirmed by HRMS and LCMS (see Figure S1−S3). As for thiophenol sensing progression, Figure 2 shows the 1H NMR spectra of probe 2, before and after the addition of 1 equiv of thiophenol. The 1H NMR spectra of compounds 3 and 4 (synthesized as standards) served as references. Notably, the peaks corresponding to a, b, c, d, e, and f for probe 2, g, h, and i for compound 3 and those corresponding to j, k, and l for compound 4 were detected when 1 equiv of thiophenol was added to probe 2. Sensing Properties of Probe 2 to Thiophenols. To gain insight into the sensitivity of probe 2 in aqueous solution, the FI change of probe 2 was studied in the presence of different concentrations of thiophenols (0−120 μM). We selected a moderate temperature like 30 °C as the working temperature throughout the whole study. It was found that the enhancement of the FI was linearly dependent on the concentration of thiophenol or p-methoxy-thiophenol at the early stage and finally reached the maximum value. In particular, the fluorescence intensity enhancement was about 700-fold and about 460-fold upon the recognition of thiophenol (Figure 3a) and p-methoxy-thiophenol (Figure 3b), respectively. Therefore, the LOD of probe 2 for sensing thiophenol was determined to be ∼4.5 nM. The improved properties are supposed to result from the anchor of N,N-diethylamino group on coumarin. In all, the above kinetic characterization demonstrated that probe 2 is the most sensitive thiophenol probe among those reported probes that could be applied in pure aqueous solution. In

marin derivatives usually involved TICT, which may affect the fluorescence; the detailed mechanism was discussed previously.37,38 Thus, we reduced the TICT by increasing the viscosity of the buffer that was prepared by a different proportion of 0.01 M phosphate buffer (pH 7.4) and glycerin, and the fluorescence of 3-amino-7-diethylamino-chromene-2one at different viscosity was measured accordingly. As anticipated, the result indicated that fluorescence increased significantly with an increase in the solvent viscosity (Figure 1a). Hence, the immobilization of the N,N-diethylamino group was expected to obtain a new probe with improved sensing properties. By using 4-diethylamino-salicyldehyde and 2, 4dinitrobenzene-1-sulfonyl chloride as the starting materials, the new probe (2) was prepared in a three-step synthetic route. The route of the synthesis is shown in Scheme 2, and the structure was well-characterized (see Figure S4−S7 in Supporting Information). Then the absorption spectra of 2 at different concentrations in 0.01 M phosphate buffer (pH 7.4) was measured, and the linear correlation between the concentration and maximum absorbance indicated the good water solubility of probe 2 (see Figure S8). The good water solubility of probe 2 is very promising for its characterization of properties in pure aqueous solution (0.01 M phosphate buffer, pH 7.4). First of all, we investigated its fluorescence without addition of thiophenol. To our delight, only probe 2 displayed near-zero fluorescence background in aqueous solution, while its fluorophore exhibits very high fluorescence intensity (see Figure S9 in Supporting Information). According to the characterization, the maximum absorption wavelength (λex) and the maximum emission wavelength (λem) are 380 and 535 nm, respectively, which exhibited a notable red shift compared to probe 1 (λex = 370 nm and λem = 515 nm). The current probe also showed very low quantum yield (Φ = 0.0014) under the excitation at 380 nm. Just a few minutes after adding thiophenol (4 equiv), it was observed that the FI was quickly and remarkably enhanced (Figure 1b). For comparison, we also performed the parallel experiment on M2 (fluorophore of probe 2) to monitor the viscosity-dependent fluorescence change, and we compared the fluorescence intensity increasement at the linear range for those two fluorophores (see Figure S10 and S11 in Supporting Information). It clearly revealed that the viscosity-dependent fluorescence increase of 3-amino-7diethylamino-chromene-2-one (fluorophore of probe 1) is 2269


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Analytical Chemistry Table 1. Summary of Fluorescent Probes for Thiophenols

addition, the recently reported fluorescent thiophenols probes were summarized in Table 1, and the comparison clearly indicated that probe 2 showed its distinguished properties. In particular, the probe developed in the current study displayed the largest FI enhancement (∼700 fold) and the second best LOD of about 4.5 nM (for comparison, the best LOD is about 1.8 nM) for detecting thiophenols. To further evaluate the selectivity of probe 2 for thiophenol detection, the interference was investigated against the commonly encountered analytes, such as different types of aliphatic thiols and many other nucleophiles. As depicted in Figure 4, probe 2 is highly selective for thiophenols (such as thiophenol and p-aminophenol or p-methoxythiophenol) over other competing amino acids. More specifically, no distinct FI enhancement was triggered by the addition of those aliphatic thiols (such as 2-mercaptoethanol, t-butylmercaptan, cysteine, and glutathione), even at high concentrations. Only four selected thiophenols induced significant fluorescence enhance-

ment when they were added into the solution containing probe 2. Among those four thiophenols, three of them (thiophenol, pamino-thiophenol and p-methoxy-thiophenol) induced remarkable fluorescence enhancement, whereas p-nitro-thiophenol produced much less fluorescence enhancement. Thus, we can draw a clear conclusion; that is, a strong electron-withdrawing group on the benzene ring sharply decreases the thiolatemediated SNAr reaction, which was in good agreement with previous literatures.20,39 The above observations suggested that probe 2 was a particularly selective probe to sense thiophenols levels without significant interference by aliphatic thiols and many other nucleophiles. Cellular Detection of Thiophenol. To determine the cell permeability of probe 2 and its capability to detect thiophenol in a cell model, an initial study in human embryonic kidney (HEK293) cells was taken by inverted fluorescence microscopy.23,42 As shown in Figure 5, the direct addition of probe 2 (20 μM) to the cells pretreated with thiophenol (50 μM) led to 2270


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Table 2. Analysis of Thiophenol Concentrations in Real Water Samples sample Changjiang River water

East Lake water

thiophenol spiked (μM)

thiophenol recovered (μM)

0 0.05 0.5 5 0 0.05 0.5 5

not detected 0.049 ± 0.013 0.51 ± 0.023 4.81 ± 0.14 not detected 0.048 ± 0.012 0.49 ± 0.017 5.03 ± 0.22

recovery (%) 98 102 96 96 98 106

filtration, the water samples were incubated with probe 2, and no significant fluorescence enhancement was observed. Then different concentrations (0.05 μM, 0.5 μM, and 5 μM) of thiophenol were spiked into water samples and measured with the newly developed probe in the current study, the resultant thiophenol recoveries were more than 96% (Table 2). These data led us to conclude that probe 2-based fluorogenic method could accurately quantify the thiophenol level in water samples with good recovery.

Figure 4. Selectivity profile of probe 2 (10 μM) to thiophenols (20 μM) and other analytes (40 μM).

a bright green fluorescence, whereas the control cells treated with either probe 2 alone or PBS alone showed no visible fluorescence. Thus, probe 2 was able to permeate into cells and exhibited good specificity to thiophenol other than biothiols and other biomolecules in mammalian cells. Besides, MTT assays indicated that probe 2 possessed low cellular cytotoxicity, because excellent cell viability has been observed for the HEK293 cells when they were treated with probe 2 (50 μM) for as long as 8 h. Water Detection of Thiophenol. To further determine its feasibility for thiophenol detection in environmental samples, we applied probe 2 to quantify thiophenol concentration in water with the standard addition method.31,40 The crude water samples were obtained from Changjiang River and East Lake in Wuhan city. After they were treated with centrifugation and

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CONCLUSION In this study, we have successfully designed a new benzoquinolizine coumarin-based, “off−on” fluorescent probe (2) for sensing thiophenol by blocking the twisting of N,Ndiethylamino group in coumarin. This new probe displayed a large Stokes shift (155 nm), quick response, excellent sensitivity, and excellent selectivity for thiophenol determination in aqueous solution without organic cosolvent. Furthermore, probe 2 showed good cell permeation, good specificity, and precise quantification for thiophenols in water samples. Thus, probe 2 demonstrated great potential to be

Figure 5. Images of living cells (HEK293). (a) Brightfield, (b) Fluorescence image of living cells pretreated with thiophenol (50 μM) for 30 min at 37 °C and further incubated with probe 2 (20 μM) for 30 min. (c) Fluorescence image of HEK293 cells treated with probe 2 (20 μM) alone for 30 min at 37 °C. (d) Merged image of (a) and (b). 2271


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applied to sense thiophenols in both biological systems and environmental samples. Compared to the probe 1 in our last report, the larger Stokes shift, higher FI enhancement (∼700fold), and improved LOD (∼4.5 nM) of the newly synthesized probe for thiophenol recognization not only provide direct evidence for the twist blockage of N,N-diethylamino group in coumarin leading to a superior probe but also offer a new approach that can be generally applied for further optimization in probe discovery.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.5b04029. The absorption spectra and fluorescence spectra of probe 2 in aqueous solution, the NMR and LC-MS spectra (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail for W.-C.Y.: [email protected]. Tel: 86-2767867706. Fax: 86-27-67867141. *E-mail for G.-F.Y.: [email protected]. Tel: 86-2767867800. Fax: 86-27-67867141. Author Contributions §

These authors contributed equally (Q.S. and S.-H.Y.).

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful for the financial support from the National Natural Science Foundation of China (Grant No. 21372094). REFERENCES

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