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Feb 12, 2016 - Heon Joo Park,. §,∥ and Keun-Hyeung Lee*,†. †. Bioorganic Chemistry Laboratory, Center for Design and Applications of Molecular Catalysts, ...
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Selective and Sensitive Detection of Heavy Metal Ions in 100% Aqueous Solution and Cells with a Fluorescence Chemosensor Based on Peptide Using Aggregation-Induced Emission Lok Nath Neupane,† Eun-Taex Oh,‡,§ Heon Joo Park,§,∥ and Keun-Hyeung Lee*,† †

Bioorganic Chemistry Laboratory, Center for Design and Applications of Molecular Catalysts, Department of Chemistry and Chemical Engineering, Inha University, Incheon 402-751, South Korea ‡ Department of Biomedical Sciences, Inha University College of Medicine, Incheon 402-751, South Korea § Hypoxia-related Disease Research Center, College of Medicine, Inha University, Incheon 402-751, South Korea ∥ Department of Microbiology, Inha Research Institute for Medical Science, College of Medicine, Inha University, Incheon 402-751, South Korea S Supporting Information *

ABSTRACT: A fluorescent peptidyl chemosensor for the detection of heavy metal ions in aqueous solution as well as in cells was synthesized on the basis of the peptide receptor for the metal ions using an aggregation-induced emission fluorophore. The peptidyl chemosensor (1) bearing tetraphenylethylene fluorophore showed an exclusively selective turn-on response to Hg2+ among 16 metal ions in aqueous buffered solution containing NaCl. The peptidyl chemosensor complexed Hg2+ ions and then aggregated in aqueous buffered solution, resulting in the significant enhancement (OFF-On) of emissions at around 470 nm. The fluorescent sensor showed a highly sensitive response to Hg2+, and about 1.0 equiv of Hg2+ was enough for the saturation of the emission intensity change. The detection limit (5.3 nM, R2 = 0.99) of 1 for Hg2+ ions was lower than the maximum allowable level of Hg2+ in drinking water by EPA. Moreover, the peptidyl chemosensor penetrated live cells and detected intracellular Hg2+ ions by the turn-on response. highly desirable to develop fluorescent chemosensors that can selectively and sensitively detect mercury ions in aqueous solutions by a turn-on response. In recent years, some propeller-like organic fluorophores such as tetraphenylethylene (TPE) and silole were reported to show unusual fluorescence properties; when they aggregated, they showed significant emission, but they did not show emission in the solution state.31 Restriction of intramolecular rotation of the benzyl ring of the fluorophores was proposed as the main reason for the enhancement of the emission. When the intramolecular rotations of the benzyl moiety of the fluorophores were restricted by physical constraint such as aggregations, these restrictions may prevent the nonradiative pathway, resulting in the enhancement of emission. This aggregation-induced emission (AIE) phenomenon is not only of academic interest but also of practical values for the detection of various analytes. Various fluorescent chemosensors

T

he detection and quantification of low contamination of heavy and transition metal (HTM) ions are especially important because these metal ions played important roles in living system and have had extremely toxic impact on the environment.1,2 Among HTM ions concerned, mercury ions are one of the most toxic metal ions for human health and environments. Thus, various types of fluorescent chemosensors for Hg2+ ions have been developed because fluorescence has provided a sensitive and efficient analytical way.1−4 On the other hand, most of the fluorescent chemosensors for Hg2+ ions displayed at least one of the shortcomings including low sensitivity, low selectivity, and interference from other heavy metal ions.5−20 In addition, as the bioaccumulation of Hg2+ was initiated mainly through water contamination, the chemosensors for mercury detection should dissolve well in aqueous solutions and detect Hg2+ ions in aqueous solutions; however, only a few reported fluorescent chemosensors operated well in aqueous solution without organic solvent.21−30 Furthermore, turn-on response to Hg2+ was highly demanded because turnoff response could not be differentiated with the false signal induced by quenching of Hg2+, the precipitation of the chemosensor, or the absorbance of impurities. Thus, it is © 2016 American Chemical Society

Received: December 25, 2015 Accepted: February 12, 2016 Published: February 12, 2016 3333

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Analytical Chemistry using TPE or silole fluorophore have been reported for the detection of various analytes such as metal ions, volatile organic compounds, proteins, anions, DNA, polysaccharide, and cancer cells.32−44 Even though the chemosensors based on TPE or silole fluorophore were reported to show highly sensitive response to target analytes, most of them did not dissolve well in aqueous solution mainly due to the hydrophobic AIE fluorophores and relatively small hydrophilic receptor molecules. Thus, almost all previously reported chemosensors based on the AIE process of TPE or silole required considerably high percent of organic solvent in aqueous solutions for their proper operations.33−44 Furthermore, they might suffer from the aggregations by changing the percent of organic solvent in working solution with the addition of aqueous sample solution. We expected that peptides might be a most promising receptor of the chemosensors using a hydrophobic TPE or silole fluorophore for the detection of heavy metal ions (HTM) in aqueous solutions because peptides are highly soluble in aqueous solutions and potent binding affinities for heavy metal ions. On the other hand, there is no attempt to use a peptide as a receptor of the chemosensor using an aggregation-induced emission process for the detection of heavy metal ions in aqueous solutions. In the present study, a peptide was used as a receptor for the aggregation-induced emission process for the detection of heavy metal ions in aqueous solutions. A fluorescent peptidebased sensor (1) bearing a TPE fluorophore was synthesized in solid-phase synthesis. Interestingly, 1 using an AIE process selectively detected Hg2+ and Ag+ ions among 16 metal ions in 100% aqueous solutions and showed an exclusively selective response to Hg2+ ions in aqueous solutions containing NaCl. Compound 1 showed a highly sensitive turn on response to Hg2+, and the detection of Hg2+ was not considerably interfered by other heavy metal ion. The detection limit (5.3 nM, R2 = 0.99) for Hg2+ was lower than the maximum allowable level of Hg2+ in drinking water by EPA. Furthermore, the peptide probe penetrated cells and detected intracellular Hg2+ successfully by a turn-on response.

Scheme 1. Synthesis Scheme of 1

at room temperature for 4 h. After cleavage of the product from resin, compound 1 was triturated with diethyl ether chilled at −20 °C and then centrifuged at 3000 rpm for 10 min at −10 °C. The crude product of 1 was purified by HPLC with a Vydac C18 column using a water (0.1% TFA)−acetonitrile (0.1% TFA) gradient to give the 69% of final product. The successful synthesis of 1 was confirmed by ESI mass spectrometry (platform II, micromass, Manchester, U.K.), and the high purity (>97%) was confirmed by analytical HPLC with C18 column. Compound 1 was characterized by melting point, 1H NMR, 13 C NMR, and HRMS. Compound 1. White solid, yield: 69%, mp = 137−138 °C, 1 H NMR (400 MHz, DMSO-d6) δ 8.94 (brs, 1H), 8.36 (d, 1H, J = 8.6 Hz), 8.31 (d, 1H, J = 8.5 Hz), 7.65 (d, 2H, J = 8.5 Hz), 7.4−7.29 (m, 2H), 7.14−7.13 (m, 5H), 7.13−7.12 (m, 5H), 7.08 (d, 2H, J = 8.4 Hz), 7.10−6.98 (m, 3H), 6.97−6.92 (m, 4H), 4.51−4.40 (m, 1H), 4.39−4.30 (m, 1H), 3.70−3.6 (m, 2H), 3.28−3.18 (m, 1H), 3.30−3.28 (m, 1H). 13C NMR (100 MHz, DMSO-d6) δ 172.05, 170.68, 166.58, 146.75, 143.05, 142.92, 141.72, 139.94, 133.72, 131.80, 130.84, 130.77, 130.70, 128.18, 128.13, 128.07, 127.38, 127.06, 126.96, 116.91, 61.53, 56.65, 51.68, 26.39. ESI-mass (m/z) calcd for C36H33N5O5 [M + H]+, 600.25; found, 600.3. General Fluorescence Measurements. A stock solution of 1 at the concentration of 1.0 × 10−4 M was prepared in distilled water and stored in a cold and dark place. This stock solution was used for all fluorescence measurements. All fluorescence measurements were carried out in a 10 mM phosphate buffer solution at pH 7.4. Fluorescence emission spectrum of 1 in 10 mm path length quartz cuvette was measured using a PerkinElmer luminescence spectrometer (Model LS55). Emission spectra of the sensor in the presence of various metal ions (Ca2+, Cd2+, Co2+, Pb2+, Ag+, Mg2+, Cu2+, Mn2+, Ni2+, and Zn2+ as perchlorate anion and Hg2+, Na+, Al3+, and K+ as chloride anion) were measured by excitation with 320 nm. The slit size for excitation and emission was 12 and 5, respectively. The concentration of the probe was confirmed by UV absorbance at 309 nm for TPE fluorophore. The sizes of the particles of the compounds were determined by using a laser diffraction particle size analyzer (particle range = between 0.6 nm and 7 μM, ELSZ, Otsuka Electronics, Osaka, Japan). Determination of Detection Limit. The detection limit was calculated on the basis of the fluorescence titration. To determine the S/N ratio, the emission intensity of 1 without mercury was measured 10 times, and the standard deviation of blank measurements was determined. Three independent duplication measurements of emission intensity were performed in the presence of mercury, and each average value of the intensities was plotted as a concentration of mercury for determining the slope. The detection limit is then calculated with the following equation:



EXPERIMENTAL SECTION Reagents. Fmoc-His(Trt)−OH, and Fmoc-Ser(tBu)−OH were purchased from Bachem. Rink Amide MBHA resin, 1hydroxybenzotriazole (HOBt) and N,N″-diisopropylcarbodiimide (DIC) were purchased from Bead Tech. Trifluoroacetic acid (TFA), N,N′-dimethylformamide (DMF), Tetrahydrofuran (THF), piperidine, n-Butyllithium, and p-Toluenesulfonic acid were purchased from Sigma-Aldrich. Diphenylmethane was purchased from TCI Company, and 4-benzolbenzoic acid was purchased from Alfa Aesar. Solid-Phase Synthesis of 1. Compound 1 was synthesized in solid-phase synthesis with Fmoc chemistry.45 Fmocprotected L−His(Trt)−OH was assembled on Rink Amide MBHA resin. After deprotection of the Fmoc group of resinbound His, Fmoc−L−Ser(tBu)−OH was coupled (Scheme 1). After deprotection of the Fmoc group, TPE (4-(1, 2, 2triphenylvinyl)benzoic acid) was performed by the following procedure. TPE (112.84 mg, 0.3 mmol), HOBt (40 mg, 0.3 mmol), and DIPC (47 μL, 0.3 mmol) in DMF (3 mL) were added into the resin, and the resulting solution was mixed for 4 h at room temperature. A TPE fluorophore was synthesized according to the previous reported procedure.42 Deprotection and cleavage of the compound 1 from the resin was achieved by treatment with a mixture of TFA/TIS/H2O (95:2.5:2.5, v/v/v)

detection limit = 3σ /m 3334

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fluorescent intensity was determined using the ImageJ program (NIH, Bethesda, MD, U.S.A.). Cell Viability. The cytotoxicity was assessed by MTS solution assay (Promega, Madison, WI, U.S.A.). RKO cells were seeded into a 96-well plate at 1 × 104/well in 150 μL of DMEM with 10% FBS and 1% P/S and incubated for 16 h. The cells were treated with 1 for 24 h. After treatment, the supernatants were removed, and the cells were incubated with 100 μL of fresh DMEM media containing 20 μL of MTS solution for another 1 h. Finally, the absorbance at 490 nm was measured using a microplate reader. Nontreated cells were used as a control and incubated in the same conditions for the same time. The relative cell viability (%) was calculated by the following equation.

where σ is the standard deviation of the emission intensity of 1, and m is the slope between the emission intensity vs concentration. Determination of Association Constant (Ka). The association constant for 2:1 complex was calculated based on the titration curve of the probes with metal ions. Association constants was determined by a nonlinear least-squares fitting of the data with the following equation as referenced elsewhere.46 y=

x x×b + 2 2 2 × a × b × (1 − x)

where x is I − I0/Imax − I0, y is the concentration of metal ions, a is the association constant, and b is the concentration of probe. The dissociation constant for 1:1 complex was calculated on the basis of the titration curve of the probe with metal ion. The fluorescence signal, F, is related to the equilibrium concentration of the complex (HL) between Host (H) and metal ion (L) by the following expression:

%cell viability = (optical density of sample



RESULTS AND DISCUSSION Design, Synthesis, and Fluorescence Property of TPEBased Peptide Sensor. Among various AIE fluorophores, TPE was selected due to its easy synthesis and functionalization.42 4-(1,2,2-Triphenylvinyl)benzoic acid was synthesized by the previously reported method.42 In last two decades, various chemosensors based on peptides or amino acids for heavy metal ions have been synthesized by conjugation of dansyl, pyrene, or NBD fluorophore into the peptide or amino acid.13,26,27,50−60 Most of these chemosensors dissolved well in aqueous solutions and detected heavy metal ions due to the hydrophilic properties of the peptide receptors. The selectivity of the chemosensors for metal ions strongly depends on metal binding ability of the amino acids. In general, His and Cys were frequently found in the metal binding sites of several metalloproteins, and both amino acids were well-known metal chelation amino acids.61−66 Fluorescent chemosensors containing Cys or His in the receptor part showed sensitive responses to several HTMs such as Hg2+, Cu2+, Cd2+, Ag+, and Zn.2+13,52−57 Considering the metal-chelating ability, a peptide receptor containing His was synthesized. Ser was chosen for the high solubility of the peptide sensor with a TPE fluorophore in aqueous solutions. TPE-based peptide sensor (1) with a high purity was easily synthesized in solid-phase synthesis using Fmoc chemistry with a high yield of 69% (Scheme 1).45 The high purity of 1 (>97%) was confirmed by HPLC chromatography and ESI mass spectrum. The detailed experimental procedure for the synthesis and characterization of 1 is described in the Experimental Section (see also Figures S1−S5). As a micromolar concentration of 1 dissolved well in water, the stock solution of 1 was prepared in 100% water, and all photochemical experiments were carried out in 100% aqueous buffered solution without organic solvent. Figure 1 shows the fluorescence response of 1 in the presence of various metal ions (Ca2+, Cd2+, Co2+, Pb2+, Cu2+, Ag+, Mg2+, Mn2+, Ni2+, Zn2+, Cr3+, Fe3+ as perchlorate anion and Na+, Al3+, K+, Hg2+ as chloride anion) by excitation with 320 nm. Compound 1 showed selective fluorescent responses to Hg2+ and Ag+ among 16 metal ions in aqueous buffered solutions via turn-on response. To improve selective detection of Hg2+, a fluorescence response of 1 to HTMs was measured in aqueous buffered solution containing NaCl (1 mM). Interestingly, 1 exhibited

F = F0 + ΔF × [HL] [HL] = 0.5 × [KD + L T + HT − {( −KD − L T − HT)2 − 4L THT}1/2 ]

where F0 is the fluorescence of the probe only and ΔF is the change in fluorescence due to the formation of HL. Association constants were determined by a nonlinear least-squares fit of the data with the equation.47 Quantum Yield Measurement. Fluorescence quantum yield of the peptide sensor was measured using an anthracene (Φr = 0.27) as standard.48 The absorbance was recorded in aqueous buffered solutions containing 5% CH3CN in 10 mm cell. The fluorescence spectra of the solutions were recorded with the excitation wavelength of 320 nm and the relative fluorescence was determined by weighing the area beneath the corrected fluorescence emission spectrum. Finally, the quantum yield was calculated as a follow equation.49 Φs = Φr

/optical density of control) × 100%

Fs A × r Fr As

Where Φs and Φr are the quantum yields of sample and the reference respectively, As and Ar are the respective absorbance of the sample and the reference, Fs and Fr are the areas of emission for sample and reference, respectively. Cell Lines and Culture Conditions. RKO human colorectal cells were purchased from ATCC (Manassas, VA, U.S.A.) and maintained in 25 cm2 plastic tissue culture flasks with Dulbecco’s modified Eagle’s medium (DMEM, Hyclone Laboratories Inc., Logan, Utah, U.S.A.) supplemented with 10% fetal bovine serum (FBS, Hyclone Laboratories Inc.) and 1% penicillin/streptomycin (P/S, Hyclone Laboratories Inc.) in humidified atmosphere containing 5% CO2/95% air at 37 °C. Live Cell Imaging. For live cell imaging, the RKO cells were plated on an 8-well chamber slide (Nunc, Roskilde, Denmark) and were treated with compound 1 for 1 h at 37 °C in aqueous buffered solution containing 1% DMSO. The RKO cells were washed three times with PBS solution. Hg2+ ions were added into the medium containing the RKO cells, and then fluorescence images of the RKO cells were monitored by confocal fluorescence microscopy. Single-plane confocal picture sequences were taken every 1 min for the confocal stacks. The 3335

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Figure 2. (a) Fluorescence emission spectra of 1 (3 × 10−6 M) upon the addition of increasing concentration of Hg2+ (0−3.6 × 10−6 M) in aqueous buffered solution (10 mM phosphate, pH 7.4, 1 mM NaCl) (λex = 320 nm, slit = 12/5 nm). (b) Images of compound 1 (3 × 10−6 M) upon the addition of 0, 0.1, 0.2, 0.3...1.0 equiv of Hg2+ under UV light illumination (365 nm).

Figure 1. Fluorescence emission spectra of 1 (3 × 10−6 M) (a) in aqueous buffered solution (10 mM phosphate) at pH 7.4, (b) in aqueous buffered solution (10 mM phosphate, 1 mM NaCl) at pH 7.4, and (c) visible color images of 1 under UV light (λem = 365 nm) in the presence of various metal ions (3 × 10−6 M) except Na+, K+, Mg2+ and Ca2+, which were 1 mM in aqueous buffered solution containing NaCl (1 mM).

Hg2+, a light cyan color became more bright as the concentration of Hg2+ increased. The result indicated that the peptide sensor made it possible for the detection of Hg2+ ions by the naked eye with a UV lamp. In general, some fluorescent chemosensors for Hg2+ showed a different emission enhancement depending on the counteranions because counteranions may participate in the formation of the complex between the sensor and Hg2+. The fluorescence spectra of 1 in the presence of HgCl2, Hg(ClO4)2, Hg(OAc)2, or Hg(NO3)2 were measured to investigate the anion effect on the detection for Hg2+ (Figure S6). 1 showed a similar turn-on response to Hg2+ regardless of the species of the counteranion; however, the enhancement of the emission of 1 by HgCl2, Hg(ClO4)2, Hg(OAc)2, or Hg(NO3)2 was slightly different. Binding Stoichiometry and Binding Affinity. A Job’s analysis was carried out to determine the binding stoichiometry of the 1−Hg2+ complex by keeping the sum of the concentration of Hg2+ and 1 at 3.0 × 10−6 M. The emission intensity change at 470 nm versus the mole fraction of Hg2+ was measured. The maximum emission intensity change was observed at a mole fraction of about 0.45 (Figure S7). The florescent chemosensor based on the peptide receptor for heavy metal ions was reported to form a mixed type of the complex (1:1 and 2:1).50 The binding mode of 1 with Hg2+ was investigated by UV−vis absorption. The UV−vis titration of 1 with Hg2+ in 100% aqueous solutions exhibited the decrease and red shift of the absorbance at 320 nm (Figure S8). This suggests that after complexation of 1 with Hg2+, TPE fluorophores of 1 overlapped, resulting in the decrease of the absorbance at 320 nm. This result supported that 1 might form a 2:1 complex. Furthermore, the emission intensity change of 1 by Hg2+ was better fitted with a 2:1 complex model than a 1:1 complex model (Figure S9). Both results suggested that 1 might form a 2:1 complex with Hg2+ predominantly. Assuming the 2:1 complex formation, the association constant was calculated as 1.52 × 1014 M−2, (R2 = 0.94), which indicates that 1 has a tight binding affinity for Hg2+ in 100% aqueous buffered solution.

exclusive selectivity toward Hg2+ ions over other heavy and transition metal ions, as shown in Figure 1b. The selectivity of the sensor for Hg2+ was improved in the presence of NaCl because silver ions were precipitated by the formation of AgCl before interacting with 1 and 1 mM Na+ has no interference effect on the detection of 1. The selective response to Hg2+ ions was confirmed by visible emission color changes under UV light. As shown in Figure 1c, 1 did not exhibit considerable color changes in the presence of other metal ions but a light cyan color in the presence of Hg2+ in aqueous solution. The fluorescence response of 1 to the increasing concentrations of Hg2+ was measured in aqueous buffered solution (10 mM phosphate, pH 7.4, 1 mM NaCl). As shown in Figure 2, 1 showed negligible fluorescence emission in aqueous buffered solution without metal ions. This might be due to the high solubility of the peptide-based sensor in aqueous solutions. Upon addition of the increasing concentration of Hg2+, an enhancement of fluorescence emission was observed. The fluorescence intensity at 470 nm increased with increasing concentrations of Hg2+ and at least 30-fold enhanced emission intensity was observed. About 1.0 equiv of Hg2+ was enough for the saturation of the emission intensity change. The fluorescence quantum yield (ΦF) of free 1 was 0.010, whereas it reaches 0.254 when 1 bound with Hg2+ (anthracene as a reference, Φf = 0.27).48 The enhancement may be due to the aggregation of 1 in the presence of Hg2+. 1 bearing the peptide receptor showed a hypersensitive response to Hg2+ ions and “OFF-On” response to Hg2+ ions in 100% aqueous solutions. Furthermore, Figure 2b showed a visible emission color change of 1 in the presence of increasing concentration of Hg2+ under a UV lamp. Upon addition of increasing concentration of 3336

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Analytical Chemistry The detection limit of 1 for Hg2+ in 100% aqueous solution was calculated on the basis of the linear relationships between the maximum emission intensity at 470 nm and the concentration of Hg2+. The sensor has a detection limit of 5.3 nM (1.06 ppb, R2 = 0.99), based on 3σ/m, where σ is the standard deviation of the blank measurements, and m is the slope of the intensity versus sample concentration plot (Figure 3). The detection limit of 1 for Hg2+ is lower than the

Figure 4. Fluorescence emission response of 1 (3 × 10−6 M) in the presence of Hg2+ and additional metal ions (3 × 10−6 M) and Group I and Group II metal ions (1 mM) in aqueous buffered solution (10 mM phosphate, pH 7.4) containing 1 mM NaCl.

transition metal ions except Cu2+ and Fe3+. The emission intensity was slightly decreased in the presence of Cu2+ and Fe3+. The previously reported peptide sensors and chemosensors bearing an imidazole moiety commonly showed a considerable responses to Cu2+ because the imidazole moiety interacted well with Cu2+.13,54,57 A decrease of fluorescence emission of 1-Hg2+ in the presence of Fe3+ might be due to the quenching effect of Fe3+ as well as the absorbance of Fe3+ corresponding to the excitation absorbance of 1.68 The reversible sensing ability of 1 for Hg2+ ions was tested in 100% aqueous solutions (Figure S11). After addition of Hg2+ to the solution of 1, a strong emission was observed, indicating the formation of the complex between 1 and Hg2+. Then addition of EDTA as a chelating agent for Hg2+ ions resulted in a decrease in emission intensity. About 3 equiv of EDTA was required to return to the original metal free spectrum, which demonstrates the reversibility of 1 and the potent binding affinity of 1 for Hg2+in aqueous solutions. Effect of pH on the Fluorescence Response of 1 to Hg2+. The effect of pH on the fluorescence response of 1 to Hg2+ was examined in aqueous buffered solution (Figure 5). The emission intensity of the peptide sensor in the absence of Hg2+ ions was negligible in a wide range of pH (4−11.5), which indicates that the peptide-based sensor bearing TPE was fully soluble in aqueous solutions in a wide range of pH. Interestingly, the response of the peptide sensor to Hg2+ ions depended on pH. At acidic pH (pH≤ 5), 1 did not show a response to Hg2+ because the protonated imidazole group of 1 (pKa ≈ 6) might not interact with Hg2+ ions, which indicated that the His residue played a critical role for the interaction with Hg2+ ions. The maximum enhancement of the emission

Figure 3. Linear intensity change of 1 (2 × 10−6 M) as a function of Hg2+ in aqueous buffered solution (10 mM phosphate, 1 mM NaCl) at pH 7.4.

maximum allowable level in drinking water (10 nM, 2 ppb) by EPA.67 This confirms that 1 can be used to detect qualitatively low levels of Hg2+ in aqueous solutions. The detection limit, enhancement, and working media of 1 was compared with those of other fluorescence chemosensors, as shown in Table 1. 1 showed the lowest detection limit even in 100% aqueous buffered solutions among recently reported fluorescent chemosensors. We investigated the response time of 1 to Hg2+ because response time was an important factor for evaluating chemosensors (Figure S10). The response time to Hg(II) was very fast, and the emission change was complete in less than 2 or 3 s. This indicated that the chemosensor could be used for real-time tracking of Hg2+ in aqueous solution. Fluorescence Study in the Presence of Other Metal Ions and at Different pH. To investigate the interference effect of other metal ions on the detection ability of 1 for Hg2+, the fluorescence response of 1 to Hg2+ was measured in the presence of other metal ions. As shown in Figure 4, the Hg2+-dependent fluorescence response of 1 was not affected by the presence of Group I and II metal ions (1 mM), such as Na+, K+, Ca2+, and Mg2+. In particular, the emission intensity change of 1 by Hg2+ was not considerably changed in the presence of other heavy and

Table 1. Property of the Peptidyl Sensor (1) and Other Turn-On Fluorescent Chemosensors key ligands histidine and dithiocarbamate benzhydryl-piperazine Schiff base derivative coumarine derivative tetrathiadiazacrown ether monoaza-crown ether tryptophan azathiacrown ether dipeptide(SerHis) a

enhancement

association constant (Ka)

19-fold 10-fold 7-fold 19-fold 25-fold 29-fold 35-fold NDa 30-fold

1.25 × 10 M 1.06 × 1010 M−2 7.36 × 104 M−1 6.85 × 103 M−1 1.3 × 104 M−1 7.63 × 104 M−1 3.98 × 1013 M−2 8.19 × 103 M−1 1.52 × 1014 M−2 6

−1

detection limit −7

1.03 × 10 M 1.0 × 10−8 M 2.82 × 10−6 M 1.93 × 10−7 M NDa NDa 5.0 × 10−7 M 2.0 × 10−7 M 5.3 × 10−9 M

medium

reference

CH3OH: H2O (4:1, v/v, 1 mM HEPES, pH 7.4) CH3CN: H2O (3:7, v/v, 10 mM HEPES, pH 7.4) DMSO:H2O (7:3, v/v) CH3OH: H2O (4:1, v/v, 10 mM HEPES, pH 7.4) H2O H2O H2O (10 mM HEPES, pH 7.4) H2O (50 mM HEPES, pH 7.4) H2O (10 mM HEPES, pH 7.4)

13 15 16 17 21 22 27 28 present work

Not determined. 3337

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titration experiments were carried out in DMSO-d6/D2O (4:1, v/v) because 5 mM of 1 dissolved well in this solvent system and 1 showed a turn-on response to Hg2+ in this solvent system. Upon addition of Hg2+ ion, a significant upfield shift of proton H1 (Δ = 0.18 ppm) and H2 (Δ = 0.06 ppm) corresponding to the protons of the imidazole group were observed (Figure 7), which indicated that Hg2+ ion coordinated Figure 5. Fluorescence emission response of 1 (3 × 10−6 M) in the absence (■) and presence (●) of Hg2+ (1 equiv) in aqueous buffer solution at different pH.

intensity by Hg2+ ions was observed at pH 7.0 and 7.4, indicating that the peptide sensor might be suitable for monitoring Hg2+ ions at physiological pH. At basic pH, the intensity of 1-Hg2+ complex decreased with increasing pH. This might be due to formation of mercury hydroxide at basic pH. Particle Size of the Aggregates of 1. The fluorescence enhancement of 1 in the presence of Hg2+ may be due to the aggregation of 1 by complexation with Hg2+. The dynamic light scattering (DLS) experiment was carried out to investigate the relationship between the fluorescence emissions and the aggregate formations. The peptide sensor (5 μM) was dissolved in aqueous buffered solution (1 mM phosphate, pH 7.4) containing 1% DMSO for DLS experiment where the peptide sensor did not aggregate and showed a negligible fluorescent emission. Upon addition of increasing concentration of Hg2+, the emission of the solution increased (Figure S12). As shown in Figure 6, the average size of the aggregates increased as the concentration of Hg2+ increased. When the

Figure 7. Partial 1H NMR spectra (400 MHz) of 1 (5 mM) in the presence of increasing amounts of HgCl2 (0−1 equiv) in DMSO-d6/ D2O (4:1, v/v) containing 1 mM ammonium formate at 25 °C.

the imidazole group of 1. A very weak amide proton of 1 was appeared at δ 8.38 ppm in DMSO-d6/D2O solvent and a considerable upfield shift (Δ = 0.28 ppm) and broadening were observed in the presence of Hg2+, suggesting that the amide group of 1 might play a role for the binding with Hg2+. In the previous results of the fluorescent chemosensors based on amino acids, the C-terminal amide group of the amino acid moiety played an important role in the binding with the heavy metal ions.56,60 Thus, the C-terminal amide group of the peptide receptor of 1 may play an important role in the binding with Hg2+. The proton H(9) corresponding to the serine moiety did not shift in the presence of Hg2+ (Figure S13), suggesting that the hydroxyl group of the serine moiety may not participate in the binding with Hg2+ ions. The binding mode of 1 with Hg2+ was proposed as shown in Figure 8. The imidazole group of the peptide receptor of 1 played a critical role in the binding with Hg2+ and the Cterminal amide group of the peptide receptor might cooperate with the imidazole group to form a stable complex. The 2:1 complex might aggregate, and the intramolecular rotation of the benzyl ring of the TPE fluorophores was restricted, resulting in the enhancement of emissions. Detection of Intracellular Hg2+ with 1. As 1 exhibited a sensitive turn on response to Hg2+ in aqueous buffered

Figure 6. Particle size distributions of 1 (5 × 10−6 M) in aqueous buffered solution (1 mM phosphate, pH 7.4, 1% DMSO) in the presence of (a) 0.25 equiv of Hg2+, (b) 0.5 equiv of Hg2+, (c) 0.75 equiv of Hg2+, and (d) 1.0 equiv of Hg2+.

peptide sensor did not aggregate in the absence of Hg2+, the peptide sensor did not show considerable emissions. When the peptide aggregated in the presence of Hg2+, the size of the aggregates correlated with the increase of the emissions. This result strongly supports that the enhanced emission of 1 in the presence of Hg2+ was due to the aggregation of the complex between 1 and Hg2+. Binding Mode of 1 with Hg2+. The binding mode of 1 with Hg2+ was investigated by 1H NMR spectroscopy. 1H NMR 3338

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CONCLUSION A new type of a peptidyl chemosensor using an aggregationinduced emission process was synthesized for the detection of heavy metal ions. The peptidyl chemosensor bearing a TPE fluorophore was fully dissolved in 100% aqueous solution without organic solvent, exhibiting a negligible fluorescent emission in aqueous solution. The peptidyl chemosensor showed an exclusively selective turn-on response to Hg2+ among 16 metal ions in aqueous solution containing NaCl. More than 30-fold increase of emission intensity at 470 nm was observed, and about 1.0 equiv of Hg2+ was enough for the saturation of the emission intensity. The sensitive activation of detection for Hg2+ was not considerably affected by other heavy metal ions. The detection limit (5.3 nM, 1.06 ppb, R2 = 0.99) for Hg2+ ions was lower than the maximum allowable level of Hg2+ in drinking water demanded by EPA. The peptidyl chemosensor penetrated RKO cells in aqueous solutions containing 1% DMSO and detected intracellular Hg2+. The new type of a peptidyl chemosensor using an aggregationinduced emission process will provide not only academic interests but also practical methods for the detection of toxic heavy metal ions in aqueous solutions and cells.

Figure 8. Proposed binding mode of 1 with Hg2+.

solutions at physiological pH, we investigated whether 1 could penetrate cells and detect intracellular Hg2+ ions. 1 was incubated with RKO cells for 1 h at 37 °C in aqueous buffered solution containing 1% DMSO, and then the RKO cells were washed with aqueous buffered solution. Hg2+ ions were added into the medium containing the RKO cells, and then fluorescence images of the RKO cells were monitored by confocal fluorescence microscopy. As shown in Figure 9, a strong green color was observed in the cells in the



ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.5b04892. Experimental procedure, 1H NMR, 13C NMR, HRMS spectra, and additional spectroscopic data of compound 1 (PDF)



AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Fax: +82-32-867-5604. Phone: +8232-860-7674.

Figure 9. Confocal fluorescence images of RKO cells incubated with 10 μM of 1 (top), with 10 μM of Hg2+ (middle), and with 10 μM of 1 and 10 μM of Hg2+ (bottom), bright-field images (a, d, g), confocal fluorescent images (b, e, h) and merged images (c, f, (i). The emission intensities collected in optical windows between 490 and 550 nm (GFP range).

Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS This work was supported by a grant (2014R1A2A1A11051727) from the National Research Foundation of Korea and a grant (2015000540007) from the Korea Environmental Industry and Technology Institute.

medium supplemented with Hg2+, whereas no considerable fluorescent emissions were observed in the cells in a basal medium. The observed green spots with high emission intensity inside of cell might be due to the large aggregates induced by the complex between 1 and Hg2+. Even though the peptidebased sensor bearing a TPE fluorophore dissolved well in aqueous solutions and detected Hg2+ ions in aqueous solution without organic cosolvent, the peptide-based sensor had a cellpenetrating ability and successfully detected intracellular Hg2+ ions by turn-on response. The cytotoxicity of 1 for RKO cells was measured using an MTT assay. Even though considerable cell damage was not observed by incubation of 1 to get the fluorescence image for 1 h, MTT assay revealed that 1 showed some toxicity to the cells for long time incubation. After 24 h incubation of RKO cells with 1, the cell viability decreased as the concentration of 1 increased from 2 μM to 10 μM (Figure S14). This result showed that the peptide-based probe (1) had low cytotoxicity in 24 h incubations.



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DOI: 10.1021/acs.analchem.5b04892 Anal. Chem. 2016, 88, 3333−3340