Dual-Analyte Fluorescent Sensor Based on [5]Helicene Derivative

(1) Among the substantial heavy metal ions, copper and zinc are essential for human life. ... ailments including prion diseases and Alzheimer's diseas...
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Dual-Analyte Fluorescent Sensor based on [5]Helicene Derivative with Super Large Stokes Shift for the Selective Determinations of Cu2+ or Zn2+ in Buffer Solutions and Its Application in Living Cell Siwakorn Sakunkaewkasem, Anuwut Petdum, Waraporn Panchan, Jitnapa Sirirak, Adisri Charoenpanich, Thanasat Sooksimuang, and Nantanit Wanichacheva ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.8b00158 • Publication Date (Web): 07 May 2018 Downloaded from http://pubs.acs.org on May 8, 2018

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Dual-Analyte Fluorescent Sensor based on [5]Helicene Derivative with Super Large Stokes Shift for the Selective Determinations of Cu2+ or Zn2+ in Buffer Solutions and Its Application in Living Cell Siwakorn Sakunkaewkasem†,⊥, Anuwut Petdum†, Waraporn Panchan‡, Jitnapa Sirirak†, Adisri Charoenpanich≠, Thanasat Sooksimuang‡, and Nantanit Wanichacheva*,† †

Department of Chemistry, Faculty of Science, Silpakorn University, Nakhon Pathom, 73000, Thailand

⊥

Department of Chemistry and the Texas Center for Superconductivity, University of Houston, Houston, Texas 77204, United States

‡

National Metal and Materials Technology Center (MTEC), Pathumthani, 12120, Thailand

≠

Department of Biology, Faculty of Science, Silpakorn University, Nakhon Pathom, 73000, Thailand

KEYWORDS: zinc ion, copper ion, dual detection, [5]helicene , fluorescent sensor ABSTRACT: A new fluorescent sensor, M201-DPA, based on [5]helicene derivative was utilized as dual-analyte sensor for determination of Cu2+ or Zn2+ in different media and different emission wavelengths. The sensor could provide selective and bifunctional determination of Cu2+ in HEPES buffer containing Triton-X100 and Zn2+ in Tris buffer/methanol without the interfering from each other and other ions. In HEPES buffer, M201-DPA demonstrated the selective ON-OFF fluorescence quenching at 524 nm toward Cu2+. On the other hand, in Tris buffer/methanol, M201-DPA showed the selective OFF-ON fluorescence enhancement upon the addition of Zn2+, which was specified by the hypsochromic shift at 448 nm. Additionally, M201-DPA showed extremely large Stokes shifts up to ∼150 nm. By controlling the concentration of Zn2+ and Cu2+ in living cell, the imaging of a HepG2 cellular system was performed, in which the fluorescence of M201-DPA in the blue channel was decreased upon addition of Cu2+ and was enhanced in UV channel upon addition of Zn2+. The detection limits of M201-DPA for Cu2+ and Zn2+ in buffer solutions were 5.6 ppb and 3.8 ppb, respectively. Importantly, the Cu2+ and Zn2+ detection limits of the developed sensors were significantly lower than permissive Cu2+ and Zn2+ concentrations in drinking water of the U.S. EPA and WHO.

Contamination of heavy metal ions in biological system is generally caused by several human activities and industrial uses.1 Among the substantial heavy metal ions, copper and zinc, are the essential abundance substances in human life. However, high dosages of Cu2+ and Zn2+ can induce several detrimental diseases. Many researches have reported that the abnormal amount of Cu2+ showed some connections with critical neurodegenerative ailments including prion and Alzheimer’s disease2. The excessive concentration of Zn2+ can also cause detrimental health problems such as skin diseases, diabetes and prostate cancer.2,3 Therefore, many researchers have been attempting to detect trace amounts of Cu2+ and Zn2+ in food, drinking water, environmental and biological sources. Several techniques including atomic absorption spectrometry (AAS),4 voltammetry 5 and inductively coupled plasma optical emission spectrometry (ICP-OES) 6 can be used to detect Cu2+ and Zn2+. Nevertheless, these methods are timeconsuming, require complicated instrumentations, and have limitations for on-site determination. The alternative options for these methods are fluorescent sensors which have received great attention in recent years due to their high sensitivity, high selectivity, and their simplicity to use. In addition, fluorescent sensors can be used for detection of cations,7 anions,8 photon,9 and some other small molecules.10 However, a single probe that is capable to detect two cation species without interfering with each other and others ions is still rare.11

There are many fluorescent sensors designed for Cu2+sensing or Zn2+-sensing, however they were frequently suffered from cross-sensitivity toward either zinc (Zn2+) or copper (Cu2+) ions. This is due to the similarity in coordination number and chemical behaviors of Zn2+ and Cu2+.12 Consequently, developing of a new sensor that can distinctly determine Cu2+ without interfering of Zn2+ or vice versa is in great demand. In order to prepare fluorescent sensor with high sensitive and selective to Cu2+ and Zn2+, [5]helicene derivative, which served as a fluorophore, was connected to di-2-picolylamine (DPA), which served as the recognition portion. [5]Helicene derivatives were mostly hydrophobic and have been utilized in organic light emitting diodes (OLEDs) applications because of their high thermal stability, high emission signal in visible wavelength (500−600 nm), and high quantum yield. Moreover, they also possessed a large Stokes shift, resulted in the reduce of the effect of self-absorption as well as the interference from the light source.13 This will greatly help to reduce the cost of optical device for on-site analysis. Although, the photophysical properties of [5]helicenes as the emissive materials for OLED are promising, use of [5]helicene for selective ion sensors are rare, hence, information for [5]helicenes as a fluorophore is still limited.14 On the other hand, DPA was chosen as a metal receptor in this study due to its excellent chelating ability with heavy metal ions.15 DPA is highly polar, and water soluble which will

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enhance hydrophilicity of the sensor and could increase chance to employ our designed sensor in aqueous or buffer solutions. We expected that DPA in combination with [5]helicene derivative could provide high sensitivity and selectivity toward Cu2+ and Zn2+ with specifically coordinate toward Cu2+ and Zn2+ via favorable electrostatic interactions in the different conditions. Cu2+ and Zn2+ ions prefer the same coordination number (C.N. = 4) with donor atoms, such as nitrogen atoms. Zn2+ favored square planar geometry but Cu2+ prefers square planar or tetrahedron binding sites depending on the ligands and binding environment. Therefore, using the same sensor in the media with different dielectric constants can lead to the changes in the orientation of the sensor, and selectively control the chelating of the sensor to either Cu2+ or Zn2+. In this work, we prepared and report a new fluorescent sensor, M201-DPA, based on [5]helicene derivative that could be utilized as dual-analyte fluorescent sensor for determination of Cu2+ or Zn2+ in difference buffer media and emission wavelengths. The same sensor can provide dual-analyte sensing by selective ON-OFF detection of Cu2+ concentrations in HEPES buffer containing Triton-X100 and selective OFF-ON determination of Zn2+ in Tris buffer/methanol. Additionally, the sensor was selective toward Cu2+ or Zn2+ without interfering from other ions including Fe3+, Pb2+, Hg2+, Ni2+, Cd2+, Li+, Na+, K+, Ba2+, Ca2+, Mg2+ and Al3+. The results demonstrated that M201-DPA sensor can be used for monitoring either Cu2+ or Zn2+ in batch analysis as well as provided a great potential for the analysis of Cu2+ in biological system (a HepG-2 cellular system). Furthermore, the detection limits of the sensor for Cu2+ and Zn2+ were considerably lower than the permitted concentrations of Cu2+ and Zn2+ in drinking water for the United State Environmental Protection Agency (U.S. EPA) and World Health Organization (WHO). RESULTS AND DISCUSSION M201, the derivative of pentahelicene with two methoxy group at para-position and two carbonyl groups, was selected as a fluorophore. The methoxy groups and imide group in the structure M201 acted as donating and withdrawing groups, respectively. The orientation of these functional groups led to good flowing of electrons from the methoxy the imide groups, which resulted in high fluorescence quantum yield of the fluorophore. Therefore, our designed sensor that consisted of DPA and M201 was expected to provide high sensitivity and selectivity toward Cu2+ and Zn2+ through ion-dipole interactions in aqueous and buffer solutions. Herein, sensor M201-DPA was prepared according to the synthetic procedure in Scheme 1. M201-OH was synthesized by imidation of M201 with ethanolamine reagent. Subsequently, M201-OMs was obtained by replacing the hydroxyl group of M201-OH by the mesylate of methanesulfonyl chloride reagent. Finally, M201-DPA was obtained by N-alkylation with di-2-picolylamine in a basic condition. 1H NMR, 13C NMR and HRMS were then used to characterize the structures of these compounds. The sensing properties of M201-DPA were investigated by UV-Visible spectroscopy and fluorescence spectroscopy in aqueous miscible organic solvent and aqueous systems (aqueous acetonitrile solution, Figures S1−S2). Moreover, to improve solubility of M201-DPA, Triton-X100, which served as a surfactant, was also added into the aqueous solution system.

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Based on our result, HEPES buffer (5.0 mM, pH = 7.2, 0.02% Triton-X 100) was the optimized solvent system of M201DPA for Cu2+ detection. The pH at 7.2 was suitable for further utilization in biological samples and the sensor provided good sensitivity in the pH range of 5−8 (Figure S3). Scheme 1. Synthesis route for preparation of sensor M201DPA MeO

MeO

O

O O

+

OH

H2N

glacial acetic acid

OH N

DMF, 110 oC 24 h O

O MeO

MeO M201

M201-OH

MeO

MeO

O OMs MsCl, Et3N

N

CH2Cl2 rt. 24 h

O MeO M201-OMs

O K2CO3, KI, DPA CH3CN

N

N N N

O MeO

M201-DPA

M201-DPA provided the maximum absorption at 335 nm and 373 nm (Figure S4). As depicted in Figure 1, the maximum fluorescence emission of M201-DPA appeared at 524 nm with a very large stroke shift of ∼ 150 nm (the excitation wavelength was 373 nm). The large Stokes shift of M201DPA could prevent the self-absorption of the sensor which would reduce the extra cost from some optical devices such as band gap filter.

Figure 1. The absorption spectra of M201-DPA (0.05 mM) and the fluorescence spectra of M201-DPA (6.0 µM) in HEPES buffer (5.0 mM, pH = 7.2, 0.02% Triton-X100).

In the presence and absence of Cu2+, the sensitivity study of sensor M201-DPA was fully explored in HEPES buffer/Triton-X 100. The fluorescence titration spectra showed that bare M201-DPA exhibited strong fluorescence signal in HEPES buffer at 524 nm. Upon gradual addition of Cu2+, more than 80% turn-off response from initial fluorescence was observed (Figure 2). Additionally, fluorescence behavior of M201-DPA demonstrated rapid “ON-OFF” switching mechanism toward Cu2+ complex formation, which could be attributed to the fluorescent quenching due to the unfilled d-orbital of Cu2+ which led to drastically lower fluorescence of M201DPA sensor.16 The fluorescence quantum yield (Φf) of M201-DPA in acetonitrile was estimated to be 0.95 by using 9,10diphenylanthracene in cyclohexane with a Φf of 0.90 as a standard reference.17 Upon addition of Cu2+, the quantum yield of M201-DPA was found to be 0.21. The significant decreasing of the quantum yield was agreed well with the “ON-OFF” switching through PET mechanism for M201-DPA: Cu2+ complex formation.

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ACS Sensors separate solution method (SSM) utilized in ion-selective electrode applications was used for selectivity study of M201DPA.22 The method involved measuring fluorescences of the sensors after addition of separated heavy metal ion into the system.

(a)

Figure 2. The titration profile change of M201-DPA (6.0 µM) at 524 nm (λex = 373 nm) HEPES buffer (5.0 mM, pH = 7.2, containing TritonX100) due to Cu2+ addition. a: 0 µM, b: 0.067 µM, c: 0.13 µM, d: 0.20 µM, e: 0.27 µM, f: 0.33 µM, g: 0.40 µM, h: 0.47 µM, i: 0.60 µM, j: 0.80 µM, k: 1.20 µM, l: 2.13 µM.

According to the plot between relative fluorescence intensity and the Cu2+ concentration, the detection limit of M201-DPA for Cu2+ was obtained 18 The calculated detection limit of M201-DPA for Cu2+ was 8.86×10-8 M or 5.6 ppb, which was much lower than the maximum allowed levels of Cu2+ in drinking water of U.S. EPA (1.3 ppm) and WHO (2.0 ppm).19 Therefore, M201-DPA is capable to detect trace amount of Cu2+ in an environment. Moreover, the detection limit of M201-DPA for Cu2+ is comparable or lower than the recently reported Cu2+-sensors (Table S1).20 The Job’s plot was conducted to indicate the binding stoichiometry between M201-DPA and Cu2+. The maximum relative fluorescence intensity was shown when the mole fraction of M201-DPA was closed to 0.5, which suggested that M201DPA and Cu2+ were coordinated with 1:1 ratio (Figure S5). The association constant (Kassoc) of the M201-DPA: Cu2+ complex that was calculated by the Benesi–Hildebrand plot of the signal changes in the fluorescence titration,21 was found to be 3.34×106 M-1. The 1:1 complex formation of M201-DPA: Cu2+ from Job’s plot was supported by molecular modeling study (Figure 3). The optimized structure of M201-DPA: Cu2+ complex using Gaussian 09 program and B3LYP/ 6-311G** basis set showed that Cu2+ was coordinated with the tetrahedral-like geometry between Cu2+ and three nitrogen and oxygen atoms with a distance of 1.94 Å, 1.95 Å and 2.01 Å for nitrogen atoms and 2.05 Å for oxygen atom.

Figure 3. Optimized structure of M201-DPA and M201-DPA: Cu2+ complex.

The selectivity of M201-DPA was performed in HEPES buffer upon adding various metal ions into the system. The

(b)

(c)

Figure 4. (a) Fluorescence intensity response at 524 nm (λex = 373 nm) of M201-DPA (6.0 µM) with various metal ions (2.13 µM), (b) Normalized fluorescence intensity at 524 nm of M201-DPA (6.0 µM) as a function of metal ions concentration (c) Fluorescence images of M201-DPA (0.05 mM) under UV-light (365 nm). (M201-DPA in HEPES buffer (5.0 mM), pH = 7.2, 0.02% Triton-X100).

As shown in Figure 4a, the addition of other foreign ions, including Zn2+, Fe3+, Pb2+, Hg2+, Ni2+, Cd2+, Li+, Na+, K+, Ba2+, Ca2+, Mg2+ and Al3+ provided only insignificant effects on fluorescence responses. This result clearly displayed a high selectivity of M201-DPA toward Cu2+. The normalized fluorescence intensities in Figure 4b showed that in the presence of each metal ion, only addition of Cu2+ caused drastic decreasing of fluorescence. On the other hand, a minor decreasing of fluorescence of M201-DPA was observed after the addition of other foreign ions under identical concentration and condition. In addition, the selectivity of M201-DPA to Cu2+ over the common metal ions was clearly shown under UV light (Figure 4c). Competitive experiments were also conducted for sensor M201-DPA. Similar fluorescence changes were observed for the sensor containing 1.0 µM Cu2+ and the sensor containing 1.0 µM Cu2+ with 10 equiv. of other competitive ions, (Figure 5). The result indicated that sensor M201-DPA provided high selectivity for Cu2+ even in the presence of Zn2+ and Pb2+, which were potential competitors of Cu2+.12, 20 Herein, the results showed that the binding affinity of sensor M201-DPA to

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Cu2+ was stronger than those to other competitive ions in the selected solvent condition.

Figure 5. Fluorescence response of M201-DPA (6.0 µM) in HEPES buffer (5.0 mM, pH = 7.2, 0.02% Triton-X100) with addition of chloride salts of 1, no ions; 2, Cu2+; 3, Cu2+/Fe3+; 4, Cu2+/Cd2+; 5, Cu2+/Hg2+; 6, Cu2+/Pb2+; 7, Cu2+/Mg2+; 8, Cu2+/Zn2+; 9, Cu2+/Ba2+; 10, Cu2+/Na+; 11, Cu2+/K+; 12, Cu2+/Li+; 13, Cu2+/Ca2+; 14, Cu2+/Al3+; 15, Cu2+/Ni3+ (10.0 µM).

Sensor M201-DPA could not only selectively coordinate to Cu2+ in HEPES buffer (0.02% Triton-X100), but the sensor could also specifically coordinate to Zn2+ in the aqueous methanol solutions (Figure S6). The change in selectivity of the sensor could be attributed to the different dielectric constants of the solvent systems, which led to the changes in the molecular orientation of the sensor, and consequently change in binding affinity of the sensor to Zn2+ and Cu2+. The optimized condition of OFF-ON fluorescence enhancement for M201DPA: Zn2+ complex was observed in Tris buffer (5 mM, pH = 7.2): methanol (1:1 v/v) with the apparent hypsochromic shift emission at 448 nm. The sensor could be utilized in the pH range of 7−9 (Figure S7). The UV-Visible spectra of M201-DPA in the Tris buffer/methanol displayed the maximum absorption at 335 nm and 373 nm, however, the absorbance at 373 nm was gradually decreased in proportional to the added Zn2+ concentrations (Figure S8). The fluorescence study was carried out using the Tris buffer/methanol system, and the maximum fluorescence intensity of bare M201-DPA was monitored at 575 nm. The addition of Zn2+ to the solution of M201-DPA could result in a hypsochromic shift of the emission spectra from 575 nm to 448 nm (Figure 6a). Importantly, the “OFF-ON” hypsochromic fluorescence response was observed after gradually addition of Zn2+. The decreasing of UV absorption and fluorescence indicated an interaction between Zn2+ and M201DPA. The hypsochromic shift of the fluorescence of M201DPA in the presence of Zn2+ presumably involved the coordination of the nitrogen atoms of DPA to Zn2+, which could perturb the charge transfer23 within the [5]helicene fluorophore. In contrast, addition of other metal ions including Cu2+, Fe3+, Pb2+, Hg2+, Ni2+, Li+, Na+, K+, Ba2+, Ca2+, Mg2+ and Al3+ induced negligible fluorescence changes at 448 nm in Tris buffer: methanol (Figure 6b and 6c).

Competitive experiments for M201-DPA containing Zn2+ with 10 equiv. of other metal ions showed a relatively consistent Zn2+-induced fluorescence enhancement in the high background of competitive ions (Figure S9). In addition, the high selectivity of M201-DPA could also be observed by the fluorogenic and chromogenic changes under UV light (365 nm) with visual eye. In the solution, the color of sensor was changed from orange-yellow to blue after addition of Zn2+ (Figure 6d) under UV light. The Φf of M201-DPA in water:methanol (1:1 v/v) was estimated to be 0.01, and Φf M201-DPA with Zn2+ was increased to be 0.09. The detection limit of M201-DPA toward Zn2+ was 5.84×10-8 M or 3.8 ppb, which was much lower than the U.S. EPA regulated of allowed Zn2+ in drinking water (5 ppm).24 Additionally, M201-DPA provided a very low detection limit compared to recently reported Zn2+ sensors (Table S2).25 (a)

(b)

(c)

(d)

Figure 6. (a) The titration profile change and hypsochromic shift of M201-DPA (10µM) at 448 nm in Tris Buffer (5 mM, pH=7.2): methanol (1:1 v/v) after addition of Zn2+ a: 0 µM, b: 0.083 µM, c: 0.33 µM, d: 0.50 µM, e: 0.83 µM, f: 1.16 µM, g: 1.50 µM, h: 2.00 µM, i: 3.33 µM, j: 4.00 µM, k: 5.33 µM (λex = 373 nm), (b) Fluorescence intensity response at 448 nm (λex = 373 nm) of M201-DPA (10 µM) with various metal ions (5.53 µM), (c) Normalized fluorescence intensity at 448 nm of M201-DPA (10 µM) as a function of metal ions concentration, (d) Fluorescence change of M201-DPA (0.06 mM) in Tris Buffer: methanol (1:1 v/v) under UV-light.

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The effects of coexisting anions on the response of M201DPA for both Cu2+ and Zn2+ were observed, and insignificant effects were found when perchlorate, acetate and chloride salts were employed (Figures S10−S11). The association constant (Kassoc) of M201-DPA for Zn2+ was determined to be 5.62×105 M-1 and 1:1 stoichiometry was suggested, which was consistent with Job’s plot results (Figure S12) and molecular modeling (Figure 7). The optimized structure of M201-DPA: Zn2+ complex using Gaussian 09 program showed that Zn2+ in M201-DPA: Zn2+ complex was bound to three nitrogen atoms with distances of 2.00 Å, 2.00 Å and 2.23 Å, with the square planar-like geometry.

spectrum of M201-DPA with 1.0 equiv. of Zn2+ was different from that of M201-DPA with 0.5 equiv. of Zn2+, but similar to that of M201-DPA with 2.0 equiv. of Cu2+. This illustrated that the M201-DPA: Zn2+ stoichiometry was 1:1, which was agreed with Job’s plot results. On the other hand, the results of 1 H NMR titration of M201-DPA with Cu2+ shown in Figure S13, were more or less the same to those of M201-DPA with Zn2+. Indeed, nitrogen atoms of di-2-picolylamine moiety were also the atoms that bound with Cu2+ and 1:1 ratio was the stoichiometry for M201-DPA: Cu2+.

Figure 7. Optimized structure M201-DPA:Zn2+ complex. The B3LYP/6311G** basis set was used for all structure in Gaussian 09 program.

To understand the possibility of chelation process, the electron density of M201-DPA and its complex with Zn2+ at the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) were further investigated. The DPA in M201-DPA was expected to coordinate to Zn2+ by using the nitrogen atoms of DPA which led to perturbation of a charge transfer within the [5]helicene fluorophore. As shown in Figure 8, the electron density of bound M201DPA-Zn2+ complex exhibited less electron density at DPA molecule and the higher electron density was appeared at the position of imide compared to the unbound sensor. The result supported the proposed explanation of the charge separation perturbation within the [5]helicene fluorophore after coordination of DPA to Zn2+.

Figure 9. 1H NMR spectra of M201-DPA upon addition of Zn2+ (0 – 2.0 equiv.) in methanol-d.

To gain further insights into the potential of M201-DPA for real samples, Cu2+ and Zn2+ were added in drinking water and the visualized responses were observed. As shown in Figure 10., the concentration of Cu2+ and Zn2+ could be noticed by the fluorogenic and chromogenic changes under UV light (365 nm) with visual eye.

Figure 10. Fluorescence changes of M201-DPA (0.06 mM) in drinking water under UV-light with the spiked by Cu2+ and Zn2+ in difference concentration. Figure 8. Electron density in HOMO and LUMO of M201-DPA and its complex with Zn2+.

To deeper understand the binding mode of M201-DPA to Zn2+or Cu2+,1H NMR titration of M201-DPA with Zn2+ in methanol-d and 1H NMR titration of M201-DPA with Cu2+ in acetonitrile-d were conducted. For Zn2+, 1H NMR spectrum of M201-DPA solution was found to be different from 1H NMR spectra of M201-DPA with Zn2+. As shown in Figure 9., the di-2-picolylamine proton chemical shifts were shifted downfield, while the M201 dye proton chemical shifts were remained unchanged. This indicated that Zn2+ bound with nitrogen atoms of di-2-picolylamine moiety. Moreover, 1H NMR

Based on the excellent fluorescent detection capability of the sensor M201-DPA which provided very large Stokes shift and the emission in visible regions in buffer solutions, M201-DPA could be utilized in fluorescence imaging to detect Cu2+/Zn2+ in the living cells. Briefly, HepG2 cells were incubated in phosphate-buffered saline (PBS) that contained M201-DPA (100 µM) for 30 minutes at 37 °C. We found that M201-DPA showed similar sensing behaviors in living cells to Cu2+ as in the solutions. Upon excitation in the blue channel, there was intense intracellular fluorescence when Cu2+ was not added,

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which indicated that M201-DPA was cell-permeable (Figure. 11E).In contrast, after incubation of HepG2 cells in Cu2+ (50 µM) that contained M201-DPA, fluorescence of the intracellular area was drastically decreased (Figure 11F). The same fluorescence behavior was also observed after adding Cu2+ and Zn2+ with M201-DPA in HepG2 cells (Figure 11G). While incubation of only Zn2+ (50 µM) in HepG2 cells did not show the quenching effect in fluorescence imaging in the blue channel (Figure 11H). However, the potential of this sensor for Zn2+ could be also observed in UV channel. The result showed that M201-DPA showed similar sensing behaviors in living cells to Zn2+ as in the solutions by enhancement of the emission from the intracellular area of HepG2 cell (Figures 11K, 11L and S14). This result demonstrated that M201-DPA was sensitive to Cu2+ and Zn2+, and was potential for detection of Cu2+ and Zn2+ in biological samples.

Figure 11. Fluorescence and bright field images of HepG2 incubated with M201-DPA at 37 °C in PBS buffers: (A−D) Bright field images of living HepG2 cells, (E−H) Fluorescence images in blue channel and (I−L) Fluorescence images in UV channel, (E and I ) 0 µM, (F and J) 50 µM CuCl2, (G and K) 50 µM CuCl2 and 50 µM ZnCl2, (H and L) 50 µM ZnCl2, respectively.

CONCLUSION In conclusion, the Cu2+/Zn2+ fluorescent sensor based on [5]helicene fluorophore was successfully developed from di-2picolylamine. M201-DPA could distinguish between coordinating to either Cu2+ or Zn2+, which could be regulated by different buffer systems. The sensor could provide bifunctional detection by selective on-off fluorescence quenching detection of Cu2+ in HEPES buffer at 524 nm, and by selective off-on fluorescence enhancement determination of Zn2+ in Tris buffer/methanol at 448 nm. Moreover, the sensor could detect Cu2+ or Zn2+ without interfering from each other and other interfering ions. Sensor M201-DPA also offered very large Stokes shift, emission in visible regions in the buffer solutions, and displayed the potential for observation of Cu2+ and Zn2+ in live cells by fluorescence imaging. In addition, the detection limits of the sensor for Cu2+ and Zn2+ were much lower than the permissive concentrations in drinking water by the U.S. EPA and WHO. EXPERIMENTAL SECTION

All reactions were carried out under a dry argon atmosphere. Dimethylformamide, dichloromethane and acetonitrile were obtained by distillation. Ethanolamine and di-2picolylamine were purchased form Sigma-Aldrich. Unless otherwise mentioned, all compounds were analytical grade and received from Sigma-Aldrich, Fluka chemical Corporation, and were used as received. The 1H NMR and 13C NMR spectra were obtained by using Bruker Avance 300 spectrometer and CDCl3 was used as solvent with TMS as internal standard. UV-Vis spectra were obtained by a single beam Hewlett Packard 8453 spectrophotometer. The fluorescence spectra were obtained from Perkin Elmer Fluorescence LS 50B spectrophotometer. The slit widths were 5.0 nm for both excitation and emission. HRMS were obtained by ThermoElectron LCQDECA-XP. For computational experiment, the geometry optimization was performed at the DFT-B3LYP level with 6311G** basis set using Gaussian 09. VMD was employed to generate the structure of M201-DPA, M201-DPA: Cu2+ and M201-DPA: Zn2+. The pH measurements were determined by Mettler Toledo pH meter. M201. The compound was synthesized according to our previous report.13c M201-OH. In 25 ml round bottom flask, ethanolamine (0.099 g, 1.63 mmol) was dissolved in dimethylformamide (5.0 ml). A compound M201 (which was prepared according to our previous works)13 was added into the mixture with glacial acetic acid (0.5 ml, 8.74 mmol) as co-solvent. The reaction mixture was heated up to 110 oC for 24 h under Ar gas atmosphere. After that the solvent was removed by a rotatory evaporator, and dichloromethane (25 mL) was added to the reaction residue. The solution was extracted three times by 25 ml of deionized (D.I) water and the organic phase was collected, then it was dried over with anh. Na2SO4. The remaining solvent was removed under reduced pressure to give yellow solid as a crude product and was used without further purification. 1H NMR (CDCl3, 300 MHz) δ (ppm): 2.30-2.45 (br-s, 2H), 2.74-2.76 (m, 4H), 3.73 (s, 6H), 3.78 (s, 4H), 3.90-4.00 (br-s, 2H), 6.43 (d, J = 8.7 Hz, 2H), 6.73 (d, J = 2.7 Hz, 2H), 7.05 (d, J = 8.7 Hz, 2H); 13C NMR (CDCl3 , 75 MHz) δ (ppm): 23.23 (2CH2), 28.01 (2CH2), 39.58 (CH2), 54.20 (2CH3), 60.35 (CH2), 110.84 (2CH), 111.48 (2CH), 124.06 (2C), 125.44 (4C), 130.29 (2CH), 137.09 (2C), 139.96 (2C), 158.42 (2C), 168.55 (2C=O); HRMS calcd C28H25NNaO5+ for (M+Na)+ 478.1625 m/z, found 478.1620 m/z M201-OMs. In 25 ml round bottom flask, the compound M201-OH (0.104 g, 0.23 mmol) was dissolved in dried dichloromethane (4.0 ml), and then triethylamine (0.10 ml, 0.72 mmol) was added. The mixture was stirred for 15 minutes at 0 o C. Then, methanesulfonyl chloride (0.07 ml, 0.90 mmol) was added and the mixture was continuously stirred for 24 h at room temperature under Ar gas atmosphere. Then, the solvent was removed under reduced pressure, dichloromethane (25 mL) was added to the residue. The solution was extracted three times by 25 ml of D.I. water and the organic phase was collected, then it was dried over with anh. Na2SO4. The solvent was removed under reduced pressure to give yelloworange solid as a crude product and was used without further purification. 1H NMR (CDCl3 , 300 MHz) δ (ppm): 2.40-2.60 (br-s, 2H), 2.80-2.90 (m, 4H), 3.06 (s, 3H), 3.82 (s, 6H), 4.02 (t, J = 5.4 Hz,2H), 4.05-4.08 (br-s, 2H), 4.49 (t, J = 5.4 Hz, 2H), 6.53 (d, J = 2.7 Hz, 2H), 6.82 (d, J = 2.7 Hz, 2H), 7.15 (d, J = 8.7 Hz, 2H); 13C NMR (CDCl3 , 75 MHz) δ (ppm):

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ACS Sensors 24.29 (2CH2), 29.02 (2CH2), 36.83 (CH2), 37.91 (CH3), 55.22 (2CH3), 65.86 (CH2 ), 111.91 (2CH), 112.52 (2CH), 124.98 (2C), 126.44 (4C), 131.33 (2CH), 138.26 (2C), 140.98 (2C), 159.51 (2C), 168.60 (2C=O); HRMS calcd C29H27NNaO7S + for (M+Na)+ 556.1400 m/z, found 556.1404 m/z M201-DPA. The compound M201-OMs (0.111 g, 0.21 mmol), potassium iodide (0.079 g, 0.47 mmol), potassium carbonate (0.071 g, 0.51 mmol) and di-2-picolylamine (100 ul, 0.21 mmol) were dissolved in dried acetonitrile (4.0 ml). The reaction mixture was refluxed at 80 oC for 24 h. After that, the solvent was removed by a rotatory evaporator. Then, dichloromethane (25 mL) was added to the residue and the mixture was extracted three times by 25 ml of D.I. water. The organic phase was collected and dried over with anh. Na2SO4. After the solvent was removed under reduced pressure, the crude product was purified by preparative thin layer chromatography using methanol: dichloromethane (1:9 v/v) containing 8% of triethylamine (Et3N) as mobile phase to afford M201-DPA (0.024 g, 20 %) as green-yellow solid. 1H NMR (CDCl3, 300 MHz) δ (ppm): 2.40-2.60 (br-s, 2H), 2.80-2.90 (m, 4H), 3.84 (s, 4H), 3.87 (s, 6H), 3.96 (s, 4H), 3.90-4.10 (m, 2H), 6.53 (d, J = 2.7 Hz, 2H), 6.82 (d, 2H), 7.15 (t, J = 8.7 Hz, 2H), 7.20 (d, 2H), 7.30-7.50 (m, 4H), 8.40 (d, 2H); 13C NMR (CDCl3, 75 MHz) δ (ppm): 24.20 (2CH2), 29.13 (2CH2), 35.69 (2CH2), 51.83 (CH2), 53.39 (2CH3), 60.21 (CH2), 111.89 (2CH), 112.55 (2CH), 121.84 (2CH), 123.03 (2CH), 125.39 (2CH), 126.58 (2C), 131.31 (4C), 136.18 (2CH), 137.68 (2C), 137.80 (2C), 140.93 (2C), 148.83 (2CH), 159.46 (2C), 168.81 (2C=O); HRMS calcd C29H27NNaO7S+for (M+H)+ 637.2809 m/z, found 637.2800 m/z.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publication website UV-Vis, 1H NMR, 13C NMR, HRMS spectra of the synthetic compounds, Job’s plots, and Competitive Study

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected], [email protected]

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was supported by Faculty of Science, Silpakorn University and the Thailand Research Fund (Grant RSA 6080058). J. Sirirak was granted by the DPST Research Grant 005/2557. A. Petdum was supported by TGIST (Thailand Graduate Institute of Science and Technology, Grant TG-33-16-59-012D) from National Science and Technology Development Agency (NSTDA), Thailand.

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