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One-Pot Gram-Scale Synthesis of Nitrogen and Sulfur Embedded Organic Dots with Distinctive Fluorescence Behaviors in Free and Aggregated States Jia Zhang, Li Yang, Yue Yuan, Jun Jiang, and Shu-Hong Yu Chem. Mater., Just Accepted Manuscript • DOI: 10.1021/acs.chemmater.6b01360 • Publication Date (Web): 26 May 2016 Downloaded from http://pubs.acs.org on May 28, 2016

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Chemistry of Materials

One-Pot Gram-Scale Synthesis of Nitrogen and Sulfur Embedded Organic Dots with Distinctive Fluorescence Behaviors in Free and Aggregated States Jia Zhang,†,§ Li Yang,‡,§ Yue Yuan,† Jun Jiang,*,‡ and Shu-Hong Yu*,† †

Division of Nanomaterials & Chemistry, Hefei National Laboratory for Physical Sciences at the

Microscale, Collaborative Innovation Center of Suzhou Nano Science and Technology, Department of Chemistry, Hefei Science Centre, CAS, CAS Center for Excellence in Nanoscience, University of Science and Technology of China, Hefei 230026, China ‡

Department of Chemical Physics, Hefei National Laboratory for Physical Sciences at the Microscale,

Collaborative Innovation Centre of Chemistry for Energy Materials, Hefei Science Centre, CAS, University of Science and Technology of China, Hefei 230026, China

KEYWORDS: organic dots, multiple photoemission, push-pull emission, self-assembly, π-π stacking

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ABSTRACT We report a new strategy for the gram-scale synthesis of highly blue fluorescent nitrogen and sulfur embedded organic dots through one-pot hydrothermal condensation of citric acid (CA) with cysteamine (Cys) at 200 oC. Under such circumstance, the dehydration between CA and Cys produces a molecular fluorophore, which self-assembles to amorphous dots through hydrophobic interaction and π-π stacking. In aqueous solution, the dots exhibit a very high fluorescent quantum yield that is above those of most photoluminescent carbon dots to date, since the fluorophore is not carbonized. The intense fluorescence emission is achieved by establishing an efficient push-pull fluorophore system, as revealed by first-principles simulations. In solid phase, the fluorescence of the dots is severely attenuated. More importantly, unlike excitation-independent emission displayed in solution, the fluorescence of the organic dots in aggregated solid state is dependent on excitation wavelength, which is quite a rare and unique phenomenon. Finally, this new kind of organic dots has shown diverse applications in sensing and imaging. INTRODUCTION Over the past two decades, the researches on fluorescent sensing and imaging have expanded the library of fluorescent materials from conventional dyes and proteins to embrace such as semiconductor quantum dots, semiconducting polymer dots, metal nanoclusters, and carbon dots (Cdots). Compared to organic dyes, quantum dots exhibit higher molar extinction coefficients, larger Stokes shifts, improved photostability, and most importantly, broader excitation bands with narrower, more symmetric, and sizetunable emission spectra.1 However, the concern about the cytotoxicity of quantum dots always needs to be considered for in vivo theranostics.2 Polymer dots consisting of π-conjugated structures utilized as fluorescent tracker dated back to a few years ago, and their excellent photophysical properties coupled with nontoxic features make them a rapid development in biomedical research.3 Nevertheless, the design and preparation of polymer dots by the selection from a huge number of available semiconducting polymers is rather difficult. Metal nanoclusters, especially gold nanoclusters, attract enormous attention recently and are regarded as excellent fluorescent trackers in sensing and biological imaging.4,5


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Compared to those mentioned above, carbon dots are carbogenic nanoparticles with sizes typically below 10 nm, display size and excitation wavelength dependent photoluminescent behavior, and can be produced inexpensively and on a large scale by many ways.6 Moreover, their prominent biocompatibility makes them a strong competitor to quantum dots comprised of heavy metals in biological applications. One of the frequently used ways to prepare brightly photoluminescent Cdots is through thermal reactions of CA-based molecular precursors. By optimizing conditions for the hydrothermal condensation between CA and ethylenediamine (EDA), Cdots with the highest quantum yield (Φ) of 94% in record have been obtained.7 A recent study by Giannelis et al. shows that the Φ of Cdots is diminished greatly as the pyrolysis of CA-ethanolamine (EA) complex proceeds to higher temperature, because the strongly intense molecular fluorophore is gradually consumed as building block of the much less photoluminescent carbogenic core.8 In this sense, to keep a low temperature during pyrolysis seems to be the key to preserve the fluorescence. Herein, we report our discovery on highly blue fluorescent organic dots (N,S-dots) embedded with nitrogen and sulfur from self-assembly of a fluorophore with remarkably high Φ. We found that hydrothermal condensation of CA with cysteamine (Cys) at 200 oC produced a fluorophore with relative Φ reaching 0.88, which could self-assemble to single, separate, amorphous organic dots with relative Φ equal to 0.66 (quinine sulfate as the reference, Φ = 0.54). When we increased the pyrolysis temperature up to 240 oC, interconnected irregularly shaped carbonaceous particles with much diminished relative Φ (0.047) were obtained. This study, in line with the former one,8 provides a simple yet efficient way to fabricate bright unconjugated organic dots in a temperature-controlled manner. We notice that a few documents report the synthesis of structurally defined fluorophores from the thermal reaction of CA with amines,9-11 but to our knowledge, this work first shows an intermediate form of self-assembled organic dots between carbon dots and molecular fluorophores. EXPERIMETAL SECTION Chemicals. All chemicals were analytical grade and used without further purification.



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Synthesis of N,S-dots. In a typical procedure, 2.0 g of citric acid monohydrate (9.5 mmol) and 1.0 g of cysteamine hydrochloride (8.8 mmol) were dissolved in 5.0 mL of water. The solution was transferred to an oven at 80 oC within 12 h for water evaporation and it turned to kind of syrup, which was heated hydrothermally in a Teflon-equipped stainless-steel autoclave at 200 oC for 3.5 h. After naturally cooling down to room temperature, the solid product was rinsed with water and frozen dried or dried at 65 oC to obtain brown powder. Nearly 0.6 g of product could be finally quantitated (based on the average of five parallel experimental results). The synthesis in liquid phase was conducted without the initial water evaporation, and other syntheses in solid phase followed with the above procedure. Synthesis of Cdots-240. The synthetic procedure was the same as that for synthesis of N,S-dots, except the reaction temperature was set at 240 oC. Quantum yield measurements. The relative quantum yields of the products were measured via two approaches. One is to compare the integrated emission intensity (λex = 360 nm) and absorbance at 360 nm with those of quinine sulfate (in 0.5 M H2SO4, Φ = 0.54) using a single point of concentration. We name this one as the single point method. The other is to determine by the relative slope of the integrated emission intensity vs absorbance using five points of concentration. We name it as the slope method. To minimize the re-absorption effect, the absorbance of all solutions at 360 nm was less than 0.10. For details in the mathematic calculation of quantum yields, please refer to the Supporting Information. Detection of alcohol in water. 1 mL of water and ethanol mixed solvent with different volume ratio was added with N,S-dots (10 µg/mL) and the fluorescence spectra were recorded. The slit widths relative to excitation and emission were 1.0 and 2.5 nm, respectively. Detection of water in DMF. 1 mL of DMF and water mixed solvent with different volume ratio was added with N,S-dots (10 µg/mL) and the fluorescence spectra were recorded. The slit widths relative to excitation and emission were both 2.5 nm. Detection of Fe3+ ion. At room temperature, N,S-dots (1.0 µg/mL) in 1 mL of Tris buffer (pH = 9.0, 20 mM) was added with different concentrations of ferric ion (0, 0.5, 1, 2, 4, 6, 8, 10, 20, 30, 40, and 50 µM)


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and the fluorescence spectra were recorded in 5 min. The slit widths relative to excitation and emission were both 2.5 nm. Cell culture. LoVo human colon carcinoma cells were obtained from the Molecular Biology Laboratory of Anhui Medical University. Cells were routinely cultured in flasks containing Dulbecco’s modified Eagle medium (DMEM) (High Glucose), supplemented with 10% fetal bovine serum, 100 U mL-1 of penicillin, and 100 U mL-1 of streptomycin at 37 oC in a humidified hood filled with 5% of CO2. The medium was changed every other day. MTT assay. The cytotoxicity was assessed using the classic MTT assay with LoVo cells. Cells growing in log phase were seeded into 96-well plate at ~5 × 103 cells per well. The cells were incubated for 12 h in a humidified atmosphere at 37 oC under 5% CO2. The N,S-dots solutions at various concentrations (1.56, 3.13, 6.25, 12.5, 25, 50, and 100 µg/mL) in 100 µL of medium were added to the wells and the cells were incubated for 24 and 48 h at the above cell culture conditions. A portion of 5 mg/mL MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) dissolved in PBS (pH = 7.4) was added to each well by 10 µL per well. A solution of 10% sodium dodecyl sulfate (SDS) dissolved in 10 mM HCl was added by 100 µL per cell for an additional 4 h of incubation. The addition of SDS destructed the cell membranes, causing the liberation and solubilization of the dark blue formazan crystals. Hence the number of viable cells was directly proportional to the level of the formazan product created. The formazan concentrations were quantified using an enzyme-linked immunosorbent assay (ELISA) reader (VARIOSKAN FLASH) to measure the absorbance at 570 nm. The viability of cells was assessed by the following formula, i.e. viability (%) = (mean of absorbance value of treatment group/mean of absorbance value of control) × 100. Cell imaging. LoVo human colon carcinoma cells were plated on a 35-mm Petri dish in culture medium one day in advance for cell imaging. For in vitro study, cells were incubated in medium containing 50 µg/mL of N,S-dots for 4 h at 37 oC, then treated with DMEM for two times and PBS (pH = 7.4) for one time to wash the unabsorbed free dots. After that, the cells were fixed with paraformaldehyde



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for at least 10 min, and then imaged on an inverted fluorescence microscope by the use of fluorescence microscope immersion oil, captured under UV, blue, and green excitation. Dialysis of N,S-dots. We treated the aqueous N,S-dots solution (1 mg/mL, 5 mL) against water (1 L) in a dialysis bag (maximum weight cut-off, i.e. MWCO = 1000 Da) for 24 h (changing water every 6-8 h), collecting solution A inside the bag. We also treated the same N,S-dots solution (1 mg/mL, 1 mL) against water (20 mL) in another dialysis tube (MWCO = 100-500 Da) for 24 h, collecting solution B out of the dialysis bag. Theoretical simulations. First-principles based simulations are performed at the density functional theory (DFT) level, using the software package of GAUSSIAN09. The hybrid functional B3LYP was chosen, and the 6-31G* basis was used to model atoms. The solvent effect was considered with the polarizable continuum model (PCM). The time-dependent DFT (TDDFT) method was used to calculate the excited states, and vibrational normal mode analysis was selected to examine the atomic vibrations, with which we predicted the wavelength and probability of photo-absorption, fluorescence, and infrared absorption, as well as the related electronic transitions of molecular orbital wave functions. Characterization. UV-Vis optical absorption spectra were recorded on a Lambda 25 UV-Vis spectrometer (PerkinElmer). Photographs were taken using a smart phone. The fluorescence spectra were recorded on a Hitachi F-4600 fluorescence spectrometer. Transmission electron microscopy (TEM) and high-resolution TEM images were obtained on a JEOL-2100F transmission electron microscope operating at 200 kV. Fluorescence lifetimes of N,S-dots and Cdots-240 in aqueous solution were measured using FLS920 time-corrected single photon counting (TCSPC) system. Absolute fluorescence quantum yield of N,S-dots was obtained in a calibrated integrating sphere in Horiba JY Fluorolog-3-Tou spectrometer. Fluorescence lifetime of solid N,S-dots was measured by time-resolved fluorescence spectroscopy using Horiba JY Fluorolog-3-Tou spectrometer. 1H and 13C NMR spectra were obtained on a Bruker AV-400 MHz spectrometer. HPLC analysis was performed on an Agilent 1200 HPLC system equipped with a G1322A pump and in-line diode array UV detector using a YMC-Pack ODS-AM column with CH3CN (0.1% of TFA) and water (0.1% of TFA) as the eluent. High-resolution ESI-MS spectra


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were obtained on a GCT premier mass spectrometer (Waters). Fourier transform infrared (FTIR) spectrum was measured on a Bruker Vector-22 FTIR spectrometer from 4000 to 400 cm-1 at room temperature. X-ray photoelectron spectroscopy (XPS) study was performed on an ESCALAB 250 spectrometer (VG Scientific). Peak positions were internally referenced to the C1s peak at 284.6 eV. Xray powder diffraction (XRD) patterns were obtained on a Philips X’Pert PRO SUPER X-ray diffractometer equipped with graphite-monochromatized Cu Kα radiation. The Zeta potential measurement was performed on a Malvern Zetasizer instrument (Malvern, UK). The elemental contents of N,S-dots were measured on an Elementar Vario EL III (Germany). Both bright-field image and fluorescence microscope images were taken on an inverted fluorescent microscope (OLYMPUS IX71). RESULTS AND DISCUSSION Synthesis and characterization of the N,S-dots. Typically, the synthesis is conducted at 200 oC for 3.5 h in the absence of solvent when Cys/CA mass ratio is 1/2. After purification with water and freezedrying, we obtain light brown solid powder, which emits faint blue fluorescence under UV irradiation (Figure 1a). XPS (Figure S1, Supporting Information) confirms the existence of C, H, O, N, and S in the sample,

and

besides,

the

elemental

analysis

shows

the

atomic

ratio

of

C:H:O:N:S

is

48.78%:3.70%:24.41%:7.07%:16.13%. To elucidate the molecular structure(s) of the product, we dissolved it in ethanol and fractionated by high performance liquid chromatography (HPLC). Unexpectedly, it shows only one intense peak in the chromatogram (Figure S2), suggesting the product is of high purity. We then used electrospray ionization-mass spectrometry (ESI-MS) for further characterization. The MS spectrum (Figure S3) suggests one native molecule (M1) with m/z = 197, the assignment of which is based on the detection of the adduct ions with m/z = 198 for [M1-H+] and 395 for [2M1-H+]. In such sense, the M1 should be the condensation product of CA and Cys by the loss of four H2O molecules with its formula as C8H7O3NS, which is quite consistent with the elemental analysis result. Scheme 1 outlines the reaction mechanism. To make clear the reaction process, we specify the carbon atoms with numbers. As it shows, an intermediate molecule M0 is first formed by the loss of three water molecules, followed by intramolecular condensation to form M1. The chemical name of M1 is 5-oxo-2,3ACS Paragon Plus Environment


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dihydro-5H-thiazolo[3,2-a]pyridine-7-carboxylic acid. It should be noted that for M0, the carbon atoms relative to number 1 and 6 are identical, and so are the atoms relative to number 2 and 5. The formation of M1 is further proven by the 1H and

13

C NMR spectra (Figures S4-S5). The FTIR

spectrum (Figure S6) agrees with the molecular structure. The bands at 1718 and 1628 cm-1 are due to the C=O stretching vibrations of the carboxylic group and carbonyl group, respectively, the bands at 1235 cm-1 to the C-S and C-N stretching vibrations,12 and the bands at 1520 cm-1 to the C=C stretching vibrations.13 Last, the C 1s binding energy spectrum (Figure S1b) also confirms the presence of five carbon species (C-S-C, C=C, C-N, C=O, O-C=O) in the sample.14 Taken together, the hydrothermal reaction of CA with Cys under the present condition yields a highly pure organic phase with identified molecular structure.

Figure 1. Photographs of N,S-dots in solid phase (a) and in aqueous solution (b) under white light and UV light (365 nm). For image (b), from left to right, the N,S-dots concentrations were 1, 0.1, 0.01 mg/mL, respectively. (c, d) TEM images of N,S-dots. (e) Optical absorption spectra and (f) fluorescence spectra of N,S-dots (0.01 mg/mL) in aqueous solution.


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Due to the protonation of the carboxylic group, the molecules have low solubility in pure water, and thus we prepared the powder in phosphate buffer (pH 7.0) at concentrations of 1, 0.1, and 0.01 mg/mL. Upon UV irradiation, the three solutions emit profoundly intense blue fluorescence without discernible difference (Figure 1b). The particulate character of the product is identified by transmission electron microscope (TEM) (Figure 1c-d), showing single, spherical, separate, and amorphous dots with a mean diameter of 23 nm (Figure S7). The dynamic light scattering (DLS) result is in line with the TEM result (Figure S8). No apparent lattice fringes can be observed by the high-resolution TEM, under which circumstance the image of the dots becomes unclear. Bearing a hydrophilic subunit of carboxylic group and a hydrophobic heterocyclic subunit, M1 molecules are prone to self-assemble to small organic dots in order to reduce free energy (Scheme 1). Herein M1 does not serve as building block to form carbogenic core because of the relatively low temperature. The XRD result (Figure S9) can support this. In the XRD pattern, we only observe strong diffraction peaks relative to the fluorophore powder instead of the broad diffraction peak near 20o that we found for the product synthesized at 240 oC.

Scheme 1. Reaction of citric acid and cysteamine towards the formation of N,S-dots. To make it clear, the reaction steps are depicted in detail as contained in the dotted square.



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Figure 1e shows the optical absorption spectra of the N,S-dots (0.01 mg/mL). In contrast to CA and Cys (0.1 mg/mL) with weak absorption, the product displays two intense absorption peaks at 240 and 342 nm, the former of which is ascribed to the π-π* transition of the nitrogen heterocyclic sp2 domain and the latter to the n-π* transition of the carbonyl bond.15 Besides, there is barely absorption above 400 nm. The fluorescent spectra of the solution are shown in Figure 1f. At excitation wavelength (λex) between 230 and 380 nm, the emission spectra are featured of λex-independent fluorescence, showing single, narrow, nearly symmetric peaks at 415 nm with the maximum intensity at λex = 340 or 360 nm. At λex > 400 nm, no fluorescence can be observed. On the other hand, the excitation spectrum at 415 nm of emission manifests a plateau of intensity from 340 to 360 nm, which is consistent with the emission result. The relative Φ was measured to be 0.80 relative to the single point method and 0.86 relative to the slope method (Figure S10). Therefore, this N,S-dots seems to top most of the citric acid-based Cdots (Table S1) in terms of fluorescence quantum yield. Due to the consistence of the two methods in determining the Φ, we chose the single point method for all the other measurements. As comparison, the relative Φ of the Cdots-240 is 0.047, and the dramatic decrease of Φ is caused by the consumption of M1 fluorophore in favor of a carbogenic core.8 Unlike the separate and spherical particles for N,S-dots, the Cdots-240 shows ill-shaped interconnected particles with much better mass-thickness contrast (Figure S11). Indeed, the Cdots-240 is characteristic of Cdots, as evidenced from the excitation-dependent emission profile (Figure S12). The elemental analysis shows that the Cdots-240 has similar atomic percentages (C:H:O:N:S = 47.95%:4.36%:26.17%:6.26%:15.25%) with those of N,S-dots, but the HPLC and ESI-MS data (Figures S13-S14) indicate a complicated mixed phase of the sample by losing most of the molecular fluorophores. Time-resolved emission spectra measured at λex = 370 nm are well explained by monoexponential decay functions, yielding a lifetime of 8.9 ns for N,S-dots while 1.3 ns for Cdots-240 (Figure S15). This suggests only one deactivation pathway or a single recombination rate within both particles.16 In addition to the syrup-phase synthesis, we also tested the synthesis in aqueous solution. After reaction some light yellow crystals sediment at the bottom of the vessel. XRD pattern of the crystal resembles that of the powder prepared by syrup phase but with much more intense peaks (Figure S9a), and the relative Φ


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is also 0.80. This suggests the crystal owns identical molecular structure with the powder by syrup-phase synthesis, as confirmed by the ESI-MS result (Figure S16). Furthermore, we found insignificant difference in XRD patterns (Figure S9b-c) and quantum yields (Table S2) between products by the use of Cys from two different reagent companies or among products prepared at other times (2, 6, and 10 h). The effect of Cys/CA mass ratio on the properties of the N,S-dots was then studied. At mass ratio of 2/2 and 3/2, the optical absorption spectra and λex-independent emission spectral profiles of the products (Figure S17) are similar with those of dots prepared at mass ratio of 1/2. The relative quantum yields were measured to be 0.76 and 0.78, respectively (Table S2). Decreasing Cys/CA mass ratio to 1/5, we obtained a sticky product with relative Φ of 0.65. The fluorescence emission is located at 417 nm with excitation from 320 to 380 nm, and excitation with longer wavelengths (> 380 nm) starts to induce an excitationdependent emission (Figure S18b-c). The ESI-MS result indicates that a mixture of products is obtained, among which there is the M1 fluorophore due to the presence of the adduct ion with m/z = 198 for [M1H+] (Figure S19). When we used citric acid alone in the reaction, the product shows relative Φ of only 0.033 (Table S2) and the fluorescence emission is dependent upon excitation within the whole spectrum (Figure S18d). As it reveals, the effect of Cys/CA mass ratio is obvious, in that the Φ will be diminished and a generic feature of carbon dots in the dependence of fluorescence emission on excitation occurs with appreciably increasing the amount of CA. And one thing is definitive that nitrogen and sulfur greatly enhance the fluorescence Φ, especially sulfur. In control experiments, EDA and EA were used in place of Cys for the reaction, keeping the molar ratios of amines to CA the same as that of Cys to CA. The products, prepared both in the absence and presence of solvent, exhibit fluorescence quantum yields much improved than the Φ for the only CA-based carbon dots but less than the Φ for the N,S-dots (Table S2). This suggests that the fluorescence enhancement caused by the doping of nitrogen and sulfur may take place via a synergistic process,17,18 even though different reactivity and selectivity of chosen substrates in reaction with CA should be taken into account. Photophysical properties of the N,S-dots. Owing to the intrinsic polarity, the dots is dispersible in many hydrophilic solvents with the solubility in the order of DMSO ≈ DMF > CH3OH ≈ CH3CN > ACS Paragon Plus Environment


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C2H5OH > CH3COCH3 ≈ THF. As shown in Figure 2a, the dots (0.01 mg/mL) in buffer have the largest fluorescence intensity, followed by CH3CN, THF, CH3COCH3, CH3OH, C2H5OH (DMF), and DMSO. Based on the fluorescence quenching in the presence of ethanol, we propose a sensor based on the N,Sdots to determine the concentration of ethanol in ethanol/water biphase system (Figure S20). The ethanol by volume can be detected less than 10%. This ethanol sensor might be useful in evaluating the alcohol content in wines and other beverages or may be applied in the monitor of alcohol consumption. Apart from the sensing of ethanol in water, we also apply the dots to detect trace water in DMF since the quantification of water amount in organic solvents may be more important for industrial applications. The sensing performance is shown in Figure S21. The N,S-dots have the lowest emission intensity and Φ at pH = 1, and the intensities and quantum yields are comparable at pH > 3 (Figure S22). In the presence of metal ions, the fluorescence of N,S-dots is selectively quenched by Fe3+ ion (Figure 2b), suggesting the dots can also be a specific sensor for Fe3+ ion. It can be seen that the addition of ferric ion to the dots solution induces the fluorescence decrease, and an exponential curve can be fitted from the data (Figure S23). The sensor shows comparable sensitivity with those of other sensors based on carbon dots,19-23 and it manifests the best selectivity. The fluorescence quenching is attributed to a static process of forming a dots-Fe3+ complex and the inner filter effect induced by ferric ion itself (Figure S24). Probably it is in greater extent related to inner filter effect than complex formation. The stability of the N,S-dots in solution was considered from two aspects. Using fluorescein isothiocyanate (FITC) as the control, the fluorescence signal preserves much more for the dots than FITC upon prolonged continuous excitation (Figures S25-S26), indicating outstanding photostability of the organic dots (the fluorophore literally). After storage at ambient condition for one year, the absorption spectra and fluorescence emission spectra barely change (Figure S27), further demonstrating superior capability of the fluorophore against photobleaching.


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Figure 2. (a) Fluorescence spectra of N,S-dots (10 µg/mL) in various solvents and (b) Selectivity of N,Sdots (10 µg/mL) towards metal ions (0.1 mM) in water. Similar to the carbon dots for luminescent inks, the N,S-dots can be used as a new kind of invisible ink since it is not only highly fluorescent in aqueous solution but glows brightly after the ink is dried (Figure S28). We applied this ink for writing by an ordinary pen on different substrates, including filter paper, lime wall, and wood. No trace of marks is detected under white light, but under UV light, the words emerge with clearly blue fluorescence. Besides, seal marks attached on a filter paper by the use of this ink are also presented, and most of the details about the graphics are preserved compared to the image printed by traditional ink. After three months of storage at ambient environment, the acronym in the filter paper still fluoresces, implying the ink owns the potential for long-term anticounterfeiting utility. In vitro cytotoxicity study reveals the dots are safe to cells (Figure S29), and moreover, the development of zebrafish embryo is little affected by the presence of the dots in the culture medium compared to the control without the dots (data not shown). In this sense, the N,S-dots possess excellent biocompatibility. Application of the dots has been demonstrated in cellular imaging by multicolor fluorescence emission from the dots-treated cancer cells in the cytoplasm around the nuclei, as shown in Figure 3 (a-d, a1-d1). In addition to blue fluorescence, we were surprised to see that the dots emitted green and red fluorescence concomitantly at excitation by lights of lower energy, which is alike the behavior of carbon dots. The multicolor fluorescence of the cellular imaging was complemented by the multiple emission of as-prepared solid sample under different excitation wavelength (Figure 3, a2-d2). To understand this, we collected the absorption spectrum of the N,S-dots powder. It shows a large difference from that in



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solution with maximum absorption shifted to higher wavelength and a long energy tail extending to the visible region (Figure S30a). Corresponding to this, the fluorescence emission spectra by excitation at 370 and 415 nm both show two emission peaks at 435 and 495 nm (Figure S30b). Instead of the nanosecondscale lifetime for the sample in solution, the lifetimes are 10.3 µs and 9.83 µs for the powder at λem = 435 nm and 495 nm (Figure S30c-d). It is noted that the microsecond-scale lifetime also occurs for other aggregated nanoparticle systems.24-26

Figure 3. (a) Bright-field image and (b-d) fluorescence microscope images of LoVo cell after incubation with N,S-dots (50 µg/mL) for 4 h at 37 oC. Fluorescent images were captured under (b) UV, (c) blue, and (d) green excitation. (a1) Bright-field image and (b1, c1) fluorescent microscope images of N,S-dotsincubated LoVo cell which is fixed by paraformaldehyde to stain the cell nuclei with DAPI. Fluorescent images were captured under (b1) green and (c1) UV excitation. (d1) A merged image of (b1) and (c1). Scale bar: 20 µm. (a2-d2) Bright-field image and fluorescent microscope images of solid N,S-dots under (b2) UV, (c2) blue, and (d2) green excitation. Scale bar: 200 µm.

Photoemission mechanism. The high fluorescence of the organic dots and the multiple emissions in aggregation are both fascinating. The possibility of aggregation-induced emission (AIE) mechanism was firstly ruled out, as the fluorescence of our samples in solid phase is much attenuated with an absolute Φ of only 1.5% (λex = 370 nm). We have treated the solution sample against water in a dialysis bag (MWCO = 1000 Da) for 24 h, collecting solution A inside the bag. Solution B was prepared by collecting the solution flowing out of another dialysis bag (MWCO = 100-500 Da) for 24 h. Since the sample is of high


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purity (Figure S2), it is reasonable to attribute solution A to be suspension of N,S-dots and solution B to be composed of single M1 molecules. The optical spectra of solutions A and B show identical absorption peaks at 342 nm, while there is absorption above 400 nm only for solution A due to the light scattering of organic dots (Figure S31). Both solutions emit excitation-independent blue fluorescence (Figure S32), with relative Φ measured to be 0.66 and 0.88 for dots and fluorophore, respectively. This indicates that single M1 fluorophore is highly fluorescent while the self-assembly to dots causes the fluorescence decrease. Theoretical investigations then reveal that the M1 fluorescence in solution is in compliance with the push-pull emission mechanism.27 As in Scheme 1 and Figure 4a, the M1 molecule presents a typical donor-π-conjugated-acceptor structure, in which the sulfur-related ring group and carboxyl group are bridged by the π-conjugated hexatomic ring. First-principles simulations at the time dependent density functional theory (TDDFT) level identify almost no geometric changes from the ground state S0 to the first excited state S1, except that the perpendicular distance of the sulfur atom to the plane of central ring varies from 0.21 Å to 0.15 Å. The S0 to S1 transition for photo-absorption involves charge transfer from the sulfur ring in the highest occupied molecular orbital (HOMO) to the carboxyl group in the lowest unoccupied molecular orbital (LUMO), through the π-conjugated ring (Figure 4b). The same charge transfer behavior is observed in the S1 to S0 transition for photoemission, exhibiting the typical push-pull process. Although there are only subtle geometric differences between the ground and excited states, we notice a relatively large stokes shift from the photo-absorption peak at 366 nm to 422 nm for photoemission. This would effectively avoid secondary absorptions, and hence ensures efficient photoluminescence. The influence of aqueous solution has also been examined by comparing to the M1 molecule in gas phase, which exhibits the same push-pull behavior (Figure S33). It is also found that the solution effect could enhance the photo-absorption and emission abilities, and hypsochromically shift the emission peak. We then optimized the M1 molecules in the dimer and trimer structure in gas phase (Figure S34a), to examine the fluorescence behavior under the effect of molecular aggregations in solid state. It is found by



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simulations that M1 molecules can easily pile up together through π-π stacking interactions, with layer distances close to 3.0 Å. The simulated energy structures in Figure 4c show that the M1 aggregation leads to energy level splitting which thereby decreases the HOMO-LUMO energy gap. Consequently, the computed absorption spectra for M1 trimer and dimer have more lower-energy absorption peaks than that of the monomer structure, which explains the broadened absorption spectrum measured for our samples in solid state (Figure S34b). Correspondingly, one can expect much weaker photoemission ability in the aggregated M1 systems than the monomer in solution, as the π-π stacking and energy splitting always suppress photoemissions. Meanwhile, it is interesting to see that many vibrational modes in M1 monomer are heavily reduced in the M1 trimer structure (Figure 4d). In general, vibrations are responsible for the quick non-radiative relaxation (~fs scale) of excited electrons from higher energy levels to lower levels, resulting in the Kasha’s rule that the wavelength of molecular fluorescence/photo-emission (> ns scale) has no dependence on the excitation wavelength. Therefore, the suppression of vibrations in aggregated M1 systems explains the breaking of Kasha’s rule in our experiments when the same sample in solid state emits lights in different colors under different excitation.


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Figure 4. (a) The optimized geometry of M1 molecule in solution, in which a subtle structural difference between the ground and excited state is marked by h (the perpendicular distance of sulfur atom to the central plane). (b) The electronic transitions between the ground (S0) and excited (S1) states as reflected by wave functions of frontier orbitals (transitions between HOMO and LUMO) in the single M1 molecule in aqueous solution. (c) The energy structures as represented by molecular orbitals for the M1 monomer, dimer, and trimer structures in gas/solid phase, where the HOMO-LUMO energy gap decreases with molecular aggregations. (d) The distribution of vibrational intensities in the M1 monomer and trimer structure in gas/solid phase, in comparing with the experimentally measured infrared absorption spectrum. Note here that we only considered the vibrations of one central M1 molecule in the trimer system to ensure a reasonable comparison with the M1 monomer. CONCLUSIONS In summary, we have developed a one-pot hydrothermal approach to produce highly blue fluorescent organic dots in gram scale by the reaction of citric acid with cysteamine at relatively low temperature. The new organic dots, self-assembled from a fluorophore containing nitrogen and sulfur mainly through π-π stacking, exhibit fluorescence quantum yield in aqueous solution higher than those of most carbon dots. Theoretical simulations confirm the fluorescence of the fluorophore in solution complies with the push-pull emission mechanism. In solution the organic dots exhibit excitation-independent emission spectra, and in solid phase, the fluorescence is much attenuated, but it emits lights in different colors under different excitation due to energy splitting, suggesting the breaking of Kasha’s rule. In contrast to the nanosecond-scale fluorescence lifetime for the sample in solution, it manifests microsecond-scale lifetime in solid phase. At higher reaction temperature, the organic dots will then be converted to carbon dots, with quantum yield greatly diminished. We show that the organic dots can be used to fabricate a sensor for alcohol, water and ferric ion, as lasting invisible fluorescent ink, and for multicolor bioimaging. Since the synthesis is conducted by a simple manner without complicated post-treatment, it can be conveniently scaled up in production, paving the way for the bioapplications of these new fluorescent organic dots. ASSOCIATED CONTENT Supporting Information. Figures S1-S34 and Tables S1-S2. This material is available free of charge via the Internet at http://pubs.acs.org.



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AUTHOR INFORMATION Corresponding Author S. H. Yu (e-mail: [email protected]), J. Jiang (e-mail: [email protected]) Author Contributions §

These authors contributed equally.

Notes The authors declare no competing financial interests. ACKNOWLEDGMENT We acknowledge the funding support from the National Natural Science Foundation of China (Grants 21431006, 21407140), the Foundation for Innovative Research Groups of the National Natural Science Foundation

of

China

(Grant

21521001), the

National Basic

Research

Program

of

China

(Grants 2014CB931800, 2013CB931800), the Users with Excellence and Scientific Research Grant of Hefei Science Center of CAS (2015HSC-UE007, 2015SRG-HSC038), the Chinese Academy of Sciences (Grant KJZD-EW-M01-1), and the Fundamental Research Funds for the Central Universities (Grants WK2060190036, WK2090050027). REFERENCES (1) Michalet, X.; Pinaud, F. F.; Bentolila, L. A.; Tsay, J. M.; Doose, S.; Li, J. J.; Sundaresan, G.; Wu, A. M.; Gambhir, S. S.; Weiss, S. Quantum Qots for Live Cells, in vivo Imaging, and Diagnostics. Science 2005, 307, 538-544. (2) Winnik, F. M.; Maysinger, D. Quantum Dot Cytotoxicity and Ways to Reduce It. Acc. Chem. Res. 2013, 46, 672-680. (3) Wu, C. F.; Chiu, D. T. Highly Fluorescent Semiconducting Polymer Dots for Biology and Medicine. Angew. Chem. Int. Ed. 2013, 52, 3086-3109. (4) Shang, L.; Dong, S. J.; Nienhaus, G. U. Ultra-Small Fluorescent Metal Nanoclusters: Synthesis and Biological Applications. Nano Today 2011, 6, 401-418. ACS Paragon Plus Environment

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(5) Chen, L. Y.; Wang, C. W.; Yuan, Z.; Chang, H. T. Fluorescent Gold Nanoclusters: Recent Advances in Sensing and Imaging. Anal. Chem. 2015, 87, 216-229. (6) Li, H. T.; Kang, Z. H.; Liu, Y.; Lee, S. T. Carbon Nanodots: Synthesis, Properties and Applications. J. Mater. Chem. 2012, 22, 24230-24253. (7) Qu, D.; Zheng, M.; Zhang, L. G.; Zhao, H. F.; Xie, Z. G.; Jing, X. B.; Haddad, R. E.; Fan, H. Y.; Sun Z. C. Formation Mechanism and Optimization of Highly Luminescent N-Doped Graphene Quantum Dots. Sci. Rep. 2014, 4, 5294. (8) Krysmann, M. J.; Kelarakis, A.; Dallas, P.; Giannelis, E. P. Formation Mechanism of Carbogenic Nanoparticles with Dual Photoluminescence Emission. J. Am. Chem. Soc. 2012, 134, 747-750. (9) Kasprzyk, W.; Bednarz, S.; Bogdał, D. Luminescence Phenomena of Biodegradable Photoluminescent Poly(Diol Citrates). Chem. Commun. 2013, 49, 6445-6447. (10) Kasprzyk, W.; Bednarz, S.; Żmudzki, P.; Galica, M.; Bogdał, D. Novel Efficient Fluorophores Synthesized from Citric Acid. RSC Adv. 2015, 5, 34795-34799. (11) Song, Y.; Zhu, S.; Zhang, S.; Fu, Y.; Wang, L.; Zhao, X.; Yang, B. Investigation from Chemical Structure to Photoluminescent Mechanism: A Type of Carbon Dots from the Pyrolysis of Citric Acid and an Amine. J. Mater. Chem. C 2015, 3, 5976-5984. (12) Sun, D.; Ban, R.; Zhang, P.-H.; Wu, G.-H.; Zhang, J.-R.; Zhu, J.-J. Hair Fiber as a Precursor for Synthesizing of Sulfur- and Nitrogen-Co-Doped Carbon Dots with Tunable Luminescence Properties. Carbon 2013, 64, 424-434. (13) Ding, H.; Wei, J. S.; Xiang, H. M. Nitrogen and Sulfur Co-Doped Carbon Dots with Strong Blue Luminescence. Nanoscale 2014, 6, 13817-13823. (14) Hu, Q.; Paau, M. C.; Zhang, Y.; Gong, X.; Zhang, L.; Lu, D.; Liu, Y.; Liu, Q.; Yao, J.; Choi, M. M. F. Green Synthesis of Fluorescent Nitrogen/Sulfur-Doped Carbon Dots and Investigation of Their Properties by HPLC Coupled with Mass Spectrometry. RSC Adv. 2014, 4, 18065-18073. (15) Zhai, X. Y.; Zhang, P.; Liu, C. J.; Bai, T.; Li, W. C.; Dai, L. M.; Liu, W. G. Highly Luminescent Carbon Nanodots by Microwave-Assisted Pyrolysis. Chem. Commun. 2012, 48, 7955-7957.



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(16) Strauss, V.; Margraf, J. T.; Dolle, C.; Butz, B.; Nacken, T. J.; Walter, J.; Bauer, W.; Peukert, W.; Spiecker, E.; Clark, T.; Guldi, D. M. Carbon nanodots: Toward a Comprehensive Understanding of Their Photoluminescence. J. Am. Chem. Soc. 2014, 136, 17308-17316. (17) Dong, Y. Q.; Pang, H. C.; Yang, H. B.; Guo, C. X.; Shao, J. W.; Chi, Y. W.; Li, C. M.; Yu, T. CarbonBased Dots Co-Doped with Nitrogen and Sulfur for High Quantum Yield and Excitation-Independent Emission. Angew. Chem. Int. Ed. 2013, 52, 7800-7804. (18) Liang, J.; Jiao, Y.; Jaroniec, M.; Qiao, S. Z. Sulfur and Nitrogen Dual-Doped Mesoporous Graphene Electrocatalyst for Oxygen Reduction with Synergistically Enhanced Performance. Angew. Chem. Int. Ed. 2012, 51, 11496-11500. (19) Zhu, S. J.; Meng, Q. N.; Wang, L.; Zhang, J. H.; Song, Y. B.; Jin, H.; Zhang, K.; Sun, H. C.; Wang, H. Y.; Yang B. Highly Photoluminescent Carbon Dots for Multicolor Patterning, Sensors, and Bioimaging. Angew. Chem. Int. Ed. 2013, 52, 3953-3957. (20) Qu, K. G.; Wang, J. S.; Ren, J. S.; Qu, X. G. Carbon Dots Prepared by Hydrothermal Treatment of Dopamine as an Effective Fluorescent Sensing Platform for the Label-Free Detection of Iron(III) Ions and Dopamine. Chem. Eur. J. 2013, 19, 7243-7249. (21) Zhang, P. J.; Xue, Z. J.; Luo, D.; Yu, W.; Guo, Z. H; Wang, T. Dual-Peak Electrogenerated Chemiluminescence of Carbon Dots for Iron Ions detection. Anal. Chem. 2014, 86, 5620-5623. (22) Zhang, H. J.; Chen, Y. L.; Liang, M. J.; Xu, L. F.; Qi, S. D.; Chen, H. L; Chen, X. G. Solid-Phase Synthesis of Highly Fluorescent Nitrogen-Doped Carbon Dots for Sensitive and Selective Probing Ferric Ions in Living Cells. Anal. Chem. 2014, 86, 9846-9852. (23) Xu, Q.; Pu, P.; Zhao, J. G.; Dong, C. B.; Gao, C.; Chen, Y. S.; Chen, J. R.; Liu, Y.; Zhou, H. J. Preparation of Highly Photoluminescent Sulfur-Doped Carbon Dots for Fe(III) Detection. J. Mater. Chem. A 2015, 3, 542-546. (24) Luo, Z. T.; Yuan, X.; Yu, Y.; Zhang, Q. B.; Leong, D. T.; Lee, J. Y.; Xie, J. P. From AggregationInduced Emission of Au(I)-Thiolate Complexes to Ultrabright Au(0)@Au(I)-Thiolate Core-Shell Nanoclusters. J. Am. Chem. Soc. 2012, 134, 16662-16670.


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(25) Jia, X. F.; Li, J.; Wang, E. K. Cu Nanoclusters with Aggregation Induced Emission Enhancement. Small 2013, 9, 3873-3879. (26) Jia, X. F.; Li, J.; Wang, E. K. Supramolecular Self-Assembly of Morphology-Dependent Luminescent Ag Nanoclusters. Chem. Commun. 2014, 50, 9565-9568. (27) Beppu, T.; Tomiguchi, K.; Masuhara, A.; Pu, Y. J.; Katagiri, H. Single Benzene Green Fluorophore: Solid-State Emissive, Water-Soluble, and Solvent- and pH-Independent Fluorescence with Large Stokes Shifts. Angew. Chem. Int. Ed. 2015, 54, 7332-7335.



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One-Pot Gram-Scale Synthesis of Nitrogen and ... - ACS Publications

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