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Fabricating a fluorescence resonance energy transfer system with AIE molecular for sensitive detection of Cu(II) ions
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 225 (2020) 117604
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Fabricating a fluorescence resonance energy transfer system with AIE molecular for sensitive detection of Cu(II) ions Pengli Guan, Binsheng Yang, Bin Liu* Key Laboratory of Chemical Biology and Molecular Engineering, Ministry of Education, Institute of Molecular Science, Shanxi University, Taiyuan, 030006, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 16 January 2019 Received in revised form 21 August 2019 Accepted 6 October 2019 Available online 7 October 2019
The aggregation-induced emission (AIE) luminogens has exhibited strong potential in fabricating the fluorescence resonance energy transfer (FRET) system. In this paper one efficient FRET system was fabricated in aqueous solution based on an AIE molecular (T) and Nile Red (NiR) dyes: T acts as the energy donor and NiR acts as the energy acceptor with a ratio of 250:1. The energy-transfer efficiency from the donor to acceptor is 82.52%, and the antenna effect is 24.9. Base on this data, a very low detection limit for Cu2þ was calculated to be 35.5 pM. This method displays penitential application on fluorescence probe for small ions or molecular detection by light-harvesting system based on a simple AIE donor under physiological conditions. © 2019 Elsevier B.V. All rights reserved.
Keywords: Aggregationeinduced emission €rster resonance energy transfer Fo Fluorescence probe Cu2þ
1. Introduction Copper is third in abundance among the essential heavy metal ions in the human body and plays an important role in a variety of fundamental physiological processes [1]. The analytical methods for detecting Cu2þ has been well developed. Among which, the traditional detecting method is using rhodamine dyes with “TurneOn” effect [2,3]. In recent year, some new detecting methods for Cu2þ such as Schiff-base fluorescence probes with or without aggregationeinduced emission (AIE) activity have been put forward [4e6]. However, detection limit of most of those probes is around micromole level, works related to this area still face great challenge. Photosynthesis is the survival foundation for living creatures such as plants, algae, cyanobacteria and anoxygenic photosynthetic bacteria [7]. The photon-harvesting process takes place in chloro€rster resonance energy transfer (FRET) plast pigments through Fo mechanism. There are about 100e800 chlorophylls closely packed within each reaction center [8e10]. Inspired by this phenomenon, great progress has been made to fabricate light harvesting systems artificially through FRET mechanism [11e15]. For example, many scaffolds and dendrimers [16,17], coordination polymers [18e23] and cyclic arrays systems [24e27] have been synthesized
* Corresponding author. E-mail address: [email protected] (B. Liu). https://doi.org/10.1016/j.saa.2019.117604 1386-1425/© 2019 Elsevier B.V. All rights reserved.
successfully to fabricate the light harvesting systems. However, most of them are carried in organic solutions, and the multistep synthesis processes have precluded their widespread application to a large extent. They always exhibit very poor energy-transfer efficiency in water due to the aggregation caused quenching (ACQ) effect [28e30]. Up to now, many excellent light-harvesting systems have been fabricated to overcome the ACQ effect [31]. Recently, some self-assembly system between pillar [6]arene [32], sulfato-bcyclodextrin [33] with fluorescence dyes (Nile red or eosin Y) in aqueous environment were fabricated. However, the use of macrocyclic molecules carries such as pillar [6]arene and b-cyclodextrin in order to improve the stability of light-harvesting systems in water still set up a bigger barrier for its convenient synthesis. Moreover, the practical application of artificial light-harvesting system is still very limited. It is a very challenging work to fabricate a simpler light-harvesting system and deepen its practical applications. In this paper, one novel efficient artificial light-harvesting system based on an AIE activity compound T and the fluorescence dye Nile Red (NiR) had been successfully fabricated in aqueous solution. The primary strategy for the light-harvesting was illustrated in Scheme 1, where the AIE active T acted as the energy donor (D) and NiR acted as the accepter (A), and efficient FRET process formed between the donor and accepter. This system can be used to detect Cu2þ ion on picomolar level.
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Scheme 1. Schematic representation for aqueous light-harvesting systems based on T-Nile Red and substantially energy quenching by Cu2þ, D: donor, A: acceptor.
2. Experimental 2.1. Materials 5-bromosalicylaldehyde, oxalyldihydrazide, metal salt and organic solvent were obtained commercially and used without further purification. Fluorescence spectra were measured by Fluoromax-X spectrofluorometer and the absorption spectra were recorded on a Varian 50 Bio spectrophotometer. Element analysis was performed with a Vario EL III analyzer. Infrared spectra were measured on a Bruker TENSOR 21 FT-IR spectrophotometer as KBr pellets. 1H NMR spectra were recorded on Brukere600 MHz spectrometers. Dynamic light scattering (DLS) analysis was measured with BECKMAN COULTER DelsaTM Nano C particle analyzer. TEM was measured with TecnaiG2 F20 S-TWIN TMP. Scanning electron microscopy was measured at JEOL-JSM-6701F scanning electron microscope (SEM). The stock solution of compound T was prepared by dissolving 0.04 mmol the sample in 4.0 mL DMSO and diluting the solution to target concentration. The fluorescence intensity at the wavelength of 400e700 nm was detected with a slit width of 10 nm for excitation and emission. The UVeVis spectra were measured at the range of 300e700 nm. 2.2. Synthesis of compound T As shown in Scheme 2, Schiff-base compound T was synthesized by the direct condensation reaction of 5-bromosalicylaldehyde with oxalyldihydrazide (2:1) by one step. Simply, the mixture of oxalyldihydrazide (0.12 g, 1.0 mmol) and 5-bromosalicylaldehyde (0.40 g, 2.0 mmol) in 20 mL ethanol was refluxed. The white precipitate of T was obtained after 1.0 h (85% yields). It was confirmed by structural characterization using ESI-MS, NMR, infrared (IR), elemental analysis and scanning electronic microscopy (SEM) (Figs. S1eS5). 1H NMR (600 MHz, DMSO-d6) (ppm) 12.67 (s, H, -OH), 10.99 (s, HN-), 8.79 (s, -HC¼N-), 7.78 (s, H,), 7.46 (s, H), 6.91 (s,
H). 13C NMR (600 MHz, DMSO-d6) (ppm): 157.01, 156.49, 148.64, 134.66, 130.37, 121.85, 119.21, 111.02. Anal. Calculated for C16H12Br2N4O4 (%): C, 39.70; H, 2.50; N, 11.57. Found (%): C, 39.76; H, 3.18; N, 11.52. Selected IR data (KBr pellet, cm1): 3586 (br), 3000 (w), 1655 (s), 1616 (s), 1561 (s), 1624 (s), 1475 (s), 730 (m). ESI-MS: m/z ¼ 485.0215 [MþH]þ. 3. Results and discussion 3.1. AIE property of T The fluorescence activity of T in an EtOH/water mixed solution was first studied to investigate its solvent-dependent aggregation behaviour. As depicted in Fig. 1a, T showed a maximum emission at Emmax ¼ 523 nm when excited at lex ¼ 380 nm in 100% EtOH. As the water content gradually increased, the fluorescence intensity was decreased and red-shifted simultaneously to 560 nm when water content reached to 50%. The fluorescence intensity at 560 nm reached the maximum at 90% water solution. The inset photograph in Fig. 1a clearly showed the color change of T with the water content (10 mM), a strong orange fluorescence in water was observed at 90% water solution by 380 nm excitation. It indicated that T demonstrated an aggregation emission activity (AIE). At the same time, the UVeVis properties of T in EtOH/water mixed solution was studied to demonstrate the AIE properties. As shown in Fig. S6, T showed an absorption reduce with the water content gradually increased. When the water reached to 50%, obvious redshifted and sharp decreased of the peak were observed, and an absorption tail appeared at the range of 400 nm to 500 nm. With the continuous increase of water content, absorption intensity increased and peak behaved extensive tail. Whereafter, absorption intensity grew up with the water content gradually increased, and this property is similar to the Fig. 1a. In other words, typical AIE characteristics of T were further demonstrated by UVeVis spectra. The luminescence quantum yield also demonstrated the AIE feature of T, the relative information can be seen in Fig. S7. The relative
Scheme 2. Synthetic routes of compound T.
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Fig. 1. a)Fluorescence spectra of T (10 mM) in EtOH/water mixtures with different water fractions. Inset: photographs of T (10 mM) in EtOH/water under 365 nm UV lamp irradiation. b) DLS data of T in EtOH and aqueous solution at 25 C. Inset: The Tyndall effect of T. c) TEM image of T (10 mM) in water. d) Fluorescence microscopy image of T (10 mM) in water.
luminescence quantum yield Q is determined to be 2.3% comparing with the reference rhodamine 6G (lex ¼ 488 nm, Table S1). Meanwhile, the aggregation behaviour in different organic solution was also investigated in Figs. S8eS9. It indicated that T was not only an AIE active compound in aqueous solution, but also in most of the organic solution [34]. That is, the fluorescence intensity and wavelength is affected by the solution polarity. However, the maxima absorption T located at 350 nm, and no obvious difference in peak shape can be observed in different solvent. This phenomenon is similar with that in the literatures [35]. The red-shifted emission of fluorescence spectra is due to the excimer formation between benzene rings and higher contents of water. Moreover, the amidic acid form was more stable in tautomerization due to its complete delocalization in the whole molecule at higher amounts of water (>40%) [36,37]. In addition, the aggregation size was verified by dynamic light scattering (DLS) experiment (Fig. 1b). The average size of aggregation for AIE (T) was found to be about 232 nm in water while 35.1 nm in ethanol. The Tyndall effect (Fig. 1b inset) is well in accordance with the DLS. In general, bigger size of nano partials induces bigger red-shifted emission [38,39]. Moreover, transmission electron microscopy (TEM) image and fluorescence microimage of AIE (T) was shown in Fig. 1c and d, respectively. It indicated that they formed spherical nanoparticles with size of 200e250 nm. All of these data indicated that T was a typical AIE active compounds, and the excited state intramolecular proton transfer (ESIPT) from eOH to neighbouring N atom of eN¼Ce is responsible for the AIE properties [5]. The AIE molecular T looks to be hydrophobic. The most concern for us is whether the aggregation state of molecular can stabilize in water instead of forming precipitates. Therefore, the stability of AIE (T) system in aqueous solution must be investigated at room temperature. As shown in Fig. 2a, the stability of fluorescence intensity was continually monitored during a week period ([T] ¼ 10 mM). No substantial changes were observed for the FI of AIE (T) system during that time. Besides, the DLS were also
measured (Fig. 2b). The average size of AIE (T) system is about 234 nm and 241 nm after 1- and 5-days’ storage, respectively, which is roughly consistent with that of the fresh solution in Fig. 1. In fact, the solution remained its original clarity and consistent Tyndall effect during the whole experiment process (one weak). It clearly illustrates the high stability of this AIE system, and it is superior to the common polymers aggregates or Nano partials. Moreover, the stability and simplicity of T is superior to other AIE systems encapsulated by pillar [6]arene (WP6) or sulfato-b-cyclodextrin (SCD) in order to improve their stability in water [32,33]. Perhaps the hydrogen bond, multi-amino and substitutional groups as well as lower concentration of the molecular T prevent it from forming bigger precipitates. 3.2. Detection of Cu2þ with T The interaction of T ([T] ¼ 10 mM) with various metal ions (Al3þ, Cr , Ni2þ, Agþ, Cd2þ, Pb2þ, Fe3þ, Kþ, Ca2þ, Zn2þ, Mn2þ and Cu2þ) was investigated by fluorescence spectroscopy at room temperature. As shown in Fig. 3a, the fluorescence peak at 560 nm was quenched by Cu2þ, no obvious fluorescence change was observed upon adding other metal ions. It showed that T can selectively detect Cu2þ. The detail fluorescence titrations of Cu2þ (0e10 mM) toward T were carried out (Fig. 3b). It showed a good linear correlation between the intensity (560 nm) and the concentration of Cu2þ (0e15) mM (red line in Fig. 3d). Based on this data, the detection limit 1.28 107 M (Fig. S10) was obtained according to the literature’s method. Meanwhile, the interference experiment demonstrated that T exerted high selectivity for Cu2þ in aqueous solution (Fig. 3c). 3þ
3.3. Light-harvesting system T-NiR fabricating The wide area of AIE emission spectra (500e700 nm) of T in aqueous solution is similar to that of phycoerythrin 545 donor [40]. It is easy to imagine that the aggregation emission of AIE (T) can not
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Fig. 2. a) The fluorescence spectra stability of AIE (T) system in aqueous solution at room temperature for 1e5 days, [T] ¼ 10 mM. b) DLS data after storage for 1 and 5 days.
Fig. 3. (a) Fluorescence spectrum of T upon addition of different metal ions (10 equiv.) in water solution (lex ¼ 380 nm). [T] ¼ 10 mM, Inset: photographs of T and T þ [Cu2þ]. (b) Fluorescence spectrum changes of T (10 mM) upon Cu2þ (0e10 mM) in water solution. Inset: changes of FI at 560 nm versus [Cu2þ]. c) Fluorescent intensity of 10 mM AIE (T) with 20 equiv. various metal ions containing 10 equiv. of Cu2þ. d) Fluorescence landscape for AIE (T) in the presence of Cu2þ.
Fig. 4. a) Normalized UVevis and emission spectra of T and NiR in water, lex ¼ 380 nm. b) Fluorescence spectra of T (10 mM) with different concentrations of NiR, Inset: photographs of T, NiR and T-NiR. [T] ¼ 1.0 105 M, [NiR] ¼ 4.0 108 M.
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only provide multiple donors for per acceptor, but also can avoid the usual intramolecular fluorescence self-quenching phenomenon. As clearly shown in Fig. 4a, T exhibited a maximum emission at Emmax ¼ 560 nm (lex ¼ 380 nm), and it is largely overlapped with the absorption peak of the pure NiR dye (Absmax ¼ 590 nm). This is a prerequisite for an efficient energy transfer (ET). As a control experiment, excitation of NiR at 380 nm resulted in no emission with 500e700 nm area, indicating the minimal absorption of NiR dye at 380 nm (Fig. 4b, pink line). Therefore, T and NiR may be an appropriate candidate to fabricate an aqueous artificial lightharvesting system. Then the detail interaction of T and NiR was monitored by fluorescence spectra. As shown in Fig. 4b, with the gradual addition of NiR to T, a strong emission peak at Emmax ¼ 630 nm was appeared along with a week quenched emission peak at 560 nm, implying an efficient energy transfer from compound T to NiR dye. Finally, a maximum emission of 630 nm was obtained at the molar ratio of donor/ acceptor ¼ 250:1. More intriguingly, the emission intensity from the ET-sensitized T-NiR (lex ¼ 380 nm, Fig. S12, black line) was much higher than that from directly excited NiR (lex ¼ 560 nm, green line). It implied that an efficient light-harvesting effect happened in this T-NiR aggregation. In a word, T had contributed greatly to the acceptor NiR emission, and the energy had been efficiently transferred from T to NiR. Herein, AIE (T) was regarded as the peripheral donor and NiR was the central acceptor. To further confirm the formation of T-NiR assembly, DLS, fluorescence lifetime, TEM and fluorescence microscopy image were also measured. Firstly, the DLS results showed that T-NiR formed well-defined aggregates (molar ratio ¼ 250:1) with a narrow size, and the average diameter is about 162.8 nm (Fig. 5a). This size is smaller than that of the free T in water (232 nm). Second, the fluorescence lifetime decay curves were shown in Fig. 5b and
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Table S2. The fluorescence lifetime for the donor T or acceptor NiR at 630 nm were fitted by a single exponential decay, respectively. Fluorescence lifetimes for free T is t1 ¼ 0.6697 ns (RW1[%] ¼ 54.96, red line), while the fluorescence lifetime for T-NiR decreased to t1 ¼ 0.4212 ns (RW1[%] ¼ 92.65, pink line, lex ¼ 380 nm, lem ¼ 560 nm). It well confirmed the energy transferred from the T donor to NiR acceptor. Moreover, both the morphology by TEM illustrated in Fig. 5c and fluorescence microscopy image measurements in Fig. 5d clearly illustrated the size and morphology of T-NiR in aqueous solution. Subsequently, the energy transfer efficiency (E) and antenna effect (AE) of the formed T-NiR system were investigated. Energy transfer efficiency was estimated from the fluorescence quenching rate of donor T in the T-NiR assembly structure, which was a widely used empirical parameter (supporting Information). As a result, E was calculated to be 82.5% when the mixing molar ratio of donor/ acceptor was at 250:1 (Fig. S11). Meanwhile, the antenna effect at this mixing ratio had been calculated to be AE ¼ 24.9 (Fig. S12). Notably, the assembly T-NiR displays an excellent light harvesting antenna in aqueous environment. Generally, both of the molar ratio and the average distance (d) between the donor and acceptor can affect the ET efficiency. Therefore, the average distance was also calculated. As a result, R0 ¼ 2.2 nm and r ¼ 1.7 nm was obtained, respectively (Table S4, see the supporting Information). This distance is suitable for most of the FRET process. All of these data indicate that T-NiR is an appropriate model to mimic the natural light-harvesting process in aqueous environment. It should be noted that the system T-NiR can be stored at room temperature for three days or 0 C for one week without obvious fluorescence changes, which implies the stability of this artificial light-harvesting process in aqueous environment.
Fig. 5. a) DLS data of T-NiR antenna at 25 C. b) Fluorescence lifetime measurements of T-NiR. c) TEM image of T-NiR. d) Fluorescence microscopy image of T-NiR. [T] ¼ 1.0 105 M, [NiR] ¼ 4.0 108 M. c) TEM image of T-NiR. d) Fluorescence microscopy image of T-NiR (10 mM) in water.
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3.4. Detection of Cu2þ with T-NiR system €rster resonance energy The influence of metal ions on the Fo transfer of this light harvesting antenna was investigated in aqueous solution. The interaction of T-NiR ([T] ¼ 10 mM, [NiR] ¼ 4.0 108 M) with various metal ions was investigated by fluorescence spectroscopy at room temperature (Fig. 6a). To our surprise, significant fluorescence quenching at both 560 and 630 nm was found when 1.0 equiv. of Cu2þ was added to the solution. However, no obvious fluorescence change was observed upon adding other metal ions. It indicated that the fluorescence of T-NiR could be selectively quenched by Cu2þ ion. The fluorescence lifetimes of T-NiR þ Cu2þ shapely decreased to t1 ¼ 0.27 ns and t2 ¼ 1.24 ns when compared with that of T-NiR (Fig. S13, blue line, Table S3), which is consistent with the fluorescence changes. The detail fluorescence titrations of Cu2þ toward T-NiR system were carried out ([T] ¼ 10 mM, [NiR] ¼ 4.0 108 M). As shown in Fig. 6b, fluorescence intensity at 630 nm decreased sharply with the addition of trace Cu2þ, and a slight blue shift was observed in the process. With more and more copper ions was added, both 560 and 630 nm are quenched by Cu2þ ion obviously. Remarkably, it showed a good linear correlation between the intensity (630 nm) and the concentration of Cu2þ (0e10) nM, in Fig. 6d). Base on this data, a very low detection limit for Cu2þ was calculated to be 35.5 pM (Fig. S14, supporting Information). Obviously, the detect limit of TNiR is much higher that of T. Meanwhile, the interference experiment demonstrated that T-NiR antenna exerted a slight fluorescence decrease with the addition of 10 equiv. other metal ions but a dramatic diminishes in the presence of 1.0 equiv Cu2þ (Fig. 6c). It indicated that the T-NiR system had high selectivity for Cu2þ in aqueous solution. Moreover, if we take T-NiR as a simple compound, the coordination constant with Cu2þ can be calculated to be 12 K2þ M1 using the empirical parameters, while T-NiRþCu 1.0 10 5 K2þ M1, the fitting curves were given as Fig. S15 e TþCu is 1.0 10 S16, respectively.
To indentify the interaction mechanism of T-NiR and Cu2þ, we assume that a tightly complex structure T-NiR-Cu2þ was formed. This assumption was verified by the completion experiment. As shown in Fig. S17, the fluorescence at 630 nm was quenched by small amount of Cu2þ. Then the competitor such as S2, OH, Cit (citric acid), CopC protein or reducer NO (DEA$NONOate, NO donor) and VC (vitamin C) was added into the solution T-NiR-Cu2þ. Normally, it is regarded that the traditional ligand S2 can compete Cu2þ with the formation of CuS precipitate and NO can reduce Cu2þ to Cuþ, which induce the distraction the Cu2þ complex [41]. Besides, the apoCopC protein is proposed to be a copper chaperone protein, which guide and protect the copper ion within the cell. It has high affinity for Cu2þ and Cuþ. Our team has studied the metal ions binding properties of CopC [42,43]. Herein, the original fluorescence for both the donor and accepter can hardly be restored in the addition of these competitors and reducer to our surprise. Obviously, a very stable Cu2þ polymer T-NiR-Cu2þ have been formed! That is, efficient FRET process was formed between AIE (T) to NiR at a high molar ratio 250:1, and small amount of Cu2þ can selectively and severely affect the fluorescence intensity. AIE system enlarges the fluorescence signal of the donor T greatly, and the FRET process further sensitizes the fluorescence intensity of the acceptor to a large extent, but both the fluorescence intensity of donor and accepter was sharply quenched by small amount of Cu2þ with a very stable Cu2þ polymer T-NiR-Cu2þ formed. By which we can detect Cu2þ very sensitively when compared to the ordinary fluorescence analytical methods [44e48]. This idea was first depicted in Scheme 3. To further substantiated the existed of polymer system T-NiRCu2þ, the SEM of T-Cu2þ and T-NiR þ Cu2þ was present in Fig. S18. As can be seen in Fig. S18, a more close packing was observed intuitively in T-NiR þ Cu2þ than T-Cu2þ for these two systems. As mentioned above, T-NiR þ Cu2þ displayed a more tight complex comparing to T-Cu2þ. At the same time, the competitive experiment was carried out with EDTA as Cu2þ competitor in the system of T-
Fig. 6. a) Fluorescence spectrum of T-NiR upon addition of different metal ions (1.0 equiv.) in water solution (lex ¼ 380 nm). [T] ¼ 10 mM, [NiR] ¼ 4.0 108 M, Inset: photographs of T-NiR and T-NiR þ [Cu2þ]. b) Fluorescence spectrum changes of T-NiR (10 mM) upon Cu2þ (0e10 mM) in water solution. Inset: changes of FI at 630 nm versus [Cu2þ]. c) FI of T-NiR with 10 equiv. various metal ions containing 1.0 equiv. of Cu2þ (T ¼ 10 mM). d) Fluorescence landscape for T-NiR in the presence of Cu2þ.
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References
Scheme 3. Proposed mechanism for the AIE, FRET and sensing Cu2þ process.
NiR-Cu2þ. As shown in Fig. S19, little FI increment could be observed with the addition of 10 equiv. of EDTA, which confirmed the formation of a very tightly aggregation by T-NiR and Cu2þ. Such a tight system is finished by large amount of T-NiR and Cu2þ, which could be definited as [T-NiR-Cu2þ]n. The polymer of [T-NiR-Cu2þ]n was consist by hydrogen-bonding interaction, chelation interaction, and aromatic stacking interaction. We can infer that it is hard for EDTA to pass through the network and take away Cu2þ. 4. Conclusions In this paper, we have designed a small AIE activity compound T which could be used to fabricate a highly efficient artificial lightharvesting system in aqueous environment with NiR. Such a regular multi-donor and one-acceptor structure is similar to the natural light harvesting antenna, which is superior to the common dendrimers and polymers systems in organic solution. In this system, the presence of trace amounts Cu2þ can induce severe fluorescence quenching of the light-harvesting systems based on FRET process. Through this method we can reduce the detection limit of Cu2þ by three orders of magnitude when compared to the ordinary analytical methods. Therefore, it is well anticipated that future works on other small molecule fluorescent recognition probes utilizing AIE based artificial light-harvesting systems are promising. Declaration of competing interest The authors declare no conflict of interest. Acknowledgements We are very grateful for financial support from the National Natural Science Foundation of PR China (No. 21271122, 21571117, 21575083) and International Cooperation Research Project of Shanxi Province (2015081049). We thank the Institute of Resources and Environment Engineering and Scientific Instrument Center of Shanxi University. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.saa.2019.117604.
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Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
Fabricating a fluorescence resonance energy transfer system with AIE molecular for sensitive detection of Cu(II) ions Pengli Guan, Binsheng Yang, Bin Liu* Key Laboratory of Chemical Biology and Molecular Engineering, Ministry of Education, Institute of Molecular Science, Shanxi University, Taiyuan, 030006, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 16 January 2019 Received in revised form 21 August 2019 Accepted 6 October 2019 Available online 7 October 2019
The aggregation-induced emission (AIE) luminogens has exhibited strong potential in fabricating the fluorescence resonance energy transfer (FRET) system. In this paper one efficient FRET system was fabricated in aqueous solution based on an AIE molecular (T) and Nile Red (NiR) dyes: T acts as the energy donor and NiR acts as the energy acceptor with a ratio of 250:1. The energy-transfer efficiency from the donor to acceptor is 82.52%, and the antenna effect is 24.9. Base on this data, a very low detection limit for Cu2þ was calculated to be 35.5 pM. This method displays penitential application on fluorescence probe for small ions or molecular detection by light-harvesting system based on a simple AIE donor under physiological conditions. © 2019 Elsevier B.V. All rights reserved.
Keywords: Aggregationeinduced emission €rster resonance energy transfer Fo Fluorescence probe Cu2þ
1. Introduction Copper is third in abundance among the essential heavy metal ions in the human body and plays an important role in a variety of fundamental physiological processes [1]. The analytical methods for detecting Cu2þ has been well developed. Among which, the traditional detecting method is using rhodamine dyes with “TurneOn” effect [2,3]. In recent year, some new detecting methods for Cu2þ such as Schiff-base fluorescence probes with or without aggregationeinduced emission (AIE) activity have been put forward [4e6]. However, detection limit of most of those probes is around micromole level, works related to this area still face great challenge. Photosynthesis is the survival foundation for living creatures such as plants, algae, cyanobacteria and anoxygenic photosynthetic bacteria [7]. The photon-harvesting process takes place in chloro€rster resonance energy transfer (FRET) plast pigments through Fo mechanism. There are about 100e800 chlorophylls closely packed within each reaction center [8e10]. Inspired by this phenomenon, great progress has been made to fabricate light harvesting systems artificially through FRET mechanism [11e15]. For example, many scaffolds and dendrimers [16,17], coordination polymers [18e23] and cyclic arrays systems [24e27] have been synthesized
* Corresponding author. E-mail address: [email protected] (B. Liu). https://doi.org/10.1016/j.saa.2019.117604 1386-1425/© 2019 Elsevier B.V. All rights reserved.
successfully to fabricate the light harvesting systems. However, most of them are carried in organic solutions, and the multistep synthesis processes have precluded their widespread application to a large extent. They always exhibit very poor energy-transfer efficiency in water due to the aggregation caused quenching (ACQ) effect [28e30]. Up to now, many excellent light-harvesting systems have been fabricated to overcome the ACQ effect [31]. Recently, some self-assembly system between pillar [6]arene [32], sulfato-bcyclodextrin [33] with fluorescence dyes (Nile red or eosin Y) in aqueous environment were fabricated. However, the use of macrocyclic molecules carries such as pillar [6]arene and b-cyclodextrin in order to improve the stability of light-harvesting systems in water still set up a bigger barrier for its convenient synthesis. Moreover, the practical application of artificial light-harvesting system is still very limited. It is a very challenging work to fabricate a simpler light-harvesting system and deepen its practical applications. In this paper, one novel efficient artificial light-harvesting system based on an AIE activity compound T and the fluorescence dye Nile Red (NiR) had been successfully fabricated in aqueous solution. The primary strategy for the light-harvesting was illustrated in Scheme 1, where the AIE active T acted as the energy donor (D) and NiR acted as the accepter (A), and efficient FRET process formed between the donor and accepter. This system can be used to detect Cu2þ ion on picomolar level.
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Scheme 1. Schematic representation for aqueous light-harvesting systems based on T-Nile Red and substantially energy quenching by Cu2þ, D: donor, A: acceptor.
2. Experimental 2.1. Materials 5-bromosalicylaldehyde, oxalyldihydrazide, metal salt and organic solvent were obtained commercially and used without further purification. Fluorescence spectra were measured by Fluoromax-X spectrofluorometer and the absorption spectra were recorded on a Varian 50 Bio spectrophotometer. Element analysis was performed with a Vario EL III analyzer. Infrared spectra were measured on a Bruker TENSOR 21 FT-IR spectrophotometer as KBr pellets. 1H NMR spectra were recorded on Brukere600 MHz spectrometers. Dynamic light scattering (DLS) analysis was measured with BECKMAN COULTER DelsaTM Nano C particle analyzer. TEM was measured with TecnaiG2 F20 S-TWIN TMP. Scanning electron microscopy was measured at JEOL-JSM-6701F scanning electron microscope (SEM). The stock solution of compound T was prepared by dissolving 0.04 mmol the sample in 4.0 mL DMSO and diluting the solution to target concentration. The fluorescence intensity at the wavelength of 400e700 nm was detected with a slit width of 10 nm for excitation and emission. The UVeVis spectra were measured at the range of 300e700 nm. 2.2. Synthesis of compound T As shown in Scheme 2, Schiff-base compound T was synthesized by the direct condensation reaction of 5-bromosalicylaldehyde with oxalyldihydrazide (2:1) by one step. Simply, the mixture of oxalyldihydrazide (0.12 g, 1.0 mmol) and 5-bromosalicylaldehyde (0.40 g, 2.0 mmol) in 20 mL ethanol was refluxed. The white precipitate of T was obtained after 1.0 h (85% yields). It was confirmed by structural characterization using ESI-MS, NMR, infrared (IR), elemental analysis and scanning electronic microscopy (SEM) (Figs. S1eS5). 1H NMR (600 MHz, DMSO-d6) (ppm) 12.67 (s, H, -OH), 10.99 (s, HN-), 8.79 (s, -HC¼N-), 7.78 (s, H,), 7.46 (s, H), 6.91 (s,
H). 13C NMR (600 MHz, DMSO-d6) (ppm): 157.01, 156.49, 148.64, 134.66, 130.37, 121.85, 119.21, 111.02. Anal. Calculated for C16H12Br2N4O4 (%): C, 39.70; H, 2.50; N, 11.57. Found (%): C, 39.76; H, 3.18; N, 11.52. Selected IR data (KBr pellet, cm1): 3586 (br), 3000 (w), 1655 (s), 1616 (s), 1561 (s), 1624 (s), 1475 (s), 730 (m). ESI-MS: m/z ¼ 485.0215 [MþH]þ. 3. Results and discussion 3.1. AIE property of T The fluorescence activity of T in an EtOH/water mixed solution was first studied to investigate its solvent-dependent aggregation behaviour. As depicted in Fig. 1a, T showed a maximum emission at Emmax ¼ 523 nm when excited at lex ¼ 380 nm in 100% EtOH. As the water content gradually increased, the fluorescence intensity was decreased and red-shifted simultaneously to 560 nm when water content reached to 50%. The fluorescence intensity at 560 nm reached the maximum at 90% water solution. The inset photograph in Fig. 1a clearly showed the color change of T with the water content (10 mM), a strong orange fluorescence in water was observed at 90% water solution by 380 nm excitation. It indicated that T demonstrated an aggregation emission activity (AIE). At the same time, the UVeVis properties of T in EtOH/water mixed solution was studied to demonstrate the AIE properties. As shown in Fig. S6, T showed an absorption reduce with the water content gradually increased. When the water reached to 50%, obvious redshifted and sharp decreased of the peak were observed, and an absorption tail appeared at the range of 400 nm to 500 nm. With the continuous increase of water content, absorption intensity increased and peak behaved extensive tail. Whereafter, absorption intensity grew up with the water content gradually increased, and this property is similar to the Fig. 1a. In other words, typical AIE characteristics of T were further demonstrated by UVeVis spectra. The luminescence quantum yield also demonstrated the AIE feature of T, the relative information can be seen in Fig. S7. The relative
Scheme 2. Synthetic routes of compound T.
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Fig. 1. a)Fluorescence spectra of T (10 mM) in EtOH/water mixtures with different water fractions. Inset: photographs of T (10 mM) in EtOH/water under 365 nm UV lamp irradiation. b) DLS data of T in EtOH and aqueous solution at 25 C. Inset: The Tyndall effect of T. c) TEM image of T (10 mM) in water. d) Fluorescence microscopy image of T (10 mM) in water.
luminescence quantum yield Q is determined to be 2.3% comparing with the reference rhodamine 6G (lex ¼ 488 nm, Table S1). Meanwhile, the aggregation behaviour in different organic solution was also investigated in Figs. S8eS9. It indicated that T was not only an AIE active compound in aqueous solution, but also in most of the organic solution [34]. That is, the fluorescence intensity and wavelength is affected by the solution polarity. However, the maxima absorption T located at 350 nm, and no obvious difference in peak shape can be observed in different solvent. This phenomenon is similar with that in the literatures [35]. The red-shifted emission of fluorescence spectra is due to the excimer formation between benzene rings and higher contents of water. Moreover, the amidic acid form was more stable in tautomerization due to its complete delocalization in the whole molecule at higher amounts of water (>40%) [36,37]. In addition, the aggregation size was verified by dynamic light scattering (DLS) experiment (Fig. 1b). The average size of aggregation for AIE (T) was found to be about 232 nm in water while 35.1 nm in ethanol. The Tyndall effect (Fig. 1b inset) is well in accordance with the DLS. In general, bigger size of nano partials induces bigger red-shifted emission [38,39]. Moreover, transmission electron microscopy (TEM) image and fluorescence microimage of AIE (T) was shown in Fig. 1c and d, respectively. It indicated that they formed spherical nanoparticles with size of 200e250 nm. All of these data indicated that T was a typical AIE active compounds, and the excited state intramolecular proton transfer (ESIPT) from eOH to neighbouring N atom of eN¼Ce is responsible for the AIE properties [5]. The AIE molecular T looks to be hydrophobic. The most concern for us is whether the aggregation state of molecular can stabilize in water instead of forming precipitates. Therefore, the stability of AIE (T) system in aqueous solution must be investigated at room temperature. As shown in Fig. 2a, the stability of fluorescence intensity was continually monitored during a week period ([T] ¼ 10 mM). No substantial changes were observed for the FI of AIE (T) system during that time. Besides, the DLS were also
measured (Fig. 2b). The average size of AIE (T) system is about 234 nm and 241 nm after 1- and 5-days’ storage, respectively, which is roughly consistent with that of the fresh solution in Fig. 1. In fact, the solution remained its original clarity and consistent Tyndall effect during the whole experiment process (one weak). It clearly illustrates the high stability of this AIE system, and it is superior to the common polymers aggregates or Nano partials. Moreover, the stability and simplicity of T is superior to other AIE systems encapsulated by pillar [6]arene (WP6) or sulfato-b-cyclodextrin (SCD) in order to improve their stability in water [32,33]. Perhaps the hydrogen bond, multi-amino and substitutional groups as well as lower concentration of the molecular T prevent it from forming bigger precipitates. 3.2. Detection of Cu2þ with T The interaction of T ([T] ¼ 10 mM) with various metal ions (Al3þ, Cr , Ni2þ, Agþ, Cd2þ, Pb2þ, Fe3þ, Kþ, Ca2þ, Zn2þ, Mn2þ and Cu2þ) was investigated by fluorescence spectroscopy at room temperature. As shown in Fig. 3a, the fluorescence peak at 560 nm was quenched by Cu2þ, no obvious fluorescence change was observed upon adding other metal ions. It showed that T can selectively detect Cu2þ. The detail fluorescence titrations of Cu2þ (0e10 mM) toward T were carried out (Fig. 3b). It showed a good linear correlation between the intensity (560 nm) and the concentration of Cu2þ (0e15) mM (red line in Fig. 3d). Based on this data, the detection limit 1.28 107 M (Fig. S10) was obtained according to the literature’s method. Meanwhile, the interference experiment demonstrated that T exerted high selectivity for Cu2þ in aqueous solution (Fig. 3c). 3þ
3.3. Light-harvesting system T-NiR fabricating The wide area of AIE emission spectra (500e700 nm) of T in aqueous solution is similar to that of phycoerythrin 545 donor [40]. It is easy to imagine that the aggregation emission of AIE (T) can not
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Fig. 2. a) The fluorescence spectra stability of AIE (T) system in aqueous solution at room temperature for 1e5 days, [T] ¼ 10 mM. b) DLS data after storage for 1 and 5 days.
Fig. 3. (a) Fluorescence spectrum of T upon addition of different metal ions (10 equiv.) in water solution (lex ¼ 380 nm). [T] ¼ 10 mM, Inset: photographs of T and T þ [Cu2þ]. (b) Fluorescence spectrum changes of T (10 mM) upon Cu2þ (0e10 mM) in water solution. Inset: changes of FI at 560 nm versus [Cu2þ]. c) Fluorescent intensity of 10 mM AIE (T) with 20 equiv. various metal ions containing 10 equiv. of Cu2þ. d) Fluorescence landscape for AIE (T) in the presence of Cu2þ.
Fig. 4. a) Normalized UVevis and emission spectra of T and NiR in water, lex ¼ 380 nm. b) Fluorescence spectra of T (10 mM) with different concentrations of NiR, Inset: photographs of T, NiR and T-NiR. [T] ¼ 1.0 105 M, [NiR] ¼ 4.0 108 M.
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only provide multiple donors for per acceptor, but also can avoid the usual intramolecular fluorescence self-quenching phenomenon. As clearly shown in Fig. 4a, T exhibited a maximum emission at Emmax ¼ 560 nm (lex ¼ 380 nm), and it is largely overlapped with the absorption peak of the pure NiR dye (Absmax ¼ 590 nm). This is a prerequisite for an efficient energy transfer (ET). As a control experiment, excitation of NiR at 380 nm resulted in no emission with 500e700 nm area, indicating the minimal absorption of NiR dye at 380 nm (Fig. 4b, pink line). Therefore, T and NiR may be an appropriate candidate to fabricate an aqueous artificial lightharvesting system. Then the detail interaction of T and NiR was monitored by fluorescence spectra. As shown in Fig. 4b, with the gradual addition of NiR to T, a strong emission peak at Emmax ¼ 630 nm was appeared along with a week quenched emission peak at 560 nm, implying an efficient energy transfer from compound T to NiR dye. Finally, a maximum emission of 630 nm was obtained at the molar ratio of donor/ acceptor ¼ 250:1. More intriguingly, the emission intensity from the ET-sensitized T-NiR (lex ¼ 380 nm, Fig. S12, black line) was much higher than that from directly excited NiR (lex ¼ 560 nm, green line). It implied that an efficient light-harvesting effect happened in this T-NiR aggregation. In a word, T had contributed greatly to the acceptor NiR emission, and the energy had been efficiently transferred from T to NiR. Herein, AIE (T) was regarded as the peripheral donor and NiR was the central acceptor. To further confirm the formation of T-NiR assembly, DLS, fluorescence lifetime, TEM and fluorescence microscopy image were also measured. Firstly, the DLS results showed that T-NiR formed well-defined aggregates (molar ratio ¼ 250:1) with a narrow size, and the average diameter is about 162.8 nm (Fig. 5a). This size is smaller than that of the free T in water (232 nm). Second, the fluorescence lifetime decay curves were shown in Fig. 5b and
5
Table S2. The fluorescence lifetime for the donor T or acceptor NiR at 630 nm were fitted by a single exponential decay, respectively. Fluorescence lifetimes for free T is t1 ¼ 0.6697 ns (RW1[%] ¼ 54.96, red line), while the fluorescence lifetime for T-NiR decreased to t1 ¼ 0.4212 ns (RW1[%] ¼ 92.65, pink line, lex ¼ 380 nm, lem ¼ 560 nm). It well confirmed the energy transferred from the T donor to NiR acceptor. Moreover, both the morphology by TEM illustrated in Fig. 5c and fluorescence microscopy image measurements in Fig. 5d clearly illustrated the size and morphology of T-NiR in aqueous solution. Subsequently, the energy transfer efficiency (E) and antenna effect (AE) of the formed T-NiR system were investigated. Energy transfer efficiency was estimated from the fluorescence quenching rate of donor T in the T-NiR assembly structure, which was a widely used empirical parameter (supporting Information). As a result, E was calculated to be 82.5% when the mixing molar ratio of donor/ acceptor was at 250:1 (Fig. S11). Meanwhile, the antenna effect at this mixing ratio had been calculated to be AE ¼ 24.9 (Fig. S12). Notably, the assembly T-NiR displays an excellent light harvesting antenna in aqueous environment. Generally, both of the molar ratio and the average distance (d) between the donor and acceptor can affect the ET efficiency. Therefore, the average distance was also calculated. As a result, R0 ¼ 2.2 nm and r ¼ 1.7 nm was obtained, respectively (Table S4, see the supporting Information). This distance is suitable for most of the FRET process. All of these data indicate that T-NiR is an appropriate model to mimic the natural light-harvesting process in aqueous environment. It should be noted that the system T-NiR can be stored at room temperature for three days or 0 C for one week without obvious fluorescence changes, which implies the stability of this artificial light-harvesting process in aqueous environment.
Fig. 5. a) DLS data of T-NiR antenna at 25 C. b) Fluorescence lifetime measurements of T-NiR. c) TEM image of T-NiR. d) Fluorescence microscopy image of T-NiR. [T] ¼ 1.0 105 M, [NiR] ¼ 4.0 108 M. c) TEM image of T-NiR. d) Fluorescence microscopy image of T-NiR (10 mM) in water.
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3.4. Detection of Cu2þ with T-NiR system €rster resonance energy The influence of metal ions on the Fo transfer of this light harvesting antenna was investigated in aqueous solution. The interaction of T-NiR ([T] ¼ 10 mM, [NiR] ¼ 4.0 108 M) with various metal ions was investigated by fluorescence spectroscopy at room temperature (Fig. 6a). To our surprise, significant fluorescence quenching at both 560 and 630 nm was found when 1.0 equiv. of Cu2þ was added to the solution. However, no obvious fluorescence change was observed upon adding other metal ions. It indicated that the fluorescence of T-NiR could be selectively quenched by Cu2þ ion. The fluorescence lifetimes of T-NiR þ Cu2þ shapely decreased to t1 ¼ 0.27 ns and t2 ¼ 1.24 ns when compared with that of T-NiR (Fig. S13, blue line, Table S3), which is consistent with the fluorescence changes. The detail fluorescence titrations of Cu2þ toward T-NiR system were carried out ([T] ¼ 10 mM, [NiR] ¼ 4.0 108 M). As shown in Fig. 6b, fluorescence intensity at 630 nm decreased sharply with the addition of trace Cu2þ, and a slight blue shift was observed in the process. With more and more copper ions was added, both 560 and 630 nm are quenched by Cu2þ ion obviously. Remarkably, it showed a good linear correlation between the intensity (630 nm) and the concentration of Cu2þ (0e10) nM, in Fig. 6d). Base on this data, a very low detection limit for Cu2þ was calculated to be 35.5 pM (Fig. S14, supporting Information). Obviously, the detect limit of TNiR is much higher that of T. Meanwhile, the interference experiment demonstrated that T-NiR antenna exerted a slight fluorescence decrease with the addition of 10 equiv. other metal ions but a dramatic diminishes in the presence of 1.0 equiv Cu2þ (Fig. 6c). It indicated that the T-NiR system had high selectivity for Cu2þ in aqueous solution. Moreover, if we take T-NiR as a simple compound, the coordination constant with Cu2þ can be calculated to be 12 K2þ M1 using the empirical parameters, while T-NiRþCu 1.0 10 5 K2þ M1, the fitting curves were given as Fig. S15 e TþCu is 1.0 10 S16, respectively.
To indentify the interaction mechanism of T-NiR and Cu2þ, we assume that a tightly complex structure T-NiR-Cu2þ was formed. This assumption was verified by the completion experiment. As shown in Fig. S17, the fluorescence at 630 nm was quenched by small amount of Cu2þ. Then the competitor such as S2, OH, Cit (citric acid), CopC protein or reducer NO (DEA$NONOate, NO donor) and VC (vitamin C) was added into the solution T-NiR-Cu2þ. Normally, it is regarded that the traditional ligand S2 can compete Cu2þ with the formation of CuS precipitate and NO can reduce Cu2þ to Cuþ, which induce the distraction the Cu2þ complex [41]. Besides, the apoCopC protein is proposed to be a copper chaperone protein, which guide and protect the copper ion within the cell. It has high affinity for Cu2þ and Cuþ. Our team has studied the metal ions binding properties of CopC [42,43]. Herein, the original fluorescence for both the donor and accepter can hardly be restored in the addition of these competitors and reducer to our surprise. Obviously, a very stable Cu2þ polymer T-NiR-Cu2þ have been formed! That is, efficient FRET process was formed between AIE (T) to NiR at a high molar ratio 250:1, and small amount of Cu2þ can selectively and severely affect the fluorescence intensity. AIE system enlarges the fluorescence signal of the donor T greatly, and the FRET process further sensitizes the fluorescence intensity of the acceptor to a large extent, but both the fluorescence intensity of donor and accepter was sharply quenched by small amount of Cu2þ with a very stable Cu2þ polymer T-NiR-Cu2þ formed. By which we can detect Cu2þ very sensitively when compared to the ordinary fluorescence analytical methods [44e48]. This idea was first depicted in Scheme 3. To further substantiated the existed of polymer system T-NiRCu2þ, the SEM of T-Cu2þ and T-NiR þ Cu2þ was present in Fig. S18. As can be seen in Fig. S18, a more close packing was observed intuitively in T-NiR þ Cu2þ than T-Cu2þ for these two systems. As mentioned above, T-NiR þ Cu2þ displayed a more tight complex comparing to T-Cu2þ. At the same time, the competitive experiment was carried out with EDTA as Cu2þ competitor in the system of T-
Fig. 6. a) Fluorescence spectrum of T-NiR upon addition of different metal ions (1.0 equiv.) in water solution (lex ¼ 380 nm). [T] ¼ 10 mM, [NiR] ¼ 4.0 108 M, Inset: photographs of T-NiR and T-NiR þ [Cu2þ]. b) Fluorescence spectrum changes of T-NiR (10 mM) upon Cu2þ (0e10 mM) in water solution. Inset: changes of FI at 630 nm versus [Cu2þ]. c) FI of T-NiR with 10 equiv. various metal ions containing 1.0 equiv. of Cu2þ (T ¼ 10 mM). d) Fluorescence landscape for T-NiR in the presence of Cu2þ.
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7
References
Scheme 3. Proposed mechanism for the AIE, FRET and sensing Cu2þ process.
NiR-Cu2þ. As shown in Fig. S19, little FI increment could be observed with the addition of 10 equiv. of EDTA, which confirmed the formation of a very tightly aggregation by T-NiR and Cu2þ. Such a tight system is finished by large amount of T-NiR and Cu2þ, which could be definited as [T-NiR-Cu2þ]n. The polymer of [T-NiR-Cu2þ]n was consist by hydrogen-bonding interaction, chelation interaction, and aromatic stacking interaction. We can infer that it is hard for EDTA to pass through the network and take away Cu2þ. 4. Conclusions In this paper, we have designed a small AIE activity compound T which could be used to fabricate a highly efficient artificial lightharvesting system in aqueous environment with NiR. Such a regular multi-donor and one-acceptor structure is similar to the natural light harvesting antenna, which is superior to the common dendrimers and polymers systems in organic solution. In this system, the presence of trace amounts Cu2þ can induce severe fluorescence quenching of the light-harvesting systems based on FRET process. Through this method we can reduce the detection limit of Cu2þ by three orders of magnitude when compared to the ordinary analytical methods. Therefore, it is well anticipated that future works on other small molecule fluorescent recognition probes utilizing AIE based artificial light-harvesting systems are promising. Declaration of competing interest The authors declare no conflict of interest. Acknowledgements We are very grateful for financial support from the National Natural Science Foundation of PR China (No. 21271122, 21571117, 21575083) and International Cooperation Research Project of Shanxi Province (2015081049). We thank the Institute of Resources and Environment Engineering and Scientific Instrument Center of Shanxi University. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.saa.2019.117604.
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