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A highly sensitive turn-on fluorescent chemosensor for recognition of Zn(II) ions and its application in live cells imaging
Journal of Photochemistry & Photobiology A: Chemistry 386 (2020) 112111
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A highly sensitive turn-on fluorescent chemosensor for recognition of Zn(II) ions and its application in live cells imaging
T
Peng Wanga, , Jiang Wub ⁎
a
Chemical Synthesis and Pollution Control Key Laboratory of Sichuan Province, College of Chemistry and Chemical Engineering, China West Normal University, Shida Road 1#, Nanchong, 637009, PR China b Key Laboratory of Tibetan Medicine Research, Northwest Institute of Plateau Biology, Chinese Academy of Science, Xining, 810008, PR China
ARTICLE INFO
ABSTRACT
Keywords: Histidine Chemosensor Reversibility Zn(II) ions Cells imaging
A novel and simple fluorescent chemosensor, DH, was synthesized. As designed, DH displayed excellent selectivity exhibiting a “turn-on” response for Zn2+ over other relevant metal ions based on PET mechanism in 100% aqueous media, and the binding constant of Zn2+ binding to DH was calculated to be 7.27 × 108 M−2. Importantly, DH exhibited high sensitivity as a Zn2+ fluorescent chemosensor with a limit of detection (LOD) of 19.25 nM, which is lower than WHO guidelines (76.0 μM). Moreover, the 2:1 stoichiometry between the chemosensor DH and Zn2+ was verified by Job's plot, fluorescent titration and high resolution mass spectroscopy. In addition, the recognition process of DH toward Zn2+ was chemically reversible and recyclable by adding Na2EDTA. Moreover, due to its good biocompatibility and low cytotoxicity, DH was successfully applied to the fluorescence imaging of Zn2+ and Na2EDTA in living HK2 cells.
1. Introduction The transition metal zinc is one of the most important minerals in biological systems, and it is the second most abundant transition metal element in the human body [1–3]. Zinc also helps the immune system, endocrine system, and gastroenterological system [1–5], and numerous proteins and enzymes that Zn2+ ions have been identified [3–6]. Significantly, zinc ions are also reportedly associated with many neurological disorders, including arteriosclerosis [1], Parkinson's disease [2], coronary disease [3] and epilepsy [4]. In addition, zinc plays a crucial role in insulin secretion and apoptosis, and many biological functions related to the immune, endocrine and gastrointestinal systems [7–10]. According to biological researches, amino acids in peptides or proteins play crucial role in maintaining several biological functions by exhibiting their specific metal ion binding property. For example, Zincfinger is a finger-shaped protein fold that is formed by Histidine (His) and Cysteine (Cys) in the protein binding to a zinc ions. Various studies have reported that it is easy to detect Zn2+ ions in body because of the reasonable biochemical markers of zinc ions (proteins, enzymes and carbohydrates and so on) [5,8–10]. However, most analytical techniques for detection of Zn2+ ions are tedious and complex, which is why fluorescence detection technology is so important for analyses of both
⁎
environmental and biological systems [6–12]. Fluorescence detection technology is an important method for the qualitative and quantitative analysis of metal ions as it is simple and practical [6–12], and exhibits outstanding advantages in sensitivity, selectivity, and time resolution. Based on the unique advantages of fluorescence detection, a variety of fluorescence chemosensors for Zn2+ detection have been successfully developed [13–22], including diarylethene derivative [13], 2-Amino-3-pyridinecarboxaldehyde [15], fluorescein hydrazide [17], and quinoline containing acetyl hydrazone [21], among others. The peptide-based fluorescent chemosensor is a molecular device for chemical analysis and detection, which can selectively bind to specific analytes resulting in changes of the fluorescence spectra of the system [23–38]. Peptide fluorescence chemosensors have many unique advantages, including high selectivity to metal ions based on a variety of different amino acid functional groups [23–28]. Significantly, peptide fluorescence chemosensors can bind strongly with metal ions, producing a rapid fluorescence response [29–33]. Many chemosensors utilize toxic organic solvents, a problem that is not observed with peptide-based chemosensors, which exhibit higher biological compatibility and solubility than organic dyes based on natural amino acids necessary to the human body [34–40]. In this work, we report a new peptide-based fluorescent chemo-
Corresponding author. E-mail address: [email protected] (P. Wang).
https://doi.org/10.1016/j.jphotochem.2019.112111 Received 5 August 2019; Received in revised form 15 September 2019; Accepted 26 September 2019 Available online 30 September 2019 1010-6030/ © 2019 Elsevier B.V. All rights reserved.
Journal of Photochemistry & Photobiology A: Chemistry 386 (2020) 112111
P. Wang and J. Wu
Scheme 1. Synthesis route of DH.
Fig. 2. (a) Fluorescent spectra of DH (10.0 μM) with changing concentrations of Zn2+ ions (0–1.0 equiv.) in HEPES buffer (20.0 mM, pH 7.4) solutions. (b) Plots of the fluorescence intensity of DH as a function of Zn2+ concentration. Inset of (b): color of DH and DH-Zn under UV lamp (365 nm).
Fig. 1. (a) Fluorescence spectra of DH (10.0 μM) upon the addition of 17 metal ions (1.0 equiv.) in HEPES buffer solutions (20.0 mM, pH 7.4). (b) Competitive tests for the fluorescence responses of DH (10.0 μM) in the presence of Zn2+ ions (5.0 μM) and other competing metal ions (25.0 μM) in HEPES buffer solution (20.0 mM, pH 7.4). Error bars represent standard deviations from three repeated experiments.
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2.2. Apparatus ESI–MS spectra were acquired on a Bruker esquire 6000 spectrometer. Fluorescence spectra measurements were performed using a Cary eclipse spectrofluorometer. UV–Vis spectrophotometric studies were performed at room temperature on a Varian Cary 5000 spectrophotometer. The high resolution mass spectra (HRMS) were measured on a maXis 4 G. All pH HEPES buffer solutions were prepared using NaOH and HCl and verified using a pHS-3E digital pH meter. 1H NMR titration characterization carried by Bruker Avance III (400 MHz) in DMSO-d6. FTIR spectroscopy was tested using Fourier transform infrared spectrometer (Nicolet 6700). 2.3. Synthesis DH was synthesized efficiently by solid phase peptide synthesis (SPPS) via 9-fluorenyl methoxycarbonyl (Fmoc) chemistry (Scheme 1). The concrete synthesis method of DH is described in the Supporting Information. The purity (> 95%) of the DH was characterized by analytical HPLC (Fig. S1 and Table. S1). DH: Yellowish solid; Synthetic yield: 93.0%; m.p.:126–131℃. ESI-MS (m/z) of DH calculated value: 387.14; Observed value: 387.8 ([DH+H+]+, Fig. S2). 1H NMR (400 MHz, DMSO, ppm): δ 13.99–13.83 (m, 2 H), 8.60 (s, 1 H), 8.48–8.42 (m, 2 H), 8.14–8.06 (m, 2 H), 7.59-7.51 (m, 2 H), 7.24–7.09 (m, 4 H), 2.96–2.76 (m, 8 H) (Fig. S3). In addition, the FTIR spectroscopy (Fig. S4.) of chemosensor DH was measured. These characterization data showed that DH has been successfully synthesized.
Fig. 3. Job’s plot for determining the stoichiometry of DH and Zn2+ ions in HEPES buffer (20.0 mM, pH 7.4) solutions.
sensor DH comprised of a fluorescent dansyl group conjugated with a histidine as a recognition group for Zn2+ ions via the N atom of imidazole group. Based on photoinduced electron transfer (PET), the fluorescence intensity of DH is very weak without Zn2+ ions, but DH displayed an effectively selective “turn-on” fluorescence enhancement when Zn2+ ions were added into either the buffered aqueous solution and living cells. What’s more, the addition of Na2EDTA quenched the fluorescence of DH-Zn complex confirming that DH functions as a reversible chemosensor.
2.4. Cell culture and imaging The MTT test and cell imaging experiments were completed using Human renal tubular epithelial cells (HK2 normal cells) provided by the Second Hospital of Lanzhou University. HK2 normal cells were cultured in Dulbecco’s modified eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37 °C and 5% CO2.
2. Experimental procedures 2.1. Materials Solvents and materials for synthesis of the chemosensor DH were purchased commercially and used without further purification, including Rink Amide resin, Fmoc-L-His (Trt)−OH, 1-hydroxybenzotriazole, triisopropylsilane (TIS) and dansyl chloride (DANSYL-Cl). Perchlorate salts of 17 common metals ions (Hg2+ as a nitrate) were purchased from Sigma Aldrich. Stock solutions of metal ions (10−3 M) and DH (10−3 M) were prepared with distilled water.
3. Results and discussion 3.1. Fluorescence spectral responses From the fluorescence response of the chemosensor DH toward 17 different metal ions in 20.0 mM HEPES buffer solutions at pH 7.4 (Fig. 1a), a 5-fold increase in fluorescence intensity for DH was observed following the addition of Zn2+ ions. Conversely, no effect on the fluorescence of DH was observed following addition of other metal
Scheme 2. The proposed sensing mechanism of DH and Zn2+ ions.
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Fig. 5. The pH influence on the fluorescence intensity of DH in the absence (black line) and presence of DH-Zn (red line) in HEPES buffer (pH 2.0–12.0) solutions.
Fig. 4. (a) Fluorescence emission spectra of DH-Zn (10.0 μM) in the presence of various anions (20.0 μM) in 20 mM HEPES buffer solution at pH 7.4. (b) The cycle index of DH-Zn (10.0 μM) reacting with H2PO4− (20.0 μM).
Scheme 3. Logic circuit diagram for the chemosensor DH. Fig. 6. (a) The reversibility experiment of DH toward Zn2+ ions by adding Na2EDTA in HEPES buffer (20.0 mM, pH 7.4) solutions. (b) The cycle index of DH-Zn (10.0 μM) reacting with Na2EDTA (10.0 μM).
ions, including Ag+, Al3+, Ba2+, Ca2+, Cd2+, Co2+, Cr3+, Cu2+, Fe3+, Hg2+, K+, Mg2+, Mn2+, Na+, Ni2+ and Pb2+, indicating that the sensor is highly-selective for Zn2+ in a pure water system. Next, the effect of the presence of other cations with Zn2+ ion 4
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Zn2+ ions (0–1.0 equiv.), and the fluorescence titration curve of DH was saturated when 0.5 equiv. Zn2+ were added to the DH solutions (Fig. 2b), which exhibited 2:1 stoichiometric complexation between DH and Zn2+ ions. In addition, under UV excitation at 365 nm, the fluorescence of DH was significantly enhanced and showed bright green when Zn2+ was added to DH solutions (Inset of Fig. 2b). The stoichiometric binding of Zn2+ with the prepared chemosensor DH was investigated by Job's plot method. As seen in Fig. 3, a maximum absorbance was observed when the molar fraction of Zn2+ reached 0.33, confirming a 2:1 coordination stoichiometry of chemosensor DH with Zn2+ in 20.0 mM HEPES buffer solutions. The binding constant for DH with Zn2+ was determined to be 7.27 × 108 M−2 (R2 = 0.9978) based on the titration curve of the chemosensor DH with Zn2+ ions (Fig. S7). Based on the plot, the detection limit for Zn2+ ions was calculated to be 19.25 nM based on the definition of IUPAC (LOD = 3σ/k), which is significantly below the guideline (76.0 μM) WHO protocol (Fig. S8). Moreover, the binding stoichiometry between the chemosensor DH and Zn2+ was further confirmed by high resolution mass spectrum (HRMS), and the appearance of a peak at m/z 837.20 that was assigned to [2DH+ Zn2+ − H+]+ in the ESI-MS spectra (Fig. S9). These experimental results further confirmed that the binding ratio between chemosensor DH and Zn2+ ions was 2:1. In addition, 1H NMR studies also provide direct evidence of the interactions between DH and Zn2+ ions (Fig. S10). When 1.0 equiv. of Zn2+ ions was added, the different chemical shifts in H (a, b) were observed, the proton (a) on imidazole group almost disappeared(a’), the single nuclear magnetic peak (b) of primary amino group is divided into two peaks(b’), and the proton of dansyl have little change, which indicated that Zn2+ ions coordinates with imidazole group and primary amino group (Fig. S10). These results suggest that the donating ability of the imidazole groups in the PET quenching of the dansyl emitter. When Zn2+ ions coordinated with imidazole groups, the recovery of intensity of dansyl emitter may be induced by the blocking of electron transfer effect.
Fig. 7. The time dependence of DH (10.0 μM) on detecting Zn2+ ions (0.5 equiv) and Na2EDTA (1.0 equiv) in HEPES buffer (20.0 mM, pH 7.4) solutions.
3.3. Recognition mechanism We improved the verification experiments: when 10.0 μM dansyl chloride solution and 10.0 μM DH was added to the 20.0 mM HEPES buffer solutions respectively, we found that the fluorescence intensity of dansyl chloride was significantly higher than the fluorescence intensity of DH (Fig. S11), these results suggest that the donating ability of the imidazole groups in the PET quenching of the dansyl emitter. Then, 20.0 equiv. L-His solution and 20.0 equiv. 2-methylimidazole was added to 10.0 μM dansyl chloride solution in 20.0 mM HEPES buffer respectively, the fluorescence intensity of dansyl chloride was significantly declined(Fig. S11), the suggests that exist quenching effect of imidazole group to dansyl. The proposed mechanism for detection of Zn2+ ions by DH is illustrated in Scheme 2. Due to the photoinduced electron transfer (PET) from histidine to the dansyl group, the fluorescence of the chemosensor DH was very weak in the initial state. The efficiency of electron transfer from donor to fluorescent group was reduced following binding of Zn2+ ions to DH. As a result, the original intensity of the fluorescent cluster was restored based on the fluorescence quenching caused by PET was interrupted. The fluorescence signal of DH is highly sensitive based on PET mechanism, and can be selectively used for the detection of Zn2+ ions [39].
Fig. 8. MTT assay of DH. Error bars represent standard deviations from three repeated experiments.
detection was studied. Competitive experiments with different cations on the DH-Zn system indicated that the sensitive detection of DH to Zn2+ was not affected by the presence of other metal ions, up to 5.0 equiv. (Fig. 1b), indicating strong anti-interference by DH. Moreover, a comparison of five different Zn2+ salts demonstrated no differences in fluorescence intensity when combined with the chemosensor DH (Fig. S5.), confirming that the fluorescence response of DH was specific to Zn2+ itself, and not to its ligand anions. 3.2. Binding mode The absorption spectra of DH in HEPES buffer solutions displayed maxima at 240, 255 and 330 nm (Fig. S6), and all absorbance bands increased simultaneously upon addition of 0.5 equiv. Zn2+ in DH solutions. These results suggested that DH participated in the coordination of Zn2+ ions. Correspondingly, a significant increase in fluorescent intensity of DH was observed (Fig. 2a) with increasing concentration of
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Fig. 9. The fluorescence images of living HK2 cells. (A) Cells incubated with 20.0 μM DH for 30 min; (B) Cells were incubated with 20.0 μM DH for 30 min and subsequently treated with 10.0 μM Zn2+ ions for another 30 min; (C) Cells were incubated with 20.0 μM DH for 30 min and subsequently treated with 10.0 μM Zn2+ ions for another 30 min and incubation with 20.0 μM Na2EDTA for another 30 min; Bright-field transmission images (A1, B1, C1), Fluorescent-field transmission images (A2, B2, C2), Merged transmission images (A3, B3, C3).
3.4. Fluorescence spectra studies of DH-Zn toward H2PO4−
On the basis of the fact that the emission intensity of DH could be effectively controlled by Zn2+ and H2PO4−, the logic gate properties with two input signals as Zn2+ (In1) and H2PO4− (In2), DH had the ability to exhibit INHIBIT function as output signal (Scheme 3). The presence and absence of Zn2+ or H2PO4− were defined as ‘1′ and ‘0′ for inputs, while turn-on and turn-off as ‘1′ and ‘0′ for outputs, respectively. From the perspective of logic functions, the turn-on of 1 (output‘1′) could be observed only in the presence of Zn2+ ions (In1 = 1) and in the absence of H2PO4− (In 2 = 0).
The fluorescence spectra responses of DH-Zn toward various anions such as F−, Cl−, Br−, I−, SCN−, AcO−, NO3−, PO43−, S2O32−, OH−, HCO3−, CO32−, SO42−, HPO42− and H2PO4− (2.0 equiv. each), were also researched in 20.0 mM HEPES buffer solutions as shown in Fig. 4a, the trong emission intensity was observed when DH was treated with 0.5 equiv. of Zn2+, and the fluorescence was totally quenched by the subsequent separate addition of H2PO4−. However, the addition of other anions above mentioned induced only insignificant responses on the fluorescence spectra. Hence, the chelate DH-Zn can be utilized as a new platform to recognize H2PO4− with high selectivity over other species. In addition, The reversibility experiments of DH (10.0 μM) with Zn2+ (5.0 μM) and H2PO4− (20.0 μM) were detected in HEPES buffer solutions (20.0 mM, pH 7.4). As illustrated in Fig. 4b, the reversibility of DH were further verified during 8 cycles of titration by alternatively adding Zn2+ and H2PO4− to the DH solutions, and the circulation effect was very good.
3.5. pH effects The effects of pH change on the chemosensor DH and DH-Zn system were studied. As depicted in Fig. 5, the fluorescence spectra of the chemosensor DH exhibited no obvious change in the pH range 2.0 to 12.0. At the same time, the change of pH from 2.0 to 5.0 did not affect the fluorescence response of chemosensor DH to Zn2+ ions. However, upon addition of Zn2+ ions, the fluorescence intensity of the
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[13] [14] [15] [16] [17] [18] [19] [20] [21] [22] This work
chemosensor DH was significantly enhanced in the range of pH 6.0–14.0, which showed that the change of pH had little effect on chemosensor DH, and DH could sensitively detect Zn2+ ions over a broad range of pH values.
No Yes No Yes Yes No No Yes Yes Yes Yes
3.6. Reversibility experiment Reversibility is a prerequisite in developing novel chemosensors for practical applications. Thus, the reversibility and cyclic nature of the binding process of chemosensor DH toward Zn2+ ions was confirmed by adding the bonding agent Na2EDTA. The addition of Na2EDTA to a mixture of DH-Zn solutions resulted in diminution of the fluorescence intensity (Fig. 6a), which indicated regeneration of the chemosensor DH. Similarly, the fluorescence intensity of DH exhibited significant fluorescence enhancement when Zn2+ was added again. As seen in Fig. 6b, DH still exhibited a strong fluorescence intensity response toward Zn2+ ions and Na2EDTA after at least 10 cycles, with small switchable changes following alternating additions of Zn2+ ions and Na2EDTA. An ideal chemosensor should exhibit a fast fluorescence response. The response times of DH toward Zn2+ ions were investigated as depicted in Fig. 7, and the fluorescence intensity of DH was rapidly enhanced and quenched within 20 s when Zn2+ and Na2EDTA were added, respectively, while the fluorescence intensity of DH exhibited no change when the action time was 180 s.
7.02 × 10−8 M 11.9 μM 8.56 × 10−9 M 3.5 × 10−8 M 0.1 μM/0.5 μM 10.5 nM 1.25 × 10−7 M 1.603 × 10−10 M 89.3 nM 4.9 nM 19.25 nM THF (2.0 × 10−5mol L−1) MeCN/bis-tris buffer (95:5) CH3CN DMSO:H2O (1:1, v/v) solution CH3CN-PBS (1/99, v/v) solution HEPES buffer at pH 7.4 containing 10% (v/v) of DMSO EtOH/HEPES solution (3/1, 10.0 μM HEPES, pH 7.4) Ethanol-water (V:V = 1:1) solution EtOH/HEPES (10 mM, pH = 7.40, 3/7, v/v) solution HEPES buffer solutions (10 mM, pH = 7.4) HEPES buffer solutions (20 mM, pH = 7.4) Diarylethene derivative ((E)-1-((2-hydroxy-3-methoxybenzylidene)amino)imidazolidine-2,4-dione) 2-Amino-3-pyridinecarboxaldehyde N′-((8-hydroxy-1,2,3,5,6,7-hexahydropyrido [3,2,1-ij]guinolin-9-yl)methylene)-4-methylbenzohydrazide Fluorescein hydrazide pyrene-appended naphthalimide-dipicolylamine 6 hydroxychromone 3 carbaldehyde (rhodamine B carbonyl) hydrazine Chromene pyrazoline derivatives Quinoline containing acetyl hydrazone Dansyl-tetrapeptide Dansyl-histidine
Turn-on Turn-on Turn-on Turn-on Turn-on Ratiometric Turn-on OFF-ON Turn-on OFF-ON Turn-on
Detection limit Detection condition Zn2+ chemosensor
Table 1 Comparison of chemosensors for Zn2+ assays reported in the literature.
Detection method
cell imaging
Refs.
P. Wang and J. Wu
3.7. Application in bioimaging In order to test the possibility of applying the chemosensor DH in biological systems, the cytotoxicity test (MTT) was first carried out in HK2 cells. A range of concentrations (5.0, 10.0, 20.0, 40.0, and 80.0 μM) of chemosensor DH were added to HK2 cells, and incubated for 24 h in 5% CO2 at 37 °C. The survival rate of HK2 cells exceeded 90% (Fig. 8) even in the presence of high concentrations of DH, which indicated that DH has good biocompatibility and low toxicity, and has the potential to be used in live cell imaging. The effects of Zn2+ ions and Na2EDTA on the fluorescence intensity of DH inside HK2 cells were investigated by confocal microscopy (Fig. 9). A dim, weak fluorescence response was observed in HK2 cells incubated only with the chemosensor DH (20.0 μM) for 30 min at 37 °C, after washing three times with PBS buffer solutions (Fig. 9A). Conversely, a bright green fluorescence was observed following introduction of exogenous Zn2+ (10.0 μM) and incubation with DH (20.0 μM) for another 30 min at 37 °C (Fig. 9B). Finally, the subsequent addition of 20.0 μM Na2EDTA to the DH-Zn cells, and incubation of reaction mixtures at 37 °C for another 30 min, resulted in elimination of the fluorescence intensity in living cells (Fig. 9C). Summarily, these cell imaging studies strongly demonstrated that the chemosensor DH has potential applications in tracking Zn2+ and Na2EDTA levels in living cells. 4. Conclusion A new fluorescent chemosensor DH was readily synthesized by solid phase peptide synthesis with high yield. Compared to other chemosensors (Table 1 and Table 2), DH selectively detected Zn2+ from among various relevant metal ions in aqueous solutions at physiological pH, and a LOD of 19.25 nM was calculated for Zn2+ in the linear response range from 0 μM to 14.0 μM. On the basis of the Job’s plot, fluorescent titration and ESI-mass spectral analysis, formation of a 2: 1 (DH : Zn2+) complex was proposed. In addition, the reversible and recyclable processes of the detection of DH toward Zn2+ ions were confirmed by adding bonding agent Na2EDTA. Moreover, DH exhibited low cytotoxicity, good cell permeability and stability, and was successfully applied for imaging and monitoring of Zn2+ and Na2EDTA in live HK2 cells by using confocal laser scanning microscope. 7
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Table 2 Comparison of peptide/dansyl based fluorescence chemosensors for Zn2+ assays in the literatures. Zn2+ chemosensor
Sensing mechanism
Sensing mechanism
Detection limit
Detection method
Refs.
HEPES buffer solutions (10 mM, pH 7.4)
PET
4.9 nM
Turn-on
[22]
HEPES buffer solutions (10 mM, pH 7.4)
PET
18 nM
Turn-on
[35]
Aqueous Solutions
FRET
97 nM
Ratiometric
[36]
HEPES buffer solutions (10 mM, pH 7.4)
PET
32 nM
Turn-on
[38]
HEPES buffer solutions (20 mM, pH = 7.4)
PET
36.8 nM
Turn-on
[40]
HEPES buffer solutions (20 mM, pH = 7.4)
PET
19.25 nM
Turn-on
This work
Declaration of Competing Interest
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The authors declared that they have no conflicts of interest to this work. We declare that we do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted. Acknowledgments This work was supported by the Fundamental Research Funds of China West Normal University (NO. 416628/18B018), the Doctoral Scientific Research Start-up Foundation of China West Normal University (NO. 412662/17E048), major project funds of Chemical Synthesis and Pollution Control Key Laboratory of Sichuan Province (NO. CSPC2018ZD03) and the Science and Technology Planning Project of QingHai Department of Science and Technology (NO. 2018-ZJ-919). Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.jphotochem.2019. 112111. References [1] C.J. Frederickson, J.Y. Koh, A.I. Bush, The neurobiology of zinc in health and disease, Nat. Rev. Neurosci. 6 (2005) 449–462. [2] A.I. Bush, W.H. Pettingell, G. Multhaup, M. Paradis, J.P. Vonsattel, J.F. Gusella, K. Beyreuther, C.L. Masters, R.E. Tanzi, Rapid induction of Alzheimer A beta amyloid formation by zinc, Science 265 (1994) 1464–1467. [3] X. Xie, T.G. Smart, A physiological role for endogenous zinc in rat hippocampal synaptic neurotransmission, Nature 349 (1991) 521–524. [4] J.M. Berg, Y. Shi, The galvanization of biology: a growing appreciation for the roles of zinc, Science 271 (1996) 1081–1085. [5] S.Y. Assaf, S.H. Chung, Release of endogenous Zn2+ from brain tissue during
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containing acetyl hydrazone: an easily accessible switch-on optical chemosensor for Zn2+, Spectrochim. Acta, Part A 188 (2018) 324–331. P. Wang, J. Wu, Highly selective and sensitive detection of Zn(II) and Cu(II) ions using a novel peptide fluorescent probe by two different mechanisms and its application in live cell imaging, Spectrochim. Acta, Part A 208 (2019) 140–149. P.K. Mehta, E.T. Oh, H.J. Park, K.H. Lee, Ratiometric fluorescent probe based on symmetric peptidyl receptor with picomolar affinity for Zn2+ in aqueous solution, Sensor Actuat. B–Chem. 245 (2017) 996–1003. P. Wang, D.G. Zhou, B. Chen, A fluorescent dansyl-based peptide probe for highly selective and sensitive detect Cd2+ ions and its application in living cell imaging, Spectrochim. Acta, Part A 207 (2019) 276–283. Y. An, P. Wang, Z.J. Yue, A sequential and reversibility fluorescent pentapeptide probe for Cu(II) ions and hydrogen sulfide detections and its application in two different living cells imaging, Spectrochim. Acta, Part A 216 (2019) 319–327. S. Jang, P. Thirupathi, L.N. Neupane, J. Seong, H. Lee, W.I. Lee, K.H. Lee, Highly sensitive ratiometric fluorescent chemosensor for silver ion and silver nanoparticles in aqueous solution, Org. Lett. 14 (2012) 4746–4749. P. Wang, Y. An, Y.W. Liao, A novel peptide-based fluorescent chemosensor for Cd (II) ions and its applications in bioimaging, Spectrochim. Acta, Part A 216 (2019) 61–68. L.N. Neupane, J.Y. Park, J.H. Park, K.H. Lee, Turn–on fluorescent chemosensor based on an amino acid for Pb (II) and Hg (II) ions in aqueous solutions and role of tryptophan for sensing, Org. Lett. 15 (2013) 254–257. R. Lohani, L.N. Neupane, J.M. Kim, K.H. Lee, Selectively and sensitively monitoring Hg2+ in aqueous buffer solutions with fluorescent sensors based on unnatural amino acids, Sensor Actuat. B-Chem. 161 (2012) 1088–1096. P. Wang, K. Chen, Y.S. Ge, Peptide-based ratiometric fluorescent probe for highly selective detection of Cd (II) and its application in bioimaging, J. Lumin. 208 (2019) 495–501. J.M. Kim, C.R. Lohani, L.N. Neupane, Y. Choi, K.H. Lee, Highly sensitive turn-on detection of Ag+ in aqueous solution and live cells with a symmetric fluorescent
peptide, Chem. Commun. (Camb.) 48 (2012) 3012–3014. [32] M.H. Yang, P. Thirupathi, K.H. Lee, Selective and sensitive ratiometric detection of Hg (II) ions using a simple amino acid based sensor, Org. Lett. 13 (2011) 5028–5031. [33] P. Wang, L.P. Duan, Y.W. Liao, A retrievable and highly selective peptide-based fluorescent probe for detection of Cd2+ and Cys in aqueous solutions and live cells, Microchem. J. 146 (2019) 818–827. [34] J.C. Yang, H. Rong, P. Shao, Y.D. Tao, J. Dang, P. Wang, Y.S. Ge, J. Wu, D. Liu, Highly selective ratiometric peptide-based chemosensors for zinc ions and applications in living cell imaging: a study for reasonable structure design, J. Mater. Chem. B Mater. Biol. Med. 4 (2016) 6065–6073. [35] P. Wang, D.G. Zhou, B. Chen, High selective and sensitive detection of Zn(II) using tetrapeptide-based dansyl fluorescent chemosensor and its application in cell imaging, Spectrochim. Acta, Part A 204 (2018) 735–742. [36] P. Wang, J. Wu, P.P. Zhou, W.S. Liu, Y. Tang, A novel peptide–based fluorescent chemosensor for measuring zinc ions using different excitation wavelengths and application in live cell imaging, J. Mater. Chem. B Mater. Biol. Med. 3 (2015) 3617–3624. [37] G.J. Park, Y.J. Na, H.Y. Jo, S.A. Lee, A.R. Kim, I. Noh, C. Kim, A single chemosensor for multiple analytes: fluorogenic detection of Zn2+ and OAc− ions in aqueous solution and an application to bioimaging, New J. Chem. 38 (2014) 2587–2594. [38] P. Wang, J. Wu, P.R. Su, C.F. Shan, P.P. Zhou, Y.S. Ge, D. Liu, W.S. Liu, Y. Tang, A novel fluorescent chemosensor based on tetra-peptides for detecting zinc ions in aqueous solutions and live cells, J. Mater. Chem. B Mater. Biol. Med. 4 (2016) 4526–4533. [39] Z.P. Liu, W.J. He, Z.J. Guo, Metal coordination in photoluminescent sensing, Chem. Soc. Rev. 42 (2013) 1568–1600. [40] P. Wang, X.H. Wu, J. Wu, Y.W. Liao, Highly selective and sensitive peptide-based fluorescent chemosensor for detection of Zinc(II) ions in aqueous medium and living cells, J. Photochem. Photobiol. A: Chem. 382 (2019) 111929.
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Journal of Photochemistry & Photobiology A: Chemistry journal homepage: www.elsevier.com/locate/jphotochem
A highly sensitive turn-on fluorescent chemosensor for recognition of Zn(II) ions and its application in live cells imaging
T
Peng Wanga, , Jiang Wub ⁎
a
Chemical Synthesis and Pollution Control Key Laboratory of Sichuan Province, College of Chemistry and Chemical Engineering, China West Normal University, Shida Road 1#, Nanchong, 637009, PR China b Key Laboratory of Tibetan Medicine Research, Northwest Institute of Plateau Biology, Chinese Academy of Science, Xining, 810008, PR China
ARTICLE INFO
ABSTRACT
Keywords: Histidine Chemosensor Reversibility Zn(II) ions Cells imaging
A novel and simple fluorescent chemosensor, DH, was synthesized. As designed, DH displayed excellent selectivity exhibiting a “turn-on” response for Zn2+ over other relevant metal ions based on PET mechanism in 100% aqueous media, and the binding constant of Zn2+ binding to DH was calculated to be 7.27 × 108 M−2. Importantly, DH exhibited high sensitivity as a Zn2+ fluorescent chemosensor with a limit of detection (LOD) of 19.25 nM, which is lower than WHO guidelines (76.0 μM). Moreover, the 2:1 stoichiometry between the chemosensor DH and Zn2+ was verified by Job's plot, fluorescent titration and high resolution mass spectroscopy. In addition, the recognition process of DH toward Zn2+ was chemically reversible and recyclable by adding Na2EDTA. Moreover, due to its good biocompatibility and low cytotoxicity, DH was successfully applied to the fluorescence imaging of Zn2+ and Na2EDTA in living HK2 cells.
1. Introduction The transition metal zinc is one of the most important minerals in biological systems, and it is the second most abundant transition metal element in the human body [1–3]. Zinc also helps the immune system, endocrine system, and gastroenterological system [1–5], and numerous proteins and enzymes that Zn2+ ions have been identified [3–6]. Significantly, zinc ions are also reportedly associated with many neurological disorders, including arteriosclerosis [1], Parkinson's disease [2], coronary disease [3] and epilepsy [4]. In addition, zinc plays a crucial role in insulin secretion and apoptosis, and many biological functions related to the immune, endocrine and gastrointestinal systems [7–10]. According to biological researches, amino acids in peptides or proteins play crucial role in maintaining several biological functions by exhibiting their specific metal ion binding property. For example, Zincfinger is a finger-shaped protein fold that is formed by Histidine (His) and Cysteine (Cys) in the protein binding to a zinc ions. Various studies have reported that it is easy to detect Zn2+ ions in body because of the reasonable biochemical markers of zinc ions (proteins, enzymes and carbohydrates and so on) [5,8–10]. However, most analytical techniques for detection of Zn2+ ions are tedious and complex, which is why fluorescence detection technology is so important for analyses of both
⁎
environmental and biological systems [6–12]. Fluorescence detection technology is an important method for the qualitative and quantitative analysis of metal ions as it is simple and practical [6–12], and exhibits outstanding advantages in sensitivity, selectivity, and time resolution. Based on the unique advantages of fluorescence detection, a variety of fluorescence chemosensors for Zn2+ detection have been successfully developed [13–22], including diarylethene derivative [13], 2-Amino-3-pyridinecarboxaldehyde [15], fluorescein hydrazide [17], and quinoline containing acetyl hydrazone [21], among others. The peptide-based fluorescent chemosensor is a molecular device for chemical analysis and detection, which can selectively bind to specific analytes resulting in changes of the fluorescence spectra of the system [23–38]. Peptide fluorescence chemosensors have many unique advantages, including high selectivity to metal ions based on a variety of different amino acid functional groups [23–28]. Significantly, peptide fluorescence chemosensors can bind strongly with metal ions, producing a rapid fluorescence response [29–33]. Many chemosensors utilize toxic organic solvents, a problem that is not observed with peptide-based chemosensors, which exhibit higher biological compatibility and solubility than organic dyes based on natural amino acids necessary to the human body [34–40]. In this work, we report a new peptide-based fluorescent chemo-
Corresponding author. E-mail address: [email protected] (P. Wang).
https://doi.org/10.1016/j.jphotochem.2019.112111 Received 5 August 2019; Received in revised form 15 September 2019; Accepted 26 September 2019 Available online 30 September 2019 1010-6030/ © 2019 Elsevier B.V. All rights reserved.
Journal of Photochemistry & Photobiology A: Chemistry 386 (2020) 112111
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Scheme 1. Synthesis route of DH.
Fig. 2. (a) Fluorescent spectra of DH (10.0 μM) with changing concentrations of Zn2+ ions (0–1.0 equiv.) in HEPES buffer (20.0 mM, pH 7.4) solutions. (b) Plots of the fluorescence intensity of DH as a function of Zn2+ concentration. Inset of (b): color of DH and DH-Zn under UV lamp (365 nm).
Fig. 1. (a) Fluorescence spectra of DH (10.0 μM) upon the addition of 17 metal ions (1.0 equiv.) in HEPES buffer solutions (20.0 mM, pH 7.4). (b) Competitive tests for the fluorescence responses of DH (10.0 μM) in the presence of Zn2+ ions (5.0 μM) and other competing metal ions (25.0 μM) in HEPES buffer solution (20.0 mM, pH 7.4). Error bars represent standard deviations from three repeated experiments.
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2.2. Apparatus ESI–MS spectra were acquired on a Bruker esquire 6000 spectrometer. Fluorescence spectra measurements were performed using a Cary eclipse spectrofluorometer. UV–Vis spectrophotometric studies were performed at room temperature on a Varian Cary 5000 spectrophotometer. The high resolution mass spectra (HRMS) were measured on a maXis 4 G. All pH HEPES buffer solutions were prepared using NaOH and HCl and verified using a pHS-3E digital pH meter. 1H NMR titration characterization carried by Bruker Avance III (400 MHz) in DMSO-d6. FTIR spectroscopy was tested using Fourier transform infrared spectrometer (Nicolet 6700). 2.3. Synthesis DH was synthesized efficiently by solid phase peptide synthesis (SPPS) via 9-fluorenyl methoxycarbonyl (Fmoc) chemistry (Scheme 1). The concrete synthesis method of DH is described in the Supporting Information. The purity (> 95%) of the DH was characterized by analytical HPLC (Fig. S1 and Table. S1). DH: Yellowish solid; Synthetic yield: 93.0%; m.p.:126–131℃. ESI-MS (m/z) of DH calculated value: 387.14; Observed value: 387.8 ([DH+H+]+, Fig. S2). 1H NMR (400 MHz, DMSO, ppm): δ 13.99–13.83 (m, 2 H), 8.60 (s, 1 H), 8.48–8.42 (m, 2 H), 8.14–8.06 (m, 2 H), 7.59-7.51 (m, 2 H), 7.24–7.09 (m, 4 H), 2.96–2.76 (m, 8 H) (Fig. S3). In addition, the FTIR spectroscopy (Fig. S4.) of chemosensor DH was measured. These characterization data showed that DH has been successfully synthesized.
Fig. 3. Job’s plot for determining the stoichiometry of DH and Zn2+ ions in HEPES buffer (20.0 mM, pH 7.4) solutions.
sensor DH comprised of a fluorescent dansyl group conjugated with a histidine as a recognition group for Zn2+ ions via the N atom of imidazole group. Based on photoinduced electron transfer (PET), the fluorescence intensity of DH is very weak without Zn2+ ions, but DH displayed an effectively selective “turn-on” fluorescence enhancement when Zn2+ ions were added into either the buffered aqueous solution and living cells. What’s more, the addition of Na2EDTA quenched the fluorescence of DH-Zn complex confirming that DH functions as a reversible chemosensor.
2.4. Cell culture and imaging The MTT test and cell imaging experiments were completed using Human renal tubular epithelial cells (HK2 normal cells) provided by the Second Hospital of Lanzhou University. HK2 normal cells were cultured in Dulbecco’s modified eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37 °C and 5% CO2.
2. Experimental procedures 2.1. Materials Solvents and materials for synthesis of the chemosensor DH were purchased commercially and used without further purification, including Rink Amide resin, Fmoc-L-His (Trt)−OH, 1-hydroxybenzotriazole, triisopropylsilane (TIS) and dansyl chloride (DANSYL-Cl). Perchlorate salts of 17 common metals ions (Hg2+ as a nitrate) were purchased from Sigma Aldrich. Stock solutions of metal ions (10−3 M) and DH (10−3 M) were prepared with distilled water.
3. Results and discussion 3.1. Fluorescence spectral responses From the fluorescence response of the chemosensor DH toward 17 different metal ions in 20.0 mM HEPES buffer solutions at pH 7.4 (Fig. 1a), a 5-fold increase in fluorescence intensity for DH was observed following the addition of Zn2+ ions. Conversely, no effect on the fluorescence of DH was observed following addition of other metal
Scheme 2. The proposed sensing mechanism of DH and Zn2+ ions.
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Fig. 5. The pH influence on the fluorescence intensity of DH in the absence (black line) and presence of DH-Zn (red line) in HEPES buffer (pH 2.0–12.0) solutions.
Fig. 4. (a) Fluorescence emission spectra of DH-Zn (10.0 μM) in the presence of various anions (20.0 μM) in 20 mM HEPES buffer solution at pH 7.4. (b) The cycle index of DH-Zn (10.0 μM) reacting with H2PO4− (20.0 μM).
Scheme 3. Logic circuit diagram for the chemosensor DH. Fig. 6. (a) The reversibility experiment of DH toward Zn2+ ions by adding Na2EDTA in HEPES buffer (20.0 mM, pH 7.4) solutions. (b) The cycle index of DH-Zn (10.0 μM) reacting with Na2EDTA (10.0 μM).
ions, including Ag+, Al3+, Ba2+, Ca2+, Cd2+, Co2+, Cr3+, Cu2+, Fe3+, Hg2+, K+, Mg2+, Mn2+, Na+, Ni2+ and Pb2+, indicating that the sensor is highly-selective for Zn2+ in a pure water system. Next, the effect of the presence of other cations with Zn2+ ion 4
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Zn2+ ions (0–1.0 equiv.), and the fluorescence titration curve of DH was saturated when 0.5 equiv. Zn2+ were added to the DH solutions (Fig. 2b), which exhibited 2:1 stoichiometric complexation between DH and Zn2+ ions. In addition, under UV excitation at 365 nm, the fluorescence of DH was significantly enhanced and showed bright green when Zn2+ was added to DH solutions (Inset of Fig. 2b). The stoichiometric binding of Zn2+ with the prepared chemosensor DH was investigated by Job's plot method. As seen in Fig. 3, a maximum absorbance was observed when the molar fraction of Zn2+ reached 0.33, confirming a 2:1 coordination stoichiometry of chemosensor DH with Zn2+ in 20.0 mM HEPES buffer solutions. The binding constant for DH with Zn2+ was determined to be 7.27 × 108 M−2 (R2 = 0.9978) based on the titration curve of the chemosensor DH with Zn2+ ions (Fig. S7). Based on the plot, the detection limit for Zn2+ ions was calculated to be 19.25 nM based on the definition of IUPAC (LOD = 3σ/k), which is significantly below the guideline (76.0 μM) WHO protocol (Fig. S8). Moreover, the binding stoichiometry between the chemosensor DH and Zn2+ was further confirmed by high resolution mass spectrum (HRMS), and the appearance of a peak at m/z 837.20 that was assigned to [2DH+ Zn2+ − H+]+ in the ESI-MS spectra (Fig. S9). These experimental results further confirmed that the binding ratio between chemosensor DH and Zn2+ ions was 2:1. In addition, 1H NMR studies also provide direct evidence of the interactions between DH and Zn2+ ions (Fig. S10). When 1.0 equiv. of Zn2+ ions was added, the different chemical shifts in H (a, b) were observed, the proton (a) on imidazole group almost disappeared(a’), the single nuclear magnetic peak (b) of primary amino group is divided into two peaks(b’), and the proton of dansyl have little change, which indicated that Zn2+ ions coordinates with imidazole group and primary amino group (Fig. S10). These results suggest that the donating ability of the imidazole groups in the PET quenching of the dansyl emitter. When Zn2+ ions coordinated with imidazole groups, the recovery of intensity of dansyl emitter may be induced by the blocking of electron transfer effect.
Fig. 7. The time dependence of DH (10.0 μM) on detecting Zn2+ ions (0.5 equiv) and Na2EDTA (1.0 equiv) in HEPES buffer (20.0 mM, pH 7.4) solutions.
3.3. Recognition mechanism We improved the verification experiments: when 10.0 μM dansyl chloride solution and 10.0 μM DH was added to the 20.0 mM HEPES buffer solutions respectively, we found that the fluorescence intensity of dansyl chloride was significantly higher than the fluorescence intensity of DH (Fig. S11), these results suggest that the donating ability of the imidazole groups in the PET quenching of the dansyl emitter. Then, 20.0 equiv. L-His solution and 20.0 equiv. 2-methylimidazole was added to 10.0 μM dansyl chloride solution in 20.0 mM HEPES buffer respectively, the fluorescence intensity of dansyl chloride was significantly declined(Fig. S11), the suggests that exist quenching effect of imidazole group to dansyl. The proposed mechanism for detection of Zn2+ ions by DH is illustrated in Scheme 2. Due to the photoinduced electron transfer (PET) from histidine to the dansyl group, the fluorescence of the chemosensor DH was very weak in the initial state. The efficiency of electron transfer from donor to fluorescent group was reduced following binding of Zn2+ ions to DH. As a result, the original intensity of the fluorescent cluster was restored based on the fluorescence quenching caused by PET was interrupted. The fluorescence signal of DH is highly sensitive based on PET mechanism, and can be selectively used for the detection of Zn2+ ions [39].
Fig. 8. MTT assay of DH. Error bars represent standard deviations from three repeated experiments.
detection was studied. Competitive experiments with different cations on the DH-Zn system indicated that the sensitive detection of DH to Zn2+ was not affected by the presence of other metal ions, up to 5.0 equiv. (Fig. 1b), indicating strong anti-interference by DH. Moreover, a comparison of five different Zn2+ salts demonstrated no differences in fluorescence intensity when combined with the chemosensor DH (Fig. S5.), confirming that the fluorescence response of DH was specific to Zn2+ itself, and not to its ligand anions. 3.2. Binding mode The absorption spectra of DH in HEPES buffer solutions displayed maxima at 240, 255 and 330 nm (Fig. S6), and all absorbance bands increased simultaneously upon addition of 0.5 equiv. Zn2+ in DH solutions. These results suggested that DH participated in the coordination of Zn2+ ions. Correspondingly, a significant increase in fluorescent intensity of DH was observed (Fig. 2a) with increasing concentration of
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Fig. 9. The fluorescence images of living HK2 cells. (A) Cells incubated with 20.0 μM DH for 30 min; (B) Cells were incubated with 20.0 μM DH for 30 min and subsequently treated with 10.0 μM Zn2+ ions for another 30 min; (C) Cells were incubated with 20.0 μM DH for 30 min and subsequently treated with 10.0 μM Zn2+ ions for another 30 min and incubation with 20.0 μM Na2EDTA for another 30 min; Bright-field transmission images (A1, B1, C1), Fluorescent-field transmission images (A2, B2, C2), Merged transmission images (A3, B3, C3).
3.4. Fluorescence spectra studies of DH-Zn toward H2PO4−
On the basis of the fact that the emission intensity of DH could be effectively controlled by Zn2+ and H2PO4−, the logic gate properties with two input signals as Zn2+ (In1) and H2PO4− (In2), DH had the ability to exhibit INHIBIT function as output signal (Scheme 3). The presence and absence of Zn2+ or H2PO4− were defined as ‘1′ and ‘0′ for inputs, while turn-on and turn-off as ‘1′ and ‘0′ for outputs, respectively. From the perspective of logic functions, the turn-on of 1 (output‘1′) could be observed only in the presence of Zn2+ ions (In1 = 1) and in the absence of H2PO4− (In 2 = 0).
The fluorescence spectra responses of DH-Zn toward various anions such as F−, Cl−, Br−, I−, SCN−, AcO−, NO3−, PO43−, S2O32−, OH−, HCO3−, CO32−, SO42−, HPO42− and H2PO4− (2.0 equiv. each), were also researched in 20.0 mM HEPES buffer solutions as shown in Fig. 4a, the trong emission intensity was observed when DH was treated with 0.5 equiv. of Zn2+, and the fluorescence was totally quenched by the subsequent separate addition of H2PO4−. However, the addition of other anions above mentioned induced only insignificant responses on the fluorescence spectra. Hence, the chelate DH-Zn can be utilized as a new platform to recognize H2PO4− with high selectivity over other species. In addition, The reversibility experiments of DH (10.0 μM) with Zn2+ (5.0 μM) and H2PO4− (20.0 μM) were detected in HEPES buffer solutions (20.0 mM, pH 7.4). As illustrated in Fig. 4b, the reversibility of DH were further verified during 8 cycles of titration by alternatively adding Zn2+ and H2PO4− to the DH solutions, and the circulation effect was very good.
3.5. pH effects The effects of pH change on the chemosensor DH and DH-Zn system were studied. As depicted in Fig. 5, the fluorescence spectra of the chemosensor DH exhibited no obvious change in the pH range 2.0 to 12.0. At the same time, the change of pH from 2.0 to 5.0 did not affect the fluorescence response of chemosensor DH to Zn2+ ions. However, upon addition of Zn2+ ions, the fluorescence intensity of the
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[13] [14] [15] [16] [17] [18] [19] [20] [21] [22] This work
chemosensor DH was significantly enhanced in the range of pH 6.0–14.0, which showed that the change of pH had little effect on chemosensor DH, and DH could sensitively detect Zn2+ ions over a broad range of pH values.
No Yes No Yes Yes No No Yes Yes Yes Yes
3.6. Reversibility experiment Reversibility is a prerequisite in developing novel chemosensors for practical applications. Thus, the reversibility and cyclic nature of the binding process of chemosensor DH toward Zn2+ ions was confirmed by adding the bonding agent Na2EDTA. The addition of Na2EDTA to a mixture of DH-Zn solutions resulted in diminution of the fluorescence intensity (Fig. 6a), which indicated regeneration of the chemosensor DH. Similarly, the fluorescence intensity of DH exhibited significant fluorescence enhancement when Zn2+ was added again. As seen in Fig. 6b, DH still exhibited a strong fluorescence intensity response toward Zn2+ ions and Na2EDTA after at least 10 cycles, with small switchable changes following alternating additions of Zn2+ ions and Na2EDTA. An ideal chemosensor should exhibit a fast fluorescence response. The response times of DH toward Zn2+ ions were investigated as depicted in Fig. 7, and the fluorescence intensity of DH was rapidly enhanced and quenched within 20 s when Zn2+ and Na2EDTA were added, respectively, while the fluorescence intensity of DH exhibited no change when the action time was 180 s.
7.02 × 10−8 M 11.9 μM 8.56 × 10−9 M 3.5 × 10−8 M 0.1 μM/0.5 μM 10.5 nM 1.25 × 10−7 M 1.603 × 10−10 M 89.3 nM 4.9 nM 19.25 nM THF (2.0 × 10−5mol L−1) MeCN/bis-tris buffer (95:5) CH3CN DMSO:H2O (1:1, v/v) solution CH3CN-PBS (1/99, v/v) solution HEPES buffer at pH 7.4 containing 10% (v/v) of DMSO EtOH/HEPES solution (3/1, 10.0 μM HEPES, pH 7.4) Ethanol-water (V:V = 1:1) solution EtOH/HEPES (10 mM, pH = 7.40, 3/7, v/v) solution HEPES buffer solutions (10 mM, pH = 7.4) HEPES buffer solutions (20 mM, pH = 7.4) Diarylethene derivative ((E)-1-((2-hydroxy-3-methoxybenzylidene)amino)imidazolidine-2,4-dione) 2-Amino-3-pyridinecarboxaldehyde N′-((8-hydroxy-1,2,3,5,6,7-hexahydropyrido [3,2,1-ij]guinolin-9-yl)methylene)-4-methylbenzohydrazide Fluorescein hydrazide pyrene-appended naphthalimide-dipicolylamine 6 hydroxychromone 3 carbaldehyde (rhodamine B carbonyl) hydrazine Chromene pyrazoline derivatives Quinoline containing acetyl hydrazone Dansyl-tetrapeptide Dansyl-histidine
Turn-on Turn-on Turn-on Turn-on Turn-on Ratiometric Turn-on OFF-ON Turn-on OFF-ON Turn-on
Detection limit Detection condition Zn2+ chemosensor
Table 1 Comparison of chemosensors for Zn2+ assays reported in the literature.
Detection method
cell imaging
Refs.
P. Wang and J. Wu
3.7. Application in bioimaging In order to test the possibility of applying the chemosensor DH in biological systems, the cytotoxicity test (MTT) was first carried out in HK2 cells. A range of concentrations (5.0, 10.0, 20.0, 40.0, and 80.0 μM) of chemosensor DH were added to HK2 cells, and incubated for 24 h in 5% CO2 at 37 °C. The survival rate of HK2 cells exceeded 90% (Fig. 8) even in the presence of high concentrations of DH, which indicated that DH has good biocompatibility and low toxicity, and has the potential to be used in live cell imaging. The effects of Zn2+ ions and Na2EDTA on the fluorescence intensity of DH inside HK2 cells were investigated by confocal microscopy (Fig. 9). A dim, weak fluorescence response was observed in HK2 cells incubated only with the chemosensor DH (20.0 μM) for 30 min at 37 °C, after washing three times with PBS buffer solutions (Fig. 9A). Conversely, a bright green fluorescence was observed following introduction of exogenous Zn2+ (10.0 μM) and incubation with DH (20.0 μM) for another 30 min at 37 °C (Fig. 9B). Finally, the subsequent addition of 20.0 μM Na2EDTA to the DH-Zn cells, and incubation of reaction mixtures at 37 °C for another 30 min, resulted in elimination of the fluorescence intensity in living cells (Fig. 9C). Summarily, these cell imaging studies strongly demonstrated that the chemosensor DH has potential applications in tracking Zn2+ and Na2EDTA levels in living cells. 4. Conclusion A new fluorescent chemosensor DH was readily synthesized by solid phase peptide synthesis with high yield. Compared to other chemosensors (Table 1 and Table 2), DH selectively detected Zn2+ from among various relevant metal ions in aqueous solutions at physiological pH, and a LOD of 19.25 nM was calculated for Zn2+ in the linear response range from 0 μM to 14.0 μM. On the basis of the Job’s plot, fluorescent titration and ESI-mass spectral analysis, formation of a 2: 1 (DH : Zn2+) complex was proposed. In addition, the reversible and recyclable processes of the detection of DH toward Zn2+ ions were confirmed by adding bonding agent Na2EDTA. Moreover, DH exhibited low cytotoxicity, good cell permeability and stability, and was successfully applied for imaging and monitoring of Zn2+ and Na2EDTA in live HK2 cells by using confocal laser scanning microscope. 7
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Table 2 Comparison of peptide/dansyl based fluorescence chemosensors for Zn2+ assays in the literatures. Zn2+ chemosensor
Sensing mechanism
Sensing mechanism
Detection limit
Detection method
Refs.
HEPES buffer solutions (10 mM, pH 7.4)
PET
4.9 nM
Turn-on
[22]
HEPES buffer solutions (10 mM, pH 7.4)
PET
18 nM
Turn-on
[35]
Aqueous Solutions
FRET
97 nM
Ratiometric
[36]
HEPES buffer solutions (10 mM, pH 7.4)
PET
32 nM
Turn-on
[38]
HEPES buffer solutions (20 mM, pH = 7.4)
PET
36.8 nM
Turn-on
[40]
HEPES buffer solutions (20 mM, pH = 7.4)
PET
19.25 nM
Turn-on
This work
Declaration of Competing Interest
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