Benzothiazole-based fluorescence chemosensors for rapid recognition and “turn-off” fluorescence detection of Fe3+ ions in aqueous solution and in living cells

Microchemical Journal 152 (2020) 104351

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Benzothiazole-based fluorescence chemosensors for rapid recognition and “turn-off” fluorescence detection of Fe3+ ions in aqueous solution and in living cells

T

Xue Gonga, Xin Dingb, Nan Jianga, Tianyuan Zhonga, Guang Wanga,

⁎

a b

Faculty of Chemistry, Northeast Normal University, Changchun 130024, PR China Faculty of Chemistry and Chemical Engineering, Inner Mongolia University for Nationalities, Tongliao 028043, PR China

ARTICLE INFO

ABSTRACT

Keywords: Benzothiazole Fluorescence sensors Fe3+ ions Cell imaging

Two new benzothiazole-based fluorescence sensors (sensor 1 and sensor 2) have been successfully synthesized and their structures have been well characterized by 1H NMR, 13C NMR and mass spectrometry. Sensor 1 and sensor 2 displayed a selective fluorescence quenching response towards Fe3+ ions in aqueous solutions with good anti-interference ability. Sensor 1 and sensor 2 exhibited lower detection limits for Fe3+, 8.43 μM for sensor 1 and 5.86 μM for sensor 2, respectively. The Job's plot, mass spectrometry, 1H NMR titration and DFT calculations proved that sensors formed 1:1 complexes with Fe3+ ions. In addition, sensor 1 and Sensor 2 had low cell toxicity and could be used to monitor the existence of Fe3+ ions in living cells.

1. Introduction

on the nitrogen and sulfur atoms, which provides the complexing possibility with different metal ions [34–36]. In this work, we designed and prepared two novel fluorescence chemosensors (sensor 1 and sensor 2) by combining the benzothiazole molecules on 5-formylsalicylic acid. The synthesized sensors displayed selective “on-off” fluorescence response towards Fe3+ ions in aqueous solution. Their molecular structures were well characterized with 1H NMR, 13C NMR, CE-MS and FT-IR spectra. Fluorescence and UV–vis absorbance spectra demonstrated the highly selective response of sensor 1 and sensor 2 towards Fe3+ions in aqueous solution. The Job's plot, CE-MS, 1H NMR titration and DFT calculations demonstrated that the fluorescence quenching of sensors were caused by the formation of 1:1 complexes between sensors and Fe3+ ions. In addition, sensor 1 and sensor 2 displayed low cytotoxicity and could monitor the existence of Fe3+ ions in living cells via fluorescence images.

Iron is an important mineral needed by the human body and is an essential element in the formation of human cells. Iron ions play critical role in many biochemical processes, for example, iron ions play an important role in oxygen transfer in hemoglobin, iron element also contributes to the storage and use of oxygen in muscles. In addition, iron ions are involved in the formation of enzymes [1–5]. Therefore, if the content of iron ions in the human body is out of balance, it will lead to various physiological diseases. Insufficient iron ions in the human body will lead to iron deficiency anemia, decline in immunity and slow liver development. Excess iron in human body, which was mainly caused by environmental pollution, can cause hemochromatosis and diabetes etc. [6–10]. Therefore, in order to maintain the balance of iron content in environment and human body, it is of great important to develop methods with high selectivity, sensitivity and simple operation to monitor and detect iron ions in environment and most organisms. In recent years, fluorescence chemosensors have been rapidly developed due to their advantages of high selectivity and sensitivity, insitu monitoring, low cost and simple operation [11–19]. So far, various types of fluorescence chemosensors for detecting iron ions have been reported, such as Schiff bases [20–22], coumarin derivatives [23–29], oxadiazole derivatives [30–33], etc. However, sensors based on benzothiazole are rarely reported. The benzothiazole molecule not only has high fluorescence quantum yield, but also has the lone pair of electrons ⁎

2. Experimental details 2.1. Materials and apparatus All of the solvents and chemicals used in this work were supplied by local commercial suppliers and used without further purification. 5formylsalicylic acid used in the experiment was purchased from Alpha chemical company. Thin layer chromatography (TLC) was performed on silica coated alumina plates. Ultra-pure water used in test was

Corresponding author. E-mail address: [email protected] (G. Wang).

https://doi.org/10.1016/j.microc.2019.104351 Received 24 June 2019; Received in revised form 18 September 2019; Accepted 19 October 2019 Available online 22 October 2019 0026-265X/ © 2019 Elsevier B.V. All rights reserved.

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prepared using a Milli-Q water purification system. 1 H NMR and 13C NMR spectra were obtained on Varian Unity Inova Spectrometer 600 MHz. CE-MS spectra were obtained on CESI 8000 Plus – Triple TOF4600 mass spectrometer. UV–vis absorption spectra and the fluorescence spectra were recorded on a Varian Cary 500 Spectrophotometer and a Cary Eclipse Fluorescence Spectrophotometer at room temperature, respectively. IR spectra were obtained on Nicolet 6700 FT-IR. Fluorescent photographs were obtained under irradiation of 365 nm UV light. The pH values of solutions were measured using a digital pH meter model PHS-2 F of Ray Magnetic Company.

prepared in acetonitrile and DMF, respectively. During testing, the stock solutions were diluted with Tris–HCl (10 mM, pH = 7.4) buffer solution to a concentration of 1 × 10−5 M (sensor 1) and 5 × 10−6 M (sensor 2), respectively. Stock solutions of metal ions (2 × 10−2 M) in distilled water were prepared using AgNO3, KNO3, Cd(NO3)2, Co (NO3)2•6H2O, Zn(NO3)2•6H2O, Mg(NO3)2•6H2O, Al(NO3)3•9H2O, Cr (NO3)3•9H2O, Fe(NO3)3•9H2O, Ni(NO3)3•6H2O, CuCl2•2H2O, NaCl, HgCl2, PbCl2, CaCl2, MnCl2•4H2O, BaCl2•2H2O, and FeSO4•7H2O, respectively. During the selective test, 3 ml of the sensor solution and 30 μl of the metal ion solution were sequentially added to the cuvette, after shaking for 60 s, the UV–vis and fluorescence spectra were measured at room temperature. The measurement conditions of fluorescence spectra for the two sensors: For sensor 1, the excitation wavelength was 319 nm, both excitation and emission slits were 5 nm; for sensor 2, the excitation wavelength was 345 nm, the slits of excitation and emission were 2.5 nm and 10 nm, respectively.

2.2. Synthesis 2.2.1. Synthesis of 5-benzothiazolyl salicylic acid (sensor 1) 5-formylsalicylic acid (0.5976 g, 3.6 mmol), o-aminothiophenol (0.45 g, 3.6 mmol) and sodium pyrosulfite (0.57 g, 3 mmol) were dissolved in 10 ml dry DMF and stirred 2 h at 110 °C. The reaction solution turned red and poured into cold water after cooled down to room temperature. The red precipitate was filtered and redissolved in dichloromethane. The organic solution was dried with anhydrous sodium sulfate and then purified by column chromatography (eluent: ethyl acetate: petroleum ether, 1:18, v:v) to give 5-benzothiazolyl salicylic acid (sensor 1) as white solid 0.64 g in 65.95% yield. Mp 328 °C;1H NMR (600 MHz, DMSO, ppm) δ 8.42 (s, 1 H), 8.09 (d, J = 8.0 Hz, 1 H), 8.04 (d, J = 8.0 Hz, 1 H), 7.99 (d, J = 8.0 Hz, 1 H), 7.50 (t, J = 7.2 Hz, 1 H), 7.40 (t, J = 7.3 Hz, 1 H), 6.94 (d, J = 8.7 Hz, 1 H);13C NMR (600 MHz, DMSO, ppm) δ 154.15, 129.79, 127.00, 125.50, 122.85, 122.68, 118.51. CE-MS calcd for C14H9NO3S 271.0303 (sensor 1+, 100%), found 271.9958 m/z ([sensor 1 + H+]+, calcd 272.0383).

2.4. Job's plot measurements The buffer solution of acetonitrile-Tris (10 mM, pH = 7.4, 4:1, v/v) for sensor 1 / Fe3+ and DMF-Tris (10 mM, pH = 7.4, 1:1, v/v) for sensor 2 / Fe3+were prepared to implement the Job's plot experiments. The sum of concentration of sensor and Fe3+ were unchanged (1 × 10−5 M for sensor 1 and 5 × 10−6 M for sensor 2, respectively), the fluorescence intensities were measured under the changing concentration ratio of the sensor molecule and Fe3+ ions. The fluorescence intensity was plotted against the molar fraction of the sensor molecule. The molar ratio corresponding to the highest point or inflection point on the Job's plot was the coordination ratio of the sensor to the Fe3+ions.

2.2.2. Synthesis of ethyl 5-benzothiazolyl-2-hydroxybenzoate (compound 1) 5-benzothiazolyl salicylic acid (2.7103 g, 10 mmol) and p-toluenesulfonic acid (0.3252 g, 1.7 mmol) were dissolved in 10 ml ethanol and stirred 4 h at 80 °C. The mixture was poured into 50 ml ice water and a large amount of white precipitate appeared. The solution was adjusted to neutral with sodium carbonate powder and was extracted with dichloromethane for three times. The organic solution was dried with anhydrous sodium sulfate, the purified product was obtained by column chromatography with ethyl acetate: petroleum ether (1:2, v:v) to obtain ethyl 5-benzothiazolyl-2-hydroxybenzoate (compound 1), 1.27 g, 42.47% yield. Mp 127–128 °C;1H NMR (600 MHz, DMSO, ppm) δ 10.98 (s, 1 H), 8.48 (d, J = 2.4 Hz, 1 H), 8.19 (d, J = 8.7, 2.4 Hz, 1 H), 8.14 (d, J = 7.9 Hz, 1 H), 8.05 (d, J = 8.1 Hz, 1 H), 7.55 (s, 1 H), 7.46 (s, 1 H), 7.19 (d, J = 8.7 Hz, 1 H), 4.44 (q, J = 7.1 Hz, 2 H), 1.40 (t, J = 7.1 Hz, 3 H).

3. Results and discussion 3.1. Synthesis The synthetic routes for sensor 1 and sensor 2 were shown in Scheme 1. Firstly, sensor 1 was prepared via the reaction of 5-formylsalicylic acid with o-aminothiophenol in DMF solution. Then, the sensor 1 and ethanol were esterified to prepare the compound 1. Finally, the compound 1 and Tris were stirred and refluxed together in ethanol to prepare the sensor 2. The molecular structures of sensor 1 and sensor 2 were well characterized by 1H NMR, 13C NMR, CE-MS and FT-IR. All spectra dates were displayed in the supporting information. 3.2. Spectral properties of sensor 1 and sensor 2 towards Fe3+ ions The selectivity of fluorescence response towards target metal ions is first considered to evaluate the sensor's performance. Generally, the factors such as polarity, protophilia, solubility and pH value of solvents affect the selective response. Therefore, the following solvent systems, including C2H5OH:H2O (1:1 and 4:1, v:v), CH3CN:H2O (1:1 and 4:1, v:v), DMF:H2O (1:1, v:v) and pure water solvent at pH = 7.4, were selected to optimize the response selectivity (As shown in Fig. S8). Finally, the following optimized solvents provided satisfactory fluorescence selectivity, CH3CN: Tris (10 mM, pH = 7.4, 4:1, v:v) for sensor 1 and DMF: Tris (10 mM, pH = 7.4, 1:1, v:v) for sensor 2, respectively. Sensor 1 and sensor 2 possessed the same conjugated unit of benzene-benzothiazole, so they presented the similar fluorescence spectra.

2.2.3. Synthesis of 5-benzothiazolyl-N-(1, 3-dihydroxy-2-(hydroxymethyl) propan)- 2-hydorxybenzamide (sensor 2) Compound 1 (1.49 g, 5 mmol) and Tris (0.60 g, 5 mmol) were added to 10 ml ethanol and stirred at 80 °C for 8 h. The mixed solution was concentrated under vacuum and further purified by column chromatography with dichloromethane: methanol (6:1, v:v) to obtain 5-benzothiazolyl-N-(1, 3-dihydroxy-2- (hydroxymethyl) propan)-2-hydorxybenzamide (sensor 2) as light yellow solid, 0.89 g, 47.59% yield. Mp 238–239 °C;1H NMR (600 MHz, DMSO, ppm) δ 8.57 (s, 1 H), 8.09 (d, J = 7.9 Hz, 1 H), 8.01 (d, J = 8.0 Hz, 1 H), 7.97 (d, J = 8.2 Hz, 1 H), 7.50 (t, J = 7.7 Hz, 1 H), 7.40 (t, J = 7.4 Hz, 1 H), 6.99 (s, 1 H), 5.00 (s, 3 H), 3.67 (s, 6 H);13C NMR (600 MHz, DMSO, ppm) δ 172.62, 159.00, 139.29, 124.62, 170.33, 67.76, 65.71, 64.31. CE-MS calcd for C18H18N2O5S 374.0936 (sensor 2+, 100%), found 375.0261 m/z ([sensor2 + H+]+, calcd 375.1016). 2.3. UV–vis absorbance and fluorescence spectra measurements

Scheme 1. Synthesis of sensor 1 and sensor 2.

The stock solutions (1 × 10−3 M) of sensor 1 and sensor 2 were 2

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Fig. 1. (A) Fluorescence intensity after adding 20 equiv. of different metal ions to sensor 1 solution (1 × 10−5 M, CH3CN:H2O, 4:1, v:v, Tirs–HCl 10 mM, pH = 7.4) (λex = 319 nm). (B) Fluorescence intensity after adding 40 equiv. of different metal ions to sensor 2 solution (5 × 10−6 M, DMF: H2O, 1:1, v: v, Tirs-HCl 10 mM, pH = 7.4), (λex = 345 nm).

Fig. 2. (A) Fluorescence photographs of sensor 1 (CH3CN:H2O, 4:1, v:v, Tirs–HCl 10 mM, pH = 7.4) and (B) sensor 2 (DMF:H2O, 1:1, v:v, Tirs-HCl 10 mM, pH = 7.4) in the presence of 20 and 40 equiv. of different metal ions.

As shown in Fig. 1, both sensor 1 (1 × 10−5 M) and sensor 2 (5 × 10−6 M) exhibited strong fluorescence emission centered at 411 nm and 421 nm, respectively. The little different of peak values were caused by the substitution groups on their molecular structures. After the addition of different metal ions including Al3+, Fe2+, Mn2+, Zn2+, Hg2+, Na+, Mg2+, Cr3+, Ca2+, Co2+, K+, Ag+, Ni3+, Ba2+, Cu2+, Pb2+, Cd2+and Fe3+ to sensor solutions, only Fe3+ ions caused the obvious fluorescence quenching of both sensor 1 and sensor 2, which indicated that sensor 1 and sensor 2 had good fluorescence response towards Fe3+ over other metal ions. In addition, the fluorescence intensities of sensor 1 and sensor 2 decreased rapidly to the equilibrium point within 1 min after adding Fe3+ ions, which indicated that sensor 1 and sensor 2 could meet the requirement of real-time detection. The quenching effect of Fe3+ towards the fluorescence of two sensors was intuitively seen by fluorescent photographs under 365 nm UV illumination, shown in Fig. 2. The fluorescent photographs showed that iron ions displayed the obvious fluorescence quenching effect on both sensor 1 and sensor 2, comparatively, this effect on sensor 2 was stronger than that of sensor 1 due to the stronger blue fluorescence of sensor 2. The fluorescence quenching of sensor 1 and sensor 2 was caused by their selective complexing effect with Fe3+ ions. The changes of UV–vis absorbance spectra of sensor 1 and sensor 2

upon the additions of the above mentioned metal ions also indicated that both sensor 1 and sensor 2 had selective coordination property towards Fe3+ ions, as shown in Fig. S9. The solutions of sensor 1 and sensor 2 exhibited the similar absorbance bands in the range 260–400 nm centered at 346 nm. After the addition of Fe3+ ions, the absorbance bands were significantly enhanced and displayed obvious blue-shifts. On the contrary, the other above mentioned metal ions did not induce the obvious absorbance changes. Here, there were two special cases, the addition of Pb2+ and Ag+ ions caused the remarkable increasing of absorbance band, which was caused by the appearance of the white precipitates in the solutions. This reason could be confirmed by the increasing of the absorbance bands of the blank solutions without sensors after the additions of Pb2+ and Ag+ ions (Fig. S9). The selective complexation of sensor 1 and sensor 2 with Fe3+ ions was due to the suitable atomic radius, charge and binding energy of Fe3+ ions with sensor 1 and 2 over the other metal ions. The Job's plot could be used to assist in proving the existence of complexation reaction and measuring the complexation stoichiometry. The Job’ plots of sensor 1 and sensor 2 with Fe3+ ions were shown in Fig. 3. The I0/I were plotted as a function of the mole fraction of sensor 1 or sensor 2 at a constant total concentration of sensor and metal ions. The maximum values appeared at 0.5 of the abscissa indicating the 1:1 3

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Fig. 3. Job's plots of sensor 1 (A) and sensor 2 (B). Fluorescence intensities of sensor 1 at 411 nm and sensor 2 at 421 nm were plotted as a function of the molar ratio [S] / ([S] + [Fe3+]) ([S] represents the concentration of sensor 1 or sensor 2).

binding mode of both sensor 1 and sensor 2 with Fe3+ ions. Competitive experiments are often used to assess the interference effect of the other concomitant metal ions on the performance of chemosensors. An equal amount of competing ions and Fe3+ ions (20 equiv. for sensor 1 and 40 equiv. for sensor 2) were simultaneously added into sensor solutions to compare the change of fluorescence intensities. As shown in Fig. 4, the addition of Cu2+, Pb2+ and Ag+ ions brought some interference in the fluorescent detection, which was due to the paramagnetic property of copper ion and appearance of turbidity in solution after the addition of Pb2+ and Ag+ ions (refer to the above explanation about UV–vis spectra changes and Fig.S9). The other competing ions just displayed the negligible influence on the fluorescence quenching effect of Fe3+ ions on sensor 1 and sensor 2. The above results illustrated that sensor 1 and sensor 2 had strong anti-interference ability and can be used as fluorescence sensors to recognize Fe3+ ions in actual samples. The pH has a great influence on the fluorescence performance of sensors. To investigate the effect of pH on the fluorescence of sensors, the series of the solution of sensors in the presence and absence of Fe3+ ions at different pH values (3–12) were prepared. For sensor 1 series, the solutions of sensor 1 (1 × 10−5 M) and sensor 1 + Fe3+ ions (1 × 10−5 M sensor 1 + 20 equiv. of Fe3+ ions) with different pH values were prepared in CH3CN:H2O solvent (4:1, v:v, Tirs-HCl 10 mM); for sensor 2 series, the solutions of sensor 2 (5 × 10−6 M) and sensor 2 + Fe3+ ions (5 × 10−6 M sensor 2 + 40 equiv. of Fe3+ ions) with

different pH values were prepared in DMF:H2O solvent (1:1, v:v, TirsHCl 10 mM). And after, their fluorescence spectra were recorded and their fluorescence intensity at 411 nm for sensor 1 and 421 nm for sensor 2 was plotted as functions of pH values, as shown in Fig. 5. The pH values exerted different influence on the fluorescence of sensor 1 and sensor 2. The fluorescence intensity of sensor 1 was little affected by pH values in the test pH range. On the contrary, the fluorescence intensity of sensor 2 obviously increased with the increasing of pH and reached to the equilibrium point (Fig. 5A and Fig. 5B). After addition of Fe3+ ions, the fluorescence of sensor 1 and sensor 2 at all test pH values was markedly quenched. The quenching effect in acidic range was larger than that in the alkaline range. Especially, the fluorescence of sensor 2 was almost quenched in the acidic range. The relatively small quenching effect of Fe3+ on the fluorescence of sensors in the alkaline range may be caused by the partly formation of iron hydroxide precipitation and further reduced the binding ability with sensors. Based on the comprehensive consideration of the above results, pH = 7.4 was finally selected as the best test pH value. Fluorescence titration experiments were performed to assess the sensing property of sensors towards Fe3+ ions. Upon the incremental addition of Fe3+ ions into the solutions of sensor 1 and sensor 2, all the fluorescence of sensor 1 and sensor 2 were gradually quenched, the results were shown in Fig. 6. The fluorescence emission was quenched by 82% for sensor 1 in the presence of 25 equiv. Fe3+ and 80% for sensor 2 in the presence of 60 equiv. Fe3+, which indicated that the two

Fig. 4. (A) Fluorescence intensity at 411 nm of sensor 1 (in CH3CN:H2O solution, 4:1, v:v, Tirs–HCl 10 mM, pH = 7.4) containing 20 equiv. of iron ions and 20 equiv. of other competing ions. (B) Fluorescence intensity at 421 nm of sensor 2 (in DMF:H2O solution, 1:1, v:v, Tirs–HCl 10 mM, pH = 7.4) containing 40 equiv. of iron ions and 40 equiv. of other competing ions. 4

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Fig. 5. Fluorescence intensity (A) at 411 nm of sensor 1 (1 × 10−5 M in CH3CN: H2O, 4:1, v:v, Tirs–HCl 10 mM, pH = 7.4) and (B) at 421 nm of sensor 2 (5 × 10−6 M in DMF:H2O, 1:1, v:v, Tirs–HCl 10 mM, pH = 7.4) in the presence and absence of Fe3+ (20 equiv.) and (40 equiv.) at different pH values, respectively.

of Fe3+ ions. The Benesi-Hildebrand plots were drawn using 1 / (F0-F) as function of 1 / [Fe3+]. As shown in Fig. 8, the fitting curves based on 1:1 stoichiometry displayed good linear pattern with high correlation coefficients, 0.9963 for sensor 1 and 0.9929 for sensor 2, which further confirmed the 1:1 complex ratio between sensor 1 and sensor 2 with Fe3+. The binding constants of sensor 1 and sensor 2 with Fe3+ ions were calculated as 3659 M−1 and 4156 M−1 respectively by the slope and intercept of the line. The larger correlation coefficients indicated that both sensor 1 and sensor 2 possessed higher affinity with iron ions.

sensors had different binding abilities to iron ions. The calibration curves of fluorescence intensities of sensor 1 and sensor 2 at peak positions versus the concentration of Fe3+ ions were plotted [37], as shown in Fig. 7. Both the fluorescence intensity of sensor 1 and sensor 2 displayed good linear relationship with the concentration of Fe3+ ions in the concentration range 50–400 μM with the large correlation coefficient, 0.9107 for sensor 1 and 0.9865 for sensor 2, respectively. The detection limits were calculated according to these calibration curves using the formula of 3σ ⁄ κ to be 8.43 μM for sensor 1 and 5.86 μM for sensor 2. Here, σ is the standard deviation of blank solutions and κ is the slope of calibration curve. Therefore, sensor 1 and sensor 2 could be applied to detect of Fe3+ ions in the wide liner range in actual samples. The several typical features of sensor 1 and sensor 2 were compared with other reported sensors for detecting iron ions (Table 1). As shown in the Table, sensor 1 and sensor 2 possessed better water solubility and low cytotoxicity, so they could be used as probes for the detection of iron ions in live cells. The binding constants of sensor 1 and sensor 2 with Fe3+ ions were determined by Benesi–Hildebrand equation according to the 1:1 stoichiometry [38]:

1 F0

F

=

1 K a × (F0

Fmin ) × C

+

3.3. Study on the combination of sensor 1 and sensor 2 with Fe3+ ions According to above results such as Job's plots and BenesiHildebrand plots, the fluorescence quenching of sensors was due to the formation of 1:1 complexes with Fe3+. To further prove the above conclusions, CE-MS, FT-IR, 1H NMR titrations and DFT calculations were carried out. The mass spectra of the complexes between sensor 1 and sensor 2 with iron ions were tested to demonstrate the 1:1 binding mode. The sensors and excess iron salts were dissolved in ethanol. After sufficient reaction, the supernatant liquid was naturally volatilized in air to obtain solid complexes for testing. The obtained spectra were shown in Fig. S10 and S11: mass spectra gave a molecular-ion peak at m/ z = 343.9916 belonged to [sensor 1+Fe3++H2O-H+]+ species (calcd 343.9678) and m/z = 475.1223 belonged to [sensor

1 F0

Fmin

where F0 and F are the fluorescence intensities in absence and presence of Fe3+ ions, Fmin is the minimum fluorescence intensity in the presence

Fig. 6. (A) The change of the fluorescence spectra of sensor 1 (1 × 10−5 M in CH3CN: H2O, 4:1, v:v, Tirs-HCl 10 mM, pH = 7.4) and (B) sensor 2 (5 × 10−6 M in DMF:H2O, 1:1, v:v, Tirs–HCl 10 mM, pH = 7.4) upon gradual addition of Fe3+ ions. 5

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Fig. 7. The calibration curves of fluorescence intensity of sensor 1 at 411 nm (A) and sensor 2 at 421 nm (B) against [Fe3+]. Table 1 comparison of type of iron sensors and their detection limits. Solvent system

Dissolved in water

Cell imaging

Response

DMF-HEPES(1:1,v/v) THF-HEPES(3:7,v/v) Methanol Na2HPO4-citric acid buffer solution Methanol DMSO-H2O

no no no yes no no

yes yes no yes no yes

turn-on turn-on turn-off turn-off turn-on turn-on

Methanol DMSO-H2O THF-H2O(1:1,v/v) DMSO CH3CN-H2O(4:1,v/v,sensor 1) DMF-H2O(1:1,v/v,sensor 2)

no no no no yes yes

no yes no yes yes yes

turn-on turn-off turn-on turn-off turn-off turn-off

LOD

Reference −6

4.8 × 10 M 1.83 × 10−7 M 4.09 × 10−8 M 3 × 10−7 M -–6.6 × 10−8 M(RDI-1) 4.45 × 10−8 M(RDI-2) 4.8 × 10−8 M 5.11 × 10−7 M 3.88 × 10−7 M 7.6 × 10−7 M 8.43 × 10−6 M 5.86 × 10−6 M

[3] [4] [5] [10] [12] [19] [20] [22] [28] [29] Present work Present work

Fig. 8. Benesi–Hildebrand plots of sensor 1 (A) and sensor 2 (B) upon gradual addition of Fe3+.

2+Fe3++C2H5OH-H+]+ species (calcd 475.0624). The above results further demonstrated 1:1 complexation pattern of sensors with iron ions. The samples for IR testing were prepared as described above. IR spectra of sensors and complexes were shown in Fig. 9. After sensor 1 was combined with Fe3+, the characteristic absorption peaks obviously shifted. The peaks of phenolic hydroxyl group at 3453 cm−1 and 3061 cm−1 shifted to 3318 cm−1 and 3155 cm−1, respectively; the peak of the carbonyl at 1673 cm−1 shifted to 1682 cm−1. Similar to

sensor 1, after binding to Fe3+, the absorption peaks of phenolic hydroxyl group and carbonyl of sensor 2 were significantly shifted. The peaks of phenolic hydroxyl group at 3403 cm−1 and the carbonyl at 1634 cm−1 shifted to 3193 cm−1 and 1673 cm−1, respectively. The above experimental results fully illustrated that Fe3+ bound with oxygen in phenolic hydroxyl group and oxygen in carbonyl group to form complexes. The possible combination modes of two sensors with Fe3+ were further investigated by 1H NMR titration. Fig. 10 showed the peak shift 6

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Fig. 9. IR spectra of sensor 1 (A), sensor 1+Fe3+ (B), sensor 2 (C), sensor 2+ Fe3+ (D).

of hydrogen atoms after different amounts of Fe3+ ions were added to the solution of sensor 1. The phenyl protons (Ha and Hb) obviously shifted downfield upon the addition of Fe3+ions (Δδ: Ha, 0.154 ppm; Hb, 0.038 ppm). A new proton peak (Hc) appeared at 9.735 ppm, which was ascribed to the hydroxyl hydrogen on the carboxyl group released

after breaking of the intramolecular hydrogen bond, as shown in Fig. 10. These results illustrated that the oxygen atoms on both the carbonyl group and the phenolic hydroxyl group of sensor 1 participated in the coordination with Fe3+ ions. Similar to sensor 1, the phenyl proton hydrogens (Ha and Hb) in the

Fig. 10. 1H NMR spectra of sensor 1 upon the addition of 0 equiv., 0.5 equiv., 1 equiv., 1.2 equiv. of Fe3+ and the possible combination mode between sensor 1 and Fe3+. 7

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Fig. 11. 1H NMR spectra of sensor 2 containing 0 equiv., 0.5 equiv., 1 equiv., 1.2 equiv. of Fe3+ and the possible combination mode between sensor 2 and Fe3+.

molecule of sensor 2 also shifted downfield after the addition of Fe3+ ions (Δδ: Ha, 0.118 ppm; Hb, 0.067 ppm). After binding to Fe3+, phenolic hydroxyl proton (Hc) was also observed at 8.686 ppm due to the cleavage of intramolecular hydrogen bond (Fig. 11). The hydrogen peaks of methylene on trihydroxymethylaminomethane unit (Tris) did not shift after addition of Fe3+ ions, which indicated that its hydroxyl did not participate in the coordination of sensor 2 with Fe3+ ions. These results also confirmed that sensor 1 and sensor 2 had similar coordination modes with Fe3+ ions. The proposed possible combination mode between sensor 2 and Fe3+ ions was shown in Fig. 11. To further clarify the coordination modes of sensor 1 and sensor 2 with Fe3+ ions, density functional theory (DFT) calculations at the B3LYP/6–31 G(d) level of Gaussian 09 package were carried out to provide theoretical evidence of interaction between sensors and iron ions. Here, the molecular modes of sensor 1 and sensor 1 + Fe3+ were optimized as representative to illustrate aforesaid coordination mode. The molecular structures of sensor 1 and its complex with Fe3+ ions were shown in Fig. 12, which fully demonstrated that sensor 1 formed

1:1 complex via the coordination of the oxygen atoms of the phenolic hydroxyl and the carbonyl of sensor 1 with Fe3+ ions. The orbital energy levels (HOMO and LUMO) also provided the formation evidences of sensor 1-Fe3+complex. The band gap between the highest occupied orbit (HOMO) and the lowest unoccupied orbit (LUMO) of the complex was significantly lower than that of individual sensor 1, which proved that the formed complex was more stable. After the formation of the complex, regardless of the ɑ orbits or β orbits, all the orbital energy levels of HOMO and LUMO remarkably reduced compared to the free sensor 1, which further demonstrated the stable structure of complex. The above calculation results were in good agreement with IR spectra and the 1H NMR titration spectra. 3.4. Analytical applications of sensor 1 and sensor 2 3.4.1. Test strip of sensor 2 for recognition of Fe3+ In order to prove the practical application value of the sensor, we used sensor 2 to prepare test strips for detecting Fe3+ ions. First, the

Fig. 12. Optimized ground-state geometries and frontier orbital shapes of sensor 1 and sensor 1+Fe3+complex.Color code: C (gray), N (blue), H (white), S (yellow), O (red), Fe (lavender). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) 8

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sensors had good cell permeability and can be used as fluorescence chemosensors for intracellular detecting of iron ions. 4. Conclusions In summary, the new benzothiazole-based fluorescence chemosensors (sensor 1 and sensor 2) have been synthesized and they displayed selective fluorescence quenching response towards Fe3+ ions. Job's plots, mass spectra, 1H NMR titration and DFT calculations demonstrated 1:1 complex formation between two sensors with iron ions. Sensor 1 and sensor 2 also showed fast fluorescence response and low detection limits for Fe3+ ions to be 8.43 μM and 5.86 μM, respectively. The sensor 2 displayed detection ability for iron ions in test strip mode. In addition, sensor 1 and sensor 2 had low cytotoxicity and good cell permeability and they displayed recognition ability for Fe3+ ions in living cells via the fluorescence cell imaging.

Fig. 13. (A) Filter paper strip for comparison. (B) Filter paper strip containing the solution of sensor 2.

Declaration of Competing Interest The authors confirm that there are no known conflicts of interest associated with this publication and that there has been no financial support for this work that could have influenced its outcome. Acknowledgement This work was supported by the National Natural Science Foundation of China (no. 50873019) and Research Fund for the Doctoral Program of Higher Education of China (20120043110007). Supplementary materials

Fig. 14. Fluorescence image of HeLa cells. A) pre- incubated with 20 μM sensor 1; B) pre-incubated with 20 μM sensor 1 and further with 40 equiv. of Fe3+ ions; C) pre-incubated with 20 μM sensor 2; D) pre-incubated with 20 μM sensor 2 and further with 40 equiv. of Fe3+ ions.

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.microc.2019.104351. Reference

filter paper strips were immersed in the solution of sensor 2 (5 × 10−6 M) and PVP (0.1 g / ml) in DMF: H2O (1:1, v: v, Tirs–HCl 10 mM, pH = 7.4) for 2 min. Then, the filter paper strips were removed from solution and dried in the air. Finally, we wrote ‘Fe’ on the filter paper with a cotton swab dipped in aqueous solution of Fe3+ ions (2 × 10−2 M). The filter paper without sensor 2 did not present fluorescence under 365 nm light (Fig. 13A). On the contrary, the sensor 2containing filter paper displayed bright fluorescence (Fig. 13B), the fluorescence of written ‘Fe’ disappeared and ‘Fe’ letter was clearly observed. This result indicated sensor 2 can be used as test strips to monitor the existence of Fe3+ ions.

[1] M. Deng, S. Wang, C. Liang, A fret fluorescent nanosensor based on carbon dots for ratiometric detection of Fe3+ in aqueous solution, RSC Adv. 6 (2016) 26936–26940. [2] Y. Zhang, B. Yan, A ratiometric fluorescent sensor with dual response of Fe3+/Cu2+ based on europium post-modified sulfone-metal-organic frameworks and its logical application, Talanta 197 (2019) 291–298. [3] B. Zhao, T. Liu, Y. Fang, A new selective chemosensor based on phenanthro[9,10- d] imidazole-coumarin with sequential “on-off-on” fluorescence response to Fe3+, and phosphate anions and its application in live cell, Sens. Actuator B-Chem. 246 (2017) 370–379. [4] C. Fan, X. Huang, L. Han, Novel colorimetric and fluorescent off–on enantiomers with high selectivity for Fe3+ imaging in living cells, Sens. Actuator B-Chem. 224 (2016) 592–599. [5] G. Liao, C. Zheng, D. Xue, A diarylethene-based fluorescent chemosensor for the sequential recognition of Fe3+ and cysteine, RSC Adv. 6 (2016) 34748–34753. [6] N.R. Chereddy, K. Suman, P.S. Korrapati, Design and synthesis of rhodamine based chemosensors for the detection of Fe3+ ions, Dyes Pigments 95 (2012) 606–613. [7] W. Lin, L. Long, L. Yuan, A novel ratiometric fluorescent Fe3+ sensor based on a phenanthroimidazole chromophore, Anal. Chim. Acta. 634 (2009) 262–266. [8] L. Huang, F. Hou, J. Cheng, Selective off–on fluorescent chemosensor for detection of Fe3+ ions in aqueous media, Org. Biomol. Chem. 10 (2012) 9634–9638. [9] R. Bai, G. Wei, S. Bai, A novel fluorescent Fe3+ sensor based on a europium complex, Opt. Mater. (Amst) 35 (2013) 833–836. [10] D. En, Y. Guo, B.T. Chen, Coumarin-derived Fe3+-selective fluorescent turn-off chemosensors: synthesis, properties, and applications in living cells, RSC Adv. 4 (2013) 248–253. [11] Z. Guo, Q. Niu, T. Li, Highly sensitive oligothiophene-phenylamine-based dualfunctional fluorescence “turn-on” sensor for rapid and simultaneous detection of Al3+and Fe3+ in environment and food samples, Spectrochim. Acta. A Mol. Biomol. Spectrosc. 200 (2018) 76–84. [12] Y. Hu, J. Wang, L. Long, A ratiometric fluorescence sensor for Fe3+ based on fret and pet processes, Luminescence 31 (2016) 16–21. [13] L. He, C. Liu, J.H. Xin, A novel turn-on colorimetric and fluorescent sensor for Fe3+ and Al3+ with solvent-dependent binding properties and its sequential response to carbonate, Sens. Actuator B-Chem. 213 (2015) 181–187. [14] S. Li, D. Zhang, M. Wang, Synthesis and properties of a novel FRET-Based ratiometric fluorescent sensor for Cu2+, J. Fluoresc. 26 (2016) 769–774. [15] G.J. Shree, G. Sivaraman, A. Siva, Anthracene-and pyrene-bearing imidazoles as turn-on fluorescent chemosensor for aluminum ion in living cells, Dyes Pigments

3.4.2. Fluorescence imaging for living cells of sensor 1 and sensor 2 Cell imaging experiments were performed to further elevate the application value of sensor 1 and sensor 2. As shown in Fig. S12, the toxicity tests proved that sensor 1 and sensor 2 had low cytotoxicity and can be used to detect iron ions in living cells. Sensors (20 μM) and iron salts (40 equiv. of sensors) were dissolved in cell culture medium to prepare test solutions, respectively. First, HeLa cells were incubated with the solutions of sensor 1 and sensor 2 (20 μM) for 90 min, respectively. Then, after cleaning out the extra sensors, the cells containing sensors were incubated with the solution of iron ions for 90 min. Finally, after washing the cells three times with PBS solution, the cells were fixed, stained and loaded onto glass slides. The fluorescence images were taken under the excitation wavelength at 405 nm, the emission band was located at the wavelength range of 430 nm–470 nm. The cell images were showed in Fig. 14, the HeLa cells cultured only with sensor 1 and sensor 2 showed strong blue fluorescence. On the contrary, the fluorescence of HeLa cells further incubated with iron ions was significantly quenched. These results proved that two synthesized 9

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X. Gong, et al. 163 (2019) 204–212. [16] P. Chinna, J. Shanmugapriya, S. Singaravadivel, Anthracene-Based highly selective and sensitive fluorescent “Turn-on” chemodosimeter for Hg2+, ACS Omega 3 (2018) 12341–12348. [17] S.A.A. Vandarkuzhali, S. Natarajan, S. Jeyabalan, Pineapple peel-derived carbon dots: applications as sensor, molecular keypad lock, and memory device, ACS Omega 3 (2018) 12584–12592. [18] G.G.V. Kumar, R.S. Kannan, C.K. Yang, An efficient “Ratiometric” fluorescent chemosensor for the selective detection of Hg2+ ions based on phosphonates: its live cell imaging and molecular keypad lock applications, Anal. Methods 11 (2019) 901–916. [19] G. Sivaraman, V. Sathiyaraja, D. Chellappa, Turn-on fluorogenic and chromogenic detection of Fe (III) and its application in living cell imaging, J. Lumin. 145 (2014) 480–485. [20] B. Li, J. Tian, D. Zhang, A novel colorimetric fluorescence sensor for Fe3+, based on quinoline Schiff base, Luminescence 32 (2017) 1567–1573. [21] P. Ju, Q. Su, Z. Liu, A salen-based covalent organic polymer as highly selective and sensitive fluorescent sensor for detection of Al3+, Fe3+ and Cu2+ ions, J. Mater. Sci. 54 (2019) 851–861. [22] L. Wang, X. Yang, X. Chen, A novel fluorescence probe based on triphenylamine Schiff base for bioimaging and responding to pH and Fe3+, Mater. Sci. Eng. C 72 (2017) 551–557. [23] N. Roy, A. Dutt, P. Mondal, Coumarin based fluorescent probe for colorimetric detection of Fe3+and fluorescence turn on-off response of Zn2+and Cu2+, J. Fluoresc. 27 (2017) 1307–1321. [24] L. Wang, W. Li, W. Zhi, A new coumarin Schiff based fluorescent-colorimetric chemosensor for dual monitoring of Zn2+, and Fe3+ in different solutions: an application to bio- imaging, Sens. Actuator B-Chem. 26 (2018) 243–254. [25] N. Roy, A. Dutta, P. Mondal, A new coumarin based dual functional chemosensor for colorimetric detection of Fe3+ and fluorescence turn-on response of Zn2+, Sens. Actuator B-Chem. 236 (2016) 719–731. [26] Olimpo García-Beltrán, B. Cassels, Claudio Pérez, Coumarin-Based fluorescent probes for dual recognition of copper (II) and iron (III) ions and their application in bio-imagin, Sensors 14 (2014) 1358–1371. [27] E.N. Kaya, F. Yuksel, G.A. Özpınar, 7-Oxy-3-(3,4,5-trimethoxyphenyl)coumarin

[28] [29] [30] [31] [32] [33] [34] [35] [36] [37] [38]

10

substituted phthalonitrile derivatives as fluorescent sensors for detection of Fe3+ ions: experimental and theoretical study, Sens. Actuator B-Chem. 194 (2014) 377–388. Z. Li, Y. Zhou, K. Yin, A new fluorescence “turn-on” type chemosensor for Fe3+based on naphthalimide and coumarin, Dyes Pigments 105 (2014) 7–11. S. Warrier, P.S. Kharkar, Highly selective on-off fluorescence recognition of Fe3+ based on a coumarin derivative and its application in live-cell imaging, Spectrochim. Acta. A Mol. Biomol. Spectrosc. 188 (2017) 659–665. L. Wang, Q.J. Bing, J. Li, A new “ON-OFF” fluorescent and colorimetric chemosensor based on 1, 3, 4-oxadiazole derivative for the detection of Cu2+ions, J. Photoch. Photobio. A. 360 (2018) 86–94. L. Zhu, C. Gu, He. Yi, G. Wang, Study on a highly selective fluorescent chemosensor for Cu2+ and its direct sensing for proton based on 1,3,4-oxadiazole, J. Lunim. 153 (2014) 439–445. L. Wang, X. Gong, Q.J. Bing, G. Wang, A new oxadiazole-based dual-mode chemosensor: colorimetric detection of Co2+ and fluorometric detection of Cu2+ with high selectivity and sensitivity, Microchem. J. 142 (2018) 279–287. X. Gong, H.Y. Zhang, N. Jiang, Oxadiazole-based ‘on-off’ fluorescence chemosensor for rapid recognition and detection of Fe2+ and Fe3+ in aqueous solution and in living cells, Microchem. J. 145 (2019) 435–443. G. Dhaka, N. Kaur, J. Singh, Luminescent benzothiazole-based fluorophore of anisidine scaffoldings: a “Turn-On” fluorescent probe for Al3+ and Hg2+ ions, J. Fluoresc. 27 (2017) 1943–1948. Y. Chen, T. Wei, Z. Zhang, A benzothiazole-based fluorescent probe for ratiometric detection of Al3+ in aqueous medium and living cells, Ind. Eng.Chem. Res. 56 (2017) 12267–12275. J. L, Y. Chen, T. Chen, A benzothiazole-based fluorescent probe for efficient detection and discrimination of Zn2+ and Cd2+, using cysteine as an auxiliary reagent, Sens. Actuator B-Chem. 268 (2018) 446–455. Y. Li, K. Li, J. He, A “turn-on” fluorescent chemosensor for the detection of Zn (II) in aqueous solution at neutral pH and its application in live cells imaging, Talanta 153 (2016) 381–385. H.A. Benesi, J.H.J. Hildebrand, A spectrophotometric investigation of the interaction of iodine with aromatic hydrocarbons, J. Am. Chem. Soc. 71 (1949) 2703–2707.