A Benzothiazole-Based Fluorescent Probe for Ratiometric Detection of Al3+ and Its Application in Water Samples and Cell Imaging

International Journal of Molecular Sciences

Article A Benzothiazole-Based Fluorescent Probe for Ratiometric Detection of Al3+ and Its Application in Water Samples and Cell Imaging

Zhen-Nan Tian, Ding-Qi Wu, Xue-Jiao Sun, Ting-Ting Liu and Zhi-Yong Xing *

Department of Applied Chemistry, College of Arts and Sciences, Northeast Agricultural University, Harbin 150030, China; [email protected] (Z.-N.T.); [email protected] (D.-Q.W.); [email protected] (X.-J.S.); [email protected] (T.-T.L.) * Correspondence: [email protected]

Received: 29 October 2019; Accepted: 26 November 2019; Published: 28 November 2019

Abstract: An easily prepared benzothiazole-based probe (BHM) was prepared and characterized by general spectra, including 1H NMR, 13C NMR, HRMS, and single-crystal X-ray diffraction. Based on the synergistic mechanism of the inhabitation of intramolecular charge transfer (ICT), the BHM 3+ displayed high selectivity and sensitivity for Al in DMF/H2O(v/v, 1/1) through an obvious blue-shift in the fluorescent spectrum and significant color change detected by the naked eye, respectively. The binding ratio of BHM with Al3+ was 1:1, as determined by the Job plot, and the binding details were investigated using FT-IR, 1H NMR titration, and ESI-MS analysis. Furthermore, the BHM was successfully applied in the detection of Al3+ in the Songhua River and on a test stripe. Fluorescence imaging experiments confirmed that the BHM could be used to monitor Al3+ in human stromal cells (HSC).

Keywords: benzothiazole; ratiometric; ﬂuorescent probe; Al3+

1. Introduction Aluminum, as one of the most abundant metals in the earth, is widely used in daily life, including in kitchen utensils, pharmaceutical packaging, and food additives [1–4]. These activities will inevitably induce the accumulation of Al3+, which is the most ionic style of aluminum in existence in environmental water and the human body. However, as a non-necessary element for humans, excess accumulation of Al3+ in the human body might be associated with diseases, including Alzheimer’s disease, Parkinson’s disease, amyotrophic sclerosis, and encephalopathy [5–10]. According to the regulations of the World Health Organization (WHO), the maximum concentration of Al3+ is 7.4 µM in drinking water. Hence, it is necessary to construct a simple and effective method with the function of qualitative and quantitative analysis in Al3+ detection. The development of fluorescent probes for detection and special analysis has attracted many researchers, and numerous excellent probes for Al3+ have been reported [11–27]. Among them, the ratiometric probe is one of the most popular types of sensors due to the inherent advantage that it can eliminate the interference of the external environment through self-calibration of its response signals either in emission or absorbance intensities at two different wavelengths. Some ratiometric Al3+ probes were developed using different fluorophores, including rhodamine–naphthalene [17], rhodamine-benzothiazole [18], 40-methyl-3-hydroxyflavone [19], coumarin-quinoline [20], rhodamine- chromone [21], carbazole-rhodamine [22], imidazoquinazoline [23], benzothiazole [24], benzimidazole [25], naphthalimide [26], and quinoline–coumarin [27]. However, only a few of these were benzothiazole- based fluorescent probes for the ratiometric detection of Al3+ [24]. Moreover, benzothiazole, an excellent

Int. J. Mol. Sci. 2019, 20, 5993; doi:10.3390/ijms20235993 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 2 of 11 stability and easy structural modification, had been used in the construction of chemosensors for the Int. J. Mol. Sci. 2019, 20, 5993 2 of 12 Int.detection J. Mol. Sci. of 2019 various, 20, x FORions PEER [28– REVIEW34]. Moreover, this type of structure, in which the hydroxyl group2 ofacts 11 as an electron donating unit in the para-position of the benzothiazolyl group used as the electron stabilitywithdrawing and easy group, structural was facile modif forication the ICT, had-based been mechanism used in the byconstruction increasing of the chemosensors push-pull electron for the fluorophore due to its distinguished merits, such as excellent photo stability and easy structural detectioneffect [35]. of various Given ions the [28 –34above]. Moreover, statements, this type aof structure novel , eas in ilywhich prepared the hydroxyl probe group BHM acts modification, had been used in the construction of chemosensors for the detection of various ions [28–34]. as(5- (anbenzo[d]thiazol electron donat-2ing-yl) -unit2-hydroxy in the -para3-methoxybenzaldehyde-position of the benzothiazolyl) was synthesized group used, and as the its electron stereo Moreover, this type of structure, in which the hydroxyl group acts as an electron donating unit in the withdrawingstructure was group,confirmed was by facile single for crystal the ICT X-ray-based diffraction mechanism (Scheme by increasing 1). The BHM the showedpush-pull an electronobvious para-position of the benzothiazolyl group used as the electron withdrawing group, was facile for the effectselectivity [35]. and Given sensitivity the to Alabove3+ with statements, a significant ablue novel-shift both eas ily in theprepared emission probespectrum BHM and ICT-based mechanism by increasing the push-pull electron effect [35]. Given the above statements, (absorbance5-(benzo[d]thiazol spectrum.-2-yl Its)-2 application-hydroxy-3 -methoxybenzaldehydein real water samples was) was investigated. synthesized Moreover,, and its the stereoBHM a novel easily prepared probe BHM (5-(benzo[d]thiazol-2-yl)-2-hydroxy-3-methoxybenzaldehyde) structurewas successful was confirmed in imaging by the single human crystal stromal X-ray cell diffraction line (HSC (Scheme). 1). The BHM showed an obvious was synthesized, and its stereo structure was confirmed by single crystal X-ray diffraction (Scheme1). selectivity and sensitivity to Al3+ with a significant blue-shift both in the emission spectrum and The BHM showed an obvious selectivity and sensitivity to Al3+ with a significant blue-shift both in the absorbance2. Results and spectrum. Discussion Its application in real water samples was investigated. Moreover, the BHM emission spectrum and absorbance spectrum. Its application in real water samples was investigated. was successful in imaging the human stromal cell line (HSC). Moreover,2.1. Synthesis the of BHM BHM was successful in imaging the human stromal cell line (HSC). 2. Results and Discussion

2.1. Synthesis of BHM

Scheme 1. Synthetic route of the benzothiazole-based probe (BHM). Scheme 1 Synthetic route of the benzothiazole-based probe (BHM). 2. ResultsThe BHM and Discussion was facile synthesized through a Duff reaction using the starting material of 4-(benzo[d]thiazol-2-yl)-2-methoxyphenol (3), which was synthesized via a condensation reaction 2.1. Synthesis of BHM between 2-aminobenzenethiolScheme 1 Synthetic and 4 -routehydroxy of the-3 -benzothiazolemethoxybenzaldehyde-based probe ([36].BHM The). single crystal of BHMThe was BHM obtained was, and facile its molecular synthesized structure through is depicteda DuDuffﬀ reactionreaction in Figure using using 1. The the selective starting crystal material data are of 44-(benzo[d]thiazol-2-yl)-2-methoxyphenoldisplayed-(benzo[d]thiazol in Table- S12-yl and)-2- methoxyphenolTable S2. ( (3),3), which which was was synthesized via via a condensation reaction between 2-aminobenzenethiol2-aminobenzenethiol and 4-hydroxy-3-methoxybenzaldehyde4-hydroxy-3-methoxybenzaldehyde [[36].36]. The The single crystal of BHM was obtained,obtained, andand itsits molecularmolecular structurestructure isis depicteddepicted inin FigureFigure1 1.. The The selective selective crystal crystal data data areare displayed in TablesTable S1 S1 and and Table S2. S2.

Figure 1. X-ray crystal structure of BHM (heteroatoms N, O and S were labeled in different color).

2.2. Spectra Performance Investigation of the BHM to Metal Ions TFigureheFigure selectivity 1. XX-ray-ray ofcrystal crystal BHM structure structure (10 μM of of) toBHM BHM different (h (heteroatomseteroatom metals ionsN, O (and Na +S , Kwere +, Mg labeled 2+, Ca 2+in , different diAgfferent+, F e 2+ color).color), Co2+. , Ba2+, Mn2+, Cu2+, Zn2+, Cd2+, Hg2+, Ni2+, Pb2+, Cr3+, Fe3+, and Al3+) was firstly determined using UV-Vis 2.2. Spectr Spectraa Performance Performance Investigation of the BHM to Metal Ions spectrum in a solution of DMF:H2O (1:1, v/v) (Figure 2). The UV-Vis absorbance of BHM showed + + 2+ 2+ + 2+ 2+ almostTThehe no selectivity change after of BHM the addition( (1010 μMµM)) to toof different dithffe erenttested metal metal metal ions ions ions ( (NaNa, except+, ,KK+, Mg , for Mg2+ Al, Ca,3+ Ca 2+in, Ag DMF:H,+ Ag, F e,2+2 FO, Co e (1:1,2+,, Co Ba v/v2+),, 2+ 2+ 2+ 2+ 2+ 2+ 2+ 2+ 3+ 3+ 3+ MnBa 2+,, Mn Cu2+, , Zn Cu2+, , Cd Zn2+, Hg, Cd2+, Ni, Hg2+, Pb, Ni2+, Cr,3+ Pb, Fe,3+ Cr, and, Fe Al3+,) andwas Al firstly) was determined firstly determined using UV using-Vis spectrumUV-Vis spectrum in a solution in asolution of DMF:H of2 DMF:HO (1:1, 2v/vO) (1:1, (Figurev/v) 2 (Figure). The 2UV).- TheVis UV-Visabsorbance absorbance of BHM of showed BHM almost no change after the addition of the tested metal ions, except for Al3+ in DMF:H2O (1:1, v/v)

Int. J. Mol. Sci. 2019, 20, 5993 3 of 12 showed almost no change after the addition of the tested metal ions, except for Al3+ in DMF:H O Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 3 of 11 2 (1:1, v/v) (Figure2a). As shown in Figure2a, upon the addition of Al 3+, an obvious blue-shift from an absorbance(Figure peak2a). As at 359 shown to 321 in nmFigure was 2a, observed upon the compared addition toof that Al3+, of an the obvious BHM blueitself,-shift which from indicated an the existenceabsorbance of apeak strong at 359 interaction to 321 nm betweenwas observed the BHM compared and Alto 3that+. Furthermore, of the BHM itself, the which absorbance indicated titration resultthe (Figure existence2b) showed of a strong the absorbance interaction between centered the at 359BHM nm and was Al gradually3+. Furthermore, decreased the absorbancewhile thepeak at 321titration nm was result gradually (Figure 2 mergedb) showed with the absorbance the isosbestic centered point atat 359 335 nm nm, was indicatinggradually decreased the formation while of a the peak3+ at 321 nm was gradually merged with the isosbestic point at 335 nm, indicating the BHM-Al complex. A good relationship was detected between the absorbance ratios (A307:A359) formation of a BHM-Al3+ complex. A good relationship was detected between the absorbance ratios 3+ µ (Figure S4) and the Al concentration3+ (0–12 M) with a limit of detection (LOD) as low as 99 nM, (A307:A359) (Figure S4) and the Al concentration3+ (0–12 μM) with a limit of detection (LOD) as low as explaining99 nM, ratiometricexplaining ratiometric detection detection by BHM by of BHM Al . of Al3+.

FigureFigure 2. (a 2.) UV-Vis (a) UV-Vis absorbance absorbance spectra spectra of of BHMBHM (10(10 μµMM)) in in the the absence absence and and presence presence of tested of tested metal metal ions (5 equiv.) in DMF/H2O (1/1, v/v); (b) Absorbance titration of BHM to Al33++. Inset: the absorbance ions (5 equiv.) in DMF/H2O (1/1, v/v); (b) Absorbance titration of BHM to Al . Inset: the absorbance of of BHM as a function of Al3+ concentration. BHM as a function of Al3+ concentration. Moreover, the selectivity of BHM (10 μM) to the different metal ions mentioned above was also Moreover, the selectivity of BHM (10 µM) to the different metal ions mentioned above was also determined by the fluorescence spectrum in the solution of DMF:H2O (1:1, v/v) (Figure 3). The result determined by the fluorescence spectrum in the solution of DMF:H O (1:1, v/v) (Figure3). The result showed that some ions, including Ni2+, Pb2+, Hg2+, Fe2+, Fe3+, Cr2 3+, and Cu2+, decreased the 2+ 2+ 2+ 2+ 3+ 3+ 2+ showedfluorescence that some intensity ions, of including BHM to different Ni , Pbextent,s Hg. However,, Fe the, Fe addition, Cr of, Al and3+ caused Cu a, significant decreased the 3+ fluorescenceblue-shift intensity from 527 ofto BHM478 nm to, accompanied different extents. by a color However, change the from addition green to of deep Al skycaused-blue a(Figure significant blue-shift3a). T fromhis result 527 indicated to 478 nm, that accompanied the BHM had by high a color selectivity change to fromAl3+. F greenluorescent to deep titration sky-blue (Figure (Figure 3b) 3a). 3+ Thisthrough result indicated the gradual that addition the BHM of Al had3+ into high the selectivity BHM solution to Al DMF:H. Fluorescent2O (1:1, v/v) titration showed (Figure that a 3b) 3+ throughsignificant the gradual blue-shift addition of 47 nm of was Al observed,into the which BHM might solution be attributed DMF:H to 2theO depression (1:1, v/v) showed of the ICT that a significantprocess blue-shift after coordination of 47 nm was with observed, the Al3+ [3 which7]. M mightoreover, be the attributed good relationship to the depression between of the the ICT 3+ processfluorescent after coordination intensity (E withm = 478 the nm Al) 3of+ BHM[37]. Moreover,(10 μM) and the the good Al relationshipconcentration between(12–28 μM the) (Fig fluorescenture S5) achieved a LOD (limit of detection was calculated3+ using 3σ/k, where σ is the standard deviation intensity (Em = 478 nm) of BHM (10 µM) and the Al concentration (12–28 µM) (Figure S5) achieved of the blank measurements, and k is the slope of the intensity ratio versus the sample concentration a LOD (limit of detection was calculated using 3σ/k, where σ is the standard deviation of the blank plot) as low as 4.39 μM calculated according to previous methods [35,38]. measurements, and k is the slope of the intensity ratio versus the sample concentration plot) as low as 4.39 µM calculated according to previous methods [35,38]. A competition experiment was conducted to verify the ability of BHM under conditions of disturbance coming from other co-existing metal ions. As illustrated in Figure4, only metal ions (including Cr3+ and Mn2+) disturbed the detection of Al3+.

Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 3 of 11

(Figure 2a). As shown in Figure 2a, upon the addition of Al3+, an obvious blue-shift from an absorbance peak at 359 to 321 nm was observed compared to that of the BHM itself, which indicated the existence of a strong interaction between the BHM and Al3+. Furthermore, the absorbance titration result (Figure 2b) showed the absorbance centered at 359 nm was gradually decreased while the peak at 321 nm was gradually merged with the isosbestic point at 335 nm, indicating the formation of a BHM-Al3+ complex. A good relationship was detected between the absorbance ratios (A307:A359) (Figure S4) and the Al3+ concentration (0–12 μM) with a limit of detection (LOD) as low as 99 nM, explaining ratiometric detection by BHM of Al3+.

Figure 2. (a) UV-Vis absorbance spectra of BHM (10 μM) in the absence and presence of tested metal ions (5 equiv.) in DMF/H2O (1/1, v/v); (b) Absorbance titration of BHM to Al3+. Inset: the absorbance of BHM as a function of Al3+ concentration.

Moreover, the selectivity of BHM (10 μM) to the different metal ions mentioned above was also determined by the fluorescence spectrum in the solution of DMF:H2O (1:1, v/v) (Figure 3). The result showed that some ions, including Ni2+, Pb2+, Hg2+, Fe2+, Fe3+, Cr3+, and Cu2+, decreased the fluorescence intensity of BHM to different extents. However, the addition of Al3+ caused a significant blue-shift from 527 to 478 nm, accompanied by a color change from green to deep sky-blue (Figure 3a). This result indicated that the BHM had high selectivity to Al3+. Fluorescent titration (Figure 3b) through the gradual addition of Al3+ into the BHM solution DMF:H2O (1:1, v/v) showed that a significant blue-shift of 47 nm was observed, which might be attributed to the depression of the ICT process after coordination with the Al3+ [37]. Moreover, the good relationship between the fluorescent intensity (Em = 478 nm) of BHM (10 μM) and the Al3+ concentration (12–28 μM) (Figure S5) achieved a LOD (limit of detection was calculated using 3σ/k, where σ is the standard deviation Int.of J. Mol.the blank Sci. 2019 measurements,, 20, 5993 and k is the slope of the intensity ratio versus the sample concentration4 of 12 plot) as low as 4.39 μM calculated according to previous methods [35,38].

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Figure 3. (a) Fluorescence emission spectra of BHM (10 μM) in the presence of 5 equiv. of various

Inset: the fluorescence intensity (Em = 478 nm) of BHM as a function of Al3+ concentration. Figure 3. (a) Fluorescence emission spectra of BHM (10 µM) in the presence of 5 equiv. of various 3+ metalA c ionsompetition in DMF /H experiment2O (1/1, v/v ).was Inset: conducted the color changes to verify of BHMthe ability (10 µM) of inBHM the presence under conditionsof Al ions of 3+ disturbance(5 equiv.) incoming DMF/H from2O (1 / other1, v/v) co under-exist UVing light metal of 365 ions. nm; As (b illustrated) Fluorescence in Fig titrationure 4, of only BHM metal to Al ions. 3+ 2+ 3+ 3+ (includingInset: the Cr ﬂuorescence and Mn intensity) disturb (Eedm the= 478 detection nm) of BHMof Al as. a function of Al concentration.

FigureFigure 4. 4.Competition Competition selectivity selectivity of of BHM BHM (5(5 µμMM)) toward Al3+3+ (5(5 equiv. equiv.)) in in the the presence presence of ofother other metal metal ionsions (5 ( equiv.)5 equiv. (E) (mEm= =478 478 nm).nm). Error bar is is represented represented as as the the mean mean ± standardstandard deviation, deviation, n = n3.= 3. ± 2.3.2.3. Sensing Sensing Mechanism Mechanism of of the the BHM BHMto to AlAl33++

A JobA Job plot plot was was firstly firstly carried carried out out to to determine determine the the binding binding ratio ratio between between BHM BHM and and Al Al3+3+ (Figure(Figure 5). The5). result The resultshowed showed that the that fluorescence the fluorescence intensity intensity (recorded (recorded at 478 nm) at 478 of BHM nm) of reached BHM reached a maximum a whenmaximum the proportion when the of proportion BHM in the of totalBHM concentration in the total concentration of BHM and ofAl 3BHM+ was and 0.5, Al indicating3+ was 0.5, that theindicating binding ratiothat the was binding 1:1 between ratio was BHM 1:1 andbetween Al3+ .BHM This and result Al3+ was. This further result supportedwas further by supported the HRMS analysisby the displayedHRMS analysis in Figure displayed6. Peaks in Figure at m/z 6.446.0606 Peaks at and m/z m 446/z.0606 464.0116 and werem/z 464.0116 attributed were to [BHM-attributed H+ + 3to+ [BHM- H+ + Al3+ ++ NO3− + DMF]+ (Calcd: m/z 446.0603) and+ [BHM3+- H+ + Al3+ + NO3− + DMF ++ H2O]+ Al + NO3− + DMF] (Calcd: m/z 446.0603) and [BHM- H + Al + NO3− + DMF + H2O] (Calcd: (Calcd: m/z 464.0708), respectively. Moreover, the binding constants of BHM and3+ Al3+ were 1.88 × 4104 1 m/z 464.0708), respectively. Moreover, the binding constants of BHM and Al were 1.88 10 M− M−1 (Figure S6) and 7.493 × 1013 M−1 (Figure S7) based on the Benesi–Hilderbrand plot [39,40] recorded× (Figure S6) and 7.49 10 M− (Figure S7) based on the Benesi–Hilderbrand plot [39,40] recorded by by the fluorescence× and absorbance signals, respectively. the fluorescence and absorbance signals, respectively.

Figure 5. Job’s plot of BHM (Em = 478 nm) with Al3+ (the crossover point between red line and x-axis is 0.5).

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Figure 3. (a) Fluorescence emission spectra of BHM (10 μM) in the presence of 5 equiv. of various

metal ions in DMF/H2O (1/1, v/v). Inset: the color changes of BHM (10 μM) in the presence of Al3+ ions (5 equiv.) in DMF/H2O (1/1, v/v) under UV light of 365 nm; (b) Fluorescence titration of BHM to Al3+. Inset: the fluorescence intensity (Em = 478 nm) of BHM as a function of Al3+ concentration.

A competition experiment was conducted to verify the ability of BHM under conditions of disturbance coming from other co-existing metal ions. As illustrated in Figure 4, only metal ions (including Cr3+ and Mn2+) disturbed the detection of Al3+.

Figure 4. Competition selectivity of BHM (5 μM) toward Al3+ (5 equiv.) in the presence of other metal ions (5 equiv.) (Em = 478 nm). Error bar is represented as the mean ± standard deviation, n = 3.

2.3. Sensing Mechanism of the BHM to Al3+

A Job plot was firstly carried out to determine the binding ratio between BHM and Al3+ (Figure 5). The result showed that the fluorescence intensity (recorded at 478 nm) of BHM reached a maximum when the proportion of BHM in the total concentration of BHM and Al3+ was 0.5, indicating that the binding ratio was 1:1 between BHM and Al3+. This result was further supported by the HRMS analysis displayed in Figure 6. Peaks at m/z 446.0606 and m/z 464.0116 were attributed to [BHM- H+ + Al3+ + NO3− + DMF]+ (Calcd: m/z 446.0603) and [BHM- H+ + Al3+ + NO3− + DMF + H2O]+ (Calcd: m/z 464.0708), respectively. Moreover, the binding constants of BHM and Al3+ were 1.88 × 104 MInt.−1 J. ( Mol.Figure Sci. S62019) and, 20, 59937.49 × 103 M−1 (Figure S7) based on the Benesi–Hilderbrand plot [39,40] recorded5 of 12 by the fluorescence and absorbance signals, respectively.

3+3+ Figure 5. JobJob’s’s plot of BHM (E(Em = 478478 nm nm)) with with Al Al (the(the crossover crossover point point between between red red line and x-axisx-axis Int. J. Mol.is 0.5).0.5) Sci.. 2019, 20, x FOR PEER REVIEW 5 of 11

FigureFigure 6.6. TheThe ESI-MSESI-MS spectrumspectrum ofof thethe BHM-AlBHM-Al33++ complexcomplex in in DMF. DMF.

For explicitexplicit bindingbinding sitessites ofof BHMBHM withwith AlAl33++,, we we measured measured the FT FT-IR-IR spectrum of free BHM andand complex BHM–AlBHM–Al33++ ((FigureFigure 7),), respectively.respectively. TheThe typicaltypical stretchingstretching vibrationvibration peakspeaks ofof thethe BHMBHMitself itself 1 1 locatedlocated atat 33033303 cmcm−−1 andand 1655 1655 cm cm−1− werewere designated designated to to the the hydroxyl hydroxyl group group ( (-OH)-OH) and and carbonyl carbonyl group (-C(-C=O=O),), respectively. For the FT-IRFT-IR spectrumspectrum ofof thethe BHM–AlBHM–Al33++,, the the stretc stretchinghing bands of -OH-OH turnedturned 1 intointo aa broadbroad peak,peak, andand thethe stretchingstretching bandband ofof CC=O=O decreaseddecreased signiﬁcantlysignificantly andand shiftedshiftedfrom from 1655 1655 cm cm−−1 1 3+ toto 16241624 cmcm−−1.. T Thesehese results results indicated indicated that the coordination ofof AlAl3+ withwith oxygen oxygen atoms came from the hydroxyl groupgroup andand carbonylcarbonyl group,group, respectively.respectively. 1 HNMR titration experiments were measured in DMSO-d6 to determine the binding sites of BHM with Al3+ further. The proton signals of the BHM itself were analyzed and are shown in 3+ Figure8. After the addition of Al , the proton signal of hydroxyl (Ha) located at 10.96 ppm gradually disappeared, suggesting the deprotonation on hydroxyl during the combination of BHM with Al3+, in accordance with the result obtained by the FT-IR analysis.

Figure 7. The FT-IR spectrum for the probe BHM and Al3+complex.

1HNMR titration experiments were measured in DMSO-d6 to determine the binding sites of BHM with Al3+ further. The proton signals of the BHM itself were analyzed and are shown in Figure 8. After the addition of Al3+, the proton signal of hydroxyl (Ha) located at 10.96 ppm gradually disappeared, suggesting the deprotonation on hydroxyl during the combination of BHM with Al3+, in accordance with the result obtained by the FT-IR analysis.

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Figure 6. The ESI-MS spectrum of the BHM-Al3+ complex in DMF.

For explicit binding sites of BHM with Al3+, we measured the FT-IR spectrum of free BHM and complex BHM–Al3+ (Figure 7), respectively. The typical stretching vibration peaks of the BHM itself located at 3303 cm−1 and 1655 cm−1 were designated to the hydroxyl group (-OH) and carbonyl group (-C=O), respectively. For the FT-IR spectrum of the BHM–Al3+, the stretching bands of -OH turned into a broad peak, and the stretching band of C=O decreased significantly and shifted from 1655 cm−1 −1 3+ to 1624Int. J.cm Mol.. Sci.These2019, 20results, 5993 indicated that the coordination of Al with oxygen atoms came 6from of 12 the hydroxyl group and carbonyl group, respectively.

Int. J. Mol. Sci. 2019, 20, x FigureFORFigure PEER 7. 7.The REVIEWThe FT FT-IR-IR spectrum spectrum for thethe probeprobe BHM BHM and and Al 3Al+complex.3+complex. 6 of 11

1 1 3+ 3+ FigureFigure 8. 8.H NMRH NMR titration titration spectra spectra of BHM inin the the presence presence of of Al Alin in DMSO-d DMSO6.-d6.

1 Hence,Hence, according according to to the the experimentexperiment results results (including (including the Job the plot, JobHNMR plot, titration,1HNMR and titration, HRMS) and HRMSmentioned) mentioned above, above, the probable the probable sensing sensing mechanism mechanism is illustrated is illustrated in Scheme 2in. Scheme 2.

Scheme 2 Proposed mechanism of BHM for Al3+ (the symbol in blue means Excitation, the symbol in green and deep sky-blue all mean Emission).

3. Materials and Methods

3.1. Materials and Apparatus All analytical reagent grade chemicals and solvents employed for the synthesis and characterization were procured from commercial sources and used as received without any treatment. Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker 600 MHz (Beijing, China) system, and the chemical shifts were reported in ppm with Me4Si as the internal standard. High-resolution mass spectroscopy (HRMS) was carried out with negative ion modes on a Waters Xevo UPLC/G2-SQ Tof MS spectrometer (Shanghai, China). The FT-IR spectra were recorded on a Bruker ALPHA-T (Beijing, China) by dispersing samples in KBr disks, in the range of 4000–400 cm−1. The UV–Vis absorption and fluorescence spectra of the samples were measured on a Pgeneral TU-2550 UV-Vis Spectrophotometer (Suzhou, Jiangsu, China) and Perkin Elmer LS55 fluorescence spectrometer (Shanghai, China), respectively.

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Figure 8. 1H NMR titration spectra of BHM in the presence of Al3+ in DMSO-d6.

Int. J. Mol.Hence, Sci. 2019 according, 20, 5993 to the experiment results (including the Job plot, 1HNMR titration,7 ofand 12 HRMS) mentioned above, the probable sensing mechanism is illustrated in Scheme 2.

Scheme 2.2 ProposProposeded mechanism mechanism of BHM for Al33++ (t(thehe symbol symbol in in blue blue means means Excitation Excitation,, t thehe symbol in green and deepdeep sky-bluesky-blue allall meanmean Emission).Emission).

3.3. Materials and Methods

3.1. Materials and Apparatus 3.1. Materials and Apparatus All analytical reagent grade chemicals and solvents employed for the synthesis and characterization All analytical reagent grade chemicals and solvents employed for the synthesis and were procured from commercial sources and used as received without any treatment. Nuclear magnetic characterization were procured from commercial sources and used as received without any resonance (NMR) spectra were recorded on a Bruker 600 MHz (Beijing, China) system, and the chemical treatment. Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker 600 MHz (Beijing, shifts were reported in ppm with Me4Si as the internal standard. High-resolution mass spectroscopy China) system, and the chemical shifts were reported in ppm with Me4Si as the internal standard. (HRMS) was carried out with negative ion modes on a Waters Xevo UPLC/G2-SQ Tof MS spectrometer High-resolution mass spectroscopy (HRMS) was carried out with negative ion modes on a Waters (Shanghai, China). The FT-IR spectra were recorded on a Bruker ALPHA-T (Beijing, China) by dispersing Xevo UPLC/G2-SQ Tof MS spectrometer (Shanghai, China). The FT-IR spectra were recorded on a samples in KBr disks, in the range of 4000–400 cm 1. The UV–Vis absorption and fluorescence spectra Bruker ALPHA-T (Beijing, China) by dispersing samples− in KBr disks, in the range of 4000–400 cm−1. of the samples were measured on a Pgeneral TU-2550 UV-Vis Spectrophotometer (Suzhou, Jiangsu, The UV–Vis absorption and fluorescence spectra of the samples were measured on a Pgeneral China) and Perkin Elmer LS55 fluorescence spectrometer (Shanghai, China), respectively. TU-2550 UV-Vis Spectrophotometer (Suzhou, Jiangsu, China) and Perkin Elmer LS55 fluorescence 3.2.spectrometer General Information (Shanghai, China), respectively. Stock solutions of the metal ions (Al3+, Fe3+, Cr3+, Ca2+, Pb2+, Cd2+, Cu2+, Co2+, Zn2+, Fe2+, Mn2+, Mg2+, Ni2+, Hg2+, Na+,K+, Ag+) from the nitrate, chloride, or perchlorate salts were prepared with ultrapure water. BHM (0.1 mM) was dissolved in DMF, which was then diluted by adding ultrapure water to 10 µM. The diluted solution DMF:H2O (1:1, v/v) was used for the spectra measurement in UV-Vis and fluorescence. The excitation wavelength (Ex = 380 nm) was used for the fluorescence experiments, and the slits, including excitation and the emission, were all set to 10 nm.

3.3. Synthesis Compounds 3 and BHM were synthesized according to the methods described in References [34,36].

3.3.1. Synthesis of Probe BHM (5-(benzo[d]thiazol-2-yl)-2-hydroxy-3-methoxybenzaldehyde) The mixture of compound 3 (500 mg, 1.94 mmol) and hexamethylenetetramine (1.37 g, 9.75 mmol) in trifluoroacetic acid (15 mL) was refluxed for 8 h. After the completion of the reaction, distilled water (30 mL) was added and then stirred for 1 h. The precipitate was filtered off and further purified by silica gel column chromatography using CH2Cl2:CH3OH (20:1, v/v) as eluent to obtain the compound BHM (359 mg, 1.26 mmol). Yield: 65%. m.p.: 219.8–220.5 ◦C. 1 H NMR (600 MHz, DMSO-d6) (Figure S1) δ (ppm) 10.96 (s, 1H), 10.37 (s, 1H), 8.13 (d, J = 7.8 Hz, 1H), 8.04 (d, J = 8.4 Hz, 1H), 7.88 (s, 1H), 7.86 (s, 1H), 7.54 (t, J = 7.2 Hz, 1H), 7.52 (t, J = 7.8 Hz, 1H), 13 4.02 (s, 3H). C NMR (151 MHz, DMSO-d6) (Figure S2) δ (ppm) 191.08, 166.95, 153.96, 153.90, 149.69, Int. J. Mol. Sci. 2019, 20, 5993 8 of 12

134.79, 127.12, 125.83, 124.67, 123.11, 123.08, 122.75, 119.56, 114.47, 56.86. HRMS (m/z) (TOF MS ES-, - + - Negative Ion Modes) (Figure S3): calcd for C15H10NO3S : 284.0381 [M-H ] , found: 284.0385.

3.3.2. Preparation of the Crystal BHM BHM (285 mg, 0.1 mmoL) was dissolved in ethanol (10 mL), and the mixture was refluxed for 1 h. After cooling to room temperature, the solution was filtered, and the filtrate was kept at room temperature for 3 days to obtain single crystals of BHM that were suitable for X-ray analysis.

3.4. Cell Culture and Staining The fibroblast cell line, human stromal cell line (HSC), was purchased from ATCC (CRL-4003). For maintained HSC, the cells were routinely cultured in mixture medium (DMEM: F-12 = 1:1) that was supplemented with 10% heat-inactivated FBS, 100 U/mL penicillin, 100 µg/mL streptomycin, and 1 mM sodiumpyruvate at 37 ◦C, 5% CO2. The HSC was placed in 6-well plates with sterile cover glass at a concentration of 105 cells/well; and after 48 h, the media was changed to being without FBS or antibiotics for chemical treatment. These cells were incubated for 2 h using different amounts of Al3+ (10 and 100 µM). Then, fibroblast cells were washed with D-Hank’s salt solution 3 times. Before staining with BHM, the cells were fixed using a standard paraformaldehyde fixation protocol and then rinsed with 5:5 mixture solutions of DMF and water. Then, the cells were stained by incubating for 2 h with BHM (1 10 5 M). Lastly, the cover glass was mounted over slide glass with an anti-fluorescence × − quenching agent and imaged using fluorescence microscopy.

3.5. X-Ray Diffraction Studies The data collections of the single crystal X-ray diffraction of BHM (0.24 0.23 0.2 mm) were × × carried out on a Rigaku AFC-10/Saturn 724 + CCD diffractometer with graphite-monochromated Mo Kα radiation (λ = 0.71073 Å), using the multi-scan technique. The structures were determined by direct methods using SHELXS-2014 and refined by full-matrix least-squares procedures on F2 with SHELXL-2014. A total of 9673 integrated reflections were collected, and 2896 were unique in the range with Rint = 0.0434 and Rsigma = 0.0409. The maximum/minimum residual electron density = 0.396/ 0.428 eÅ 3. Structural information was deposited with the Cambridge Crystallographic Data − − Center (CCDC 1902058).

4. Applications

4.1. Application in Water Samples BHM for the determination of Al3+ was carried out in a real water sample to verify its practical application. The water samples were collected from the campus of our university and the Songhua River in Heilongjiang Province. Then, Al3+ at different levels (15–20 µM) was mixed with the water samples. The fluorescence responses of the BHM were recorded for sensing Al3+ at 478 nm (Figure8). The results showed that with the increase in the concentration of Al3+ in the tested samples (including ultrapure water, tap water, and Songhua water), the fluorescence intensity increased gradually, and good linearity was found between the fluorescence intensity and Al3+ over the concentration range of 15–20 µM (Figures S9–S11). The desirable recovery and relative standard deviation (RSD) values (Table1) indicated that BHM could be applied in the quantitative analysis of real water samples for the detection of Al3+. Int. J. Mol. Sci. 2019, 20, 5993 9 of 12

Table 1. Determination of Al3+ in water samples.

Total Al3+ Recovery of Amount of Standard Al3+ RSD Relative Error Water Samples Found (n = 3) Al3+ (n = 3) Added (µmol/L) (%) (%) (µmol/L) Added (%) 15 15.08 100.54 1.54 0.69 16 16.18 101.12 2.33 1.41 17 16.95 99.72 2.39 0.34 Ultrapure water − 18 17.66 98.10 1.20 2.31 − 19 19.03 100.19 2.25 0.23 20 20.16 100.81 1.67 0.97 15 15.30 102.00 2.33 2.55 16 15.63 97.67 0.20 2.91 − Tap water (Department of 17 16.98 99.93 1.42 0.07 − Chemistry) 18 17.90 99.47 1.90 0.64 − 19 18.81 99.01 2.84 1.19 − 20 19.83 99.18 2.83 0.96 − 15 14.82 98.82 0.22 1.15 − 16 15.94 99.60 2.67 0.39 − 17 17.33 101.99 2.14 1.95 Songhua River water 18 17.91 99.47 2.84 0.51 − 19 18.99 99.97 1.51 0.02 − 20 19.86 99.32 1.36 0.66 −

4.2. Application in Cell Imaging Monitoring metal ions in biological systems is an important aspect in the evaluation of a probe’s application ability. Therefore, human stromal cells (HSC) were employed for incubation using different amounts of Al3+ and BHM (10 µM) for a certain time and then imaged using fluorescence microscopy. As shown in Figure9A, the cells displayed green fluorescence after incubation with BHM (10 µM) for 2 h. However, after incubation with Al3+ (50 µM) and BHM (10 µM), weak blue fluorescence was detected (Figure9B). The blue fluorescence intensity increased when the cells were treated with a higher concentration of Al3+ (100 µM) in the presence of the same concentration of BHM (10 µM) 3+ (FigureInt. J. Mol.9 C).Sci. 2019 These, 20 results, x FOR PEER indicated REVIEW that BHM could be used as an Al monitor in biological systems.9 of 11

FiguFigurere 9. The incubationincubation ofof cells cells with with BHM BHM (10 (10µM) μM for) for 2 h 2 ( Ah );(A incubation); incubation of the of cellsthe cells with with Al3+ Al(503+µ (M)50 μMand) BHMand BHM (10 µ (M)10 μM (B);) incubation(B); incubation of the of cells the cells with with Al3+ Al(1003+ (10µM)0 μM and) and BHM BHM (10 (µ10M) μM (C)). ( ScaleC). Scale bar bar for forthe the images images is 10 isµ 10m. μm.

5. Conclusions In summary, an easily prepared benzothiazole-based probe BHM was prepared and characterized. In summary, an easily prepared benzothiazole-based probe BHM was prepared and 3+ The BHM displayed high selectivity and sensitivity for Al in DMF:H2O (1:1, v/v) through an characterized. The BHM displayed high selectivity and sensitivity for Al3+ in DMF:H2O (1:1, v/v) throughobvious blue-shiftan obvious in blue the ﬂuorescent-shift in the spectrum fluorescent and spectrum signiﬁcant and color significant change detectedcolor change by the detected naked eye, by respectively. The binding ratio of BHM with Al3+ was 1:1, as determined by the Job plot, where the the naked eye, respectively. The binding ratio of BHM with Al3+ was 1:1, as determined by the Job plot, where the binding details were investigated by FT-IR, 1H NMR titration, and ESI-MS analysis. Furthermore, BHM was successfully applied in the detection of Al3+ in the Songhua River, on a test stripe, and in human stromal cells (HSC).

Supplementary Materials: The following are available online at www.mdpi.com/xxx/s1. Author Contributions: Conceptualization, Z.-Y.X.; methodology, Z.-Y.X.; X.-J.S.; and T.-T.L.; formal analysis, Z.-N.T. and D.-Q.W.; writing—original draft preparation, Z.-N.T. and X.-J.S.; writing—review and editing, Z.-Y.X and T.-T.L. Acknowledgments: This work was supported by the Students’ Innovation and Entrepreneurship Training Program of the Northeast Agricultural University (No. 201910224325) and the Postdoctoral Scientific Research Developmental Fund of Heilongjiang Province (No. LBH-Q14023).

Conflicts of Interest: The authors declare no conflict of interest.

References

1. Azadbakht, R.; Talebi, M.; Karimi, J.; Golbedaghi, R. Synthesis and characterization of a new organic nanoparticle as fluorescent chemosensor for aluminum ions. Inorg. Chim. Acta 2016, 453, 728–734. 2. Yokel, R.A.; Hicks, C.L.; Florence, R.L. Aluminum bioavailability from basic sodium aluminum phosphate, an approved food additive emulsifying agent, incorporated in cheese. Food Chem. Toxicol. 2008, 46, 2261–2266. 3. Wang, C.X.; Wu, B.; Zhou, W.; Wang, Q.; Yu, H.; Deng, K.; Li, J.M.; Zhuo, R.X.; Huang, S.W. Turn-on fluorescent probe-encapsulated micelle as colloidally stable nano-chemosensor for highly selective detection of Al3+ in aqueous solution and living cell imaging. Sensors Actuators B: Chem. 2018, 271, 225–238. 4. Sont, M.G.; White, S.M.; Flamm, W.G.; Burdock, G.A. Safety evaluation of dietary aluminum. Regul. Toxicol. Pharmacol. 2001, 33, 66–79. 5. Filippini, T.; Tancredi, S.; Malagoli, C.; Cilloni, S.; Malavolti, M.; Violi, F.; Vescovi, L.A. Bargellini, M. Vinceti, Aluminum and tin: Food contamination and dietary intake in an Italian Population. J. Trace Elem. Med. Biol. 2019, 52, 293–301. 6. Levesque, L.; Mizzen, C.A.; McLachlan, D.R.; Fraser, P.E. Ligand specific effects on aluminum incorporation and toxicity in neurons and astrocytes. Brain Res. 2000, 877, 191–202.

Int. J. Mol. Sci. 2019, 20, 5993 10 of 12 binding details were investigated by FT-IR, 1H NMR titration, and ESI-MS analysis. Furthermore, BHM was successfully applied in the detection of Al3+ in the Songhua River, on a test stripe, and in human stromal cells (HSC).

Supplementary Materials: The following are available online at http://www.mdpi.com/1422-0067/20/23/5993/s1. Author Contributions: Conceptualization, Z.-Y.X.; methodology, Z.-Y.X.; X.-J.S.; and T.-T.L.; formal analysis, Z.-N.T. and D.-Q.W.; writing—original draft preparation, Z.-N.T. and X.-J.S.; writing—review and editing, Z.-Y.X. and T.-T.L. Acknowledgments: This work was supported by the Students’ Innovation and Entrepreneurship Training Program of the Northeast Agricultural University (No. 201910224325) and the Postdoctoral Scientific Research Developmental Fund of Heilongjiang Province (No. LBH-Q14023). Conflicts of Interest: The authors declare no conflict of interest.

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