A Simple Turn-Off Schiff Base Fluorescent Sensor for Copper (II) Ion and Its Application in Water Analysis

molecules

Article A Simple Turn-off Schiff Base Fluorescent Sensor for Copper (II) Ion and Its Application in Water Analysis

Xing Zhang 1 , Ling-Yi Shen 2, Qi-Long Zhang 1,2,*, Xian-Jiong Yang 2, Ya-Li Huang 2, Carl Redshaw 3 and Hong Xu 1,2,*

1 The key Laboratory of Environmental Pollution Monitoring and Disease Control, School of Public Health, Ministry of Education, Guizhou Medical University, Guiyang 550004, China; [email protected] 2 School of Basic Medical Science, Guizhou Medical University, Guiyang 550004, China; [email protected] (L.-Y.S.); [email protected] (X.-J.Y.); [email protected] (Y.-L.H.) 3 Department of Chemistry, University of Hull, Cottingham Road, Hull, Yorkshire HU6 7RX, UK; [email protected] * Correspondence: [email protected] or [email protected] (Q.-L.Z.); [email protected] (H.X.); Fax: +86-851-8817-4017 (H.X.)

Abstract: An aniline-functionalized naphthalene dialdehyde Schiff base fluorescent probe L with aggregation-induced enhanced emission (AIEE) characteristics was synthesized via a simple one-step condensation reaction and exhibited excellent sensitivity and selectivity towards copper(II) ions in aqueous media with a fluorescence “ turn-off ” phenomenon. The detection limit of the probe is 1.64 × 10−8 mol·L−1. Furthermore, according to the results of the UV-vis/fluorescence titrations, Job’s plot method and 1H-NMR titrations, a 1:2 stoichiometry was identified. The binding constant 2+ 3 between L and Cu was calculated to be Ka = 1.222 × 10 . In addition, the AIEE fluorescent probe L could be applied to detection in real water samples with satisfactory recoveries in the range 99.10–102.90% in lake water and 98.49–102.37% in tap water.

Citation: Zhang, X.; Shen, L.-Y.; Keywords: synthesis; fluorescent probe; copper(II) ion; AIEE; water detection Zhang, Q.-L.; Yang, X.-J.; Huang, Y.-L.; Redshaw, C.; Xu, H. A Simple Turn-off Schiff Base Fluorescent Sensor for Copper (II) Ion and Its 1. Introduction Application in Water Analysis. In recent years, fluorescent probes have been widely used in the field of rapid detection Molecules 2021 26 , , 1233. https:// (food, metal ion, anion, biomarker, etc.) due to advantages such as ease of operation, low doi.org/10.3390/molecules26051233 cost, high selectivity, and sensitivity [1–4]. Copper is an earth abundant element which plays an important role in many domains like industrial material, [5] bactericide, and Received: 1 February 2021 herbicide [6,7], etc. On the other hand, copper ions are important environmental pollutants. Accepted: 20 February 2021 Published: 25 February 2021 It not only produces toxic effects on the growth of animals and plants, but also lead to risks to human health via the food chain [8–10]. Lack or excess of copper will cause

Publisher’s Note: MDPI stays neutral health damage. with regard to jurisdictional claims in In order to protect human health, the WHO stipulates that the limit of copper ions published maps and institutional affil- in drinking water should not exceed 31.4 µM[7]. Thus, the development of technologies iations. for copper ion detection has become an important focus in environmental and food nu- tritional fields. Traditional methods such as atomic absorption spectroscopy (AAS), gas chromatography-mass spectrometry (GC-MS), inductively coupled plasma mass spectrometry (ICP-MS) and high performance liquid chromatography (HPLC) have been widely utilized for detection copper ion [3,11], but these systems also have their limitations, e.g., Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. large and expensive instruments, complex modes of operation and high detection costs. In This article is an open access article recent years, methods based on fluorescent probes have emerged as the more cutting-edge distributed under the terms and technology in the field of metal ion detection due to convenient procedures, super sensi- conditions of the Creative Commons tivity, and fast response times [12,13]. Many copper(II) probes have been reported due to Attribution (CC BY) license (https:// their low detection limit and remarkable optical changes [14,15]. creativecommons.org/licenses/by/ However, most traditional fluorescent materials exhibit an aggregation caused quench- 4.0/). ing (ACQ) effect, which means they are highly emissive in solution, but a quenching affect

Molecules 2021, 26, 1233. https://doi.org/10.3390/molecules26051233 https://www.mdpi.com/journal/molecules Molecules 2021, 26, x FOR PEER REVIEW 2 of 12

However, most traditional fluorescent materials exhibit an aggregation caused Molecules 2021, 26, 1233 quenching (ACQ) effect, which means they are highly emissive in solution, but a quench-2 of 11 ing affect exists in the aggregation state. In the case of dyes, the ACQ effect can greatly limit their practical applications in material science [16]. In contrast, an abnormal photophysical phenomenon was observed by Tang’s group in 2001 [2,17], namely, aggregation exists in the aggregation state. In the case of dyes, the ACQ effect can greatly limit their induced emission (AIE) and aggregation-induced emission enhancement (AIEE), practical applications in material science [16]. In contrast, an abnormal photophysical whereby the fluorescent molecules show non-/weak emission in solution but enhanced phenomenon was observed by Tang’s group in 2001 [2,17], namely, aggregation induced fluorescence in the aggregation state. Molecules emit energy through intramolecular ro- emission (AIE) and aggregation-induced emission enhancement (AIEE), whereby the flu- orescenttation and molecules vibrational show motions non-/weak in the solution, emission while in solution in the but aggregation enhanced state fluorescence the non-ra- in thediative aggregation transition state. is blocked, Molecules and emit, in turn energy, energy through is lost intramolecular through the radiative rotation andtransition vibra- tionalchannel motions [18]. in the solution, while in the aggregation state the non-radiative transition is blocked,Naphthalene and, in turn, and energy its derivatives is lost through have thebeen radiative extensively transition applied channel in the [ 18assembly]. of lightNaphthalene-emitting devices and and its derivatives chemical sensors have been due extensivelyto their excellent applied optical in the properties assembly and of light-emittingunique chemical devices stability and [19 chemical–21]. In sensorsthis research, due to we their have excellent developed optical a small properties molecular and uniqueprobe Lchemical possessing stability AIEE characteristics [19–21]. In this via research, a simple we one have-step developed reaction, which a small has molec- good ularrecognition probe L performancepossessing AIEE and characteristics anti-interference via ability a simple for one-step copper ion reaction, detection which under has goodaqueous recognition conditions. performance and anti-interference ability for copper ion detection under aqueous conditions. 2. Results and Discussion 2.2.1. Results Synthesis and Discussion 2.1. Synthesis A new Schiff base probe L was obtained from 2,6-dihydroxynaphthalene-1,5-dialde- hydeA and new aniline Schiff by base a simple probe L onewas-step obtained reaction. from The 2,6-dihydroxynaphthalene-1,5-dialdehyde molecular structure was characterized andby 1H aniline-NMR by spectroscopy, a simple one-step HRMS reaction., and single The molecular crystal X-ray structure diffraction. was characterized The Schiff base by 1 probeH-NMR L exhibited spectroscopy, excellent HRMS, solubility and single in common crystal X-ray solvents diffraction. (such as The dichloromethane, Schiff base probe tet-L exhibitedrahydrofuran, excellent DMSO, solubility etc.) and in common possessed solvents good (suchacid and as dichloromethane, alkali-resistance tetrahydrofu-over the pH ran,range DMSO, 3–11 within etc.) and 1440 possessed min (Table good S2). acid This and work alkali-resistance provides a new over strategy the pH for range extending 3–11 withinthe practical 1440 min application (Table S2).s of This small work molecular provides probes a new strategyin the heavy for extending metal detection the practical field. applications(Scheme 1) of small molecular probes in the heavy metal detection field. (Scheme1).

SchemeScheme 1.1. Synthetic route toto Schiff-baseSchiff-base LL..

2.2.2.2. AIEE Properties TheThe probe LL emitsemits glaring glaring red red light light in inthe the solid solid state state but butthe theemission emission decreases decreases dra- dramaticallymatically in the in dissolved the dissolved state, state, so we so propose we propose that this that probe this may probe exhibit may exhibitAIEE proper- AIEE properties.ties. To investigate To investigate our hypothesis, our hypothesis, a good solvent a good tetrahydrofuran solvent tetrahydrofuran (THF) and (THF) a bad andsol- avent bad water solvent were water selected were as selected the testing as thesystems. testing As systems. shown in As Figure shown 1, the in Figurecompound1, the L compoundemitted orangL emittede mission orange with mission λmax em with = 565λmax nm em in =pure 565 THF nm in solution. pure THF As solution. the water As frac- the watertion (f fractionw) gradually (f w) graduallyincreased increasedfrom 0% to from 50%, 0% the to fluorescence 50%, the fluorescence intensity barely intensity changed. barely changed.Subsequently, Subsequently, when the when water the fraction water fraction (fw) reached (f w) reached60%, some 60%, particles some particles could be could ob- beserved observed in the in mixture, the mixture, and the and mixture the mixture exhibited exhibited bright bright orange orange light light under under 365 365nm nmUV UVirradiation. irradiation. The Thefluorescence fluorescence spectrum spectrum also also corresponded corresponded with with this thisphenomenon: phenomenon: the theflu- f fluorescenceorescence intensity intensity at at60% 60% was was much much higher higher than than that that of of fww = 0 0–50%.–50%. When the waterwater fraction reached 70%, the fluorescence intensity of the solution attained the maximum value (807 a.u.) with an approximate 40.31-fold increase versus that in the pure solution. In addition, we also tested the quantum yield of L in the solid state and in pure THF solvent. The quantum yield in the solid state (Φ f = 18.4%) is higher than that in THF solution (Φ f = 0.5%). Thus, the Schiff-base L is a chromophore with aggregation-induced Molecules 2021, 26, x FOR PEER REVIEW 3 of 12

fraction reached 70%, the fluorescence intensity of the solution attained the maximum value (807 a.u.) with an approximate 40.31-fold increase versus that in the pure solution. Molecules 2021, 26, 1233 3 of 11 In addition, we also tested the quantum yield of L in the solid state and in pure THF solvent. The quantum yield in the solid state (Φ f = 18.4%) is higher than that in THF solution (Φ f = 0.5%). Thus, the Schiff-base L is a chromophore with aggregation-induced enhanced emissionenhanced characteristics. emission characteristics. As the pro Asbe the polymerizes probe polymerizes at fw = 60%, at f w referring = 60%, referring to the experi- to the mentalexperimental conditions conditions of similar of similar probes probes [20, [2220],,22 we], we choose choose the the mixture mixture of of THF/water THF/water (VTHF/V/VH2OH2O = 4:1)= 4:1) as asthe the recognition recognition environment. environment.

Figure 1. (A(A) )Fluorescence Fluorescence spectra spectra of of L (40L (40 μM)µM) in tetrahydrofuran in tetrahydrofuran (THF (THF)/water)/water mixtures mixtures with with different water fractions (λex/λem = 428 nm/565 nm, slit: 5/5 nm, voltage: 660 v). (B) Plots of fluores- different water fractions (λex/λem = 428 nm/565 nm, slit: 5/5 nm, voltage: 660 v). (B) Plots of cence intensity at 565 nm. (C) photographs in THF/water mixtures with different water fractions ﬂuorescence intensity at 565 nm. (C) photographs in THF/water mixtures with different water taken under 365 nm UV irradiation. fractions taken under 365 nm UV irradiation.

2.3. Stability of Detection TThehe luminescent and recognition behavior of Schiff base compounds are are often af- fected by many factors (such asas pH,pH, time,time, etc.).etc.). If thethe recognitionrecognition environmentenvironment tendstends toto acidic/alkaline, this this can can induce induce C=N C=N fracture, fracture, which which may may affe affectct the optical properties and recognition ability ability of of these these compounds compounds [23 [23,24,24].]. In In order order to to test test the the luminescent luminescent stability stability of probeof probe L, Lthe, the pH pH 3– 3–1111 range range was was selected selected as as the the detection detection environment. environment. In In addition, the pre-experimentalpre-experimental resultsresults showedshowed thatthat thethe probeprobe LL hadhad thethe abilityability toto recognizerecognize CuCu2+2+,, soso thethe mixed solution of probe L-Cu-Cu2+ waswas also also selected selected for for determination determination at at the the same same time. time. As shown in Figure 22A,A, thethe emissionemission intensityintensity ofof probeprobe L at 565 nm barely changed, which indicated that this probe possessed good acid andand alkali-resistancealkali-resistance over the pHpH range 3–11.3–11. When adding Cu2+ toto the the mixture, mixture, a a significant significant fluorescent fluorescent quenching phenomenon was observed over the pH range from 8 to 11, whilst little fluorescencefluorescence changechange was found over the pH range 3 toto 7.7. TheThe acidicacidic conditionsconditions can induce dissociation of the L--CuCu2+ complexcomplex because because of of the the protonation protonation of ofprobe, probe, and and excessive excessive H+ in H +thein solution the solution may pushmay pushthe coordination the coordination equilibrium equilibrium to the to left the [25 left]. As [25 ].for As higher for higher pH conditions, pH conditions, the alka- the linealkaline recognition recognition environment environment can provide can provide sufficient sufficient hydroxyl hydroxyl ions to ionsconsume to consume H+, and Hthe+, 2+ ionizedand the phenolic ionized phenolic groups interact groupsed interacted more easily more with easily copper with ions copper to form ions L to-Cu form2+ complexL-Cu [complex26,27]. These [26,27 may]. These be the may reasons be the why reasons pro whybe L probeexhibitedL exhibited better recognition better recognition ability abilityunder alkalineunder alkaline conditions. conditions. We also We tested also the tested time the-dependent time-dependent optical stability optical stability of probe of L probeand theL and the L-Cu2+ mixture, and the results showed that L and the L-Cu2+ complex were stable over a certain period of time.

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Molecules 2021, 26, 1233 4 of 11 L-Cu2+ mixture, and the results showed that L and the L-Cu2+ complex were stable over a certain period of time.

FigureFigure 2. TheThe fluorescence ﬂuorescence intensity intensity stability stability of the of theL andL andL-CuL2+-Cu systems2+ systems versus versus different different (A) pH ( Avalues) pH and values (B) reaction and (B) reactiontime. time.

2.4. Cation Sensing Study SchiffSchiff basebase compoundscompounds areare widelywidelyused used in in the the recognition recognition of of rare-earth rare-earth metals, metals, transi- tran- tionsition metals metals and and alkali alkali metals metals as as a a result result of of their their high high binding binding affinityaffinity [[2828––3030]].. ToTo testtest thethe detecting abilityability toward toward metal metal ions, ions, probe probeL (40 L (µ40M) μ wasM) was exposed exposed to many to many metal meta ionsl (such ions 2+ 2+ + 2+ + 3+ 2+ 2+ 3+ 3+ 2+ 2+ 2+ + + + as(such Cd as, CuCd2+,,K Cu,2+ Pb, K+,, Pb Li 2+,, FeLi+, ,Fe Mg3+, Mg, Co2+, Co, Cr2+, Cr, Al3+, Al,3+ Ba, Ba,2+ Ni, Ni2+, Zn, Zn2+,, Ag Ag+,, NaNa+,, HgHg+, n+ µ − − − − 2− − − 2− [M][M]n+ == 200 200 μM)M) and and anions anions (such (such as as A AcOcO−, ClO, ClO4−, 4Br,−, BrNO,2− NO, CO2 32,−, CO I−, 3F−, SO,I 42,F−, Cl,− SO, PO4 43−,, − 3− 2− 2− − − n− µ ClC2O,42 PO−, SO4 32,C−, HSO2O4 3−, ,HCO SO3 3−,, [A] HSOn− 3= 200, HCO μM)3 in, [A] mixtures= 200 of THFM) in and mixtures water (V ofTHF THF/VH2O and = water4/1, pH (V =THF 8.00)./V H2O = 4/1, pH = 8.00). As shown in in Figure Figure 3,3, on on adding adding the the metal metal ions ions to tothe the solvent solvent containing containing L, onlyL, onlyCu2+ 2+ Cucause causethe solution the solution colour colourto change to via change naked via-eye naked-eye observation observation (Figure S3). (Figure The absorption S3). The absorptionspectra and spectrafluorescence and fluorescence spectra of L– spectracation mixture of L–cation indicated mixture that indicated probe L exhibits that probe goodL 2+ exhibitsselectivity good toward selectivity Cu2+, while toward other Cu cations, while or other anions cations (Figure or anions S4) had (Figure little impact S4) had on little the L impactoptical b onehavior the optical of probe behavior L. On ofother probe hand,. On under other a 365 hand, nm under UV lamp, a 365 only nm the UV L lamp,-Cu2+ only the L-Cu2+ mixture led to the emission light quenching dramatically (Figure S3). mixture led to the emission light quenching dramatically (Figure S3). Furthermore, com- Furthermore, competitive experiments were also performed to investigate the selectivity petitive experiments were also performed to investigate the selectivity of the probe to- of the probe toward Cu2+. When Cu2+ was present in the solution, the emission of the ward Cu2+. When Cu2+ was present in the solution, the emission of the mixture at λem = 565 mixture at λ = 565 nm was quenched, while without Cu2+, the emission barely changed nm was quenched,em while without Cu2+, the emission barely changed (Figure 4), which (Figure4), which suggested that the coexisting ions/anions had a limited impact on the suggested that the coexisting ions/anions had a limited impact on the detection of Cu2+. Molecules 2021, 26, x FOR PEER REVIEWdetection of Cu2+. Thus, the interference experiments indicated that the probe displays5 of 12 a Thus, the interference experiments indicated that the probe displays a high specificity and high specificity and selectivity for detecting Cu2+ ions. selectivity for detecting Cu2+ ions.

FigureFigure 3. 3. ((AA)) The The UV UV–vis–vis and and ( (BB)) Fluorescence Fluorescence spectra spectra of of the the fluorescence ﬂuorescence probe probe LL interactinginteracting with with different different metal metal ions ions (λex/λem = 428/565 nm, slit: 5/5 nm, voltage: 900 v). (λex/λem = 428/565 nm, slit: 5/5 nm, voltage: 900 v).

Figure 4. Bar diagram of the competitive experiments of various metal cations (A) and anions (B) on the fluorescence intensity of the probe/Cu2+ complex in buffer solution.

2.5. Job’s Plot and Binding Constant Based on the above experimental conditions, the fluorescence titration experiments were performed with progressive addition of Cu2+ and are presented in Figure 5. As the figure demonstrates, the fluorescence intensity of probe L at λmax em = 565 nm gradually decreased as the Cu2+ ions were added. In addition, when the concentration of probe L change from 0 to 72μM (Figure S5), there exist a good linear relationship between the probe and the copper(II) ions (y = 432.59482 − 218.198x, R2 = 0.99276). Herein, the detection limit was calculated by utilizing the data of the fluorescence titration experiments following the IUPAC method (IUPAC Compendium of Analytical Nomenclature): 10 groups of blank samples were tested in the absence of copper under the same conditions, and then the standard deviation (SD) was calculated from the emission peak at 565 nm. After that, following the formula: the detection limit = 3SD/S, where S is the slope of the linear relationship during the fluorescence titration, the detection limit of probe L for Cu2+ is calculated to be 16.4 nM. Comparing with other Cu2+ probes (Table S3), the probe L has the advantages of lower detection limit and simpler synthetic route.

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Figure 4. Bar diagram of the competitive experiments of various metal cations (A) and anions (B) on the ﬂuorescence intensity of the probe/Cu2+ complex in buffer solution.

2.5. Job’s Plot and Binding Constant Based on the above experimental conditions, the fluorescence titration experiments were performed with progressive addition of Cu2+ and are presented in Figure5. As the figure demonstrates, the fluorescence intensity of probe L at λmax em = 565 nm gradually decreased as the Cu2+ ions were added. In addition, when the concentration of probe L change from 0 to 72µM (Figure S5), there exist a good linear relationship between the probe and the copper(II) ions (y = 432.59482 − 218.198x, R2 = 0.99276). Herein, the detection limit was calculated by utilizing the data of the fluorescence titration experiments following the IUPAC method (IUPAC Compendium of Analytical Nomenclature): 10 groups of blank samples were tested in the absence of copper under the same conditions, and then the standard deviation (SD) was calculated from the emission peak at 565 nm. After that, following the formula: the detection limit = 3SD/S, where S is the slope of the linear relationship during the fluorescence titration, the detection limit of probe L for Cu2+ is Molecules 2021, 26, x FOR PEER REVIEWcalculated to be 16.4 nM. Comparing with other Cu2+ probes (Table S3), the probe L6 ofhas 12

the advantages of lower detection limit and simpler synthetic route.

2+ Figure 5. ((AA)) Fluorescence Fluorescence spectra spectra on addition addition of Cu2+ toto the the probe; probe; (B (B) )Job’s Job’s plot plot for for the the determination determination of of the the stoichiometry stoichiometry of L and Cu2+ in the mixture of THF/H2O (VTHF:VH2O = 4/1), the total concentration of L and Cu2+ was2+ 40 μM. of L and Cu in the mixture of THF/H2O (VTHF:VH2O = 4/1), the total concentration of L and Cu was 40 µM.

Further,Further, the the binding mode of probe L and Cu2+ waswas investigated investigated by by a a Job’s Job’s plot plot by 2+2+ controllingcontrolling the total concentration ofof thethe probeprobe andand Cu Cu at 40 µμMM inin aa mixturemixture ofof THF/HTHF/H2O (V(VTHFTHF:V:VH2OH2O = 4/1).= 4/1). The The results results indicated indicated that that the the binding binding stoichiometry stoichiometry between between the the probe probe andand Cu Cu2+2+ waswas 1:2. 1:2. On On the the other other hand, hand, a a Benesi Benesi–Hildebrand–Hildebrand (Figure (Figure 66)) curve basedbased onon thethe above experiments could been obtained and the binding constant calculated. According to the formula [31]:

F − F0 = △F = [Cu2+](Fmax − F0)/(1/Ka + [Cu2+]) (1)

1 Here, F and F0 are the corresponding fluorescence intensity at 598 nm in the presence and absence of Cu2+, and Fmax is the fluorescence intensity when the probe has completely complexed with cations. Based on the data of fluorescence titration and the slop of the correlation curve, the binding constant (Ka) was calculated to be Ka = 1.222 × 103.

Figure 6. The Benesi–Hildebrand plot of 1/(F − F0) versus 1/[Cu2+].

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above experiments could been obtained and the binding constant calculated. According to the formula [31]:

2+ 2+ F − F0 = 4F = [Cu ](Fmax − F0)/(1/Ka + [Cu ]) (1)

2+ Figure 6. The Benesi–Hildebrand plot of 1/(F − F0) versus 1/[Cu ].

Here, F and F0 are the corresponding fluorescence intensity at 598 nm in the presence 2+ and absence of Cu , and Fmax is the fluorescence intensity when the probe has completely complexed with cations. Based on the data of fluorescence titration and the slop of the 3 correlation curve, the binding constant (Ka) was calculated to be Ka = 1.222 × 10 .

2.6. A Possible Mechanism for Detection Cu2+ To better understand the recognition mechanism of L for detecting copper ions, 1H- NMR spectroscopic titration experiments in d-DMSO: D2O solution were performed. As shown in Figure7, when there did not exist any copper ions in the solution, one single peak (δ = 9.69 ppm) and one doublet (δ = 8.66–8.68 ppm) were observed corresponding to the -OH and -CH=N, respectively. The proton peaks at δ = 7.16–7.59 ppm were from the naphthalene ring and aniline ring of the probe L. When copper ions were added into the mixture, the proton signals at 9.69 ppm and 8.66 ppm were weakened and split, and new proton peaks at 10.72–10.74 ppm, 9.00–9.03 ppm, and 9.12–9.14 ppm could be observed at the same time. This indicated that in the presence of Cu2+, the O atom from the hydroxyl and the N atom from the -CH=N bond coordinated with Cu2+ ﬁrstly. On L 2+ 1 the other hand, as the initial content of probe exceeded that of the Cu in the mixture, there existed two coordination sites in one L molecule, and the probe molecule adopted

a pattern with partially complexed with Cu2+. Thus, the proton environment of the -OH and -CH=N exhibited differences at different ratios of L and Cu2+, respectively. Similarly, the proton signals from the naphthalene and benzene rings also undergo a high ﬁeld shift from 7.16–7.59 ppm to 7.09–7.57 ppm, indicating that the probe L does interact with Cu2+. On further increasing the amount of Cu2+ (1eq–2.5eq), the proton signals of -OH and Molecules 2021, 26, x FOR PEER REVIEW 7 of 12

the same time. This indicated that in the presence of Cu2+, the O atom from the hydroxyl and the N atom from the -CH=N bond coordinated with Cu2+ firstly. On the other hand, as the initial content of probe L exceeded that of the Cu2+ in the mixture, there existed two coordination sites in one L molecule, and the probe molecule adopted a pattern with partially complexed with Cu2+. Thus, the proton environment of the -OH and -CH=N exhib- 2+ Molecules 2021, 26, 1233 ited differences at different ratios of L and Cu , respectively. Similarly, the proton signals7 of 11 from the naphthalene and benzene rings also undergo a high field shift from 7.16–7.59 ppm to 7.09–7.57 ppm, indicating that the probe L does interact with Cu2+. On further increasing the amount of Cu2+ (1eq–2.5eq), the proton signals of -OH and -CH=N became 2+ -CH=Nmuch weaker became and much tend weaker to be stable and tend after to the be content stable after of Cu the2+ contentreached of 2.0 Cu eq, suggestedreached 2.0 that eq, 2+ thesuggested probe L that and the Cu probe2+ mayL adoptand Cu a 1:2may coordination adopt a 1:2 mode, coordination consistent mode, with consistent the results with ob- tainedthe results via the obtained Job’s plot via themethod. Job’s plot method.

Figure 7. 1H-NMR spectroscopic titration spectra of L-Cu2+2+ on increasing concentrations of Cu2+2+ in d-DMSO/D2O solution. Figure 7. H-NMR spectroscopic titration spectra of L-Cu on increasing concentrations of Cu in d-DMSO/D2O solution.

The data from the single crystal X-rayX-ray diffraction analysis verify that two benzenebenzene rings at the ends of the probe are parallel to each other, but the naphthalenenaphthalene ring is not parallel to the benzene rings. As As shown shown in in Figure 8 8,, the the dihedral dihedral angle angle between between the the N1/NN1/N##(connecting (connecting with with the the plane plane of of benzene) benzene) and and the the double double bond bond C (connecting with the plane of naphthalene) is 167.14167.14°,◦, so probe L is a nonplanar nonplanar molecule. Furthermore, the hydrogen atoms on two hydroxyl groups form a stable six membered ring structure with the N atoms from imineimine groupsgroups viavia thethe O-HO-H······N intramolecular hydrogen bonds. As the 2,62,6-dihydroxynaphthalene-1,5-dialdehyde-dihydroxynaphthalene-1,5-dialdehyde reac reactsts with with aniline aniline to to form the -CH=N-CH=N bond, this functional group bridges the naphthalene and benzene ring leading to the probes probes conjugated structure.structure. WhenWhen probeprobe molecules molecules are are excited excited by by light, light, an an electron electron ﬂow flow from from the thenaphthalene naphthalene ring ringto the to imine the iminegroup groupcan easily can occur; easily on occur; the other on the hand, other in the hand, THF/H in the2O THF/Hsolution,2O there solution, exist athere fast structureexist a fast convert structure from convert enol form from to ketoenol formform into theketo molecule form in [ 32the], moleculethe intramolecular [32], the intramolecular hydrogen bonds hydrogen limit the bonds nonradiative limit the transition; nonradiative thus, transition the probe; thusL in, 1 the probe dissolved L in state the dissolved emits orange state light emits [33 orange–35]. According light [33–35] to.the AccordingH-NMR to spectroscopic the 1H-NMR 2+ spectroscopictitration experiments, titration theexperiments, probe coordinates the probe with coordinates Cu via with the N Cu atom2+ via of the imine N atom and the of iminO atome and of the -OH O toatom form of - aOH stable to form complex. a stable At complex. this time, At the this lone time, pair the electronslone pair onelectrons the N and O atoms will transfer to the empty orbitals of the copper ion, which blocks the initial excited electron transfer process (Figure9)[ 36], resulting in ﬂuorescence quenching of the probe solution. Molecules 2021, 26, x FOR PEER REVIEW 8 of 12 Molecules 2021, 26, x FOR PEER REVIEW 8 of 12

Molecules 2021, 26, 1233 on the N and O atoms will transfer to the empty orbitals of the copper ion, which block8 of 11s on the N and O atoms will transfer to the empty orbitals of the copper ion, which blocks the initial excited electron transfer process (Figure 9) [36], resulting in fluorescence the initial excited electron transfer process (Figure 9) [36], resulting in fluorescence quenching of the probe solution. quenching of the probe solution.

FigureFigure 8. 8. TheThe X-rayX-ray single crystal diffraction image image of of probe probe L L (Dashed (Dashed lines lines represent represent hydrogen hydrogen bonds,Figure Symmetry8. The X-ray code: single# = −crystalx, 1 − y diffraction, 1 − z). image of probe L (Dashed lines represent hydrogen bonds,bonds, Symmetry Symmetry code:# code:# = =− −xx,, 11 −− yy,, 1 1 −− zz).). 428nm 565nm 428nm ESIPT 565nm ESIPT

N N N N Cu2+ LMCT Cu2+ OH LMCT O OH Cu2+ O Cu2+ LMCT HO O LMCT HO 2+CuO 2+ N Cu N N N

fluorescence on fluorescence off fluorescence off fluorescence on Figure 9. The possible recognition mechanism process for detection of copper ions. FigureFigure 9. 9.The The possible possible recognition recognition mechanism mechanism process process for for detection detection of of copper copper ions. ions. 2.7.2.7. ApplicationsApplications 2.7. Applications InIn orderorder toto furtherfurther evaluate the potential potential application application of of probe probe LL forfor the the detection detection of In order to further evaluate the potential application of probe L for the detection of ofCu Cu2+ in2+ realin real specimens, specimens, water water samples samples from froman artificial an artificial lake (at lake Guizhou (at Guizhou Medical Medical Univer- Cu2+ in real specimens, water samples from an artificial lake (at Guizhou Medical Univer- University)sity) and running and running water water(at our (at laboratory) our laboratory) have been have collected been collected for testing. for testing. The specific The sity) and running water (at our laboratory) have been collected for testing. The specific specificexperimental experimental process processis as follows: is as 7. follows:6 mL THF 7.6 solution, mL THF 4 solution,00 μL probe 400 stockµL probe soluti stockon (40 experimental process is as follows: 7.6 mL THF solution, 400 μL probe stock solution (40 solutionμM), 1 mL (40 bufferµM), 1 solution mL buffer, and solution, 1 mL water and 1 sample mL water (had sample been filtered) (had been were filtered) added were into μM), 1 mL buffer solution, and 1 mL water sample (had been filtered) were added into addedone volumetric into one volumetricflask and the flask mixture and theshaken mixture well. shaken At the same well. time, At the another same time, water another sample one volumetric flask and the mixture shaken well. At the same time, another water sample waterwas processed sample was with processed the same with steps the sameand an steps appropriate and an appropriate amount of amount standard of standardsubstance was processed with the same steps and an appropriate amount of standard substance substance(Cu(NO3) (Cu(NO2) was added3)2) was in addedit. After in it.standing After standing for 30 min, for 30the min, fluorescence the fluorescence intensity intensity of the (Cu(NO3)2) was added in it. After standing for 30 min, the fluorescence intensity of the ofsample the sample at 565 at nm 565 had nm been had beenrecorded recorded for further for further calculations. calculations. As shown As shown in Table in Table 1, the1, sample at 565 nm had been recorded for further calculations. As shown in Table 1, the therecoveries recoveries of ofthe the probe probe were were calculated calculated in in the the range range of of 99.10 99.10–102.90%–102.90% in lakelake waterwater andand recoveries of the probe were calculated in the range of 99.10–102.90% in lake water and 98.49–102.37%98.49–102.37% inin tap tap water.water. These These results results suggest suggest that thatL Lis is a a sensitive sensitive and and selective selective probe probe 98.49–2+102.37% in tap water. These results suggest that L is a sensitive and selective probe forfor Cu Cu2+ monitoring in environmental water samples. for Cu2+ monitoring in environmental water samples.

Table 1. The detail data for Cu2+ detection in real water samples.

Measured Added Detected Recovery RSD Sample (µmol·L−1)(µmol·L−1)(µmol·L−1)(n = 3, %) (n = 3, %) Running water 8.99 5.00 14.03 99.71 0.93 10.00 19.28 98.49 2.01 20.00 28.32 102.37 1.15 Artiﬁcial lake 15.92 5.00 21.11 99.10 1.48 10.00 25.19 102.90 1.72 20.00 35.99 99.81 2.07

Molecules 2021, 26, 1233 9 of 11

3. Experimental Section 3.1. Materials and Characterization Unless otherwise stated, all of the starting materials were commercially available and used without further purification. The solution of the metal ions and anions were prepared from their nitrate and sodium salts, respectively. 1H-NMR spectra (400 MHz) were recorded on a Inova-400 Bruker AV 400 spectrometer (Bruker, Karlsruhe, Germany) using d-DMSO solvent and tetramethylsilane as the internal reference. J-values are given in Hz. High resolution mass spectra (HRMS) were recorded on a GCT premier CAB048 mass spectrometer (Waters Corp., Milford, MA, USA) operating in a MALDI-TOF mode. UV-vis absorption spectra were obtained on a UV-2600 Milton Ray Spectrofluorometer (Shimadzu, Kyoto, Japan). PL spectra were recorded on a Cary Eclipse Hitachi 4500 spectrofluorometer (Hitachi, Tokyo, Japan). The crystal structure was determined by Bruker Smart Apex single crystal diffractometer (Bruker, Karlsruhe, Germany).

3.2. Synthesis of the Compound L Firstly, 43.2 mg (0.2 mmol) 2,6-dihydroxynaphthalene-1,5-dialdehyde was dissolved in 30 mL methanol, and then 50 µL (95%, 5.44 mmol) aniline was added into the mixture and stirred for 10 min in room temperature. After that, adding 10 µLH2SO4 into the mixture and stirring for another 5 h. The precipitate was ﬁltered and washed with hot methanol for three times, and dried in vacuum to obtain the target red compound (63 mg, 0.17 mmol, yield 86.12%). An appropriate amount of precipitate was dissolved in a mixture of tetrahydrofuran and methanol (2:1), and then evaporated. After 4 days, red acicular crystals suitable for single crystal diffraction analysis were obtained. 1H-NMR (400 MHz, DMSO, TMS) δ 9.69 (s, 2H), 8.67 (d, J = 9.4 Hz, 2H), 7.58 (d, J = 7.8 Hz, 4H), 7.47 (t, J = 7.7 Hz, 4H), 7.30 (t, J = 7.3 Hz, 2H), 7.17 (d, J = 9.4 Hz, 2H),. HRMS calculated: 398.11, found 398.15.

3.3. X-ray Crystallography Crystallographic data for ligand L was collected on a Bruker APEX 2 CCD diffractometer with graphite-monochromated Mo Kα radiation (λ = 0.71073 Å) in the ω scan mode [37]. The structure was solved by a charge ﬂipping algorithm and reﬁned by full-matrix least- squares methods on F2 [38]. All relative standard deviation were estimated using the full covariance matrix. Further details are presented in Table S1. CCDC: 2059923, L.

3.4. General Methods for Optical Tests Firstly, 3.7 mg (10µM) of probe L was dissolved in 10.00 mL THF solution to prepare 1mM stock solution. Then the nitrate of metal ions and the sodium salt of anions (Ag+, Al3+, Ba2+, Cd2+, CO2+, Cr3+, Na+, Cu2+,K+, Ni2+, Pb2+, Zn2+, Hg2+, Fe3+, Mg2+, Li+, AcO−, − − 2− 2− − − 2− − − 3− 2− 2− 2− ClO4 , Br , NO , CO3 ,I ,F , SO4 , Cl ,H2PO4 , PO4 , HPO4 ,C2O4 , SO3 , − − HSO3 , HCO3 ) were accurately weighed and dissolved in 10.00 mL Tris-HCl buffer to form 10 mM ion stock solution. The preparation method of Tris-HCl buffer solution (2 mM) was as follows: 121.20 mg of trimethylol aminomethane was dissolved in 1.00 L ultrapure water, then its pH was adjusted to 8.00 with 0.10 M HCl solution and 0.10 M NaOH solution.

4. Conclusions In summary, we have developed a new simple AIEE ﬂuorescent probe based on two aniline units attached to a naphthalene dialdehyde core. The ﬂuorescence intensity of the probe in good solvents and poor solvents can differ by 40.31 times. Furthermore, in the presence of copper(II) ions, the probe solution showed an obvious colour change from yellow to brown under daylight and from bright to dark under UV lamp irradiation with a detection limit as low as 1.64 × 10−8 mol·L−1. This indicates that the probe L has the potential to be used for the detection of Cu2+ by naked eye and via instrumentation. Based on the titration experiments, a good linear relationship was found which may apply the Molecules 2021, 26, 1233 10 of 11

probe to the quantitative and qualitative detection of Cu2+ in real samples. We believe this work not only provides a new example of a small molecular probe for ion detection, but these results may inform research in broader ﬁelds such as biometrics and photodynamic therapy, and such research is going-on in our laboratory.

Supplementary Materials: The following are available online, Figure S1: 1H-NMR of probe L title, Figure S2: HRMS spectrum of L, Figure S3: Photographs of probe L-cation complex under natural light and 365 nm UV lamp, Figure S4: UV-vis and Fluorescence spectra of the fluorescence probe L interacting with different anions; Photograph of probe L-anions complex in THF/water (VTHF/Vwater = 4/1, pH = 8.00) solution under 365 nm UV lamp, Figure S5: The absorbance and 2+ fluorescence spectra on addition of Cu to the probe (40 µM, VTHF:VH2O = 4/1, λex = 428 nm, slit: 5/5 nm, voltage: 900 v). The fluorescence intensity change plots at 565 nm on addition of Cu2+. Insert: when [Cu2+]/[probe] is in the range of 0–1.8 ratio, the fluorescence intensity of the probe has a good linear relationship with Cu2+ (y = 432.59482 − 218.198x, R2 = 0.99276), Table S1: Summary of crystal data of probe L, Table S2: The fluorescence intensity(a.u.) of probe L and L-Cu2+ complex versus different pH value within 1440 min (565 nm), Table S3: Comparison data with reported Cu2+ sensors. Author Contributions: X.Z. and L.-Y.S. carried out the experiments; H.X. and Y.-L.H. analyzed the experiment data; L.-Y.S. and X.-J.Y. analyzed the X-ray structure; and C.R. and Q.-L.Z. wrote the paper. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Data Availability Statement: No new data were created or analyzed in this study. Data sharing is not applicable to this article. Acknowledgments: This work was supported by and the National Natural Science Foundation of China (22065009, 22066007), the Guizhou Provincial Natural Science Foundation (grant number [2019] 2792, grant number [2018] 5779-14). CR thanks the EPSRC for an Overseas Travel Grant (EP/R023816/1). Conflicts of Interest: The authors declare no conflict of interest.

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