A Simple Schiff Base Probe for Quintuplicate-Metal Analytes with Four Emission-Wavelength Responses

A versatile mono-Schiff compound consisting of o-aminobenzene-hydroxyjulolidine (ABJ-MS) has been easily synthesized using a one-step reaction. ABJ-MS displays four diverse fluorescence responses to the addition of Zn2+/Al3+/Fe3+/Ag+, with the maximum fluorescence emission at 530 nm undergoing a hypsochromic shift to 502/490/440/430 nm, synchronously with the discriminating fluorescence enhancement being 10.6/22.8/2.6/7.1-fold, respectively. However, the addition of Cu2+ into ABJ-MS leads to an opposite behavior, namely, fluorescence quenching. Meanwhile, ABJ-MS also displays distinct absorption changes after adding these five metal ions due to different binding affinities between them and ABJ-MS, which gives ABJ-MS quite a versatile detecting nature for Cu2+/Zn2+/Al3+/Fe3+/Ag+. Moreover, ABJ-MS can mimic a series of versatile AND/OR/INH-consisting logic circuits on the basis of the Cu2+/Zn2+/Al3+/Fe3+/Ag+-mediated diverse optical responses. These will endow the smart ABJ-MS molecule and potential applications in the multi-analysis chemosensory and molecular logic material fields.


Introduction
As is well known, metal ions play a quite important role in diverse physiology and industry processes, although they could also cause some serious diseases in excessive/deficient conditions [1,2].For example, Zn 2+ as an essential element has taken part in some physiological activities.Yet, the insufficiency/surfeit of zinc ions could cause Friedreich's ataxia, Alzheimer's and Parkinson's diseases [3,4].Similarly, excessive/deficient Cu 2+ could bring about certain neurological sickness and gastrointestinal disturbances, Fe 3+ may induce liver and kidney diseases as well as metabolism maladjustment, while Al 3+ can cause some damage to human s central nervous and immune systems [5][6][7][8].Ag + accumulation could also cause serious consequences, such as grayish-blue skin discolorations [9].Furthermore, excessive metal ions in domestic/industrial wastewater could induce bad environmental pollution.Thus, developing highly effective methods to detect these metal ions is extraordinarily necessary.
Among numerous analytic techniques, fluorescent probes, particularly those with detecting functions for some important metal ions, are being paid growing attention to due to their rapid responsiveness, on-site real monitoring, and high sensitivity [10][11][12].However, most fluorescent probes concentrate on one-to-one detections, while only a few studies have focused on one-to-multi-sensing, either simultaneously or separately [13][14][15][16][17]. Nevertheless, the single molecular probes for multi-analytes could not only shorten the preparation procedure for multiple probes but also exhibit a high detecting efficiency compared to single-analyte detecting sensors [18][19][20][21].Thus, it is extremely desirable to explore and develop single fluorescent probes for multiple targets through diverse optical responses.To actualize the versatile monomolecular detection for multiple targets, generally, more than one luminophore or receptor is combined into the single-molecular probe through a laborious multistep organic synthetic procedure [22,23].Suzuki and coworkers prepared a probe consisting of tris-fluorophores to detect Fe 3+ /Pb 2+ /Al 3+ /Cu 2+ based on the diverse complexing modes between receptors and metal ions [24].Akkaya et al. synthesized a Bodipy probe for Zn 2+ /Ca 2+ /Hg 2+ , consisting of three receptors [25], while the Jiang group prepared a one-receptor-bridging porphyrin-Bodipy dyad probe for Fe 2+ /Hg 2+ [26].Meanwhile, few investigations have been conducted on probes consisting of single-fluorophore-receptors for multi-analytes associated with diverse detecting mechanisms, etc. [27][28][29][30].However, to the best of our knowledge, there still appears to be no exploration into a molecular probe with only one luminophore and a single receptor, except for quintuplicate-metal ions, especially with the easy-to-synthesize process.
Meanwhile, ortho-hydroxy keto-imines are receiving increasing attention owing to their wide application [31,32].Among them, the salicylaldazine moiety has been one of the most promising construction units for fluorescent probes due to its excellent metal complexing affinity, its unique enol-keto tautomerization, and C=N isomerization, which could induce sensitive optical changes [33][34][35].In addition, the metal-binding affinity and optical-signal sensibility can be easily improved by the introduction of a third binding site onto the salicylaldazine moiety [36,37].Herein, we designed and synthesized a new three-in-receptor mono-Shiff molecule (ABJ-MS) by introducing an o-aminobenzene unit onto the salicylaldazine moiety based on the mono-condensation reaction between o-diaminobenzene and 9-formyl-8-hydroxyjulolidine, Scheme 1.As expected, ABJ-MS displays five discriminating fluorescence behaviors with four emission wavelengths after adding Zn 2+ , Cu 2+ , Al 3+ , Fe 3+ , or Ag + , which are simultaneously accompanied by the significantly different absorption responses, owing to different binding affinities between these five metal ions and ABJ-MS.In addition, a series of complicated logic circuits consisting of AND/OR/INH gates could be mimicked for the ABJ-MS on the basis of the Cu 2+ /Zn 2+ /Al 3+ /Fe 3+ /Ag + -mediated diverse optical responses.These will endow the ABJ-MS molecule and the potential applications in the versatile multi-analysis chemoprobe and the molecular logic material fields.
Molecules 2023, 28, x FOR PEER REVIEW 2 of 15 compared to single-analyte detecting sensors [18][19][20][21].Thus, it is extremely desirable to explore and develop single fluorescent probes for multiple targets through diverse optical responses.To actualize the versatile monomolecular detection for multiple targets, generally, more than one luminophore or receptor is combined into the single-molecular probe through a laborious multistep organic synthetic procedure [22,23].Suzuki and co-workers prepared a probe consisting of tris-fluorophores to detect Fe 3+ /Pb 2+ /Al 3+ /Cu 2+ based on the diverse complexing modes between receptors and metal ions [24].Akkaya et al. synthesized a Bodipy probe for Zn 2+ /Ca 2+ /Hg 2+ , consisting of three receptors [25], while the Jiang group prepared a one-receptor-bridging porphyrin-Bodipy dyad probe for Fe 2+ /Hg 2+ [26].Meanwhile, few investigations have been conducted on probes consisting of single-fluorophore-receptors for multi-analytes associated with diverse detecting mechanisms, etc. [27][28][29][30].However, to the best of our knowledge, there still appears to be no exploration into a molecular probe with only one luminophore and a single receptor, except for quintuplicate-metal ions, especially with the easy-to-synthesize process.Meanwhile, ortho-hydroxy keto-imines are receiving increasing attention owing to their wide application [31,32].Among them, the salicylaldazine moiety has been one of the most promising construction units for fluorescent probes due to its excellent metal complexing affinity, its unique enol-keto tautomerization, and C=N isomerization, which could induce sensitive optical changes [33][34][35].In addition, the metal-binding affinity and optical-signal sensibility can be easily improved by the introduction of a third binding site onto the salicylaldazine moiety [36,37].Herein, we designed and synthesized a new threein-receptor mono-Shiff molecule (ABJ-MS) by introducing an o-aminobenzene unit onto the salicylaldazine moiety based on the mono-condensation reaction between o-diaminobenzene and 9-formyl-8-hydroxyjulolidine, Scheme 1.As expected, ABJ-MS displays five discriminating fluorescence behaviors with four emission wavelengths after adding Zn 2+ , Cu 2+ , Al 3+ , Fe 3+ , or Ag + , which are simultaneously accompanied by the significantly different absorption responses, owing to different binding affinities between these five metal ions and ABJ-MS.In addition, a series of complicated logic circuits consisting of AND/OR/INH gates could be mimicked for the ABJ-MS on the basis of the Cu 2+ /Zn 2+ /Al 3+ /Fe 3+ /Ag + -mediated diverse optical responses.These will endow the ABJ-MS molecule and the potential applications in the versatile multi-analysis chemoprobe and the molecular logic material fields.

Results
The aimed mono-Schiff probe consisting of o-aminobenzene-hydroxyjulolidine and a three-in-receptor (ABJ-MS) (Scheme 1) was obtained by the mono-condensation of the o-diaminobenzene with the 8-hydroxyjulolidine-9-carboxaldehyde (1:1 equiv.), in ethanol and under a refluxing condition.The ABJ-MS compound was characterized by NMR, elementary analysis, and mass spectroscopies, as shown in Figures S1 and S2 (Supporting Information).

Results
The aimed mono-Schiff probe consisting of o-aminobenzene-hydroxyjulolidine and a three-in-receptor (ABJ-MS) (Scheme 1) was obtained by the mono-condensation of the odiaminobenzene with the 8-hydroxyjulolidine-9-carboxaldehyde (1:1 equiv.), in ethanol and under a refluxing condition.The ABJ-MS compound was characterized by NMR, elementary analysis, and mass spectroscopies, as shown in Figures S1 and S2 (Supporting Information).

The Selectivity Ability
To understand the optical properties of ABJ-MS, the electronic absorption and fluorescence emission behaviors of this compound (20 µM) in a DMSO-H 2 O mixture with different H 2 O fractions (f w , the volume percentage of H 2 O in DMSO-H 2 O mixtures) were studied.As shown in Figure S3 (Supporting Information), when the water fraction f w was below 60%, the electronic absorption of ABJ-MS undergoes minimal changes.Whereas, when f w was further increased from 60 to 95%, the maximum absorption of ABJ-MS becomes slightly broader and red-shifted, and in the meantime, the level-off tail gradually appears in the long-wavelength region, accompanied by a slight decrease in the absorbance, which is possibly due to the Mie light scattering, and suggests the formation of aggregated nanoparticles.Meanwhile, along with the increase in f w from 0-60% to 60-5%, the weak fluorescence emission of ABJ-MS was observed to be firstly enhanced and then decreased, probably due to the formation of aggregated nanoparticles, which inhibit the intramolecular rotation motion and enhance the intermolecular π-π stacking interaction, thereby resulting in aggregation-caused quenching in the high f w conditions [38].Thus, the sensing properties of ABJ-MS were investigated in a mixture of DMSO/H 2 O (v:v = 1:4).
To identify the detecting capability of this compound for metal ions, the optical properties of ABJ-MS (20 µM) were investigated upon the addition of metal ions, such as Zn 2+ , Cu 2+ , Al 3+ , Fe 3+ , Ag + , Hg 2+ , Pb 2+ , Co 2+ , Mn 2+ , Ni 2+ , Cd 2+ , Ca 2+ , Ba 2+ , Mg 2+ , Li + , Na + , or K + (10 equiv.), in DMSO/H 2 O (4:1).As can be found from Figure 1, the absorption and fluorescent spectra of ABJ-MS remained almost unchanged after adding the above metal ions, except for Zn 2+ , Al 3+ , Fe 3+ , Ag + , or Cu 2+ .After adding Zn 2+ /Al 3+ /Fe 3+ /Ag + , the weak fluorescence emission of ABJ-MS displayed diverse responses: The maximum emission wavelength of ABJ-MS (530 nm) underwent a hypsochromica shift to 502/490/440/430 nm, which was synchronously accompanied by a discriminating 10.6/22.8/2.6/7.1-foldincrease in fluorescence emission, respectively, alongside an increase in the relative fluorescence quantum yield from 0.005 of free ABJ-MS to 0.040, 0.107, 0.010, and 0.024 upon the addition of Zn 2+ , Al 3+ , Fe 3+ , and Ag+, respectively [39].On the contrary, the addition of Cu 2+ induces the fluorescence quenching, Figure 1A.Meanwhile, the addition of these five metal ions into ABJ-MS also causes obviously different changes in absorption spectra.After adding Zn 2+ ions, the absorption at 427 nm from ABJ-MS undergoes a bathochromic shift to 437 nm following a slight enhancement in absorbance.This is also true for the addition of Cu 2+ , although the maximum absorption appears at 441 nm.Differently, after adding Al 3+ , the absorption of ABJ-MS at 427 nm decreased simultaneously with a bathochromic shift to 436 nm as well as a slightly weak shoulder band appearing around 376 nm.The addition of Ag + also induced a similar change but with the maximum absorption appearing at 373 nm, thereby resulting in the varying ratio of A 427 /A 373 from 2.8 to 0.7.After adding Fe 3+ , the above-mentioned maximum absorption for ABJ-MS decreased simultaneously alongside an obvious increase at 377 nm, which led to a broad absorption in the range of 310-490 nm, Figure 1B.These results endow ABJ-MS as a quite versatile detecting nature for Zn 2+ /Cu 2+ /Al 3+ /Fe 3+ /Ag + that is associated with five discriminating fluorescence and absorption dual-responses, which is quite rare in reported fluorescence probes.

The Responsive Metal Sensing
Zn 2+ /Cu 2+ sensing: The fluorescent titration experiment for ABJ-MS (20 µM) was carried out by increasing the Zn 2+ content in the DMSO/H 2 O (4:1) solution.As can be seen in Figure 2A, along with the increase of Zn 2+ quantity from 0 to 1.5 equiv., the fluorescence emission from ABJ-MS gets gradually increased with a hypsochromic shift from 530 nm to 502 nm, whereas the strong absorption by this compound is found to undergo a bathochromic shift to 437 nm.Subsequently, the fluorescent emission and absorption spectra remain almost unchanged upon the continuous increase in Zn 2+ to 10 equiv., Figure S4 (Supporting Information).Furthermore, the fluorescence emission (F − F min ) of ABJ-MS raised linearly along with the increase in Zn 2+ from 0 to 1.5 equiv., on the basis of the Benesi-Hildebrand equation, Figure 2B, suggesting a probable 1:1 complexing between ABJ-MS and Zn 2+ , where the approximate association constant (K a ) was 3.04 × 10 4 [40], which is further affirmed in the fluorescent Job's plot, Figure 2B.In addition, the ESI-mass spectrometry for ABJ-MS upon the addition of Zn 2+ (10 equiv.)produces a peak at m/z = 388.42,which is assignable to [ABJ-MS + Zn 2+ + H 2 O + H] + (calculated at 388.1), Figure S5 (Supporting Information); thus, again confirming the 1:1 complexing between ABJ-MS and Zn 2+ .Notably, despite the fluorescence quenching response following the addition of Cu 2+ , the fluorescent titration experiment of ABJ-MS (20 µM) with Cu 2+ (0-10 equiv.)also displays a similar 1:1 binding stoichiometry for the ABJ-MS-Cu 2+ system, yet with an approximate K a of 1.2 × 10 5 , Figure 2C,D.The detection limits of ABJ-MS for Zn 2+ and Cu 2+ ions were determined as 3.21 × 10 −7 and 1.06 × 10 −7 M under the present condition, which was lower than those applied to the potable water by the WHO [41], suggesting that ABJ-MS could be a highly sensitive probe for Zn 2+ and Cu 2+ using the diverse fluorescence off-on and on-off signals, respectively.Fe 3+ /Al 3+ sensing: As shown in Figure 3A, the fluorescent titration test for ABJ-MS with Al 3+ was also studied by successively increasing Al 3+ (0-10 equiv.) in the DMSO/H2O mixture (4:1).Alongside increasing the Al 3+ amount from 0 to 4 equiv., the weak fluorescence emission from ABJ-MS gets gradually increased with a synchronous hypsochromic shift from 530 to 490 nm, resulting in a fluorescence intensity ratio of 25:1 for F490/F530.Meanwhile, the changes in the absorption for ABJ-MS mainly focus on the added Al 3+ amount being 0-4 equiv., Figure S6 (Supporting Information).Furthermore, the maximum intensity in the fluorescent Job's plot for ABJ-MS with Al 3+ was observed when the molar fraction of (ABJ-MS) vs. (Al 3+ ) + (ABJ-MS) was 0.66, suggesting the possible 2:1 complexing of ABJ-MS with Al 3+ , Figure 3B [42].Based on the 2:1 binding stoichiometry and fluores- Fe 3+ /Al 3+ sensing: As shown in Figure 3A, the fluorescent titration test for ABJ-MS with Al 3+ was also studied by successively increasing Al 3+ (0-10 equiv.) in the DMSO/H 2 O mixture (4:1).Alongside increasing the Al 3+ amount from 0 to 4 equiv., the weak fluorescence emission from ABJ-MS gets gradually increased with a synchronous hypsochromic shift from 530 to 490 nm, resulting in a fluorescence intensity ratio of 25:1 for F 490 /F 530 .
Meanwhile, the changes in the absorption for ABJ-MS mainly focus on the added Al 3+ amount being 0-4 equiv., Figure S6 (Supporting Information).Furthermore, the maximum intensity in the fluorescent Job's plot for ABJ-MS with Al 3+ was observed when the molar fraction of (ABJ-MS) vs. (Al 3+ ) + (ABJ-MS) was 0.66, suggesting the possible 2:1 complexing of ABJ-MS with Al 3+ , Figure 3B [42].Based on the 2:1 binding stoichiometry and fluorescence titration experiments of ABJ-MS with Al 3+ , the binding constant for this compound with Al 3+ was estimated to be 9.52 × 10 3 , by plotting the Benesi-Hildebrand equation lg[(F l − F 0 )/(F − F 0 )] against lg(1/(Al 3+ )) [43,44].Similarly, the fluorescence titration experiment and fluorescent Job's plot for ABJ-MS with Fe 3+ also showed a similar 2:1 binding mode between ABJ-MS and Fe 3+ , with an approximate K a of 8.72 × 10 4 , Figure 3C,D  Ag + sensing: As shown in Figure 4A, the fluorescence emission by ABJ-MS at 430 nm gradually increases as the Ag + content increases from 0 to 10 equiv., and the corresponding 1/(F − Fmin) raises linearly against the alteration in 1/(Ag + ) (0-12 equiv.),according to the Benesi-Hildebrand equation, Figure 4B.Moreover, the absorption titration for ABJ-MS with Ag + shows that both absorptions 1/(A − Amin) and 1/(Amax − A) at 373 and 427 nm, respectively, by ABJ-MS also increased linearly against the change of 1/(Ag + ) (0-10 equiv.), with an approximate Ka of 1.6 × 10 3 M −1 , Figure 4C,D [45].These results indicate a possible 1:1 stoichiometry between ABJ-MS and Ag + , which is again approved by the fluorescent Job's plot of ABJ-MS towards Ag + , Figure 4A.The detecting limitation by ABJ-MS for Ag + is 1.03 × 10 −5 M, suggesting an excellent detecting function for ABJ-MS towards Ag + through the fluorescence off-on and absorption dual-channels.Ag + sensing: As shown in Figure 4A, the fluorescence emission by ABJ-MS at 430 nm gradually increases as the Ag + content increases from 0 to 10 equiv., and the corresponding 1/(F − F min ) raises linearly against the alteration in 1/(Ag + ) (0-12 equiv.),according to the Benesi-Hildebrand equation, Figure 4B.Moreover, the absorption titration for ABJ-MS with Ag + shows that both absorptions 1/(A − A min ) and 1/(A max − A) at 373 and 427 nm, respectively, by ABJ-MS also increased linearly against the change of 1/(Ag + ) (0-10 equiv.), with an approximate K a of 1.6 × 10 3 M −1 , Figure 4C,D [45].These results indicate a possible 1:1 stoichiometry between ABJ-MS and Ag + , which is again approved by the fluorescent Job's plot of ABJ-MS towards Ag + , Figure 4A.The detecting limitation by ABJ-MS for Ag + is 1.03 × 10 −5 M, suggesting an excellent detecting function for ABJ-MS towards Ag + through the fluorescence off-on and absorption dual-channels.

Competition Experiment
The competition experiments were carried out in DMSO/H2O (4:1) mixtures to further study the detecting abilities for ABJ-MS of Zn 2+ /Cu 2+ /Al 3+ /Fe 3+ /Ag + .As can be observed in Figures 5A and S7 (Supporting Information), the fluorescent emission of the ABJ-MS-Fe 3+ (1:10) complex at 440 nm is quenched after adding Cu 2+ (10 equiv.);meanwhile, the absorption spectrum is observed to be similar to the ABJ-MS-Cu 2+ complex, suggesting the substitution of Fe 3+ by Cu 2+ in the ABJ-MS-Fe 3+ composite, which is also true for the ABJ-MS-Zn 2+ /Al 3+ /Ag + complexes.Similarly, the addition of Fe 3+ into the ABJ-MS-Zn 2+ /Al 3+ /Ag + systems also results in the displacement of metal in these systems by Fe 3+ as well as the consequential fluorescence response, Figure 5B.In a similar manner, Zn 2+ can replace the metal ion in the ABJ-MS-Al 3+ /Ag + complex and Al 3+ can replace the metal ion in the ABJ-MS-Ag + complex, thereby inducing the corresponding fluorescence and absorption responses, Figures 5C,D, S8, and S9 (Supporting Information).By contrast, after adding other metal ions, the fluorescence emission of the ABJ-MS-Cu 2+ /Fe 3+ /Zn 2+ /Al 3+ /Ag + systems remained almost unchanged, Figures S10-S14 (Supporting Information).These results show the excellent binding affinity between ABJ-MS and these five metal ions, in the order of Cu 2+ > Fe 3+ > Zn 2+ > Al 3+ > Ag + , which is in good agreement with their association constant results mentioned, thereby revealing the favorable selectivity of ABJ-MS for Cu 2+ /Fe 3+ /Zn 2+ /Al 3+ /Ag + over other tested metal ions.In addition, the selectivity of ABJ-MS toward anions was also investigated through the addition of sodium salts and different anions, including F − , Cl − , Br − , I − , HSO3 − , OAc − , S −2 , SO3 −2 , SO4 −2 , CO3 −2 , HPO4 −2 , and H2PO4 − .Notably, the electronic absorption and fluorescence emission spectra of ABJ-MS made either a slight or no change upon the addition of 100 equiv. of each anion, meaning little effect was shown by these anions on the sensing properties of ABJ-MS, Figure S15.Thus, ABJ-MS could be utilized as a versatile molecular probe for Cu 2+ /Fe 3+ /Zn 2+ /Al 3+ /Ag +

Competition Experiment
The competition experiments were carried out in DMSO/H 2 O (4:1) mixtures to further study the detecting abilities for ABJ-MS of Zn 2+ /Cu 2+ /Al 3+ /Fe 3+ /Ag + .As can be observed in Figure 5A and Figure S7 (Supporting Information), the fluorescent emission of the ABJ-MS-Fe 3+ (1:10) complex at 440 nm is quenched after adding Cu 2+ (10 equiv.);meanwhile, the absorption spectrum is observed to be similar to the ABJ-MS-Cu 2+ complex, suggesting the substitution of Fe 3+ by Cu 2+ in the ABJ-MS-Fe 3+ composite, which is also true for the ABJ-MS-Zn 2+ /Al 3+ /Ag + complexes.Similarly, the addition of Fe 3+ into the ABJ-MS-Zn 2+ /Al 3+ /Ag + systems also results in the displacement of metal in these systems by Fe 3+ as well as the consequential fluorescence response, Figure 5B.In a similar manner, Zn 2+ can replace the metal ion in the ABJ-MS-Al 3+ /Ag + complex and Al 3+ can replace the metal ion in the ABJ-MS-Ag + complex, thereby inducing the corresponding fluorescence and absorption responses, Figure 5C,D, Figures S8 and S9 (Supporting Information).By contrast, after adding other metal ions, the fluorescence emission of the ABJ-MS-Cu 2+ /Fe 3+ /Zn 2+ /Al 3+ /Ag + systems remained almost unchanged, Figures S10-S14 (Supporting Information).These results show the excellent binding affinity between ABJ-MS and these five metal ions, in the order of Cu 2+ > Fe 3+ > Zn 2+ > Al 3+ > Ag + , which is in good agreement with their association constant results mentioned, thereby revealing the favorable selectivity of ABJ-MS for Cu 2+ /Fe 3+ /Zn 2+ /Al 3+ /Ag + over other tested metal ions.In addition, the selectivity of ABJ-MS toward anions was also investigated through the addition of sodium salts and different anions, including F − , Cl − , Br − , I − , HSO 3 − , OAc − , S −2 , SO 3 −2 , SO 4 −2 , CO 3 −2 , HPO 4 −2 , and H 2 PO 4 − .Notably, the electronic absorption and fluorescence emission spectra of ABJ-MS made either a slight or no change upon the addition of 100 equiv. of each anion, meaning little effect was shown by these anions on the sensing properties of ABJ-MS, Figure S15.Thus, ABJ-MS could be utilized as a versatile molecular probe for Cu 2+ /Fe 3+ /Zn 2+ /Al 3+ /Ag + through dual-mode optical signals associated with immediate interactions between these five metal ions and ABJ-MS as well as the diverse metal-displacement of the ABJ-MS-M(Ag + /Al 3+ /Zn 2+ /Fe 3+ ) complexes by Cu 2+ /Fe 3+ /Zn 2+ /Al 3+ , representing the quite rare example of smart single molecular probe for quintuple-metal analysis using the sensitive fluorescence and absorption dual-signals.
through dual-mode optical signals associated with immediate interactions between these five metal ions and ABJ-MS as well as the diverse metal-displacement of the ABJ-MS-M(Ag + /Al 3+ /Zn 2+ /Fe 3+ ) complexes by Cu 2+ /Fe 3+ /Zn 2+ /Al 3+ , representing the quite rare example of smart single molecular probe for quintuple-metal analysis using the sensitive fluorescence and absorption dual-signals.

The Sensing Mechanism
As is well known, there is usually an equilibrium between the enol-imine and ketoenamine isomers for salicylaldazine moiety in solution due to the enol-keto isomerization, while the equilibrium moves easily under trace amounts of acid catalysis [46,47].To understand the sensing mechanism between ABJ-MS and the corresponding metal ions, the optical behaviors of ABJ-MS after sequentially adding HCl were investigated under the same measuring requirement as mentioned above.As can be found from Figure S16 (Supporting Information), the increase in HCl content from 0 to 1 equiv., caused a bathochromic shift in the strong absorption of ABJ-MS from 427 to 438 nm with a little decrease in the absorbance.Then, continuously increasing the HCl amount, even to 100 equiv., caused the maximum absorption to decrease with a distinct enhancement in the absorption band at 368 nm.At the same time, the fluorescent emission for ABJ-MS at 530 nm was simultaneously decreased alongside the appearance of a new strong emission band at 434 nm along with the increasing HCl amount.The optical responses for ABJ-MS to the addition of HCl can be rationalized as follows: ABJ-MS may coexist as a mixture of enol-imine and keto-enamine isomers, although favors the keto-enamine isomer under a neutral environment, which is approved by the proton band at 13.52 ppm and is associated with an intramolecular hydrogen band (O…H-N) in the 1 H NMR for ABJ-MS [4,30].Upon the addition of HCl, the H + firstly coordinates with the oxygen atom in the C=O unit from the keto-enamine isomer, which inhibits the intramolecular H-bonding interaction, promotes

The Sensing Mechanism
As is well known, there is usually an equilibrium between the enol-imine and ketoenamine isomers for salicylaldazine moiety in solution due to the enol-keto isomerization, while the equilibrium moves easily under trace amounts of acid catalysis [46,47].To understand the sensing mechanism between ABJ-MS and the corresponding metal ions, the optical behaviors of ABJ-MS after sequentially adding HCl were investigated under the same measuring requirement as mentioned above.As can be found from Figure S16 (Supporting Information), the increase in HCl content from 0 to 1 equiv., caused a bathochromic shift in the strong absorption of ABJ-MS from 427 to 438 nm with a little decrease in the absorbance.Then, continuously increasing the HCl amount, even to 100 equiv., caused the maximum absorption to decrease with a distinct enhancement in the absorption band at 368 nm.At the same time, the fluorescent emission for ABJ-MS at 530 nm was simultaneously decreased alongside the appearance of a new strong emission band at 434 nm along with the increasing HCl amount.The optical responses for ABJ-MS to the addition of HCl can be rationalized as follows: ABJ-MS may coexist as a mixture of enol-imine and keto-enamine isomers, although favors the keto-enamine isomer under a neutral environment, which is approved by the proton band at 13.52 ppm and is associated with an intramolecular hydrogen band (O. ..H-N) in the 1 H NMR for ABJ-MS [4,30].Upon the addition of HCl, the H + firstly coordinates with the oxygen atom in the C=O unit from the keto-enamine isomer, which inhibits the intramolecular H-bonding interaction, promotes the conversion from the keto-enamine isomer to the enol-imine isomer, and results in a bathochromic shift in the maximum absorption from ABJ-MS [48,49].Then, the nitrogen atom from the imine unit is protonated along with the increasing HCl amount, and the photoinduced electron transition is restrained, which in turn induces fluorescence enhancement by ABJ-MS.
Notably, the addition of Al 3+ /Fe 3+ into ABJ-MS has similar optical responses to those for ABJ-MS under the same acid conditions mentioned above.The responsive optical changes in the ABJ-MS-Al 3+ /Fe 3+ system may be attributed to the hydrolysis of Al 3+ /Fe 3+ , which results in an acidic environment, promotes the equilibrium shift to the enol-imine form, and increases the complexing of ABJ-MS with Al 3+ /Fe 3+ .These changes, in combination with the 1:2 stoichiometry for the ABJ-MS-Al 3+ /Fe 3+ complex, imply a possible binding mode between ABJ-MS and Al 3+ /Fe 3+ ions, Figure 6A [50,51].For the ABJ-MS-Ag + complex, the quite similar optical responses to the ABJ-MS-HCl system, together with the 1:1 complexing, suggest a possible coordination mode between ABJ-MS and the Ag + ions, Figure 6B.By contrast, upon the addition of Zn 2+ /Cu 2+ , there exist diverse changes in the absorption spectra of ABJ-MS, despite the similar red-shift in the maximum absorption, which could be attributed to the different binding mode for the ABJ-MS-Zn 2+ /Cu 2+ complex, Figure 6C.The increase in fluorescence by ABJ-MS upon the addition of Zn 2+ /Al 3+ /Fe 3+ /Ag + could be attributed to the restraint of both the C=N isomerization and the excited-state proton transfer (ESPT), in addition to the photoinduced electron transition process [52,53].However, possibly due to the different characteristic outermost electronic structures between these responsive metal ions, the binding of ABJ-MS to Zn 2+ /Al 3+ /Fe 3+ /Ag + induces a diverse fluorescence emission, whereas its coordination with Cu 2+ results in fluorescent quenching, which is probably associated with its usual paramagnetic fluorescence quenching property, meaning that the ABJ-MS is quite a versatile multi-responsive molecular probe for Cu 2+ /Fe 3+ /Zn 2+ /Al 3+ /Ag + .
the conversion from the keto-enamine isomer to the enol-imine isomer, and results in a bathochromic shift in the maximum absorption from ABJ-MS [48,49].Then, the nitrogen atom from the imine unit is protonated along with the increasing HCl amount, and the photoinduced electron transition is restrained, which in turn induces fluorescence enhancement by ABJ-MS.
Notably, the addition of Al 3+ /Fe 3+ into ABJ-MS has similar optical responses to those for ABJ-MS under the same acid conditions mentioned above.The responsive optical changes in the ABJ-MS-Al 3+ /Fe 3+ system may be attributed to the hydrolysis of Al 3+ /Fe 3+ , which results in an acidic environment, promotes the equilibrium shift to the enol-imine form, and increases the complexing of ABJ-MS with Al 3+ /Fe 3+ .These changes, in combination with the 1:2 stoichiometry for the ABJ-MS-Al 3+ /Fe 3+ complex, imply a possible binding mode between ABJ-MS and Al 3+ /Fe 3+ ions, Figure 6A [50,51].For the ABJ-MS-Ag + complex, the quite similar optical responses to the ABJ-MS-HCl system, together with the 1:1 complexing, suggest a possible coordination mode between ABJ-MS and the Ag + ions, Figure 6B.By contrast, upon the addition of Zn 2+ /Cu 2+ , there exist diverse changes in the absorption spectra of ABJ-MS, despite the similar red-shift in the maximum absorption, which could be attributed to the different binding mode for the ABJ-MS-Zn 2+ /Cu 2+ complex, Figure 6C.The increase in fluorescence by ABJ-MS upon the addition of Zn 2+ /Al 3+ /Fe 3+ /Ag + could be attributed to the restraint of both the C=N isomerization and the excited-state proton transfer (ESPT), in addition to the photoinduced electron transition process [52,53].However, possibly due to the different characteristic outermost electronic structures between these responsive metal ions, the binding of ABJ-MS to Zn 2+ /Al 3+ /Fe 3+ /Ag + induces a diverse fluorescence emission, whereas its coordination with Cu 2+ results in fluorescent quenching, which is probably associated with its usual paramagnetic fluorescence quenching property, meaning that the ABJ-MS is quite a versatile multi-responsive molecular probe for Cu 2+ /Fe 3+ /Zn 2+ /Al 3+ /Ag + .

Application of ABJ-MS in Zn 2+ /Al 3+ /Fe 3+ Analysis in Water Samples
Two kinds of water samples, such as from a river (Yongding River) and artificial water, were employed to explore the practical application potential of ABJ-MS.After performing a simple filtrate, there were no Zn 2+ /Al 3+ /Fe 3+ according to the atomic absorption spectrometry.The water samples were spiked with a Zn 2+ /Al 3+ /Fe 3+ solution and analyzed using the ABJ-MS molecular probe.As can be seen from Table 1, the results indicated the satisfying recovery and R.S.D. values for the tested samples using this newly prepared probe [54], endowing ABJ-MS with an improved potential application in sensing Zn 2+ , Al 3+ , and Fe 3+ .

Application of ABJ-MS in Zn 2+ /Al 3+ /Fe 3+ Analysis in Water Samples
Two kinds of water samples, such as from a river (Yongding River) and artificial water, were employed to explore the practical application potential of ABJ-MS.After performing a simple filtrate, there were no Zn 2+ /Al 3+ /Fe 3+ according to the atomic absorption spectrometry.The water samples were spiked with a Zn 2+ /Al 3+ /Fe 3+ solution and analyzed using the ABJ-MS molecular probe.As can be seen from Table 1, the results indicated the satisfying recovery and R.S.D. values for the tested samples using this newly prepared probe [54], endowing ABJ-MS with an improved potential application in sensing Zn 2+ , Al 3+ , and Fe 3+ .

Chemicals and Instruments
All reactants were commercially available and used without further purification. 1H and 13 C NMR spectra ( 1 H-400 MHz and 13 C-100 MHz) were recorded on a Bruker DPX 400 MHz spectrometer in CDCl3 with shifts referenced to SiMe4 (0.00 ppm).MALDI-TOF mass spectra were recorded by a Bruker BIFLEX III ultra-high-resolution Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer using an alpha-cyano-4-hydroxycinnamic acid as the matrix.Elemental analyses were performed using an Elementar Vavio El Ш. Electronic absorption spectra were recorded by a U-4100 spectrophotometer.Steady-state fluorescence spectroscopic studies were performed by a F4600 (Hitachi) with a slit width of 5 nm and a photon multiplier voltage of 700 V for emission.The relative fluorescence quantum yields for ABJ-MS and its metal complexes were obtained by comparing the area under the corrected emission spectrum of the test sample with the solution of [N,N-Bis(salicylidene)-1,2-phenylenediamine]zinc(II) in DMSO with an excitation wavelength of 400 nm, which has a quantum efficiency of 0.008, according to the literature [39].

Chemicals and Instruments
All reactants were commercially available and used without further purification. 1H and 13 C NMR spectra ( 1 H-400 MHz and 13 C-100 MHz) were recorded on a Bruker DPX 400 MHz spectrometer in CDCl 3 with shifts referenced to SiMe 4 (0.00 ppm).MALDI-TOF mass spectra were recorded by a Bruker BIFLEX III ultra-high-resolution Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer using an alpha-cyano-4hydroxycinnamic acid as the matrix.Elemental analyses were performed using an Elementar Vavio El III.Electronic absorption spectra were recorded by a U-4100 spectrophotometer.Steady-state fluorescence spectroscopic studies were performed by a F4600 (Hitachi) with a slit width of 5 nm and a photon multiplier voltage of 700 V for emission.The relative fluorescence quantum yields for ABJ-MS and its metal complexes were obtained by comparing the area under the corrected emission spectrum of the test sample with the solution of [N,N-Bis(salicylidene)-1,2-phenylenediamine]zinc(II) in DMSO with an excitation wavelength of 400 nm, which has a quantum efficiency of 0.008, according to the literature [39].

Preparation of Mono-Schiff Probe Consisting of o-Aminobenzene-Hydroxyjulolidine (ABJ-MS)
The compound of o-diaminobenzene (54 mg, 0.5 mmol) was dissolved in anhydrous ethanol (15 mL) before adding 9-formyl-8-hydroxyjulolidine (109 mg, 0.5 mmol), and glacial acetic acid (200 µL).The reacting mixture was heated to reflux under N 2 protection for 5 h before being cooled to room temperature.The orange-yellow suspended substance was filtrated and washed three times in ethanol before the pure ABJ-MS samples were obtained

Figure 6 .
Figure 6.Subfigure A proposed coordination modes between ABJ-MS and Al 3+ or Fe 3+ ; Subfigure B proposed coordination modes between ABJ-MS and Ag + ; Subfigure C proposed coordination modes between ABJ-MS and Cu 2+ or Zn 2+ , with the other coordinated solution molecule or anion being omitted.

Figure 6 .
Figure 6.Subfigure (A) proposed coordination modes between ABJ-MS and Al 3+ or Fe 3+ ; Subfigure (B) proposed coordination modes between ABJ-MS and Ag + ; Subfigure (C) proposed coordination modes between ABJ-MS and Cu 2+ or Zn 2+ , with the other coordinated solution molecule or anion being omitted.

Figure 7 .
Figure 7.The truth value for ABJ-MS (20 µM) logic gate (A) and two complicated logic circuits (B,C) with 10 equiv. of Ag + , Fe 3+ , Al 3+ , and Zn 2+ as four inputs (In1, In2, In3, and In4), respectively, the corresponding maximum fluorescence emission intensity as output, and the threshold level of fluorescence intensity being 3000 and 1500.Dotted lines represent the threshold levels for fluorescence outputs.

Figure 7 .
Figure 7.The truth value for ABJ-MS (20 µM) logic gate (A) and two complicated logic circuits (B,C) with 10 equiv. of Ag + , Fe 3+ , Al 3+ , and Zn 2+ as four inputs (In1, In2, In3, and In4), respectively, the corresponding maximum fluorescence emission intensity as output, and the threshold level of fluorescence intensity being 3000 and 1500.Dotted lines represent the threshold levels for fluorescence outputs.