A lysosome-targetable fluorescent probe for imaging trivalent cations Fe3+, Al3+ and Cr3+ in living cells

A lysosome-targetable fluorescent probe for imaging trivalent cations Fe3+, Al3+ and Cr3+ in living cells

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 222 (2019) 117242 Contents lists available at ScienceDirect Spectrochimica Acta ...

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Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 222 (2019) 117242

Contents lists available at ScienceDirect

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa

A lysosome-targetable fluorescent probe for imaging trivalent cations Fe3 , Al3 and Cr3 in living cells Fei Ye, Nan Wu, Ping Li, Yu-Long Liu, Shi-Jie Li, Ying Fu ⁎ Department of Applied Chemistry, College of Science, Northeast Agricultural University, Harbin 150030, PR China College of Life Science, Northeast Agricultural University, Harbin 150030, PR China

a r t i c l e

i n f o

Article history: Received 23 April 2019 Received in revised form 4 June 2019 Accepted 5 June 2019 Available online 07 June 2019 Keywords: Lysosome-targeting Naphthalimide-morpholine derivative Trivalent cations Turn-on Live cell imaging

a b s t r a c t An effective morpholine-type naphthalimide chemsensor, N-p-chlorophenyl-4-(2-aminoethyl)morpholine-1,8naphthalimide (CMN) has been developed as a lysosome-targeted fluorometric sensor for trivalent metal ions (Fe3 , Al3 and Cr3 ). Upon the addition of Fe3 , Al3 or Cr3 ions, the probe CMN showed an evident naked-eye color changes which pale yellow solution of CMN turned deepened and it displayed turn-on fluorescence response in methanol. CMN showed a significant selective and sensitive toward Fe3 , Al3 or Cr3 ions, while there was no obvious behavior to other monovalent or divalent metal ions from the UV–vis and fluorescence spectrum. Based on the Job's plot analyses the 1:1 coordination mode of CMN with Fe3 , Al3 or Cr3 was proposed. The limit of detection (LOD) observed were 0.65, 0.69 and 0.68 μM for Fe3 , Al3 and Cr3 ions, respectively. The N-atom of morpholine directly involved in complex formation, CMN emitted fluorescence through inhibition of photoinduced electron transfer (PET). This probe exhibited excellent imaging ability for Fe3 , Al3 and Cr3 ions in living cells with low cytotoxicity. Significantly, the cellular confocal microscopic research indicated that the lysosome-targeted group of morpholine moiety was introduced which realized the capability of imaging lysosomal trivalent metal ions in living cells for the first time. © 2019 Elsevier B.V. All rights reserved.

1. Introduction Exploring of the new type of chemosensors for trivalent metal ions (Fe3 , Al3 and Cr3 ), is a significant research filed because they are indispensable for various biological systems and directly involved in the function of cell [1–4]. Iron (III) exist in multiple enzymes and proteins in cellular system and involve in electron transport and oxygen transfer. It is critical for most organisms and has a central physiological signification. The overload or deficiency of Fe3 ions in the human body will perturb the cellular environmental homeostasis which result in various kinds of diseases, including kidney and liver dysfunction, diabetes mellitus, Alzheimer's disease, and malignant tumor [5–8]. As the most abundant metallic element existed in the earth's crust, aluminium is extensively used in our daily life, including food additives, water purification reagents and cookware which lead the concentration of Al3 moderate increase in food. However, excessive Al3 result in drinking water contamination which is toxic to human body. Alzheimer's disease, osteoporosis, rickets, anaemia and memory loss are all possible as a result of aluminium toxicity [8–10]. Chromium is one of the most essential trace elements in human nutrition. It has the ability of activate ⁎ Corresponding author at: College of Life Science, Northeast Agricultural University, Harbin 150030, PR China. E-mail address: [email protected] (Y. Fu).

https://doi.org/10.1016/j.saa.2019.117242 1386-1425/© 2019 Elsevier B.V. All rights reserved.

certain enzymes, stabilize nucleic acids and proteins, play a decisive role in metabolism of fats, carbohydrates, proteins and nucleic acids. Excessive Cr3 has affected the level of glucose, the structure and function of cell, and the metabolism of lipid, while the deficiency of Cr3 may result in both diabetes mellitus and cardiovascular disease [11–14]. Therefore, it is significant to track Fe3 , Al3 and Cr3 in environmental and biological systems. To determine Fe3 , Al3 and Cr3 , a number of the conventional analytical techniques were available, such as chromatography, inductively coupled plasma atomic emission spectrometry (ICP-AES), flame atomic absorption spectroscopy (FAAS), inductively coupled plasma mass spectrometry (ICP-MS) and accelerator mass spectroscopy (AMS) etc. [15–18]. However, these techniques involve sophisticated instrument, tedious sample preparation procedures and inconvenient for sensitive real-time monitoring of Fe3 , Al3 and Cr3 , which limit their practical applications. To overcome these drawbacks, fluorescence technique has provided a promising approach to detect Fe3 , Al3 and Cr3 ions even could be applied in cellular environment has greatly benefited from its simplicity, rapidness, sensitivity detection of analytes even in low concentration [19–22]. The research of fluorescence turn-on response for Fe3 , Al3 and Cr3 ions has been recognized difficult compared with other metal cations, because Fe3 and Cr3 has the property of paramagnetic quenching and the coordinating ability of Al3 ions is weakly [23–25]. Even though

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there has large quantity of fluorescent probes for the individual detection of Fe3 , Al3 or Cr3 were available [26–28], the single chemosensor to detected the trivalent metal ions (Fe3 , Al3 or Cr3 ) was still lacking [29,30], meanwhile no organelle-targeted Fe3 , Al3 and Cr3 ions fluorescent probes have been discovered up to now. Lysosomes exist in most of the eukaryotic cells, it is a sort of the important organelles. It participates in cellular apoptosis, immunological stress reaction and enzymeprocessing, plays vital roles in the metabolism of living cells [31]. Consequently, it was significant to exploit the lysosome-targeting turn-on fluorescent probe for the detection of Fe3 , Al3 and Cr3 ions in this subcellular organelle. Naphthalimide moiety features as strong yellow-green fluorescence and high fluorescence quantum yield, it has been diffusely applied to fluorescent dyes and bioimaging makers [32–35]. The morpholine group has been confirmed that it possesses a capability of lysosometargeting and can be used as a lysosomal localization group [36–38]. Inspired by these findings, the lysosome-targeted fluorescent probe for trivalent metal cations (Fe3 , Al3 and Cr3 ), N-p-chlorophenyl-4-(2aminoethyl)morpholine-1,8-naphthalimide (CMN) was designed and synthesized (Scheme 1) [39–41]. Interestingly, morpholine moiety plays dual roles, the nitrogen atom of morpholine also acts as receptors which transferring lone pair of electrons to the naphthalimide by photoinduced electron transfer (PET) mechanism [38,42,43]. CMN showed significant colorimetric change and selectively turn-on fluorescence behavior in the presence of trivalent metal ions (Fe3 , Al3 and Cr3 ) over the other monovalent as well as divalent cations in methanol solution. The proposed sensor was applied in the fluorescence imaging of Fe3 , Al3 and Cr3 ions detection, it was able to target lysosome as a result of the introduction of morpholine moiety. As far as know, CMN is the first lysosome-targetable fluorescent probe which could selective imaging trivalent metal ions (Fe3 , Al3 and Cr3 ) in live cells. 2. Experimental 2.1. Material and methods The solvents and reactants were used without further to purified and all commercially obtainable. The melting points were measured used the Shanghai Inesa melting point apparatus (WRS-3), were uncorrected. The IR spectra were measured used the Bruker ALPHA-T spectrometer (KBr, Bruker, Germany), 1H NMR and 13C NMR spectra were measured used the Bruker AVANVE 400 MHz nuclear magnetic resonance spectrometer (Bruker, Germany) and the internal standard was TMS. The high-resolution mass spectrometry (HRMS) was measured used the FTMS Ultral Apex MS spectrometer (Bruker Daltonics Inc.,

USA). Fluorescence spectra were recorded on PerkinElmer LS55 fluorescence spectrometer (PerkinElmer, UK). Absorption spectra were recorded on UV-2550 ultraviolet spectrophotometer (Shimadzu, Japan). The absorbance for CCK-8 testing was recorded on a multi-function microplate reader (Tecan, Switzerland) at 450 nm. Cell images were taken on Nikon A1 confocal laser microscopy system (Nikon, Japan). 2.2. Synthesis 2.2.1. Synthesis of N- (p-chlorophenyl) -4-bromo-1,8-naphthalimide (CN) The synthetic method was shown in Scheme 2. N-(p-chlorophenyl)4-bromo-1,8-naphthalimide was synthesized on the basis of the method reported [32]. 4-Bromo-1,8-naphthalic anhydride (2.4 g, 7.4 mmol) was dissolved in 40 mL of acetic acid and the 4chlorobenzamine (1.08 g, 8.8 mmol) was added, the solution were refluxed with continuous stirring at 80 °C. After refluxing for 12 h, the solution of reaction mixture was decanted in ice water. The precipitate was filtered under reduced pressure and dry, then recrystallized from acetone to get N-(p-chlorophenyl)-4-bromo-1,8-naphthalimide as beige powder. m.p. 210.4–211.0 °C; yield: (2.23 g) 72%. IR (KBr) cm−1: 1699 (νC=O); 1H NMR (400 MHz, DMSO d6): δ ppm 7.45–8.65 (m, 9H, ArH). 13C NMR (100 MHz, DMSO d6,): δ ppm 163.62, 163.46, 135.21, 133.44, 133.31, 132.12, 131.91, 131.50, 130.46, 129.74, 129.44, 129.44, 129.37, 129.31, 123.85, 123.08. 2.2.2. Synthesis of N- (p-chlorophenyl)-4-(2-aminoethyl)morpholine-1,8naphthalimide (CMN) N-(p-chlorophenyl)-4-(2-aminoethyl)morpholine-1,8naphthalimide was synthesized by improving a previously reported method [35]. N-(p-chlorophenyl)-4-bromo-1,8-naphthalimide (0.92 g 2.4 mmol) and 4-(2-aminoethyl)morpholine (1.3 mL, 9.6 mmol) were dissolved in 30 mL dry DMSO, the solution was refluxed overnight at 90 °C. Subsequently, the mixture was removed in ice water, a yellow precipitate was isolated by filtration. The crude product was further purified by silica gel preparation column chromatography used CH2Cl2/ MeOH (v/v, 50/1) as eluent to get yellow solid. m.p. 229.6–230.0 °C; yield: (0.53 g) 53%. IR (KBr) cm−1: 3353 (νN-H), 2830, 2901 (νC-H), 1682 (νC=O); 1H NMR (400 MHz, DMSO d6,): δ ppm 6.85–8.73 (m, 9H, ArH), 3.56–3.62 (m, CH2CH2, 4H), 3.53–3.54 (d, J = 4.0 Hz, 2H, CH2), 2.66–2.70 (t, J = 8.0 Hz, 2H, CH2). 13C NMR (100 MHz, DMSO d6,): δ ppm 164.39, 163.51, 151.24, 151.24, 134.83, 132.83, 131.69, 131.69, 129.29, 129.29, 124.82, 124.82, 123.35, 122.76, 120.72, 120.49, 108.32, 104.40, 66.70, 66.70, 56.98, 56.98, 53.81, 53.81. HRMS (ESI): m/z calcd for C24H23ClN3O3 ([M H] ): 436.1422, found 436.1418.

Scheme 1. Design of the effective lysosome-targeted Fe3 , Al3 and Cr3 ions fluorescent probe.

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Scheme 2. The synthetic route of the compound CMN.

2.3. Spectrophotometric studies The stock solution of probe CMN (10−3 M) was prepared in methanol. The stock solutions of metal ions (10−2 M) were prepared from KCl, NaCl, AgNO3, MgCl2, ZnCl2, CuCl2, NiCl2, BaCl2, CaCl2, Hg(OAc)2, PbCl2, MnCl2, CoCl2, FeCl3, AlCl3, CrCl3, FeCl2, AuCl3, K2Cr2O7 and InCl3 with deionized water. The probe CMN stock solution were diluted to 10.0 mL with methanol solution for the UV–visible and fluorescence measurement. The probe CMN stock solution (100 μL) was mixed with gradual incremental Fe3 , Al3 and Cr3 solution separately and diluted to 10.0 mL to form methanol solutions for the titration experiments. The excitation wavelength was fixed at 438 nm, the slit width was set at 10 nm. 2.4. Measurement of fluorescence quantum yields The fluorescence quantum yield was calculated used the following equation

Ф unk ¼ Ф std

   Iunk =Aunk ηunk 2 Istd =Astd ηstd

where Фunk and Фstd are the radiative quantum yields of the sample and standard, Iunk and Istd are the respective integrated emission intensities of the corrected spectra for the sample and standard, Aunk and Astd are the respective absorbances of the sample and standard, and ηunk and ηstd are the indices of refraction of the corresponding solvents of the sample and standard solutions. 2.5. Cytotoxicity assay The cytotoxicity of CMN in C2C12 cells were determined by using the Cell Counting Kit-8 (CCK-8, a commercially available cell viability dye). 5000 cells per well were seeded in a 96 well cell culture cluster and incubated for 24 h at 37 °C in a humidified incubator containing 5% CO2 in air. Cells were washed with DMEM once before treatments with various concentrations of related dyes, the precise concentration of related dyes are 10, 20 and 100 μM. The treated cells were incubated for 24 h at 37 °C in a humidified incubator containing 5% CO2 in air. Cells were washed with serum-free DMEM once, 100 μL serum-free DMEM containing 10% CCK-8 were added to each well and incubated for 1 h. The absorbance was measured at 450 nm on a plate reader. Cell viability rate was calculated according to the equation: Cell viability ¼

AS−Ab Ac−Ab

where As is the absorbance of the experimental group, Ac is the absorbance of the control group, and Ab is the absorbance of the blank group (no cells). 2.6. Cell incubation The C2C12 cells and human stromal cell line (HSC) were purchased from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China) with 20% fetal bovine serum, 100 μg/mL penicillin, and 100 μg/mL streptomycin in a 5% CO2 incubator at 37 °C before the cell imaging experiments. For imaging of Fe3 , Al3 and Cr3 ions, C2C12 cells were pretreated with CMN (20 μM) for 30 min at 37 °C, washed three times with prewarmed PBS, and then incubated with Fe3 , Al3 and Cr3 ions (40 μM) for 30 min at 37 °C. Cell imaging was then carried out after washing the cells with prewarmed PBS buffer. For imaging of Fe3 , Al3 and Cr3 ions in lysosomes, the living HSC cells were firstly maintained at 37 °C for 30 min after introduction of CMN (20 μM) and then treated with Fe3 , Al3 and Cr3 ions for 30 min. Next, Lyso-Tracker Red (70 nM) was incubated for another 0.5 h. The medium was then carefully removed and each dish was washed three times with PBS. Then the fluorescence of Lyso-Tracker Red and CMN was collected by Nikon A1 confocal laser microscopy system (Nikon, Japan). 3. Results and discussion 3.1. Synthesis The 4-bromo-1,8-naphthalic anhydride was refluxed in acetic acid with 4-chlorobenzamine to afford the intermediate N-(pchlorophenyl)-4-(2-aminoethyl)morpholine-1,8-naphthalimide. The reaction of N-(p-chlorophenyl)-4-bromo-1,8-naphthalimide with 4(2-aminoethyl)morpholine in dry DMSO provided the desired probes in 54% yield. The structures of the compounds were characterized by IR, 1H NMR, 13C NMR, and ESI–HRMS spectroscopies (Figs. S1–S7 in Electronic Supplementary information). IR spectrum revealed that the presence of a carbonyl group at 1680 cm−1, a distinct imine N\\H stretching band at 3350 cm−1. From the 1H NMR, the signals from 6.84 to 8.64 ppm indicated that was Ar\\H in the structure. The structure of CMN also was identified by ESI-HRMS with the peak at m/z 436.1418 corresponding to [CMN H] . 3.2. Spectral characteristics toward various metal cations To investigate the ability of detecting capability of CMN (10 μM), the UV–visible and fluorescence spectra were measured in the presence of various metal cations like K , Na , Ag , Mg2 , Zn2 , Cu2 , Ni2 , Ba2 , Ca2 , Hg2 , Pb2 , Mn2 , Co2 , Cr3 , Al3 , Fe3 , Au3 , Fe2 , Cr6 and In3 in methanol. Free

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Fig. 1. Change in absorption spectra of CMN (10 μM) in the absence and presence of various metal cations (20 μM) in methanol. Inset: The photograph of the solution of CMN in the absence and presence of Fe3 , Al3 and Cr3 in methanol under lamplight.

Fig. 4. Fluorescence spectra of probe CMN (10 μM) in the presence of incremental concentrations of Fe3 in methanol. Inset: Fluorescence intensity at 509 nm versus the number of equiv. of Fe3 added.

solution of CMN turned deepened, which provided a sensitive nakedeye detective way of trivalent cation (Fe3 , Al3 and Cr3 ) (Fig. 1). Under the identical conditions, the absorption spectral and the color of solution not was induced any effective change after the addition of the other monovalent and divalent metal ions. The absorption spectral was observed blue shift due to Fe3 , Al3 and Cr3 binding to CMN. The rigid structure of CMN were enhanced after complexation. As shown in Fig. 2, free CMN was nearly non-fluorescent emission at 509 nm, among the various metal cations, the most significant fluorescence enhancement was caused by Fe3 , Al3 and Cr3 . Other metal ions did not cause any obvious intensity variation. The fluorescence enhancement as a result of the inhibition of the PET, the N-atom of morpholine directly involved in the formation of CMN\\Fe3 , CMN\\Al3 and CMN\\Cr3 complexes. Based on the changes in the absorbance and fluorescence spectra it could be identify that CMN has good selectivity to trivalent metal ions (Fe3 , Al3 and Cr3 ). Fig. 2. Change in fluorescence spectra of CMN (10 μM) in the absence and presence of various metal cations (20 μM) in methanol. Inset: The photograph of the solution of CMN in the absence and presence of Fe3 , Al3 and Cr3 in methanol under 365 nm UV lamp.

3.3. Competitive experiment studies

CMN revealed a maximum peak in UV–vis at 438 nm. Upon the addition of 2 equiv. of various metal cations, the absorption spectrum of CMN changed obviously as well as selectively in the presence of trivalent metal ions (Fe3 , Al3 and Cr3 ), was observed underwent a blue shift of 23, 18 and 18 nm, respectively. At the same time, the pale-yellow

The competitive experiments of CMN toward Fe3 , Al3 and Cr3 ions in the presence of other metal ions were carried out in order to further certify the ability of CMN resisted the interference from the other competing metal ions (Fig. 3). It was noticeable that CMN could be applied to detect Fe3 and Al3 ions in the presence of the other excess metal ions except Hg2 . However, Cr3 could be detected in the presence of other metal ions except Cr6 . The experiment results indicated that

Fig. 3. Fluorescence intensity at 509 nm of CMN (10 μM) toward Fe3 , Al3 and Cr3 (20 μM) in the presence of different metal cations (40 μΜ) in methanol.

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values of trivalent metal ions chemosensors, the LOD of CMN presented here was much lower than others (Table 1). Based on the fluorescence titration experiments, the binding constant K of the three metal ions was calculated by Stern-Volmer equation, I0/(I0\\I) = 1/A 1/KA·1/[Q], where I0 is the fluorescence intensity of the blank probe CMN and I is the fluorescence intensity of the CMNcomplex, Q is [Fe3 ], [Al3 ] or [Cr3 ], A is a constant, K is a binding constant. The binding constants were calculated for Fe3 , Al3 and Cr3 were 53,400 M−1, 33,200 M−1, 49,900 M−1, respectively (Fig. S8). There values for trivalent cations are within those (103–107) previously reported. These results clearly indicated that Fe3 had more binding affinity than Al3 and Cr3 ions toward CMN. In this paper, the fluorescence quantum yields of CMN and its complex with trivalent cations have been determined. The quantum yield of the standard (here Rhodamine-6G in methanol) is 0.94 [54]. Quantum yield (ФF) of CMN increased from 0.01 to 0.33, 0.32 and 0.32 in the presence of Fe3 , Al3 and Cr3 , respectively. Fig. 5. Fluorescence spectra of probe CMN (10 μM) in the presence of incremental concentrations of Al3 in methanol. Inset: Fluorescence intensity at 509 nm versus the number of equiv. of Al3 added.

probe CMN displayed an excellent selectivity toward Fe3 , Al3 and Cr3 ions in the presence of the monovalent and divalent metal ions. 3.4. CMN for quantitative determination of trivalent cation The fluorescence titration experiments were studied in order to identify the sensing properties of CMN toward trivalent metal ions (Fe3 , Al3 and Cr3 ). The gradual incremental concentrations of Fe3 , Al3 and Cr3 were added have effect on the fluorescence characteristics was investigated. As shown in Figs.4–6, upon the addition of Fe3 (0 to 30 μM) ions to CMN in methanol, the fluorescence intensity of CMN was gradually enhanced and reached the maximum level until the addition of 20 μM of Fe3 . With the amount of Fe3 increased, the maximal fluorescence intensity of CMN was linearly increased in the concentration range of 0–20 μM of Fe3 (R2 = 0.986). The gradual incremental concentrations of Al3 or Cr3 ions were added even obtained analogous results. Further, the limit of detection (LOD) for Fe3 , Al3 or Cr3 were evaluated to be 6.5 × 10−7, 6.9 × 10−7 and 6.8 × 10−7 M, respectively (the equation is LOD = 3σ/S, where σ is the standard deviation of the blank solution, and S is the slope of the calibration curve). Compared with the previously reported

3.5. Response time Rapid response was another requirement for a chemosensor except high sensitivity and selectivity. The fluorescence of CMN was affected by time with and without Fe3 , Al3 and Cr3 was researched. Based on the time course study, the fluorescence intensity of CMN maintained steady at least for 30 min. The result indicated that CMN was an excellent chemosensor for the determination of Fe3 , Al3 and Cr3 due to its good fluorescence stability (Fig. S9). The fluorescence intensity of CMN was synchronously enhanced and accomplished the maximum within 10 s in the present of Fe3 , Al3 and Cr3 ions which indicated a quick respond time of CMN for Fe3 , Al3 and Cr3 . Additionally, the enhanced fluorescence intensity kept stability in the followed 30 min, which demonstrated CMN has the distinct characteristic of high reactivity with trivalent cation. There satisfactory results indicated that turn-on fluorescence respond of CMN was a steady and real-time approach to detected trivalent cation (Fe3 , Al3 and Cr3 ). 3.6. Reversibility studies applied as logic circuit devices The reversibility experiment was carried out, by the addition of ethylene diamine tetraacetic acid (EDTA) to CMN-M3 solutions to investigate the coordination reversibility of the CMN-M3 complex. As shown in Fig. 7, the fluorescence was quenched obviously after the addition of EDTA (2 equiv.) to the solutions of CMN-M 3 complex. The fluorescence spectra almost recovered to the original state which indicated the regeneration of CMN. The results demonstrated that CMN was a chemically reversible chemosensor for Fe3 , Al3 and Cr3 ions. On the basis of the reversible property of CMN, INHIBIT logic gate has been constructed. In this logic gate, used M3 and EDTA as two input signals and the fluorescence emission of CMN at 509 nm as an output signal, which could be symbolized as a combination of AND and NOT gates, the logic scheme and corresponding truth table was shown in Fig. 8. The presence and absence of chemical inputs was defined as 1 (on-state) and 0 (off-state), the high fluorescence emission of CMN at 509 nm as 1 (on-state), the low fluorescence emission as 0 (off-state). The low signal output of fluorescence emission of CMN was caused when in the absence of the used M3 as inputs. However, the high signal fluorescence emission was caused only when both of the inputs were high simultaneously and the AND logic gate was mimicked. The above results demonstrated that the fluorescence emission at 509 nm was high in the presence of M3 and in the absence of EDTA. 3.7. Binding mechanism

Fig. 6. Fluorescence spectra of probe CMN (10 μM) in the presence of incremental concentrations of Cr3 in methanol. Inset: Fluorescence intensity at 509 nm versus the number of equiv. of Cr3 added.

The stoichiometry of CMN-complex was calculated using Job plot analysis. The maximum fluorescence intensity was reproducibly

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Table 1 Comparison of CMN with previously reported Fe3 , Al3 and Cr3 chemosensors. Probes

Working media

LOD (Fe3 Al3 Cr3 )

Linear ranges (Fe3 Al3 Cr3 )

Application

Ref

MeOH/H2O

0.80 μM 1.60 μM 2.66 μM

0–50 μM 0–100 μM 0–150 μM

Cell imaging

[20]

CH3CN/HEPES buffer

20 μM 23 μM 25 μM

0–80 μM 0–80 μM 0–80 μM

No statement

[44]

MeOH/H2O

2.90 μM 0.00174 μM 2.36 μM

0–26 μM 0–20 μM 0–20 μM

Test strips

[45]

CH3CN

0.0503 μM 2.16 μM 0.0127 μM

0.05–0.2 μM 0–9 μM 0–0.2 μM

Molecular logic gate

[46]

EtOH

0.669 μM 0.646 μM 1.15 μM

0–20 μM 0–20 μM 0–20 μM

No statement

[47]

DMSO/HEPES buffer

3.0 μM 2.88 μM 1.89 μM

0–300 μM 0–300 μM 0–300 μM

Paper strip

[48]

CH3CN

0.9834 μM 2.434 μM 0.9923 μM

0–20 μM 0–20 μM 0–20 μM

Cell imaging

[49]

CH3CN/H2O

0.106 μM 0.117 μM 0.111 μM

0–20 μM 0–20 μM 0–20 μM

Cell imaging

[50]

MeOH/H2O

0.004 μM 0.001 μM 0.001 μM

0–45 μM 0–45 μM 0–55 μM

Molecular logic gate

[51]

CH3CN/H2O

0.19 μM 0.28 μM 0.25 μM 2.09 μM 2.10 μM 4.16 μM

1.5–5 μM 1–4 μM 1–4.5 μM 0–120 μM 1–100 μM 0–200 μM

Molecular logic gate; Real sample analysis

[52]

Cell imaging

[53]

0.65 μM 0.69 μM 0.68 μM

0–20 μM 0–20 μM 0–20 μM

Molecular logic gate; Organelle targetable imaging

This work

H2O

MeOH

observed at the molar ratio was 0.5 which demonstrated the binding stoichiometry of the CMN-M3 complex was 1:1(Fig. 9). In order to better conclude the binding mechanism of CMN with Fe3 , Al3 and Cr3 ions, the IR spectroscopic analysis was recorded before and after the addition of these trivalent metal cations separately (Fig. 10). With the introduction of Fe3 , Al3 or Cr3 , the sharp peak disappeared between 3300 cm−1 in IR spectra, which suggested the Natom of amines might participate in complex formation. For the

changes of fluorescence emission, it was predicted CMN emitted fluorescence through PET mechanism. It was possible that an electron transfers from lone pair electron of nitrogen atom of morpholine to naphthalimides fluorophore while the coordination between trivalent metal ions (Fe 3 , Al3 and Cr 3 ) and nitrogen atoms prohibited the electron transfer [38,42,43]. Based on these observations, the possible binding mode of Fe3 , Al3 and Cr3 with CMN was shown in Scheme 3.

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Fig. 7. Fluorescence spectral changes of CMN (10 μM) after the sequential addition of M3 and EDTA (2 equiv.) in MeOH.

Fig. 8. The INHIBIT type logic gate scheme and the truth table.

3.8. Cell imaging Since low cell toxicity is a key feature for living cell imaging, the cytotoxicity of CMN was first examined by CCK-8 testing. As shown in

Fig. S10, even the living cells were incubated with 20 μM of CMN for 24 h, the cells still kept high survival rate. Cell viability was affected only when excessive concentrations of CMN were added. This result suggested that CMN was suitable for applications in biological systems.

Fig. 9. Job's plot for calculate the stoichiometry of CMN and trivalent metal ions (Fe3 , Al3 and Cr3 ).

Fig. 10. IR spectra of CMN in the absence (black) and presence of Fe3 (red), Al3 (Orange Yellow) and Cr3 (green) ions.

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Scheme 3. Possible sensing mechanism of CMN.

To explore this potential, imaging of Fe3 , Al3 and Cr3 with CMN in living cells were investigated. In order to research the capability of CMN detected Fe3 , Al3 and Cr3 in biological systems, fluorescence imaging preliminary experiment was carried out in C2C12 (Mouse myoblast) cells. There was no obvious fluorescence signal was detected in C2C12 cells incubated with 20 μM blank probe CMN at excitation wavelength at 438 nm, which indicated that the probe could maintain its fluorescence-off form in living cells. In contrast, the C2C12 cells pre-incubated with probe CMN were further treated with 40 μM Fe3 , Al3 and Cr3 separately, in which remarkable fluorescence signal could be observed. There results demonstrated that CMN could be effectively used for cell imaging and detected the presence of Fe3 , Al3 and Cr3 in vivo (Fig. 11). The co-localization experiment was carried out, in order to further investigate the subcellular distribution of CMN used the Lyso-Tracker Deep Red, a commercially available lysosomal tracker (Fig. 12). The fibroblast cell line, HSC (human endometrium) cells were incubated with Fe3 , Al3 and Cr3 ions for 30 min, then the cells were incubated with CMN (20 μM) for 30 min and further were incubated with the

Lyso-Tracker Deep Red (70 nM) for 30 min. As shown in Fig. 12D, the emission of probe CMN was treated with Fe3 , Al3 or Cr3 (Fig. 12B) as well as Lyso-Tracker Deep Red (Fig. 12C) revealed remarkable overlaid. Furthermore, the intensity scatter plot of the probe CMN (green channel) and Lyso-Tracker Deep Red (red channel) indicated the fluorescence emissions correlate closely (Fig. 12E). Furthermore, the variation in the intensity profile of linear regions of interest trend toward synchronization (Fig. 12F). The Pearson's colocalization coefficient for Fe3 , Al3 and Cr3 was 0.83, 0.81, 0.89 and the Mander's overlap coefficient was 0.89, 0.86, 0.90, respectively. These results demonstrated that the effective probe exhibited an excellent imaging capacity for lysosomes Fe3 , Al3 and Cr3 in living cells as designed. 4. Conclusion In summary, based on morpholine-naphthalimide, an effective turnon fluorescent probe CMN was design and synthesized for sensing trivalent metal ions (Fe3 , Al3 and Cr3 ) with significant biological as well as environmental relevance in methanol solution. CMN exhibited

Fig. 11. The C2C12 cells confocal microscopic images. (a): the C2C12 cells were incubated with 20 μM of CMN for 30 min and washed with PBS for three times to remove residual CMN, (b)(d): the cells were treated with 40 μM of Fe3 , Al3 and Cr3 , respectively. and with 20 μM of CMN for 30 min, the fluorescence emission was collected at 520–560 nm (λex = 438 nm). (e)(h): Bright-field images of cells.

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Fig. 12. Fluorescence images of HSC cells co-stained with CMN (20 μM) and Lyso-Tracker Deep Red (70 nM) in the presence of Fe3 (A1-F1), Al3 (A2-F2), Cr3 (A3-F3), respectively. (A) Bright field images. (B) Fluorescence of CMN the emission was collected at 520–560 nm (λex = 438 nm). (C) The fluorescence emission of Lyso-Tracker Deep Red (70 nM) was collected at 570–620 nm (λex = 577 nm). (D) Merge images of A-C. (E) The intensity scatter plot of green channel and red channel. (F) The fluorescence intensity profiles of the region of interest across the cells co-stained with CMN and Lyso-Tracker Deep Red.

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excellent selectivity and sensitivity for trivalent metal ions (Fe3 , Al3 and Cr3 ) in the presence of other monovalent and divalent metal ions and naked-eye detected color changes. It has shown 1:1 chelation between CMN and Fe3 , Al3 and Cr3 from Job's plot analysis and the LOD in the micro molar range. CMN could be successfully applied to build an INHIBIT type molecular logic gate. In this work, we provide an effective strategy utilizing the morpholine-naphthalimide platform for targeting and imaging Fe3 , Al3 and Cr3 ions in lysosome. Declaration of Competing Interest All authors don't have conflicts of interest to declare. Acknowledgements The work was supported by the Natural Science Foundation of Heilongjiang Province (LH2019B002), Postdoctoral Foundation of Heilongjiang Province (LBH-Z17014) and Young Talent Plan of Northeast Agricultural University (17QC24). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.saa.2019.117242. References [1] S.Y. Ma, Z. Yang, M.Y. She, W. Sun, B. Ying, P. Liu, S.Y. Zhang, J.L. Li, Design and synthesis of functionalized rhodamine based probes for specific intracellular fluorescence imaging of Fe3 , Dyes Pigments 115 (2015) 120–126, https://doi.org/10. 1016/j.dyepig.2014.12.014. [2] L. Wang, X.P. Liu, L.H. Yao, Y.Z. Ci, S.Y. Chang, P. Yu, G.F. Gao, Y.Z. Chang, Hepcidin and iron regulatory proteins coordinately regulate ferroportin 1 expression in the brain of mice, J. Cell. Physiol. 234 (2019) 7600–7607, https://doi.org/10.1002/jcp.27522. [3] J.B. Vincent, Recent advances in the nutritional biochemistry of trivalent chromium, Proc. Nutr. Soc. 63 (2004) 41–47, https://doi.org/10.1079/PNS2003315. [4] J.W. Lee, J.D. Helmann, The PerR transcription factor senses H2O2 by metal-catalysed histidine oxidation, Nature 440 (2006) 363–367, https://doi.org/10.1038/ nature04537. [5] M. Regenboog, A.E. Bohte, E.M. Akkerman, J. Stoker, C.E.M. Hollak, Iron storage in liver, bone marrow and splenic Gaucheroma reflects residual disease in type 1 Gaucher disease patients on treatment, Br. J. Haematol. 179 (2017) 635–647, https://doi.org/10.1111/bjh.14915. [6] G.T. Sucak, Z.A. Yegin, Z.N. Ozkurt, S.Z. Aki, T. Karakan, G. Akyol, The role of liver biopsy in the workup of liver dysfunction late after SCT: is the role of iron overload underestimated? Bone Marrow Transplant. 42 (2008) 461–467, https://doi.org/10. 1038/bmt.2008.193. [7] S. Altamura, M.U. Muckenthaler, Iron toxicity in diseases of aging: Alzheimer's disease, Parkinson's disease and atherosclerosis, J. Alzheimers Dis. 16 (2009) 879–895, https://doi.org/10.3233/JAD-2009-1010. [8] S.W. Zhang, J.X. Li, X.Y. Zeng, J.Z. Xu, X.K. Wang, W.P. Hu, Polymer nanodots of graphitic carbon nitride as effective fluorescent probes for the detection of Fe3 and Cu2 ions, Nanoscale 6 (2014) 4157–4162, https://doi.org/10.1039/c3nr06744k. [9] N. Narayanaswamy, T. Govindaraju, Aldazine-based colorimetric sensors for Cu2 and Fe3 , Sensors Actuators B Chem. 161 (2012) 304–310, https://doi.org/10.1016/ j.snb.2011.10.036. [10] S.V. Verstraeten, L. Aimo, P.I. Oteiza, Aluminium and lead: molecular mechanisms of brain toxicity, Arch. Toxicol. 82 (2008) 789–802, https://doi.org/10.1007/s00204008-0345-3. [11] A.K. Manna, J. Mondal, K. Rout, G.K. Patra, A new ICT based Schiff-base chemosensor for colorimetric selective detection of copper and its copper complex for both colorimetric and fluorometric detection of cysteine, J. Photochem. Photobiol., A 367 (2018) 74–82, https://doi.org/10.1016/j.jphotochem.2018.08.018. [12] J.R. Walton, Aluminum in hippocampal neurons from humans with Alzheimer's disease, Neurotoxicology 27 (2006) 385–394, https://doi.org/10.1016/j.neuro. 2009.06.010. [13] Y. Liu, A.Y. Bi, T. Gao, X.Z. Cao, F. Gao, P.F. Rong, W. Wang, W.B. Zeng, A novel selfassembled nanoprobe for the detection of aluminum ions in real water samples and living cells, Talanta 194 (2019) 38–45, https://doi.org/10.1016/j.talanta.2018. 09.104. [14] R.R. Crichton, D.T. Dexter, R.J. Ward, Metal based neurodegenerative diseases from molecular mechanisms to therapeutic strategies, Coord. Chem. Rev. 252 (2008) 1189–1199, https://doi.org/10.1016/j.ccr.2007.10.019. [15] J. Sulejmanovic, E. Sabanovic, S. Begic, M. Memic, Molybdenum (VI) oxide-modified silica gel as a novel sorbent for the simultaneous solid-phase extraction of eight metals with determination by flame atomic absorption spectrometry, Anal. Lett. 52 (2019) 588–601, https://doi.org/10.1080/00032719.2018.1481418.

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