English

• 0.   Introduction

  Compounds containing heavy metals such as Fe³⁺ and Cu²⁺ are widely used in our daily lives and extensively employed in industrial and agricultural processes such as metal etching^[1], textile dyeing^[2], and insecticide production^[3]. During these processes, heavy metal residues are inevitably generated, increasing the risk of environmental pollution^[4]. Additionally, iron and copper are essential micronutrients in living organisms. For humans, adequate daily intake of copper and iron is necessary to maintain cellular respiration, iron homeostasis, and antioxidant defense^[5-8]. However, excessive intake of Fe³⁺ and Cu²⁺ can lead to toxicity^[9-13]. Therefore, detecting these ions in a timely and specific way is crucial to mitigate their hazards.

  To achieve effective detection of heavy metal ions like Fe³⁺ and Cu²⁺, various detection methods have been developed, including inductively coupled plasma mass spectrometry^[14-15], atomic absorption spectroscopy^[16-17], and voltammetry^[18-19]. However, these detection methods are complex and time-consuming, limiting their further application. In recent years, fluorescent probes have attracted increasing attention due to their simple and rapid operation, high sensitivity, and good selectivity^[20-23]. Currently, several fluorescent probes for Cu²⁺ and Fe³⁺ have been developed and applied in both in vitro and in vivo based on different fluorophores such as pyrene^[24-25], rhodamine^[26-27], fluorescein^[28-29], methylene blue^[30], and 1, 8-naphthalimide^[31-32]. Among those fluorophores, 1, 8-naphthalimide stands out with high fluorescence quantum yield, large Stokes shift, and excellent photostability^[31-32]. In addition to its high electron affinity and excellent photostability, naphthalimide is favored by researchers for its ease of modification, making it an ideal fluorophore for constructing fluorescent probes. In fact, based on the naphthalimide structure, various types of fluorescent probes have been developed and applied in the detection of heavy metals such as Fe³⁺, Cu²⁺, reactive oxygen species (ROS), and biological enzymes^[33-35]. However, a comparative analysis of the testing conditions of these probes in vitro reveals significant differences in testing conditions, especially for heavy metal ions. These ions themselves are significantly influenced by different solvent conditions^[36-43]. For example, currently, probes for Cu²⁺ use buffer solutions such as HEPES (2-[4-(2-hydroxyethyl)piperazin‑1‑yl]ethane sulfonic acid), Tris (tris (hydroxymethyl)aminomethane), and PBS (phosphate buffered saline) in in vitro testing (Table S1, Supporting information). The suitability of solvent conditions for accurately evaluating probe performance remains unclear, which often leads to inaccurate characterization of many probes.

  In response to this issue, by using the naphthalimide structure as the fluorophore, we selected the amino urea structure as the metal recognition site and constructed the fluorescent probe DHU-NP-4 (Scheme 1). Systematic studies using DHU-NP-4 illustrated that solvent conditions have a significant impact on the probe′s performance, especially buffer solutions containing organic components such as HEPES and Tris, which are not suitable for investigating the response behavior of the probe with heavy metals in vitro. Additionally, the pH of the testing system may significantly affect the performance of the probe, suggesting that attention should be paid to the effect of pH changes on the probe′s structure when developing fluorescent probes for heavy metals. Furthermore, we found that by limiting the pH of the solution, the probe can achieve quantitative detection of Fe³⁺ and Cu²⁺ in solutions.

  Scheme 1

  

  Scheme 1.  Synthetic route of DHU-NP-4

  下载: 全尺寸图片 幻灯片

  1.   Experimental

  1.1   Materials and instruments

  All starting materials were obtained from commercial suppliers and used without further purification. Double-distilled water was used in all procedures. All experiments were performed in compliance with the relevant laws and institutional guidelines. ¹H NMR (400 MHz) and ¹³C NMR (100 MHz) were obtained on a Bruker AV400 NMR spectrometer using CDCl₃ or DMSO-d₆ as the solvent. Proton chemical shifts are reported in parts per million downfield from tetramethyl silane (TMS), with tetramethyl silane (δ=0.0) or DMSO-d₆ (δ=39.52 for ¹³C) as the chemical shift standard. High-resolution mass spectra (HRMS) were carried out on a Bruker Micro TOF Ⅱ instrument with the electrospray ionization (ESI) technique and direct injection method. UV-Vis spectra were performed using a UV-2600 spectrophotometer (Shimadzu, Japan). Fluorescence spectra were obtained by FLS 1000 photoluminescence spectrometer (Edinburgh, England).

  1.2   Synthesis and characterization of the probes

  2-Butyl-6-(dimethylamino)-1H-benzo[de]isoquinoline-1, 3(2H)-dione (DHU-NP-1) was prepared according to the literature^[44].

  1.2.1   Synthesis of compound 1

  4-Bromo-1, 8-naphthalic anhydride (1.39 g, 5 mmol, 1 eq.) and butylamine (520 μL, 5.25 mmol, 1.05 eq.) with 15 mL ethanol were added to a 100 mL flask and then heated to 85 ℃ under nitrogen atmosphere. After stirring for 8 h, the mixture was cooled with an ice-water bath. Light wheat-coloured precipitation was collected by vacuum filtration, washed with 4 mL cold methanol, and dried in vacuum to give compound 1 (1.44 g, 87%).

  ¹H NMR (400 MHz, CHCl₃): δ 8.66 (dd, J=7.2, 1.2 Hz, 1H), 8.57 (dd, J=8.4, 0.8 Hz, 1H), 8.42 (d, J=8.0 Hz, 1H), 8.04 (d, J=8.0 Hz, 1H), 7.85 (dd, J=8.4, 7.2 Hz, 1H), 4.18 (t, J=7.6 Hz, 2H), 1.76-1.68 (m, 2H), 1.50-1.40 (m, 2H), 0.98 (t, J=7.4 Hz, 3H).

  1.2.2   Synthesis of DHU-NP-2

  Compound 1 (1.33 g, 4 mmol, 1 eq.), boc-2-(methylamino) ethylcarbamate (0.77g, 4.4 mmol, 1.1 eq.), TEA (840 μL, 6 mmol, 1.5 eq.) and DMF (20 mL) were added to a 100 mL flask. The mixture was heated to 110 ℃ and stirred for 10 h under a nitrogen atmosphere. After removing about 18 mL DMF under reduced pressure, 10 mL water was slowly added to the hot mixture to precipitate a yellow compound. The mixture was filtered to obtain the crude product which was washed with methanol/H₂O (8∶2, V/V) and dried to give DHU-NP-2 as a yellow solid (1.41 g, 83%).

  ¹H NMR (400 MHz, CHCl₃): δ 8.60 (d, J=7.6 Hz, 2H), 8.52 (d, J=8.0 Hz, 1H), 7.73 (t, J=8.0 Hz, 1H), 7.33 (d, J=8.0 Hz, 1H), 4.75 (s, 1H), 4.17 (t, J=7.4 Hz, 2H), 3.50 (s, 4H), 3.13 (s, 3H), 1.75-1.67 (m, 2H), 1.49-1.42 (m, 2H), 1.39 (s, 9H), 0.97 (t, J=7.2 Hz, 3H).

  ¹³C NMR (100 MHz, CHCl₃): δ 164.6, 164.1, 156.4, 155.9, 132.4, 131.2, 130.7, 130.2, 126.2, 125.5, 123.3, 116.3, 115.4, 79.7, 56.3, 42.2, 40.2, 38.2, 30.4, 28.5, 20.5, 14.0.

  HRMS (ESI, m/z) Calcd. for C₂₄H₃₁N₃O₄ ([M+H]⁺): 426.238 8; Found: 426.240 4.

  1.2.3   Synthesis of DHU-NP-3

  DHU-NP-2 (0.85 g, 2 mmol) dissolved in 10 mL of CH₂Cl₂ (DCM) in a 50 mL flask was cooled with an ice-water bath. 0.5 mL hydrochloric acid was added dropwise into the solution. The DCM phase slowly turned from yellow to colorless. After the DCM phase was completely colorless, Na₂CO₃ solution was added to the flask until the pH of the water phase was higher than 7.5. The resulting mixture was extracted with three 100 mL portions of DCM. The combined extracts were dried over anhydrous sodium sulfate concentrated on a rotary evaporator and then purified by column chromatography (V_MeOH/V_DCM=1/20) to afford DHU-NP-3 as yellow oily liquid (0.62 g, 95%).

  ¹H NMR (400 MHz, CHCl₃): δ 8.54 (d, J=7.2 Hz, 1H), 8.45 (d, J=8.0 Hz, 2H), 7.65 (t, J=7.8 Hz, 1H), 7.21 (d, J=8.0 Hz, 1H), 6.21 (s, 2H), 4.15 (t, J=7.4 Hz, 2H), 3.58 (d, J=6.0 Hz, 2H), 3.50 (d, J=5.6 Hz, 2H), 3.08 (s, 3H), 1.73-1.66 (m, 2H), 1.48-1.39 (m, 2H), 0.97 (t, J=7.2 Hz, 3H).

  ¹³C NMR (100 MHz, CHCl₃): δ 164.6, 164.1, 156.7, 132.4, 131.1, 130., 130.1, 126.2, 125.4, 123.2, 116.1, 115.2, 59.2, 42.2, 40.1, 39.5, 30.3, 20.5, 13.9.

  HRMS (ESI, m/z) Calcd. for C₁₉H₂₄N₃O₂ ([M+H]⁺): 326.186 4; Found: 326.187 5.

  1.2.4   Synthesis of DHU-NP-4

  DHU-NP-3 (0.98 g, 3 mmol, 1 eq.), sodium carbonate (1.27g, 12 mmol, 4 eq.), 10 mL DCM was added into a 50 mL flask and cooled with an ice-water bath. Under a nitrogen atmosphere, a solution of triphosgene (BTC, 1.33 g, 4.5 mmol, 1.5 eq.) in DCM was dropwise added into the flask. After slowly heating to room temperature, the reaction was completed in another 4 h indicated by TLC analysis. Then hydrazine hydrate (85%, 353 μL, 6 mmol, 2 eq.) was added into the flask. The reaction was completed in 2 h indicated by TLC analysis. The reaction mixture was poured into 150 mL of ice water while stirring, and the resulting mixture was extracted with three 100 mL portions of DCM. The combined extracts were dried over anhydrous sodium sulfate, concentrated on a rotary evaporator, and then purified by column chromatography (V_MeOH/V_DCM=1/40) to yield DHU-NP-4 as a yellow solid (0.43 g, 37%).

  ¹H NMR (400 MHz, DMSO-d₆): δ 8.55 (dd, J=8.4, 1.2 Hz, 1H), 8.46 (dd, J=7.2, 0.8 Hz, 1H), 8.34 (d, J=8.0 Hz, 1H), 7.77-7.73 (m, 1H), 7.31 (d, J=8.0 Hz, 1H), 6.98 (s, 1H), 6.55 (s, 1H), 4.05-4.01 (m, 4H), 3.45-3.39 (m, 4H), 3.08 (s, 3H), 1.64-1.56 (m, 2H), 1.39-1.30 (m, 2H), 0.92 (t, J=7.4 Hz, 3H).

  ¹³C NMR (100 MHz, DMSO-d₆): δ 163.6, 163.0, 160.2, 156.4, 132.1, 131.3, 130.5, 129.5, 125.2, 124.8, 122.3, 114.3, 113.8, 56.7, 40.9, 36.7, 29.7, 19.8, 13.7.

  HRMS (ESI, m/z) Calcd. for C₂₀H₂₆N₅O₃ ([M+H]⁺): 384.203 1; Found: 384.202 1.

  1.3   Preparation of different analytes and buffers

  The stock solutions (10 mmol·L^-1) of all analytes were prepared from CuCl₂, NaCl, MgCl₂, AlCl₃, KCl, CaCl₂, FeCl₃, Hg(ClO₄)₂·3H₂O, NiCl₂·6H₂O, NH₄Cl, CH₃COONa, Na₂CO₃, Na₂SO₄, NaF, NaNO₂, Na₂S₂O₃, and NaClO₄ using pure water. All buffers (10 mmol·L^-1) were prepared by dissolving the corresponding solid compound in pure water. 137 mmol·L^-1 NaCl was added to PB (phosphate buffer) to obtain PBS. pH of PBS and PB were adjusted by adding 2 mol·L^-1 NaOH or 2 mol·L^-1 H₃PO₄ dropwise. pH of other buffers was adjusted by adding 2 mol·L^-1 NaOH or 2 mol·L^-1 HCl dropwise.

  1.4   Measurements of fluorescence changes upon addition of analytes

  Stock solutions of DHU-NP-4 (5 mmol·L^-1) were prepared in DMF. The test solutions of probe (10 μmol·L^-1) were prepared by placing stock solutions in a cuvette, diluted with different solvents (pure water, 10 mmol·L^-1 PBS/PB buffer (pH=4‑10), Tris buffer, and HEPES buffer) followed by adding analytes. The resulting solution was shaken well and incubated at room temperature for 3 h before recording the spectra. Unless otherwise noted, for all fluorescent measurements, the excitation wavelength was 440 nm and the emission wavelength was collected from 460 to 750 nm. The stock solutions were used freshly.

  1.5   Method and setup for the theoretical calculations

  The structures of compounds were constructed by Chemd3D 20.0, and geometry optimization was finished by Gaussian 09W quantum chemistry package with density functional theory method at B3LYP/6-311G** level^[45].

  2.   Results and discussion

  2.1   Design and optimization of the probes

  After definitively selecting the naphthalimide structure as the fluorophore, we found that amino or urea bonds were prone to coordinate with metals. Therefore, we used the amino urea structure with good coordination properties as the recognition site and designed and synthesized DHU-NP-4. Considering that the naphthalimide structure had two derivative positions, we introduced a non-reactive alkyl chain into the naphthalimide core, retaining only one derivatization site for modifying the recognition group. To compare the performance of DHU-NP-4, we also synthesized reference compounds DHU-NP-1, DHU-NP-2, and DHU-NP-3 (Fig. 1a).

  Figure 1

  

  Figure 1.  (a) Chemical structure of as-synthesized compounds; (b-e) Absorbance spectra of the compounds (10 μmol·L^-1, containing 0.2% DMF as co-solvent) in PBS

  下载: 全尺寸图片 幻灯片

  The spectroscopic properties of these compounds were tested in PBS (pH=7.4, 10 mmol·L^-1 containing 0.2% DMF as co-solvent). All the compounds gave an absorption centered at around 440 nm (Fig. 1b-1e). The response behavior with Cu²⁺ revealed that only DHU-NP-4 could respond to Cu²⁺ in PBS (Fig. 2a). The analysis of the product through LC-MS (Fig. 2b) revealed that the final product was DHU-NP-3. Furthermore, we explored the response mechanism of the probe with Cu²⁺ through theoretical calculations (Fig.S1). The results showed that the amino urea structure could undergo photo-induced electron transfer (PET) with the naphthalimide structure. After responding to Cu²⁺, an intermediate complex was generated, which underwent hydrolysis in the solution, eventually forming compound DHU-NP-3 (Fig. 2c). PET was thus interrupted, leading to the fluorescence recovery of the probe. Therefore, we chose DHU-NP-4 for subsequent testing.

  Figure 2

  

  Figure 2.  (a) Fluorescent intensity of as-synthesized compounds (10 μmol·L^-1) at 556 nm in the presence of 50 μmol·L^-1 Cu²⁺; (b) LC-MS analysis of mixture of DHU-NP-4 (4 μmol·L^-1) and Cu²⁺ (6 μmol·L^-1) compared with DHU-NP-3 (4 μmol·L^-1) standard peak; (c) Proposed reaction mechanism of DHU-NP-4 towards Cu²⁺

  下载: 全尺寸图片 幻灯片

  2.2   Influence of solvent conditions

  As mentioned, solvent conditions significantly influence the response of the probe to metals. To obtain deeper insights into the impact of solvent condition variations on its response behavior, we tested the response of DHU-NP-4 in different types of buffer solutions. As shown in Fig. 3a, DHU-NP-4 could respond to both Fe³⁺ and Cu²⁺ in pure water. However, when HEPES buffer (pH=7.4) or PBS (pH=7.4) was used as the solution, the probe displayed excellent selectivity to Cu²⁺. Meanwhile, under the same testing conditions, DHU-NP-4 exhibited superior responsiveness in PBS compared to HEPES. This might be attributed to the organic components of the HEPES buffer (2‑[4‑(2‑hydroxyethyl)piperazin-1-yl]ethane sulfonic acid) coordinating with metals, such as Cu²⁺, thereby interfering with the experiment. Meanwhile, similar phenomena were observed when conducting related tests using Tris buffer solution (Fig. 3a). In Tris buffer solution, the probe did not respond to Fe³⁺ either (Fig. 3a). These data further demonstrated that buffers containing organic components are not suitable for studying the performance of metal fluorescent probes.

  Figure 3

  

  Figure 3.  (a) Fluorescent intensity of DHU-NP-4 (10 μmol·L^-1) in different solvents at 556 nm after treated with Cu²⁺ and Fe³⁺ (50 μmol·L^-1); (b) Fluorescent intensity of DHU-NP-4 (10 μmol·L^-1) after treated with Cu²⁺ and Fe³⁺ (50 μmol·L^-1) in PBS at different pH values; (c) Proposed mechanism of DHU-NP-4 towards Fe³⁺ in acid condition; (d) Fluorescent intensity of DHU-NP-4 (10 μmol·L^-1) after treated with Cu²⁺ and Fe³⁺ (50 μmol·L^-1) in PB at different pH values; (e) Fluorescent intensity of DHU-NP-4 (10 μmol·L^-1) after treated with Cu²⁺ and Fe³⁺ (50 μmol·L^-1) in NaCl solution with different concentrations

  下载: 全尺寸图片 幻灯片

  pH was also an important factor for the detecting of metal ions by the fluorescence probes, which might be the reason for the difference between the probe′s performance in neutral PBS and pure water (Fig. 3a, 3b, and S2). The pH of pure water is around 5.5, under which condition the probe may undergo structural change, making it more prone to coordinate with Fe³⁺ (Fig. 3c). To verify this hypothesis, we used PBS as a buffer solution to study the response behavior of the probe under different pH conditions. The results showed that the response capability of the probe to Cu²⁺ increased with increasing pH under alkaline conditions (Fig. 3b). In contrast, as the system gradually became acidic, the response capability of the probe to Fe³⁺ was increased. The selectivity difference in acidic and alkaline conditions was further validated using other types of buffer systems primarily composed of inorganic salts. The results were shown in Fig. 3d, DHU-NP-4 responded to Cu²⁺ under alkaline conditions and to Fe³⁺ under acidic conditions in PB. This suggests that in the development of fluorescent probes for heavy metals, attention should be paid to the pH variations that may affect the probe′s structure and, consequently, its response behavior. Meanwhile, we observed variations in response behavior between PBS and PB buffer systems. While the probe exhibited consistent response trends in both buffers, its performance in PBS was notably superior. Furthermore, the addition of chloride ions in PB affected the probe′s response to Cu²⁺ (Fig. 3e), which is ascribed to the participation of Cl^- in DHU-NP-4-Cu complex, improving solubility of the complex and reducing the non-radiative energy transfer from DHU-NP-4 to Cu^{2+ [46]}.

  2.3   Application of DHU-NP-4 in the solution

  Using PBS as the solvent, we systematically explored the detecting performance of the probe in solution. The results are depicted in Fig. 4. Under strongly acidic conditions, the probe exhibited a specific response to Fe³⁺ (Fig. 4a), while under alkaline conditions, it displayed specificity towards Cu²⁺ (Fig. 4b and 4c) with a low detection limit (14.95 nmol·L^-1) determined by 3σ/k (σ: standard deviation, k: slope). For the quantitative detecting application, we conducted additional tests to assess its performance in solutions using an added method. We added a certain amount (5, 30, and 55 μmol·L^-1) of Cu²⁺ or Fe³⁺ ions to acidic and alkaline PBS solutions and determined the recovery rate using DHU-NP-4. As shown in Table 1, the probe demonstrated good recovery rates when detecting ions. Therefore, we predict that DHU-NP-4 could quantitatively detect Cu²⁺ or Fe³⁺ in real water samples with appropriate pH, holding potential practical value.

  Figure 4

  

  Figure 4.  (a-c) Fluorescent intensity of DHU-NP-4 (10 μmol·L^-1) at 556 nm after treated with different cations (50 μmol·L^-1) at different pH values; (d) Fluorescent intensity of DHU-NP-4 (10 μmol·L^-1) at 556 nm after treated with different concentrations of Cu²⁺ (0, 0.2, 0.4, 0.8, 1.2, 1.6 μmol·L^-1)

  From A to R: blank, Al³⁺, Ca²⁺, Cd²⁺, Co²⁺, Cu²⁺, Fe²⁺, Fe³⁺, Hg²⁺, K⁺, Mg²⁺, Na⁺, NH⁴⁺, Ni²⁺, Pb²⁺, Sn²⁺, Ti⁴⁺, Zn²⁺.

  下载: 全尺寸图片 幻灯片

  Table 1

  

  Table 1.  Recovery of Cu²⁺ and Fe³⁺ tested by DHU-NP-4 in PBS

  下载: 导出CSV

  Ion	Concentration of ion / (μmol·L-1)		Recovery / %
  	Added	Found
  Cu2+	0.45	0.431 9±0.006 9	95.56
  	1.05	1.051 5±0.001 0	100.14
  	1.55	1.554 3±0.013 1	100.28
  Fe3+	5	4.94±0.04	98.9
  	30	30.99±0.40	103.3
  	55	55.92±0.54	101.6

  3.   Conclusions

  A naphthalimide-based fluorescent probe, DHU-NP-4, was constructed to detect Fe³⁺ or Cu²⁺ specifically under defined conditions. Using DHU-NP-4, we determined that buffer solutions containing organic components such as HEPES and Tris were unsuitable for exploring the response behavior of heavy metals in vitro. Instead, PBS was found to be the preferred solution. Moreover, the pH of the solution significantly affected the detecting performance of the probe. This probe enabled quantitative detection of Fe³⁺ under strongly acidic conditions and Cu²⁺ under alkaline conditions.

  
  Acknowledgments: We gratefully acknowledge the financial support from NSFC (Grants No. 22371038, 22177019). Supporting information is available at http://www.wjhxxb.cn
  
• 1. [1]

     STEINBRÜCHEL C. Patterning of copper for multilevel metallization: Reactive ion etching and chemical-mechanical polishing[J]. Appl. Surf. Sci., 1995, 91(1/2/3/4):  139-146.

  2. [2]

     RAMAN C D, KANMANI S. Textile dye degradation using nano zero valent iron: A review[J]. J. Environ. Manage., 2016, 177:  341-355. doi: 10.1016/j.jenvman.2016.04.034

  3. [3]

     LIU Z L, XIE J Y, DENG Z R, WANG M L, DANG D D, LUO S, WANG Y F, SUN Y J, XIA L Q, DING X Z. Enhancing the insecticidal activity of new Bacillus thuringiensis X023 by copper ions[J]. Microb. Cell Fact., 2020, 19(1):  195. doi: 10.1186/s12934-020-01452-8

  4. [4]

     RUYTERS S, SALAETS P, OORTS K, SMOLDERS E. Copper toxicity in soils under established vineyards in Europe: A survey[J]. Sci. Total Environ., 2013, 443:  470-477. doi: 10.1016/j.scitotenv.2012.11.001

  5. [5]

     TAHIR N, ASHRAF A, WAQAR S H B, RAFAE A, KANTAMNENI L, SHEIKH T, KHAN R. Copper deficiency, a rare but correctable cause of pancytopenia: A review of literature[J]. Expert Rev. Hematol., 2022, 15(11):  999-1008. doi: 10.1080/17474086.2022.2142113

  6. [6]

     BALAMURUGAN K, SCHAFFNER W. Copper homeostasis in eukaryotes: Teetering on a tightrope[J]. Biochim. Biophys. Acta‒Mol. Cell Res., 2006, 1763(7):  737-746. doi: 10.1016/j.bbamcr.2006.05.001

  7. [7]

     MILNE D B. Copper intake and assessment of copper status[J]. Am. J. Clin. Nutr., 1998, 67(5):  1041S-1045S. doi: 10.1093/ajcn/67.5.1041S

  8. [8]

     KRASNOVSKAYA O, NAUMOV A, GUK D, GORELKIN P, EROFEEV A, BELOGLAZKINA E, MAJOUGA A. Copper coordination compounds as biologically active agents[J]. Int. J. Mol. Sci., 2020, 21(11):  3965. doi: 10.3390/ijms21113965

  9. [9]

     HSU C C, SENUSSI N H, FERTRIN K Y, KOWDLEY K V. Iron overload disorders[J]. Hepatol. Commun., 2022, 6(8):  1842-1854. doi: 10.1002/hep4.2012

  10. [10]

      D'MELLO S R, KINDY M C. Overdosing on iron: Elevated iron and degenerative brain disorders[J]. Exp. Biol. Med., 2020, 245(16):  1444-1473. doi: 10.1177/1535370220953065

  11. [11]

      ODAI T, TERAUCHI M, SUZUKI R, KATO K, HIROSE A, MIYASAKA N. Severity of subjective forgetfulness is associated with high dietary intake of copper in Japanese senior women: A cross-sectional study[J]. Food Sci. Nutr., 2020, 8(8):  4422-4431. doi: 10.1002/fsn3.1740

  12. [12]

      DAMERON C T, HARRISON M D. Mechanisms for protection against copper toxicity[J]. Am. J. Clin. Nutr., 1998, 67(5):  1091S-1097S. doi: 10.1093/ajcn/67.5.1091S

  13. [13]

      SUBRAMANIYAM V, SUBASHCHANDRABOSE S R, THAVAMANI P, CHEN Z L, KRISHNAMURTI G S R, NAIDU R, MEGHARAJ M. Toxicity and bioaccumulation of iron in soil microalgae[J]. J. Appl. Phycol., 2016, 28(5):  2767-2776. doi: 10.1007/s10811-016-0837-0

  14. [14]

      KIM P, ZHANG C C, THORÖE-BOVELETH S, WEISKIRCHEN S, GAISA N T, BUHL E M, STREMMEL W, MERLE U, WEISKIRCHEN R. Accurate measurement of copper overload in an experimental model of Wilson disease by laser ablation inductively coupled plasma mass spectrometry[J]. Biomedicines, 2020, 8(9):  356. doi: 10.3390/biomedicines8090356

  15. [15]

      SOLOVYEV N, ALA A, SCHILSKY M, MILLS C, WILLIS K, HARRINGTON C F. Biomedical copper speciation in relation to Wilson's disease using strong anion exchange chromatography coupled to triple quadrupole inductively coupled plasma mass spectrometry[J]. Anal. Chim. Acta, 2020, 1098:  27-36. doi: 10.1016/j.aca.2019.11.033

  16. [16]

      WEINSTOCK N, UHLEMANN M. Automated determination of copper in undiluted serum by atomic absorption spectroscopy[J]. Clin. Chem., 1981, 27(8):  1438-1440. doi: 10.1093/clinchem/27.8.1438

  17. [17]

      SOFIKITIS A M, COLIN J L, DESBOEUFS K V, LOSNO R. Iron analysis in atmospheric water samples by atomic absorption spectroscopy (AAS) in water-methanol[J]. Anal. Bioanal. Chem., 2004, 378(2):  460-464. doi: 10.1007/s00216-003-2282-6

  18. [18]

      DASTANGOO H, MAJIDI M R, HORMOZI M K. Stripping voltammetry on palladized aluminum: A novel sensing platform for trace analysis of copper[J]. Microchem. J., 2023, 187:  108404. doi: 10.1016/j.microc.2023.108404

  19. [19]

      INAUDI P, ABOLLINO O, ARGENZIANO M, MALANDRINO M, GUIOT C, BERTINETTI S, FAVILLI L, GIACOMINO A. Advancements in portable voltammetry: A promising approach for iron speciation analysis[J]. Molecules, 2023, 28(21):  7404. doi: 10.3390/molecules28217404

  20. [20]

      QUE E L, DOMAILLE D W, CHANG C J. Metals in neurobiology: Probing their chemistry and biology with molecular imaging[J]. Chem. Rev., 2008, 108(5):  1517-1549. doi: 10.1021/cr078203u

  21. [21]

      WU Z T, GUO Y, JIANG W W, YANG Y Q, WEI P, YI T. Recent process in organic small molecular fluorescent probes for tracking markers of tumor redox balance[J]. Trac‒Trends Anal. Chem., 2024, 170:  117461. doi: 10.1016/j.trac.2023.117461

  22. [22]

      WEN Y, JING N, ZHANG M, HUO F J, LI Z Y, YIN C X. A space-dependent 'enzyme-substrate' type probe based on 'carboxylesterase-amide group' for ultrafast fluorescent imaging orthotopic hepatocellular carcinoma[J]. Adv. Sci., 2023, 10(8):  2206681. doi: 10.1002/advs.202206681

  23. [23]

      WEN Y, LONG Z Q, HUO F J, YIN C X. Novel strategy for accurate tumor labeling: Endogenous metabolic imaging through metabolic probes[J]. Sci. China Chem., 2022, 65(12):  2517-2527. doi: 10.1007/s11426-022-1372-y

  24. [24]

      ZHANG Z X, TAN W J, ZHANG R N, GUAN S W, MIAO J, ZHANG M. A dendrimer consisting of a pyrene core and a 9-phenylcarbazole periphery as a multi-functional fluorescent probe for iodide, iron(Ⅲ)and mercury(Ⅱ)[J]. Microchim. Acta, 2019, 186(8):  586. doi: 10.1007/s00604-019-3661-9

  25. [25]

      SUN S H, HU W T, GAO H F, QI H L, DING L P. Luminescence of ferrocene-modified pyrene derivatives for turn-on sensing of Cu²⁺ and anions[J]. Spectroc. Acta Pt. A‒Molec. Biomolec. Spectr., 2017, 184:  30-37. doi: 10.1016/j.saa.2017.04.073

  26. [26]

      QIN Z H, SU W W, LIU P, MA J M, ZHANG Y R, JIAO T F. Facile preparation of a rhodamine B derivative-based fluorescent probe for visual detection of iron ions[J]. ACS Omega, 2021, 6(38):  25040-25048. doi: 10.1021/acsomega.1c04206

  27. [27]

      GAUTHAMA B U, NARAYANA B, SAROJINI B K, KODLADY S N, SANGAPPA Y, KUDVA A K, RAGHU S V. A versatile rhodamine B-derived fluorescent probe for selective copper(Ⅱ) sensing[J]. Inorg. Chem. Commun., 2022, 141:  109501. doi: 10.1016/j.inoche.2022.109501

  28. [28]

      ARON A T, LOEHR M O, BOGENA J, CHANG C J. An endoperoxide reactivity-based FRET probe for ratiometric fluorescence imaging of labile iron pools in living cells[J]. J. Am. Chem. Soc., 2016, 138(43):  14338-14346. doi: 10.1021/jacs.6b08016

  29. [29]

      CHEN B X, WANG L L, ZHAO Y F, NI Y, XIN C Q, ZHANG C W, LIU J H, GE J Y, LI L, HUANG W. Photocontrollable fluorogenic probes for visualizing near-membrane copper(Ⅱ) in live cells[J]. RSC Adv., 2017, 7(49):  31093-31099. doi: 10.1039/C7RA03559D

  30. [30]

      CHEN Y, LONG Z Q, WANG C C, ZHU J J, WANG S S, LIU Y, WEI P, YI T. A lysosome-targeted near-infrared fluorescent probe for cell imaging of Cu²⁺[J]. Dyes Pigment., 2022, 204:  110472. doi: 10.1016/j.dyepig.2022.110472

  31. [31]

      GOPALA L, CHA Y, LEE M H. Versatile naphthalimides: Their optical and biological behavior and applications from sensing to therapeutic purposes[J]. Dyes Pigment., 2022, 201:  110195. doi: 10.1016/j.dyepig.2022.110195

  32. [32]

      CHEVALIER A. The how and why of naphthalimide/heterocycle-fused hybrid dyes: An overview of the latest developments in the quest for dyes with innovative optical properties[J]. Org. Biomol. Chem., 2023, 21(37):  7498-7510. doi: 10.1039/D3OB01035J

  33. [33]

      DONG H Q, WEI T B, MA X Q, YANG Q Y, ZHANG Y F, SUN Y J, SHI B B, YAO H, ZHANG Y M, LIN Q. 1, 8-Naphthalimide-based fluorescent chemosensors: Recent advances and perspectives[J]. J. Mater. Chem. C, 2020, 8(39):  13501-13529. doi: 10.1039/D0TC03681A

  34. [34]

      KAUR G, SINGH I, TANDON N, TANDON R, BHAT A A. 1, 8-Naphthalimide-based chemosensors: A promising strategy for detection of metal ions in environmental and biological systems[J]. ChemistrySelect, 2023, 8(44):  e202301661. doi: 10.1002/slct.202301661

  35. [35]

      HAN C, SUN S B, JI X, WANG J Y. Recent advances in 1, 8-naphthalimide-based responsive small-molecule fluorescent probes with a modified C4 position for the detection of biomolecules[J]. Trac‒Trends Anal. Chem., 2023, 167:  117242. doi: 10.1016/j.trac.2023.117242

  36. [36]

      SARAVANAN A, SUBASHINI G, SHYAMSIVAPPAN S, SURESH T, KADIRVELU K, BHUVANESH N, NANDHAKUMAR R, MOHAN P S. A selective fluorescence chemosensor: Pyrene motif Schiff base derivative for detection of Cu²⁺ ions in living cells[J]. J. Photochem. Photobiol. A‒Chem., 2018, 364:  424-432. doi: 10.1016/j.jphotochem.2018.06.021

  37. [37]

      WEI J H, SUN H, JIANG Y, MIAO B X, HAN X E, ZHAO Y, NI Z H. A novel 1, 8-naphthalimide-based Cu²⁺ ion fluorescent probe and its bioimaging application[J]. Spectroc. Acta Pt. A‒Molec. Biomolec. Spectr., 2021, 261:  120037. doi: 10.1016/j.saa.2021.120037

  38. [38]

      LI X Y, GUO Y F, XU T T, FANG M, XU Q W, ZHANG F, WU Z Y, LI C, ZHU W J. A highly sensitive naphthalimide-based fluorescent probe for detection of Cu²⁺ via selective hydrolysis reaction and its application in practical samples[J]. J. Chin. Chem. Soc., 2020, 67(6):  1070-1077. doi: 10.1002/jccs.201900315

  39. [39]

      DALBERA S, KULOVI S, DALAI S. Pyrene-based Schiff base as selective chemosensor for copper(Ⅱ) and sulfide ions[J]. ChemistrySelect, 2018, 3(23):  6561-6569. doi: 10.1002/slct.201801205

  40. [40]

      WANG H, CUI J J, FANG X H, ZHANG W B, WANG J J, CHEN S Y, QIAN J H. Fluorescent detection of copper ions with acylhydrazine-based probes: Effects of substitute and its position[J]. Dyes Pigment., 2022, 197:  109954. doi: 10.1016/j.dyepig.2021.109954

  41. [41]

      MENG Z Y, WANG Z L, LIANG Y Y, ZHOU G C, LI X Y, XU X, YANG Y Q, WANG S F. A naphthalimide functionalized chitosan-based fluorescent probe for specific detection and efficient adsorption of Cu²⁺[J]. Int. J. Biol. Macromol., 2023, 239:  124261. doi: 10.1016/j.ijbiomac.2023.124261

  42. [42]

      PARK S Y, KIM W, PARK S H, HAN J, LEE J, KANG C, LEE M H. An endoplasmic reticulum-selective ratiometric fluorescent probe for imaging a copper pool[J]. Chem. Commun., 2017, 53(32):  4457-4460. doi: 10.1039/C7CC01430A

  43. [43]

      WANG S C, SHENG Z H, YANG Z G, HU D H, LONG X J, FENG G, LIU Y B, YUAN Z, ZHANG J J, ZHENG H R, ZHANG X J. Activatable small-molecule photoacoustic probes that cross the blood-brain barrier for visualization of copper(Ⅱ) in mice with Alzheimer's disease[J]. Angew. Chem.‒Int. Edit., 2019, 58(36):  12415-12419. doi: 10.1002/anie.201904047

  44. [44]

      WEI Y J, WANG N N, LI D L, WANG G, HE Y. Study on the fluorescence modulation of benzimidazole through energy transfer and photochromic isomerization in the pillar(5)arene-based supermolecular system[J]. React. Funct. Polym., 2019, 144:  104351. doi: 10.1016/j.reactfunctpolym.2019.104351

  45. [45]

      FRISCH M J, TRUCKS G W, SCHLEGEL H B, SCUSERIA G E, ROBB M A, CHEESEMAN J R, SCALMANI G, BARONE V, PETERSSON G A, NAKATSUJI H, LI X, CARICATO M, MARENICH A V, BLOINO J, JANESKO B G, GOMPERTS R, MENNUCCI B, HRATCHIAN H P, ORTIZ J V, IZMAYLOV A F, SONNENBERG J L, WILLIAMS-YOUNG D, DING F, LIPPARINI F, EGIDI F, GOINGS J, PENG B, PETRONE A, HENDERSON T, RANASINGHE D, ZAKRZEWSKI V G, GAO J, REGA N, ZHENG G, LIANG W, HADA M, EHARA M, TOYOTA K, FUKUDA R, HASEGAWA J, ISHIDA M, NAKAJIMA T, HONDA Y, KITAO O, NAKAI H, VREVEN T, THROSSELL K, MONTGOMERY JR. J A, PERALTA J E, OGLIARO F, BEARPARK M J, HEYD J J, BROTHERS E N, KUDIN K N, STAROVEROV V N, KEITH T A, KOBAYASHI R, NORMAND J, RAGHAVACHARI K, RENDELL A P, BURANT J C, IYENGAR S S, TOMASI J, COSSI M, MILLAM J M, KLENE M, ADAMO C, CAMMI R, OCHTERSKI J W, MARTIN R L, MOROKUMA K, FARKAS O, FORESMAN J B, FOX D J. Gaussian 16, Revision C. 01[CP]. Gaussian, Inc., Wallingford CT, 2016.

  46. [46]

      SAID A I, STANEVA D, ANGELOVA S, GRABCHEV I. A multi-channel rhodamine-pyrazole based chemosensor for sensing ph, Cu²⁺, CN^- and Ba²⁺ and its function as a digital comparator[J]. J. Photochem. Photobiol. A‒Chem., 2022, 433:  114218. doi: 10.1016/j.jphotochem.2022.114218

• 

  Scheme 1  Synthetic route of DHU-NP-4

  下载: 全尺寸图片 幻灯片
  

  Figure 1  (a) Chemical structure of as-synthesized compounds; (b-e) Absorbance spectra of the compounds (10 μmol·L^-1, containing 0.2% DMF as co-solvent) in PBS

  下载: 全尺寸图片 幻灯片
  

  Figure 2  (a) Fluorescent intensity of as-synthesized compounds (10 μmol·L^-1) at 556 nm in the presence of 50 μmol·L^-1 Cu²⁺; (b) LC-MS analysis of mixture of DHU-NP-4 (4 μmol·L^-1) and Cu²⁺ (6 μmol·L^-1) compared with DHU-NP-3 (4 μmol·L^-1) standard peak; (c) Proposed reaction mechanism of DHU-NP-4 towards Cu²⁺

  下载: 全尺寸图片 幻灯片
  

  Figure 3  (a) Fluorescent intensity of DHU-NP-4 (10 μmol·L^-1) in different solvents at 556 nm after treated with Cu²⁺ and Fe³⁺ (50 μmol·L^-1); (b) Fluorescent intensity of DHU-NP-4 (10 μmol·L^-1) after treated with Cu²⁺ and Fe³⁺ (50 μmol·L^-1) in PBS at different pH values; (c) Proposed mechanism of DHU-NP-4 towards Fe³⁺ in acid condition; (d) Fluorescent intensity of DHU-NP-4 (10 μmol·L^-1) after treated with Cu²⁺ and Fe³⁺ (50 μmol·L^-1) in PB at different pH values; (e) Fluorescent intensity of DHU-NP-4 (10 μmol·L^-1) after treated with Cu²⁺ and Fe³⁺ (50 μmol·L^-1) in NaCl solution with different concentrations

  下载: 全尺寸图片 幻灯片
  

  Figure 4  (a-c) Fluorescent intensity of DHU-NP-4 (10 μmol·L^-1) at 556 nm after treated with different cations (50 μmol·L^-1) at different pH values; (d) Fluorescent intensity of DHU-NP-4 (10 μmol·L^-1) at 556 nm after treated with different concentrations of Cu²⁺ (0, 0.2, 0.4, 0.8, 1.2, 1.6 μmol·L^-1)

  From A to R: blank, Al³⁺, Ca²⁺, Cd²⁺, Co²⁺, Cu²⁺, Fe²⁺, Fe³⁺, Hg²⁺, K⁺, Mg²⁺, Na⁺, NH⁴⁺, Ni²⁺, Pb²⁺, Sn²⁺, Ti⁴⁺, Zn²⁺.

  下载: 全尺寸图片 幻灯片

  Table 1.  Recovery of Cu²⁺ and Fe³⁺ tested by DHU-NP-4 in PBS

  Ion	Concentration of ion / (μmol·L-1)		Recovery / %
  	Added	Found
  Cu2+	0.45	0.431 9±0.006 9	95.56
  	1.05	1.051 5±0.001 0	100.14
  	1.55	1.554 3±0.013 1	100.28
  Fe3+	5	4.94±0.04	98.9
  	30	30.99±0.40	103.3
  	55	55.92±0.54	101.6

  下载: 导出CSV
•