A novel hydrophilic fluorescent probe for Cu2+ detection and imaging in HeLa cells

Copper is an essential element in living systems and plays an important role in human physiology; therefore, methods to detect the concentration of copper ions in living organisms are important. Herein, we report a highly water-soluble naphthalimide-based fluorescent probe that can be used for the detection of Cu2+. The probe, BNQ, has high selectivity and sensitivity. The fluorescence intensity of the probe at 520 nm was visible to the naked eye under a UV lamp; upon the gradual addition of Cu2+, there was a colour change from green to nearly colourless. Furthermore, the detection limit of BNQ for Cu2+ was 45.5 nM. The detection mechanism was investigated using a Job's plot and density functional theory (DFT) calculations. In addition, owing to great biocompatibility, we were able to successfully use BNQ to detect Cu2+ in living HeLa cells with low toxicity.


Introduction
2][3][4][5][6] The average concentration of copper in human blood is 100-150 mg dL À1 (ref.46) and the recommended intake of copper for humans is 0.9 mg each day. 7xcessive or inadequate intake of copper can cause a variety of diseases. 8,9Copper deciency leads to Menkes disease, 10,11 haematological neurological sequelae, and complications caused by malabsorption, including macrocytic anaemia, myelopathy, chronic diarrhoea, celiac disease, and inammatory bowel disease (IBD). 6,126][17] The US Environmental Protection Agency stipulates that the maximum concentration of copper in drinking water should be 1.3 mg L À1 ($1 ppm). 180][21][22][23] It has been reported that copper from drinking water and food can increase its concentration in human blood and accumulate in the kidney, cornea, and brain, which can lead to disease. 24For this reason, it is necessary to develop a test method to detect the concentration of copper ions in living organisms.
5][26] These methods require complicated preparation procedures, expensive instruments and trained professional technicians. 273][34][35][36][37][38] However, there are many unavoidable drawbacks for the probes that have been reported so far, such as weak hydrophilia, tedious synthetic steps, stringent applied conditions, and the likelihood of interference from Hg 2+ , Al 3+ , or Zn 2+ .Therefore, more research is required to develop uorescent probes with strong hydrophilia, good biocompatibility, low toxicity, high anti-interference ability, and easy preparation.
Herein, we report the design and synthesis of a new uorescent probe (E)-2-butyl-6-hydroxy-5-((quinolin-8-ylimino) methyl)-1H-benzo[de]isoquinoline-1,3(2H)-dione (BNQ), a naphthalimide Schiff base derivative, to be used for Cu 2+ detection.The BNQ probe exhibits high selectivity, high sensitivity, low toxicity, and strong hydrophilia, and was successfully used to image Cu 2+ in living cells.The detection mechanism was based on excited state double-proton transfer (ESDPT), which is not commonly used for Cu 2+ detection.This has been conrmed with Job's plot analysis and DFT studies.This study may provide a feasible plan for the specic detection of Cu 2+ in biomedicine and biological pathology.
1 H NMR and 13 C NMR spectra were recorded on a Bruker AVANCE III 400 spectrometer ( 1 H, 400 MHz; 13 C, 101 MHz) in CDCl 3 or DMSO-d 6 .Fluorescence spectra were obtained on a Hitachi F-4500 uorescence spectrophotometer (Japan) equipped with a 1 cm quartz cell, and UV-vis spectra were measured using a Hitachi U-3010 spectrometer.Mass spectra were recorded on an Agilent 1290-micr OTOF Q II mass spectrometer (US).The pH of the solution was determined using a Mettler-Toledo instrument.FT-IR spectrum was taken on a Varian Scimitar 1000 spectrometer.All measurements were performed at approximately room temperature.

Synthesis of probe BNQ
The synthesis of the probe is shown in Scheme 1.

Cell culture
The HeLa cells were cultured in DMEM medium containing 10% fetal bovine serum at 37 C in a 95% humidity under a 5% CO 2 environment.Approximately 24 h later, the cells adhered to the surface of the dish.The cells that exhibited the logarithmic growth phase were selected for the following experiments.

Cytotoxicity of BNQ probe
To evaluate the cytotoxicity of the BNQ probe, the cell viability of the HeLa cells was investigated using the standard MTT (3-(4,5dimethyl-2-thiazolyl)-2,5-diphenyl-tetrazolium bromide) assay.The HeLa cells were incubated with 0, 0.0625, 0.125, 0.25, 0.5, and 1 mM of the BNQ probe for 24 h at 37 C. Subsequently, 10 mL MTT (5 mg mL À1 ) prepared in HEPES was added and coincubated for another 4 h.We used the enzyme labeller to read the OD value at 490 nm and calculated the cell survival rate according to the following equation: Cell viability (%) ¼ OD 490 (sample)/OD 490 (control) Â 100%

Cell imaging of BNQ probe
For cell imaging, Hela cells were seeded in 24-well plates (2 Â 10 5 cells per well) and allowed to adhere for 24 h.The cells were incubated with 1 mM probe for 30 min, and then treated with 10 mM Cu 2+ for another 30 min.Confocal microscopic imaging was

Effect of pH
The effect of pH on the emission intensity of BNQ and BNQ-Cu 2+ was measured (Fig. 1).When the pH was lower than 4, the uorescence intensity of the BNQ probe was minimal, which may be due to the reaction of hydrogen ions in the solution with the nitrogen on the probe.The uorescence intensity increased gradually from pH 6.0 to 8.0 and then rapidly increased from pH 8.0 to 10.0.Aer the addition of Cu 2+ , the uorescence intensity decreased gradually from pH 4.0 to 12.0, possibly due to the reaction of the hydroxide ions in the solution with the phenolic hydroxyl group.However, the uorescence intensity remained high when the pH was greater than 9.0 because of the reaction of the hydroxide with Cu 2+ to Cu(OH) 2 .Therefore, the pH range 6.0-8.0 was considered optimal, because the uorescence of the BNQ probe was stable, and there was a signicant change in uorescence upon adding Cu 2+ in this range.pH ¼ 6.0 was selected as the test condition of this experiment because the uorescence reduction was most signicant.

Probe response time to Cu 2+
Subsequently, time-dependent uorescence intensity studies were carried out for Cu 2+ (10 mM) determination by BNQ (5 mM) in DMSO/Tris (1/9, v/v) (Fig. 2).Aer adding Cu 2+ , the uorescence intensity of BNQ dropped sharply within the rst 2 min and then reached saturation, which indicated that the probe reacted with Cu 2+ completely in 2 min.

Fluorescent response of BNQ probe in the presence of Cu 2+
We carried out UV-vis absorption studies on the BNQ probe in the presence of Cu 2+ (5 mM, DMSO/HEPES ¼ 1/9, v/v, pH ¼ 6.0).With increasing Cu 2+ in the solution, the absorption peak between 300 nm and 500 nm gradually increased (Fig. S6 †).The UV-vis absorption remained constant aer 1.5 equivalents Cu 2+ were added to the solution.Along with the spectral changes, a notable colour change from bright yellow to colourless was easily observed by the naked eye (Fig. S8 †).
To further understand the sensing properties of the BNQ probe for Cu 2+ detection, uorescence titration was performed by increasing the concentration of Cu 2+ in a DMSO/HEPES (1/9, v/v) solution.As shown in Fig. 3a, with the increase in Cu 2+ , the uorescence decreased gradually from 520 nm and reached the minimum when 1.5 equivalents of Cu 2+ were added.The colour changed from green to colourless under a UV lamp, which indicated that Cu 2+ can be detected because of the complexation of phenolic hydroxyl and imide structures on the BNQ probe with copper ions.The regression equation y ¼ 0.78635-0.0925xwas obtained from the uorescence titration data, and a good linear relationship (R 2 ¼ 0.99297) between the uorescence intensity and Cu 2+ concentration was observed (Fig. 3b).The detection limit of the BNQ probe was calculated to be 45.5 nM based on the following equation: 40 DL ¼ KS bl /S where K ¼ 3; S bl is the standard deviation of the blank solution; and S is the slope of the calibration curve.The limit of detection (LOD) for Cu 2+ was calculated to be 45.5 nM, which is signicantly lower than that for Cu 2+ limit (20.5 mM) set by the World Health Organization. 41g. 1 Fluorescence intensity of BNQ (5 mM) with and without Cu 2+ in solutions of different pH.Test condition: Cu 2+ (10 mM), DMSO/HEPES (1/9, v/v), l ex ¼ 390 nm, slits: 2.5/2.5.

Fluorescence response to various metal ions
As shown in Fig. 4, we also performed selectivity experiments and anti-interference ability experiments for the BNQ probe.A high uorescence intensity of the BNQ probe (5 mM) at 520 nm was detected immediately before the addition of any metal ions, and the uorescence spectrum did not change signicantly even aer the addition of 15 equivalents of other metal ions (Ni 2+ , Mn 2+ , Mg 2+ , Fe 2+ , Pb 2+ , Ca 2+ , Ba 2+ , Zn 2+ , Cd 2+ , Hg 2+ , Sn 4+ , Cr 3+ , Fe 3+ , Al 3+ , Na + and K + ) (Fig. S9 and S10a †), However, aer adding Cu 2+ (1.5 equivalents) to the mixed solutions of the BNQ probe (5 mM) and 15 equivalents of every interference metal ion mentioned above, the uorescence intensity decreased signicantly, changing from green to colourless when observed under the UV lamp (Fig. S10b †).The results demonstrated that the BNQ probe can detect Cu 2+ with excellent selectivity and antiinterference ability.

Possible mechanism
The stoichiometry of the obtained BNQ-Cu 2+ complex was determined to be 2 : 1 (BNQ : Cu 2+ ) using Job's method and the Benesi-Hildebrand method (Fig. S11 and S12 †).And according to previous literature reports, 42,43 Cu 2+ is a powerful uorescence emission quencher due to the paramagnetism.In addition, C]O and C]N groups can be involved in complexation with Cu 2+ .Thus, we hypothesised that the reaction mechanism would proceed as follows (Scheme 2): an unstable seven-membered ring forms Subsequently, the steric hindrance of the molecule decreases and excited state intramolecular proton transfer (ESIPT) occurs.The hydrogen transfers from the phenolic hydroxyl group to the quinoline nitrogen.These changes disrupt the conjugated structure of naphthalimide, leading to uorescence quenching.In addition, the geometric conguration of the BNQ probe implies that it more easily coordinates with Cu 2+ rather than the other ions due to the different ionic radius.To further study the effect of nitrogen in quinoline on the reaction mechanism, BNQ2 was synthesised and its anti-interference ability was studied.The results (Fig. S13 †) showed that the absence of nitrogen on the naphthalene ring resulted in lower selectivity of the probe.

Theoretical calculation
Density functional theory (DFT) calculations were performed using the Gaussian 16 program 44,45 to understand the complexation structures and electronic properties of BNQ + Cu 2+ (Fig. 5).The DFT optimisation of each structure was conducted with the CAM-B3LYP functional and 6-31G(d)/Lanl2dz basis sets.For Cu, we used the LANL2DZ basis set, and treated all other atoms using the 6-31G(d) basis set.As shown in Fig. 5a, we suggest that there are four binding sites for each dimer, which involve imine N and hydroxyl O atoms coordinated to one Cu 2+ ion.The hydrogens from the hydroxyl phenols were transferred to nitrous oxide by intramolecular Hbonding simultaneously.The highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital

Biological research
Before cell imaging, the cytotoxicity of BNQ was investigated to ensure safe application in living organisms.As shown in Fig. S15, † the cell viability was maintained above 90% aer incubation with different concentrations of BNQ/Cu 2+ /BNQ + Cu 2+ at 37 C for 4 h (DMSO/HEPES ¼ 1/9, v/v, pH ¼ 6.0), indicating that the BNQ probe has good biocompatibility and low cytotoxicity in cells.Subsequently, we further examined the ability of the probe to monitor Cu 2+ in HeLa cells.As shown in Fig. 6, a strong green uorescence could be observed when HeLa cells were only treated with 1 mM of the BNQ probe.The uorescence disappeared aer subsequent treatment with 10 mM of Cu 2+ for another 30 min, indicating that the BNQ probe can monitor Cu 2+ in living cells with excellent membrane permeability.

Comparison with previous probes
3][34][35][36][37][38] As shown in Table 1, the probe BNQ not only has a relatively lower detection limit, but also can detect Cu 2+ with shorter time and better water solubility.Therefore, the probe BNQ is a promising candidate to detect Cu 2+ in vivo and in vitro.In addition, the probe BNQ detects Cu 2+ using a rather rare mechanism of ESDPT.

Conclusions
In summary, we have designed and synthesised a naphthalimide-derived uorescence probe with high selectivity, excellent hydrophilia, and a quick response time, to be used for the detection of Cu 2+ .The detection limit of the BNQ probe was 45.5 nM.The mechanism is based on the ESDPT reaction of naphthalimide in which hydrogen transfers from the phenolic hydroxyl group to the quinoline nitrogen twice aer reaction with Cu 2+ .As a result, the uorescence of the BNQ probe was quenched.During detection, our hypothesised BNQ-Cu 2+ -BNQ model was conrmed by Job's plot analysis and DFT studies.In addition, the BNQ probe was successfully used to image Cu 2+ in living cells, demonstrating its potential as a tool for monitoring trace Cu 2+ in biological and pathological processes.

Scheme 1
Scheme 1 Synthesis of probes BNQ and BNQ2.

Table 1
Comparison of the present work with other previous works for Cu 2+ detection Hela cells ESDPT This work Building Projects of Jilin Province (No. 2019C007), the Department of Education of Jilin Province (No. JJKH20190200KJ), the National Natural Science Foundation of China (No. 21174052), Natural Science Foundation of Jilin Province of China (No. 20170101105JC), and Department of Science and Technology of Jilin Province (No. 20180201085GX).