Development of a Fluorescein-Based Probe with an “Off–On” Mechanism for Selective Detection of Copper (II) Ions and Its Application in Imaging of Living Cells

Copper is a common metallic element that plays an extremely essential role in the physiological activities of living organisms. The slightest change in copper levels in the human body can trigger various diseases. Therefore, it is important to accurately and efficiently monitor copper ion levels in the human body. Recent studies have shown that fluorescent probes have obvious advantages in bioimaging and Cu2+ detection. Therefore, a novel Cu2+ probe (N2) was designed and synthesized from fluorescein, hydrazine hydrate and 5-p-nitrophenylfurfural that is sensitive to and can detect Cu2+ within 100 s. The response mechanism of the N2 probe to Cu2+ was studied by several methods such as Job’s plots and MS analysis, which showed that the Cu2+ and the N2 probe were coordinated in a complexation ratio of 1:1. In addition, compared with other cations investigated in this study, the N2 probe showed excellent selectivity and sensitivity to Cu2+, exhibiting distinct fluorescence absorption at 525 nm. Furthermore, in the equivalent range of 0.1–1.5, there is a good linear relationship between Cu2+ concentration and fluorescence intensity, and the detection limit is 0.10 μM. It is worth mentioning that the reversible reaction between the N2 probe and Cu2+, as well as the good biocompatibility shown by the probe in bioimaging, make it a promising candidate for Cu2+ biosensor applications.


Introduction
Copper is commonly found in various organisms and is one of the more abundant transition metals in the human body [1,2]. Compared with other transition metals, Cu has a strong stability constant of binding ligands, which makes it an ideal enzyme cofactor [3,4]. Cu can enter the human body in a variety of ways, such as through inhalation, diet and environmental intake [5]. According to some studies, Cu 2+ solutions and metallic Cu can effectively inhibit the growth of viruses and even inactivate them [6,7]. However, for human body, systematic Cu content must be intermediate at a relatively fixed level [8]. Changes in Cu levels can contribute to anemia, coronary heart disease, Wilson's disease and Alzheimer's disease and can also trigger cell carcinogenesis and apoptosis [3,[8][9][10]. Levels of Cu have been reported to be a sign of cancers, such as breast cancer, which can be measured by Cu 2+ levels, and prostate tumor proliferation, which can be inhibited by reducing the uptake of Cu [11]. Therefore, accurate detection of the content of Cu 2+ in organisms is required.
In this study, we designed and synthesized a novel fluorescein derivative of Cu 2+ fluorescence, the N2 probe. It is worth noting that this probe has a unique ability to recognize Cu 2+ in multi-ion coexistence solutions and shows a very intense fluorescence absorption peak at 525 nm, unlike other ions that are almost unchanged. Compared with other probes reported in the published literature [25,[39][40][41][42][43], this probe has a relatively low detection limit and a stabilized fluorescence response at pH 6.0-9.0. In addition, through the discussion of the response mechanism between Cu 2+ and the probe, it was revealed that Cu 2+ enabled a certain reversibility of the recognition effect of the N2 probe by opening the lactam ring of the N2 probe and complexing with it in a 1:1 coordination ratio ( Figure 1). Furthermore, cytotoxicity testing showed that the N2 probe has good biocompatibility. Bioimaging showed that N2 probe can be used as an intracellular Cu 2+ tracer sensing material.
In this study, we designed and synthesized a novel fluorescein derivative of Cu 2+ fluorescence, the N2 probe. It is worth noting that this probe has a unique ability to recognize Cu 2+ in multi-ion coexistence solutions and shows a very intense fluorescence absorption peak at 525 nm, unlike other ions that are almost unchanged. Compared with other probes reported in the published literature [25,[39][40][41][42][43], this probe has a relatively low detection limit and a stabilized fluorescence response at pH 6.0-9.0. In addition, through the discussion of the response mechanism between Cu 2+ and the probe, it was revealed that Cu 2+ enabled a certain reversibility of the recognition effect of the N2 probe by opening the lactam ring of the N2 probe and complexing with it in a 1:1 coordination ratio ( Figure 1). Furthermore, cytotoxicity testing showed that the N2 probe has good biocompatibility. Bioimaging showed that N2 probe can be used as an intracellular Cu 2+ tracer sensing material.

Chemical Reagents
Hydrochloric acid, copper sulfate, sodium hydrate, hydrazine hydrate, fluorescein, ethanol and 5-p-nitrophenyl furfural were purchased from Aladdin Reagent Co., Ltd. (Shanghai, China). All such chemical reagents were used with no additional purification.

Chemical Reagents
Hydrochloric acid, copper sulfate, sodium hydrate, hydrazine hydrate, fluorescein, ethanol and 5-p-nitrophenyl furfural were purchased from Aladdin Reagent Co., Ltd. (Shanghai, China). All such chemical reagents were used with no additional purification.

Apparatus and Instrumentation
ZF-C-type UV-vis spectrophotometer, a Bruker Tensor 27 spectrometer, a Hitachi F-4500 fluorescence spectrophotometer, a Bruker micro TOF-Q II ESI-TOF LC/MS/MS spectrometer, a Varian INOVA-400 MHz spectrometer (400 MHz), a Spectra max190 molecular devices and an Olympus FV1000 confocal microscope were used in this research.

The Synthesis of N2
According to the literature [44], the synthesis process scheme was exhibited in Scheme S1. Fluorescein hydrazine was synthesized from fluorescein and hydrazine hydrate. First 6.00 g (18.05 mmol) of fluorescein was added to a 250 mL three-necked round-bottom flask and dissolve it by adding 110 mL of anhydrous ethanol. Then, 8.0 mL of hydrazine hydrate was slowly added to the solution over the course of about 30 min. The temperature of the reaction was gradually raised to 80 • C, and the reaction solution was refluxed for 12 h. After the reaction was finished, the solution was cooled to room temperature, and the reaction solvent was evaporated under reduced pressure. Then, 500 mL of water was added, and the pH was adjusted to 4-5 with concentrated hydrochloric acid. The pH of the system was continuously adjusted to 9-10 with sodium hydroxide. The solid was filtered under reduced pressure, washed 3 times with distilled water and dried to obtain 5.88 g of light-yellow solid.
Then, 80 mL of fluorescein hydrazine (3.59 g, 10.36 mmol) dissolved in anhydrous ethanol and 50 mL of 5-p-nitrophenyl furfural (1.50 g, 6.90 mmol) dissolved in anhydrous ethanol were added to a 250 mL round-bottom flask. The solution was heated to 78 • C for reaction, and TLC, ethyl acetate and petroleum ether (v/v = 5/3) were used to monitor the reaction process. The reaction solution was refluxed for 3 h, and a large amount of solid was precipitated from the bottom of the bottle. After filtration under reduced pressure, a solid was obtained, which was washed with mother liquor 3 times and recrystallized with anhydrous ethanol. The crystallized solid was dried, and 3.65 g of an orange solid was obtained with a yield of 96.88% and a melting point of 243-245 • C. 1 13

Colorimetric Determination of Copper Ions
To facilitate the titration experiments, the N2 probe, deionized water and EtOH were prepared as a 1 mM master mix. In the titration experiment, a set concentration gradient of Cu 2+ was added to a 10 mL colorimetric tube containing 1.0 mL of 200 µM N2 probe master mix. Then, it was fixed to 10 mL with PBS solution. For the interference assay, 20 µM Cu 2+ and 1.0 mL of 200 µM N2 probe master mix were mixed with 1.0 mL of the test substance, which was two equivalents of the probe N2, and the volume was fixated with PBS to 10 mL in a colorimetric tube. In the ethylenediamine titration context, 1.0 mL of 200 µM Cu 2+ , 1.0 mL of 200 µM N2 probe master mix and different amounts of ethylenediamine were added to a 10 mL colorimetric tube with PBS. Spectroscopic analysis was performed using a 1 cm cuvette. In various tests, absorbance at 440 nm and fluorescence intensity at 525 nm were recorded separately.

Detection Limit of the Probe
The detection limits were calculated based on the measured fluorescence signals. In this study, the luminescence intensity of N2 (20.0 µM) was multiplied by 10 to determine the ratio of δ/S and the standard deviation of the blank assay. Under this condition, there was a good linear relationship between the relative luminescence intensity (525 nm) and the concentration of Cu 2+ in the range of 10.0-40.0 µM. The detection limit was determined by the following equation: detection limit = K × δ/S, where S is the gradient of the concentration and the intensity of the sample, and δ is the standard deviation of the blank determination. Fluorescence analysis showed: y = 124.41x + 111.28 (R 2 = 0.983), δ = 4.147 (N = 10), S = 124.41, K = 3; LOD = 3 × 4.147/124.41 = 0.10 µM.

Cytotoxicity Study
The CCK-8 method was carried out to analyze cytotoxicity. Different concentrations of probes (0 µM, 2.5 µM, 5 µM, 10 µM, 20 µM and 40 µM) were added into the cells, which had been cultured at 37 • C in 96-well plates for 24 h. The absorbance of the cells was measured at 450 nm, combined with CCK-8 and incubated for two hours. The above experiments were repeated three times, and the results of cytotoxicity were presented as a percentage of control cells.

Cell Culture Experiment and Cell Imaging
MCF-7 cells were digested in trypsin containing 0.25% EDTA. When a tendency of rounding and floating was observed under the microscope, DMEM complete medium (89% DMEM medium, 10% FBS, 1% penicillin-streptomycin) was added to terminate the digestion, and the supernatant was centrifuged and separated on a centrifuge. Then, DMEM complete medium was added to afford a given concentration of cell suspension, divided equally into three confocal dishes and incubated in a 5% CO 2 incubator at 37 • C for 24 h. Then, 1 mL of PBS buffer was added, and confocal microscopic imaging was performed. Then, the configured probe solution was added and incubated in the incubator for 20 min, at which time the concentration of the fluorescent probe in the confocal Petri dishes was about 40 µmol/L. After washing three times with PBS buffer, imaging was performed under confocal microscopy in a wavelength channel of 488 nm. The cells were then incubated in a copper ion solution at a concentration of approximately 40 µmol/L for 20 min, washed three times with PBS and imaged. Fluorescence field and bright-field images were acquired separately and superimposed.

Effect of pH and Response Time
The influence of pH on N2 and N2 towards Cu 2+ was evaluated in PBS buffer (10 Mm, PH = 7.4)/EtOH (1:1, v/v) (Figure 2A). It is apparent that the N2 probe has a relatively stable fluorescence response to Cu 2+ in the pH range of 6.0 to 9.0. Hence, it can be adapted for bioimaging experiments at pH 7.4. The response times were examined at 525 nm ( Figure 2B). After the addition of Cu 2+ (20.0 µM), the fluorescence intensity at 525 nm intensified and reached a smoothed level after 100 s, which means that Cu 2+ could be promptly detected by the N2 probe.

Qualitative and Quantitative Studies
Cu 2+ with different molar ratios (0-100 μM) was added to the equimolar N2 probe (5 μM) solution configured with PBS buffer (10 mM, PH = 7.4)/EtOH (1:1, v/v). Figure 4A shows that the fluorescence intensity at 525 nm increased with increased Cu 2+ concentration. When the Cu 2+ concentration reached 2.4 eq, the fluorescence intensity reached the maximum and no longer increased. There is a good linear relationship between the fluorescence intensity of the N2 probe in response to Cu 2+ and the concentration of Cu 2+ in the range of 0.1-1.5 eq ( Figure 4B). The LOD of the N2 probe for Cu 2+ was calculated as

Qualitative and Quantitative Studies
Cu 2+ with different molar ratios (0-100 µM) was added to the equimolar N2 probe (5 µM) solution configured with PBS buffer (10 mM, PH = 7.4)/EtOH (1:1, v/v). Figure 4A shows that the fluorescence intensity at 525 nm increased with increased Cu 2+ concentration. When the Cu 2+ concentration reached 2.4 eq, the fluorescence intensity reached the maximum and no longer increased. There is a good linear relationship between the fluorescence intensity of the N2 probe in response to Cu 2+ and the concentration of Cu 2+ in the range of 0.1-1.5 eq ( Figure 4B). The LOD of the N2 probe for Cu 2+ was calculated as 0.10µmol/L using the formula LOD = 3σ/K which implies that the N2 probe has good sensitivity for detection of Cu 2+ . , x FOR PEER REVIEW 6 of 11 0.10μmol/L using the formula LOD = 3σ/K which implies that the N2 probe has good sensitivity for detection of Cu 2+ .

Proposed Sensing Mechanism
Methods such as Job's plots and MS analysis were applied to further investigate the response mechanism of the N2 probe to Cu 2+ . The reversible complex reaction of the probe

Proposed Sensing Mechanism
Methods such as Job's plots and MS analysis were applied to further investigate the response mechanism of the N2 probe to Cu 2+ . The reversible complex reaction of the probe to Cu 2+ was confirmed in an ethylenediamine titration experiment ( Figure 5A), with the complexation ratio of 1:1 shown in the Job's plots ( Figure 5B). In addition, the peak position of [C31H19CuO7N3 (M + H)] + at m/z 610.34 in the mass spectrum can be matched with the signal of the N2 probe and Cu 2+ , which is consistent with the 1:1 coordination mechanism mentioned earlier ( Figure 5C). This indicates that during the process of recognizing Cu 2+ , the amide bond broke, and a new Cu-O bond was formed. In summary, the complexation mechanism of the N2 probe in response to Cu 2+ can be described by Figure5D.

Cell Imaging
Based on the excellent properties of the probe, we investigated its bioimaging properties in cells. First, the cytotoxicity of the probe to MCF-7 cells was investigated by the CCK-8 method. MCF-7 cells were cultured in probe solutions of different concentrations (0-40 μM) for 24 h. As shown in Figure 6A and Table S1  In addition, the peak position of [C 31 H 19 CuO 7 N 3 (M + H)] + at m/z 610.34 in the mass spectrum can be matched with the signal of the N2 probe and Cu 2+ , which is consistent with the 1:1 coordination mechanism mentioned earlier ( Figure 5C). This indicates that during the process of recognizing Cu 2+ , the amide bond broke, and a new Cu-O bond was formed. In summary, the complexation mechanism of the N2 probe in response to Cu 2+ can be described by Figure 5D.

Cell Imaging
Based on the excellent properties of the probe, we investigated its bioimaging properties in cells. First, the cytotoxicity of the probe to MCF-7 cells was investigated by the CCK-8 method. MCF-7 cells were cultured in probe solutions of different concentrations (0-40 µM) for 24 h. As shown in Figure 6A and Table S1 in the Supplemetary Material, the probe had low cytotoxicity. Then, the bioimaging properties of the probe were examined. MCF-7 cells were cultured in the probe solution (40 µmol/L) for 20 min. No fluorescence was detected under confocal microscopy. After being treated with Cu 2+ (40 µM) at 37 • C for 20 min, the cells showed obvious green fluorescence under the excitation of 448 nm light. Figure 6 also shows that the probe stained the cells but did not enter the nuclei. This further indicates that the N2 probe has good biocompatibility and a tracing effect on intracellular Cu 2+ .
Biosensors 2023, 13, x FOR PEER REVIEW  8 of 11 for 20 min, the cells showed obvious green fluorescence under the excitation of 448 nm light. Figure 6 also shows that the probe stained the cells but did not enter the nuclei. This further indicates that the N2 probe has good biocompatibility and a tracing effect on intracellular Cu 2+ .

Conclusions
This study describes a novel fluorescent probe (N2), the superior selectivity for Cu 2+ of which makes it suitable for Cu 2+ detection applications. Analysis of data from ethylenediamine titration experiments and mass spectrometry also revealed that the probe complexes with Cu 2+ in a 1:1 coordination ratio. Moreover, the probe achieves a reversible fluorescence response to Cu 2+ via a switching ring, which provides a potential idea for the design of reusable probes. Cell imaging shows that the probe has good biocompatibility and that the fluorescence response of the probe to Cu 2+ is relatively stable at pH 6.0-9.0. In summary, the N2 probe can be used as a highly promising Cu 2+ sensor in biological samples.

Conclusions
This study describes a novel fluorescent probe (N2), the superior selectivity for Cu 2+ of which makes it suitable for Cu 2+ detection applications. Analysis of data from ethylenediamine titration experiments and mass spectrometry also revealed that the probe complexes with Cu 2+ in a 1:1 coordination ratio. Moreover, the probe achieves a reversible fluorescence response to Cu 2+ via a switching ring, which provides a potential idea for the design of reusable probes. Cell imaging shows that the probe has good biocompatibility and that the fluorescence response of the probe to Cu 2+ is relatively stable at pH 6.0-9.0. In summary, the N2 probe can be used as a highly promising Cu 2+ sensor in biological samples.