The Monitoring and Cell Imaging of Fe3+ Using a Chromone-Based Fluorescence Probe

A new structurally simple fluorescent CP probe based on chromone was designed and synthesized, and its structure was fully characterized using various analytical techniques. The CP probe displays a high selectivity and sensitivity for sensing Fe3+ with a “turn-off” fluorescence response over other metal ions in a DMSO/H2O (4:1, v/v) solution. The experiment results show that the CP probe is stable over a wide pH range of 2.0–12.0. The detection limit for Fe3+ was calculated to be 0.044 μmol•L−1. The molar ratio method indicated that the binding mode between the CP probe and Fe3+ is a 1:1 complex formation. HR-MS and density functional theory (DFT) calculations were also performed to further confirm the recognition mechanism. Both fluorescence imaging experiments and the MTT assay demonstrated that the CP probe was suitable for detecting intracellular Fe3+ and no significant cytotoxicity in living cells.


Introduction
Iron, as one of the essential microelements [1], is widespread in living organisms and plays crucial roles in a variety of fundamental biochemical processes, like oxygen metabolism, electron transfer, and enzyme catalysis [2][3][4].In the human body, most irons are mainly stored in Fe 3+ form [5]. When the Fe 3+ content is within the proper level range, the body can function normally.Either the deficiency or the excess of Fe 3+ could induce diseases such as hemochromatosis, heart failure, diabetes, and liver fibrosis [6][7][8][9].Thus, it is very important to design a good method for the determination of Fe 3+ .
Compared with traditional detection methods, such as spectrophotometry, mass spectrometry, and atomic absorption spectroscopy [10][11][12], a method based on a fluorescent probe has attracted increasing interest due to its simple operation, high selectivity, and fast response.Currently, various fluorescent probes based on different fluorophores, such as naphthalimide, quinazolinone, rhodamine, and BODIPY, have been developed for the detection of Fe 3+ [13][14][15][16][17][18].Nevertheless, most of them were easily disrupted by other transition metal ions (e.g., Cu 2+ , Al 3+ , etc.), resulting in poor selectivity [19][20][21].Therefore, it is still necessary to develop highly sensitive and selective Fe 3+ fluorescent probes.Due to their excellent spectroscopic properties, many chromone-based derivatives have also been used as fluorescent probes.For example, Bai et al. proposed a fluorescent probe based on a chromone-dendron Schiff base to detect Mg 2+ and Zn 2+ [22].Li et al. reported a chromone-derived probe for the monitoring of Al 3+ based on a photoinduced electrontransfer mechanism [23].A Cu 2+ chromone derivative was also developed by Liu et al. by means of fluorescence quenching [24].However, few chromone-based fluorescent probes were reported for the detection of Fe 3+ .Herein, we designed and synthesized a Schiff base fluorescent CP probe based on chromone with simple structure.The CP probe can be successfully applied to detect Fe 3+ with high sensitivity and good selectivity.Meanwhile, fluorescence microscopy experiments demonstrated that the CP probe can also be used to monitor Fe 3+ in living cells.

Results and Discussion
The general procedure for the synthesis of the CP probe is shown in Scheme 1. Intermediate compound 1 was prepared according to the reported method [25].The CP probe was fully characterized by 1 H NMR, 13 C NMR, IR, and HR-MS.
Molecules 2024, 29, x FOR PEER REVIEW 2 of 9 fluorescence microscopy experiments demonstrated that the CP probe can also be used to monitor Fe 3+ in living cells.

Results and Discussion
The general procedure for the synthesis of the CP probe is shown in Scheme 1. Intermediate compound 1 was prepared according to the reported method [25].The CP probe was fully characterized by 1 H NMR, 13 C NMR, IR, and HR-MS.Scheme 1. Synthesis route for the CP probe.

Characterization of the CP Probe
The UV/visible absorption and emission spectra of the CP probe in DMSO/H2O mixed solvent is investigated, as shown in Figure 1a.The absorption maximum of CP probe is centered at approximately 345 nm due to molecular skeleton group π→π* transition.Exciting at 345 nm in the CP probe provides an emission band at 439 nm.Meanwhile, we also studied the effect of pH on the fluorescence of the CP probe (Figure 1b).In the pH range of 2.0-12.0,there was no significant change in fluorescence intensity, which demonstrated that it is insusceptible to the change of acid-base solution.

Fluorescence Detection and Selectivity of the CP Probe with Fe 3+
Next, we conducted spectrophotometric titration experiments to investigate the response of the CP probe on Fe 3+ ions in a DMSO/H2O mixed solvent, and the results are shown in Figure 2. The emission properties of the CP probe were explored using the gradual addition of Fe 3+ (0 to 3 equiv.)by exciting the solution at 345 nm.As shown in Figure 2a, upon the gradual addition of Fe 3+ to the CP solution, a decrease in fluorescence intensity at 439 nm can be observed with a virtually unchanged peak position; the intensity linearly decreased and remained unchanged after one equivalent addition of Fe 3+ .A plot of the intensity of the CP probe at 439 nm versus [Fe 3+ ] initially showed linear behavior and then remained constant, which prompted us to propose a 1:1 binding between the CP probe and Fe 3+ (Figure 2a Inset).The fluorescence response of CP toward Fe 3+ is Scheme 1. Synthesis route for the CP probe.

Characterization of the CP Probe
The UV/visible absorption and emission spectra of the CP probe in DMSO/H 2 O mixed solvent is investigated, as shown in Figure 1a.The absorption maximum of CP probe is centered at approximately 345 nm due to molecular skeleton group π→π* transition.Exciting at 345 nm in the CP probe provides an emission band at 439 nm.Meanwhile, we also studied the effect of pH on the fluorescence of the CP probe (Figure 1b).In the pH range of 2.0-12.0,there was no significant change in fluorescence intensity, which demonstrated that it is insusceptible to the change of acid-base solution.
Molecules 2024, 29, x FOR PEER REVIEW 2 fluorescence microscopy experiments demonstrated that the CP probe can also be us monitor Fe 3+ in living cells.

Results and Discussion
The general procedure for the synthesis of the CP probe is shown in Scheme 1.I mediate compound 1 was prepared according to the reported method [25].The CP p was fully characterized by 1 H NMR, 13 C NMR, IR, and HR-MS.Scheme 1. Synthesis route for the CP probe.

Characterization of the CP Probe
The UV/visible absorption and emission spectra of the CP probe in DMSO/ mixed solvent is investigated, as shown in Figure 1a.The absorption maximum o probe is centered at approximately 345 nm due to molecular skeleton group π→π* sition.Exciting at 345 nm in the CP probe provides an emission band at 439 nm.M while, we also studied the effect of pH on the fluorescence of the CP probe (Figure 1b the pH range of 2.0-12.0,there was no significant change in fluorescence intensity, w demonstrated that it is insusceptible to the change of acid-base solution.

Fluorescence Detection and Selectivity of the CP Probe with Fe 3+
Next, we conducted spectrophotometric titration experiments to investigate th sponse of the CP probe on Fe 3+ ions in a DMSO/H2O mixed solvent, and the result shown in Figure 2. The emission properties of the CP probe were explored using the g ual addition of Fe 3+ (0 to 3 equiv.)by exciting the solution at 345 nm.As shown in Fi 2a, upon the gradual addition of Fe 3+ to the CP solution, a decrease in fluorescence in sity at 439 nm can be observed with a virtually unchanged peak position; the inte linearly decreased and remained unchanged after one equivalent addition of Fe 3+ .A of the intensity of the CP probe at 439 nm versus [Fe 3+ ] initially showed linear beha and then remained constant, which prompted us to propose a 1:1 binding between th probe and Fe 3+ (Figure 2a Inset).The fluorescence response of CP toward Fe

Fluorescence Detection and Selectivity of the CP Probe with Fe 3+
Next, we conducted spectrophotometric titration experiments to investigate the response of the CP probe on Fe 3+ ions in a DMSO/H 2 O mixed solvent, and the results are shown in Figure 2. The emission properties of the CP probe were explored using the gradual addition of Fe 3+ (0 to 3 equiv.)by exciting the solution at 345 nm.As shown in Figure 2a, upon the gradual addition of Fe 3+ to the CP solution, a decrease in fluorescence intensity at 439 nm can be observed with a virtually unchanged peak position; the intensity linearly decreased and remained unchanged after one equivalent addition of Fe 3+ .A plot of the intensity of the CP probe at 439 nm versus [Fe 3+ ] initially showed linear behavior and then remained constant, which prompted us to propose a 1:1 binding between the CP probe and Fe 3+ (Figure 2a Inset).The fluorescence response of CP toward Fe 3+ is normalized as R = 1 − Ii/Imax [26], where Imax is the fluorescence response of CP without Fe 3+ , Ii is the luminescent intensity of CP with Fe 3+ .The lowest detection limit was 0.044 µmol•L −1 .A good linear relationship between the fluorescence response and concentration of Fe 3+ was given with a 0.9915 correlation coefficient (Figure 2b).
Molecules 2024, 29, x FOR PEER REVIEW normalized as R = 1 − Ii/Imax [26], where Imax is the fluorescence response of CP wi Fe 3+ , Ii is the luminescent intensity of CP with Fe 3+ .The lowest detection limit was µmol•L −1 .A good linear relationship between the fluorescence response and conc tion of Fe 3+ was given with a 0.9915 correlation coefficient (Figure 2b).The specificity of the CP probe toward Fe 3+ was also determined, and the resul shown in Figure 3. First, the effect of response time on the fluorescence emission inte of the CP probe at 439 nm with and without Fe 3+ was studied.When Fe 3+ was adde fluorescence emission intensity at 439 nm completely decreased in 1 min and becam urated for 25 min, as shown in Figure 3a.Thus, the results show that this CP probe be applied for the real-time detection of Fe 3+ .Subsequently, we investigated the fluorescence response of CP to all sorts of com metal ions, such as Na + , K + , Ca 2+ , Mg 2+ , Zn 2+ , Ni 2+ , Co 2+ , Cu 2+ , Cd 2+ , Fe 3+ , and Al 3+ , in to evaluate selectivity, which is a very important parameter for a fluorescence prob results are shown in Figure 3b.When these ions were separately added to CP The specificity of the CP probe toward Fe 3+ was also determined, and the results are shown in Figure 3. First, the effect of response time on the fluorescence emission intensity of the CP probe at 439 nm with and without Fe 3+ was studied.When Fe 3+ was added, the fluorescence emission intensity at 439 nm completely decreased in 1 min and became saturated for 25 min, as shown in Figure 3a.Thus, the results show that this CP probe could be applied for the real-time detection of Fe 3+ .
Molecules 2024, 29, x FOR PEER REVIEW 3 normalized as R = 1 − Ii/Imax [26], where Imax is the fluorescence response of CP with Fe 3+ , Ii is the luminescent intensity of CP with Fe 3+ .The lowest detection limit was 0 µmol•L −1 .A good linear relationship between the fluorescence response and concen tion of Fe 3+ was given with a 0.9915 correlation coefficient (Figure 2b).The specificity of the CP probe toward Fe 3+ was also determined, and the results shown in Figure 3. First, the effect of response time on the fluorescence emission inten of the CP probe at 439 nm with and without Fe 3+ was studied.When Fe 3+ was added fluorescence emission intensity at 439 nm completely decreased in 1 min and became urated for 25 min, as shown in Figure 3a.Thus, the results show that this CP probe co be applied for the real-time detection of Fe 3+ .Subsequently, we investigated the fluorescence response of CP to all sorts of com metal ions, such as Na + , K + , Ca 2+ , Mg 2+ , Zn 2+ , Ni 2+ , Co 2+ , Cu 2+ , Cd 2+ , Fe 3+ , and Al 3+ , in o to evaluate selectivity, which is a very important parameter for a fluorescence probe results are shown in Figure 3b.When these ions were separately added to CP Subsequently, we investigated the fluorescence response of CP to all sorts of common metal ions, such as Na + , K + , Ca 2+ , Mg 2+ , Zn 2+ , Ni 2+ , Co 2+ , Cu 2+ , Cd 2+ , Fe 3+ , and Al 3+ , in order to evaluate selectivity, which is a very important parameter for a fluorescence probe; the results are shown in Figure 3b.When these ions were separately added to CP in a DMSO/H 2 O (4:1, v/v) mixed solution, only Fe 3+ completely quenched the fluorescence of CP.Other metal ions only induced slight changes (either fluorescence enhancement or quenching) in the selected working conditions, even if their concentration was 33 times greater than that of Fe 3+ ions.Some other ions, including anions and ROS, such as SO 4 2− , and OH•, were also studied to investigate the fluorescence response of the CP probe.The results show that additions of these other ions did not cause any discernible changes (Figure S1, Supplementary Information).These observations showed that the CP probe had good selectivity for Fe 3+ ions and could be used to effectively detect them.

Sensing Mechanism of the CP Probe with Fe 3+
The molar ratio method proved that the CP probe and Fe 3+ formed a 1:1 stoichiometry complex, as shown in the inset to Figure 2 [27][28][29][30].To further prove the sensing mechanism of the CP probe toward Fe 3+ , ESI-MS spectrometry analysis and a density functional theory (DFT) calculation were applied.Subsequently, the ESI-MS spectrometry of the forming complex CP-Fe 3+ was first determined, in which the peak at m/z = 370.0169was assigned to [CP + Fe 3+ + 2H 2 O + H + ], supporting the 1:1 binding mode of the CP probe and Fe 3+ (Figure 4).The IR spectra also indicated that the CP probe and Fe 3+ formed a coordination compound, where the characteristic absorption moieties of C=O-and C=Nsituated chromones at 1641 cm −1 and 1615 cm −1 shifted to 1616 cm −1 and 1553 cm −1 , respectively (Figure S2, Supplementary Information).
DMSO/H2O (4:1, v/v) mixed solution, only Fe 3+ completely quenched the fluorescence of CP.Other metal ions only induced slight changes (either fluorescence enhancement or quenching) in the selected working conditions, even if their concentration was 33 times greater than that of Fe 3+ ions.Some other ions, including anions and ROS, such as SO4 2− , HSO3 − , S2O3 2− , SCN − , C2O4 2− , NO2 − , Cl − , Br − , I − , H2O2, and OH•, were also studied to investigate the fluorescence response of the CP probe.The results show that additions of these other ions did not cause any discernible changes (Figure S1, Supplementary Information).These observations showed that the CP probe had good selectivity for Fe 3+ ions and could be used to effectively detect them.

Sensing Mechanism of the CP Probe with Fe 3+
The molar ratio method proved that the CP probe and Fe 3+ formed a 1:1 stoichiometry complex, as shown in the inset to Figure 2 [27][28][29][30].To further prove the sensing mechanism of the CP probe toward Fe 3+ , ESI-MS spectrometry analysis and a density functional theory (DFT) calculation were applied.Subsequently, the ESI-MS spectrometry of the forming complex CP-Fe 3+ was first determined, in which the peak at m/z = 370.0169was assigned to [CP + Fe 3+ + 2H2O + H + ], supporting the 1:1 binding mode of the CP probe and Fe 3+ (Figure 4).The IR spectra also indicated that the CP probe and Fe 3+ formed a coordination compound, where the characteristic absorption moieties of C=O-and C=N-situated chromones at 1641 cm −1 and 1615 cm −1 shifted to 1616 cm −1 and 1553 cm −1 , respectively (Figure S2, Supplementary Information).A DFT calculation was carried out using Gaussian 09 [31] software with the help of the GaussianView 5.0 visualization program.The structures of the probe and the complexes were fully optimized using the B3LYP/6-311++G(d,p) basis set and Lanl2dz basis set in a DMSO/H2O mixed solution according to the experimental procedure, respectively.For this, we performed an optimization using the polarizable continuum model (PCM) method.Figure 5 shows the energy-optimized structures of the probe and its complexes.Figure 5a indicated that the structure of CP probe is planar.Because of the repulsive force between two hydrogen atoms of imine moiety, the structures of the complexes are nonplanar (Figure 5b,c).Additionally, the total energy values of the CP probe and the complex CP-Fe 3+ (Figure 5b) were −931.2326582a.u. and −2194.3728797a.u., respectively.Again, the total energy of the complex CP-Fe 3+ -2H2O (Figure 5c) was lower (−2347.3856608a.u.) than that of both CP and CP-Fe 3+ , which implied a higher stability.This result was in accordance with that of ESI-MS spectrometry analysis.A DFT calculation was carried out using Gaussian 09 [31] software with the help of the GaussianView 5.0 visualization program.The structures of the probe and the complexes were fully optimized using the B3LYP/6-311++G(d,p) basis set and Lanl2dz basis set in a DMSO/H 2 O mixed solution according to the experimental procedure, respectively.For this, we performed an optimization using the polarizable continuum model (PCM) method.Figure 5 shows the energy-optimized structures of the probe and its complexes.Figure 5a indicated that the structure of CP probe is planar.Because of the repulsive force between two hydrogen atoms of imine moiety, the structures of the complexes are nonplanar (Figure 5b,c).Additionally, the total energy values of the CP probe and the complex CP-Fe 3+ (Figure 5b) were −931.2326582a.u. and −2194.3728797a.u., respectively.Again, the total energy of the complex CP-Fe 3+ -2H 2 O (Figure 5c) was lower (−2347.3856608a.u.) than that of both CP and CP-Fe 3+ , which implied a higher stability.This result was in accordance with that of ESI-MS spectrometry analysis.
Furthermore, the energy of highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) of the CP probe and its complex CP-Fe 3+ , along with their spatial distribution, was calculated, as shown in Figure 6.For the CP probe, both HOMO and LUMO essentially resided on its entire skeleton.In the case of the complex CP-Fe 3+ , the HOMO was mainly distributed in the chromone moiety, but the LUMO was mostly situated on the pyridine moiety.It can be seen in the frontier molecular orbital diagram that the HOMO of the complex CP-Fe 3+ did not overlap with its LUMO.After the binding of Fe 3+ to CP, the HOMO-LOMO energy gap (∆E) was lowered from 4.01 eV to 1.12 eV, which further stabilized the CP-Fe 3+ system.Hence, the electron transfer from the chromone moiety to the pyridine moiety easily occurred, resulting in fluorescence quenching.The probable detection mechanism for monitoring Fe 3+ is described in Figure 7. Furthermore, the energy of highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) of the CP probe and its complex CP-Fe 3+ , along with their spatial distribution, was calculated, as shown in Figure 6.For the CP probe, both HOMO and LUMO essentially resided on its entire skeleton.In the case of the complex CP-Fe 3+ , the HOMO was mainly distributed in the chromone moiety, but the LUMO was mostly situated on the pyridine moiety.It can be seen in the frontier molecular orbital diagram that the HOMO of the complex CP-Fe 3+ did not overlap with its LUMO.After the binding of Fe 3+ to CP, the HOMO-LOMO energy gap (ΔΕ) was lowered from 4.01 eV to 1.12 eV, which further stabilized the CP-Fe 3+ system.Hence, the electron transfer from the chromone moiety to the pyridine moiety easily occurred, resulting in fluorescence quenching.The probable detection mechanism for monitoring Fe 3+ is described in Figure 7.

Practical Application of CP Probe
Next, to evaluate its practical application, the CP probe was subsequently used for living cell fluorescence imaging to detect Fe 3+ ions.As shown in Figure 8, when the HeLa  Furthermore, the energy of highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) of the CP probe and its complex CP-Fe 3+ , along with their spatial distribution, was calculated, as shown in Figure 6.For the CP probe, both HOMO and LUMO essentially resided on its entire skeleton.In the case of the complex CP-Fe 3+ , the HOMO was mainly distributed in the chromone moiety, but the LUMO was mostly situated on the pyridine moiety.It can be seen in the frontier molecular orbital diagram that the HOMO of the complex CP-Fe 3+ did not overlap with its LUMO.After the binding of Fe 3+ to CP, the HOMO-LOMO energy gap (ΔΕ) was lowered from 4.01 eV to 1.12 eV, which further stabilized the CP-Fe 3+ system.Hence, the electron transfer from the chromone moiety to the pyridine moiety easily occurred, resulting in fluorescence quenching.The probable detection mechanism for monitoring Fe 3+ is described in Figure 7.

Practical Application of CP Probe
Next, to evaluate its practical application, the CP probe was subsequently used for living cell fluorescence imaging to detect Fe 3+ ions.As shown in Figure 8, when the HeLa  Furthermore, the energy of highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) of the CP probe and its complex CP-Fe 3+ , along with their spatial distribution, was calculated, as shown in Figure 6.For the CP probe, both HOMO and LUMO essentially resided on its entire skeleton.In the case of the complex CP-Fe 3+ , the HOMO was mainly distributed in the chromone moiety, but the LUMO was mostly situated on the pyridine moiety.It can be seen in the frontier molecular orbital diagram that the HOMO of the complex CP-Fe 3+ did not overlap with its LUMO.After the binding of Fe 3+ to CP, the HOMO-LOMO energy gap (ΔΕ) was lowered from 4.01 eV to 1.12 eV, which further stabilized the CP-Fe 3+ system.Hence, the electron transfer from the chromone moiety to the pyridine moiety easily occurred, resulting in fluorescence quenching.The probable detection mechanism for monitoring Fe 3+ is described in Figure 7.

Practical Application of CP Probe
Next, to evaluate its practical application, the CP probe was subsequently used for living cell fluorescence imaging to detect Fe 3+ ions.As shown in Figure 8, when the HeLa

Practical Application of CP Probe
Next, to evaluate its practical application, the CP probe was subsequently used for living cell fluorescence imaging to detect Fe 3+ ions.As shown in Figure 8, when the HeLa cells were incubated for 10 min at 37 • C with a 5 µmol•L −1 CP probe, a significant blue emission from the intracellular region was observed upon excitation at 405 nm (Figure 8a).After the cells were treated with 50 µmol•L −1 Fe 3+ in the growth medium for 10 min at 37 • C, only weak intracellular fluorescence was observed in the microscope images (Figure 8d).Both bright field (Figure 8b,e) and overlay measurements (Figure 8c,f) with the Fe 3+ and CP probe after treatment confirmed that the cells were viable for imaging experiments.The results confirm that the CP probe can monitor Fe 3+ ions in a biological environment.
at 37 °C, only weak intracellular fluorescence was observed in the microscope image (Figure 8d).Both bright field (Figure 8b,e) and overlay measurements (Figure 8c,f) w the Fe 3+ and CP probe after treatment confirmed that the cells were viable for imagin experiments.The results confirm that the CP probe can monitor Fe 3+ ions in a biolog environment.The cell viability of the probe is also an important parameter for its practical app tion.Therefore, the MTT assay experiments were conducted to evaluate the cell via of the CP probe using three different kinds of cells, including HeLa, HepG2, and M (Figures S3 and S4, Supplementary Information).Cell viability was monitored for after treatment with the CP probe over a wide range of concentrations (0-50 µmol• The results demonstrate that the CP probe did not negatively affect cell viability u concentrations 20 µmol•L −1 for HeLa cells or 50 µmol•L −1 for HepG2 and MCF-7 which showed that this probe had no prominent cytotoxicity to cells.Therefore, th probe could be an excellent device that has great potential for detecting Fe 3+ in living

Materials and Instruments
Both pyridine-4-carboxaldehyde and chromone-3-carboxaldehyde were purch from HEOWNS Biochem Technologies LLC, Tianjin, China.Unless otherwise note other reagents and solvents were commercially available and used without further fication.NMR spectra were recorded using a Bruker Avance NEO 600 MHz spectrom The cell viability of the probe is also an important parameter for its practical application.Therefore, the MTT assay experiments were conducted to evaluate the cell viability of the CP probe using three different kinds of cells, including HeLa, HepG2, and MCF-7 (Figures S3 and S4, Supplementary Information).Cell viability was monitored for 24 h after treatment with the CP probe over a wide range of concentrations (0-50 µmol•L −1 ).The results demonstrate that the CP probe did not negatively affect cell viability up to concentrations 20 µmol•L −1 for HeLa cells or 50 µmol•L −1 for HepG2 and MCF-7 cells, which showed that this probe had no prominent cytotoxicity to cells.Therefore, this CP probe could be an excellent device that has great potential for detecting Fe 3+ in living cells.

Materials and Instruments
Both pyridine-4-carboxaldehyde and chromone-3-carboxaldehyde were purchased from HEOWNS Biochem Technologies LLC, Tianjin, China.Unless otherwise noted, all other reagents and solvents were commercially available and used without further purification.NMR spectra were recorded using a Bruker Avance NEO 600 MHz spectrometer (at 600 MHz for 1 H NMR or 150 MHz for 13 C NMR; Rheinstetten, Germany).A WQF-510A FT-IR spectrometer (Beijing Rayleigh Analytical Instrument Co., Ltd., Beijing, China) recorded the infrared spectroscopy in KBr discs in the 400-4000 cm −1 region.The melting points were measured on a Beijing Cossim X-5T micro-melting point apparatus (Beijing Century Letter Scientific instrument Co., Ltd., Beijing, China).HRMS (high-resolution mass spectra) was performed using an Aglient 7250 spectrometer (Santa Clara, CA, USA).Absorption spectra and fluorescence spectra were obtained using a TU-1901 UV-Vis spectrophotometer (Beijing Puxi General Instrument Co., Ltd., Beijng, China) and a Varian Cary Elipses spectro fluorophotometer (Palo Alto, CA, USA), respectively.

Synthesis of the CP Probe
A solution of chromone-3-carboxaldehyde in EtOH (5 mL) was added dropwise to a suspension of compound 2 (0.21 g, 1.72 mmol) EtOH (10 mL) over 5 min in an ice bath and the mixture was continually stirred for 8 h.Then, the precipitate was filtered and washed with EtOH.Finally, the solid was recrystallized from EtOAc to afford the CP probe (0.30 g, 66%) as a white solid.R f = 0.34 (V (EtOAc) :V (Petroleum ether) = 2:1), m.p. 220.5-221.

Procedures for Metal Ions Sensing
Stock solutions of various metal ions (0.1 mol•L −1 ), such as Na + , K + , Ca 2+ , Mg 2+ , Zn 2+ , Ni 2+ , Co 2+ , Cu 2+ , Cd 2+ , and Al 3+ , were prepared in deionized water.Fe 3+ (0.001 mol•L −1 ) is prepared in deionized water.For titration, the CP probe (3 mL, 10 µmol•L −1 , dimethyl sulfoxide-water mixed solution) was added to a quartz optical cell of 1 cm optical path length.Metal ion stock solution was gradually added to the CP probe through a micropipette.The test samples for the evaluation of selectivity were obtained by adding appropriate amounts of metal ion stock solution to the CP probe (3 mL, 10µmol•L −1 , dimethyl sulfoxide-water mixed solution).Fluorescence intensity was recorded at an excitation of 345 nm within 350-600 nm.

DFT Calculation
DFT calculation was performed in a binary solvent (DMSO/H 2 O, v/v, 4/1) using Gaussian 09.The calculation method is as follows.The static dielectric constant (EPS) and dynamic dielectric constant (EpsInf) of DMSO and H 2 O were calculated.The EPS and EpsInf values of DMSO were 46.8260 and 2.0079, respectively.The EPS and EpsInf values of H 2 O were 78.3553 and 1.7778, respectively.Then, according to the volume ratio, the static dielectric constant and dynamic dielectric constant of the mixed solvent with DMSO and H 2 O equal to 4:1 were determined.In the energy-optimized structure of probes and complexes, we used "scrf = (PCM, solvent = generic, read)" keywords.

Cell Culture and Fluorescence Bioimaging
The HeLa cell line was provided by Chemistry and Chemical Engineering of Shanxi University (Shanxi, China).The MTT assays for HepG2 and MCF-7 cells were conducted by Shiyanjia Lab (www.Shiyanjia.com,21 February 2024).The cells were cultured in DMEM (4.5 g of glucose/L) supplemented with 10% FBS at 37 • C and 5% CO 2 , seeded into 96-well plates, and then stored for 12 h.The cells were washed with PBS buffer before use and then incubated with a 5 µmol•L −1 CP probe in a PBS buffer for another 10 min at 37 • C. The experiments were also carried out over 10 min in the same medium supplemented with 50 µmol•L −1 FeCl 3 •6H 2 O in order to assess Fe 3+ uptake.Cell imaging was then carried out after washing the cells with PBS buffer 3 times.Confocal fluorescence imaging was performed with an Olympus FluoView FV1000 confocal microscope (Tokyo, Japan).Cells incubated with the CP probe were excited at 405 nm using a multi-line argon laser.

Conclusions
In summary, a new fluorescent CP probe was synthesized and structurally characterized.The recognition properties for Fe 3+ were also evaluated.It displayed a high sensitivity and selectivity toward Fe 3+ , no significant optical interference arose from the other examined metal ions such as Na + , K + , Ca 2+ , Mg 2+ , Zn 2+ , Ni 2+ , Co 2+ , Cu 2+ , Cd 2+ and Al 3+ .CP probe exhibited a fast fluorescence response within 1 min and a low detection limit of 0.044 µmol•L −1 for Fe 3+ .The stoichiometry ratio of CP probe binding with Fe 3+ was verified as 1:1.The recognition mechanism of fluorescence quenching was the photoin-duced electron transfer (PET) process between the CP probe and Fe 3+ and the destruction of the large π-system of the CP probe.Further investigation demonstrated that the CP probe can be applied for the bioimaging of Fe 3+ in living cells.

Figure 2 .
Figure 2. (a) Fluorescence spectra of the CP probe (10 µmol•L −1 ) upon addition of Fe 3+ in mix DMSO/H2O (4:1, v/v) solution (λex = 345 nm).(Inset) The emission intensity at 439 nm of CP changes as a function of the ratio of Fe 3+ concentration to CP probe concentration.(b) Depend of normalized signal on concentration of Fe 3+ ions.

Figure 2 .
Figure 2. (a) Fluorescence spectra of the CP probe (10 µmol•L −1 ) upon addition of Fe 3+ in mixed DMSO/H 2 O (4:1, v/v) solution (λex = 345 nm).(Inset) The emission intensity at 439 nm of CP probe changes as a function of the ratio of Fe 3+ concentration to CP probe concentration.(b) Dependence of normalized signal on concentration of Fe 3+ ions.

Figure 2 .
Figure 2. (a) Fluorescence spectra of the CP probe (10 µmol•L −1 ) upon addition of Fe 3+ in mixe DMSO/H2O (4:1, v/v) solution (λex = 345 nm).(Inset) The emission intensity at 439 nm of CP p changes as a function of the ratio of Fe 3+ concentration to CP probe concentration.(b) Depende of normalized signal on concentration of Fe 3+ ions.

Figure 6 .
Figure 6.energy level diagram of CP probe and the CP-Fe 3+ complex for the HOMO and LUMO.

Figure 7 .
Figure 7. Proposed recognition mechanism for the CP probe.

Figure 6 .
Figure 6.energy level diagram of CP probe and the CP-Fe 3+ complex for the HOMO and LUMO.

Figure 7 .
Figure 7. Proposed recognition mechanism for the CP probe.

Figure 6 .
Figure 6.Energy level diagram of CP probe and the CP-Fe 3+ complex for the HOMO and LUMO.

Figure 6 .
Figure 6.energy level diagram of CP probe and the CP-Fe 3+ complex for the HOMO and LUMO.

Figure 7 .
Figure 7. Proposed recognition mechanism for the CP probe.

Figure 7 .
Figure 7. Proposed recognition mechanism for the CP probe.

Figure 8 .
Figure 8. Confocal fluorescence, bright field images, and overlay image of the CP probe in He cells.(a) Cells incubated with 5 µmol•L −1 of CP probe for 10 min at 37 °C upon excitation at 40 nm.(d) Cells supplemented with 50 µmol•L −1 of Fe 3+ in growth media for 10 min at 37 °C upo excitation at 405 nm.(b,e) Bright field image of cells shown in the panel.(c,f) Overlay image o cells is shown in panel.

Figure 8 .
Figure 8. Confocal fluorescence, bright field images, and overlay image of the CP probe in HeLa cells.(a) Cells incubated with 5 µmol•L −1 of CP probe for 10 min at 37 • C upon excitation at 405 nm.(d) Cells supplemented with 50 µmol•L −1 of Fe 3+ in growth media for 10 min at 37 • C upon excitation at 405 nm.(b,e) Bright field image of cells shown in the panel.(c,f) Overlay image of cells is shown in panel.