A Coumarin-Based Fluorescent Probe for Ratiometric Detection of Cu2+ and Its Application in Bioimaging

The fluorescent probe L, based on naphthalimide-modified coumarin, was designed, synthesized, and characterized, which could recognize Cu2+ from other cations selectively and sensitively in HEPES buffer (10 mM, Ph = 7. 4)/CH3CN (1:4, V/V). When the probe L interacted with Cu2+, the color and the fluorescent intensity changed obviously and it provided the naked-eye detection for Cu2+. The recognition mode between them was achieved by Job's plot, IR, MS, SEM, and 1HNMR. In addition, test strips made from L could still interact with Cu2+ in tap water effectively. The limit of detection (LOD) of L was 3.5 × 10−6 M. Additionally, the density functional theory (DFT) calculation method was used to analyze the action mechanism of L toward Cu2+. Importantly, the fluorescent probe L could demonstrate favorable selectivity toward Cu2+ in Caenorhabditis elegans. Thus, L was considered to have some potential for application in bioimaging.

Formylcoumarins have been linked with aromatic amine through C=N to acquire the derivatives (Qin et al., 2016;He et al., 2019;Srivastava et al., 2019). Although few derivatives, which are connected by amide linkage, have been reported. It is obvious that the amide-modified derivatives have more potential sites to interact with Cu 2+ by amide than by C=N, which might enhance their selectivity and sensitivity .
In this paper, a naphthalimide-modified probe L (Scheme 1) based on coumarin was designed and synthesized with amide. It is interesting that probe L could distinguish Cu 2+ from other cations selectively and sensitively in HEPES buffer (10 mM, pH = 7.4)/CH 3 CN (1:4, V/V) observable by the naked-eye. In addition, test strips made from L could also detect Cu 2+ successfully. Importantly, probe L could identify Cu 2+ in Caenorhabditis elegans. From these data, probe L has potential applications in bioimaging.

Instruments and Reagents
1 H NMR and 13 C NMR spectra were both performed on a Bruker at 400 MHz using TMS as an internal standard (DMSO-d 6 as the solvents). Infrared spectra were obtained on a Nicolete 5700 FT-IR spectrophotometer. Mass spectra were carried on with a Shimadzu LCMS-IT/TOF mass spectrometer. SCHEME 1 | Synthesis of probe L.
UV-Vis absorption spectra were studied on a Shimadzu UV-1601 spectrophotometer. Fluorescence spectrum was operated on a HORIBA FLUOROMAX-4-NIR spectrometer. Biological imaging was performed on a LEICA DM 2500. All reagents used were of analytical grade.

RESULTS AND DISCUSSION
Study on Spectral Properties of the Probe L Physiological pH (e.g., in the human body) is between 7.35 and 7.45 (Lee et al., 2010), thus, pH 7.4 was used in the  subsequent study, in which Cu 2+ in adult C. elegans was detected. The effect of pH on the fluorescent signal was investigated (Supplementary Figure 10). When the solution of Cu 2+ was added into the L solution, the maximum absorption peak shifted from 412 nm to 385 nm ( Figure 1A). The solution color changed from faint yellow to colorless ( Figure 1B). While other cations didn't cause any change. It was clear from the     Frontiers in Chemistry | www.frontiersin.org competitive experiment that other cations have little impact on the selectivity of L toward Cu 2+ (Figure 2). According to UV-Vis spectroscopy, the L solution color change caused by Cu 2+ could be observed directly by the naked-eye. So, the fluorescent probe L can demonstrate favorable selectivity toward Cu 2+ among other metals.
When the probe L was excited by 412 nm, the fluorescent emission peak appeared at 465 nm. Interestingly, only Cu 2+ caused the fluorescent intensity at 465 nm to reduce when the cation solution was added ( Figure 3A). The fluorescent change induced by Cu 2+ could also be observed easily by the nakedeye under a 365 nm UV lamp ( Figure 3B). According to the competition experiment, other cations seldom interfered with the detection of L toward Cu 2+ (Figure 4). Moreover, the limit of detection for L toward Cu 2+ was calculated to be 3.5 × 10 −6 M (Supplementary Figure 8). Based on the data above, it was concluded that L might recognize Cu 2+ selectively and sensitively.

The Interaction Mode Between L and Cu 2+
In order to determine the stoichiometric ratio of L toward Cu 2+ , the molar method (Supplementary Figure 4) and the continuous variation method (Supplementary Figure 5) were both carried out. The results showed that the stoichiometric ratio was 1:1 between them. To our great joy, the result was supported by mass spectral analyses because the ion peak was detected at m/z 670.08 which was in accordance with [L+Cu 2+ +2NO − 3 ] + (Supplementary Figure 3b). On the basis of the data, it was concluded that the stoichiometric ratio between them was 1:1 when L interacted with Cu 2+ . To study how Cu 2+ changed the L aggregation morphology, a SEM experiment was performed. When L (1 equiv) combined with the Cu 2+ (2 equiv), it was discovered that the L morphology changed from the layer to the petal shape whose diameter was 1 um (Figure 5) which may be the result of the interaction between the fluorescent probe L and Cu 2+ .
To clarify the interaction mode between L and Cu 2+ , IR analyses and 1 H NMR titration were conducted. From IR (Supplementary Figure 6), the absorption band at 1,704 cm −1 assigned to the C=O stretching vibration vanished when L (1 equiv) interacted with Cu 2+ (2 equiv). The absorption band at 3,337 cm −1 assigned to the N-H stretching vibration also disappeared, were the absorption band at 1,537 cm −1 corresponding to the stretching vibration of C=N appeared. The amide group tautomerized to C=N once L associated with Cu 2+ . It is important that the conclusion from IR was in accordance with the 1 H NMR titration. As the Cu 2+ concentration increased, the chemical shift of N-H in the amide group at 8.50 disappeared by a degree (Supplementary Figure 7). From the above data, the interaction mode between L and Cu 2+ was shown as (Figure 6).

Theoretical Computations
To clarify the interaction mode between L and Cu 2+ , the orbital energy and spatial distribution levels of L and L-Cu 2+ were gained with the DFT calculation (Figure 7). The electron density for L was mainly distributed over the coumarin groups in the highest occupied molecular orbital (HOMO), where the electron density for L-Cu 2+ was focused on Cu 2+ in the highest occupied molecular orbital (HOMO). The electron density was mainly located in the naphthalimide group in the lowest unoccupied molecular orbital (LUMO) of L and L-Cu 2+ . The energy gaps of the L and L-Cu 2+ were calculated to be 2.9764 and 3.8984 eV, which were in accordance with the hypsochromic shift in the UV-Vis spectra after the Cu 2+ solution was added into the L solution. The theoretical calculation results also confirmed the interaction mode between them.

Application
To evaluate the practical application of L, test strips were made from L to detect Cu 2+ , in which the filer paper was soaked in the L solution (1 × 10 −5 M) and dried in the air. After the test strips were immersed in the Cu 2+ solution (1.0 × 10 −5 M), the test strips color change was examined directly by the naked-eye under a 365 UV lamp (Figure 8). It meant that probe L could also recognize Cu 2+ in the solid state as well. In addition, test strips were made from L to detect Cu 2+ in tap water (Supplementary Figure 9). It is interesting that only the aqueous solution containing the Cu 2+ faded and the fluorescence decreased which shows that probe L could potentially identify Cu 2+ water pollution.  To explore the application of L in the biological system, the ability of probe L to sense Cu 2+ in adult C. elegans was studied (Figure 9). Bright field and fluorescent images of the C. elegans nematodes are shown in Figures 9A,D. The nematodes cultured with L exhibited blue fluorescence (Figures 9B,E). The fluorescence reduced obviously after the nematodes were cultured with Cu 2+ (Figures 9C,E). This result showed the applicability of probe L to in vivo studies.

CONCLUSION
In summary, probe L, based on naphthalimide-modified coumarin derivatives, was designed, synthesized, and characterized. Probe L showed good selectivity and high sensitivity toward Cu 2+ while other metal ions did not cause interference. At the same time, the solution color change was observed directly by the naked-eye. The proposed interaction mode between them was confirmed by UV-Vis spectroscopy, fluorescence, Job's plot, 1 H NMR titration, ESI-MS, and SEM. In addition, probe L has a good application prospect for detecting Cu 2+ qualitatively. The LOD of L was 3.5 × 10 −6 M. Additionally, a DFT calculation method was utilized to analyze the action mechanism of L toward Cu 2+ . Furthermore, the successful detection of Cu 2+ in the living system using L also suggests its potential utilization in practical applications.

DATA AVAILABILITY STATEMENT
All datasets generated for this study are included in the article/Supplementary Material.

AUTHOR CONTRIBUTIONS
JZ and RQ designed the work and wrote the manuscript. JZ, M-YC, BW, LZ, and C-QQ carried out the experiments. R-QL performed the spectroscopic experiments. C-BB revised and edited the manuscript. All authors reviewed the manuscript and have agreed to its publication.