Design and Synthesis of a Fluorescent Probe Based on Copper Complex for Selective Detection of Hydrogen Sulfide

A novel fluorescence probe NA-LCX was rationally designed and synthesized for the sequential recognition of Cu and H2S by the combination of hydroxyl-naphthalene and diformylphenol groups. The response properties of NA-LCX for Cu ions and H2S with “on-off-on” manner were investigated by fluorescence emission spectra. A highly selective and sensitive response of complex NALCX-Cu for H2S over other competing amino acids was observed with a limit of detection at 2.79μM. The stoichiometry of NALCX toward Cu ions was determined to be 1 : 1 by the UV-Vis absorption spectrum, and the coordination configuration was calculated by density functional theory (DFT) calculations. Moreover, probe NA-LCX was applied successfully for the recognition of Cu ions and H2S in living cells.


Introduction
Hydrogen sulfide (H 2 S), the simplest biomercapto compound, is not only a rotten egg smelling gas pollutant but also the third gasotransmitter and cellular signaling molecule after CO and NO [1,2]. The endogenous H 2 S could regulate vascular smooth muscle tension and cardiac contractile function, anti-inflammatory and antioxidative stress, neurotransmitter transmission, and insulin signaling inhibition, which plays an important role in the physiological and pathological processes of the cardiovascular, nervous, immune, and digestive systems [3][4][5][6][7]. The concentrations of H 2 S in the normal metabolism often maintain dynamic equilibrium, while abnormal changes of the H 2 S level could induce serious health problems, such as heart diseases [8,9], chronic obstructive pulmonary diseases [10,11], cirrhosis [12,13], and Alzheimer [14,15]. Hence, it is crucial to exploit a highly sensitive and selective method for the detection of hydrogen sulfide in living systems.
Many conventional methods for H 2 S detection have been developed, including colorimetric method [16,17], electro-chemical analysis [18,19], liquid chromatography mass spectrometry [20,21], and fluorescence analysis [22][23][24]. Among them, fluorescence analysis is more desirable due to its simple operation, high sensitivity, wide dynamic range, high fluorescence quantum yield, good biocompatibility, noninvasiveness, and ability of in situ real-time detection in living systems [25]. In recent years, many fluorescent probes for H 2 S detection have been reported on account of different types of strategies such as reduction reactions [26,27], nucleophilic addition reactions [28,29], dinitrophenyl ether/sulfonyl ester cleavage [30,31], and metal sulfide precipitation reaction [32][33][34][35][36][37][38][39][40]. However, there are some limitations to those reaction methods as well as the products obtained via those reactions. For example, those reactions are insensitive, complex, and time-consuming; moreover, fluorescent probes prepared via those reactions are sometimes not biocompatible and sometimes unstable in the presence of biological thiols (glutathione, cysteine, etc.) [31]. The strategy by using a metal displacement approach is in high demand for its fast response and high sensitivity and selectivity. Sulfide is known to react with copper ion to form very stable CuS with a very low solubility product constant K sp = 6:3 × 10 −36 (for cyanide, K sp = 3:2 × 10 −20 ). Thus, the utilization of the higher affinity of Cu 2+ towards sulfide for designing a specific Cu 2+ sensor to sequentially identify H 2 S has received considerable attention because it can effectively eliminate the interference of other analytes in the system.
Naphthalene derivatives with an electron donor-π-acceptor (D-π-A) structure have been widely used due to good optical properties, such as high fluorescence quantum yield, good biocompatibility, and light stability. Herein, we synthesized a new fluorescent probe NA-LCX based on hydroxylnaphthalene and diformylphenol which have excellent coordination ability to metal ions. The probe showed an obvious "on-off" fluorescence quenching response toward Cu 2+ , and the NA-LCX-Cu 2+ complex showed an "off-on" fluorescence enhancement response toward H 2 S in a DMSO/HEPES (3 : 2 v/v, pH = 7:4). The photophysical capabilities of probe NA-LCX for Cu 2+ and NA-LCX-Cu 2+ for H 2 S were studied in details from fluorescence spectroscopy, absorption spectroscopy, and fluorescence images in vivo.

General
Method for Cell Imaging. Human liver cancer HepG-2 cells were cultured in a 12-well plate, and when the cell saturation exceeded 80%, ligand NA-LCX and probe NA-LCX-Cu 2+ solution were added. The mixture was then incubated for 3 hours in a CO 2 incubator and washed three times with precooled PBS, followed by the addition of 1 mL PBS. The resulting mixture was observed under a Leica DMI8 inverted fluorescence microscope.   Journal of Sensors ligand NA-LCX to Cu 2+ was determined as 1 : 1 based on the continuous changes in absorbance at 438 nm ( Figure S4).

Fluorescence Spectroscopy
Recognition of Probe NA-LCX toward Cu 2+ . The sensitivity of probe NA-LCX for Cu 2+ was investigated by fluorescence titration. Probe NA-LCX showed a strong fluorescence emission peak at 575 nm upon excitation at 312 nm. As shown in Figure 2, the fluorescence intensity of the ligand NA-LCX gradually decreased upon the addition of Cu 2+ ions and became constant until about 2 equiv. of Cu 2+ ions were added. The quenching rate was extremely high, indicating that probe NA-LCX was highly sensitive to Cu 2+ , which could be due to the photoinduced electron transfer of Cu 2+ ions and/or the d-d electron paramagnetic quenching effect [41][42][43]. Moreover, the fluorescence emission intensity showed a good linear relationship (R 2 = 0:99) with the concentration of Cu 2+ ions in the range of 1~20 μM. The quenching constant value of probe NA-LCX with Cu 2+ ions was determined from the titration plots.
The corrected Stern-Volmer fitting indicated the value of 2:6 × 10 4 mol −1 · L ( Figure S5). The fluorescence response of probe NA-LCX to other metal ions in DMSO : HEPES (3 : 2, v/v) was shown in Figure S6. It could be found that many other metal ions, such as Co 2+ , Fe 3+ . It could be found that many other metal ions, such as Co 2+ , Fe 3+ , Fe 2+ , Ni 2+ , Zn 2+ ,

Journal of Sensors
Cd 2+ , and Mn 2+ , also exhibited a similar fluorescence quenching response.

Fluorescence Spectra Response of Complex NA-LCX-Cu 2+
toward H 2 S. The complex formed by probe NA-LCX and Cu 2+ was used as a new sensor NA-LCX-Cu 2+ for sequential recognition of H 2 S. Upon the addition of Na 2 S, as shown in Figure 3, the fluorescence intensity was gradually increased and remained unchanged up to 10 equiv. The probe NA-LC-Cu 2+ released Cu 2+ ions due to the strong reaction between sulfide and copper ions, which restored the original fluorescence of the probe. Furthermore, the detection limit was 2.79 μM according to the formula LOD = 3σ/S ( Figure S7). The responses of ligand NA-LCX to other metal ions such as Co 2+ , Cd 2+ , Zn 2+ , Ni 2+ , Fe 2+ , Mn 2+ , and Fe 3+ and the subsequent addition of 10 equiv. of Na 2 S are shown in Figure S8. It could be found that the complex NA-LCX-Cu 2+ possess the highest fluorescence intensity ratio of recovery/quenching, and hence, the probe NA-LCX-Cu 2+ was chosen to pursue H 2 S detection.
To further explore whether probe NA-LCX-Cu 2+ could be used as a highly selective H 2 S sensor, the fluorescence response of probe NA-LCX-Cu 2+ (20 μM) to different amino acids was tested. As described and shown in Figure 4, only the addition of H 2 S instantly caused an obvious fluorescence enhancement. The fluorescence intensity of probe NA-LCX-Cu 2+ remains unchanged in the presence of 10 equiv. of different mercapto-amino acids such as glutathione, cysteine, N-acetyl-L-cysteine, homocysteine, and non-mercaptoamino acids. And other reactive sulfur species (S 2 O 3 2-, SO 4 2-, and HSO 3 -) also did not cause obvious fluorescence changes. The competitive experiments further showed significant fluorescent enhancement without being interfered by other amino acids and reactive sulfur species, which further indicated the good selectivity of the probe NA-LCX-Cu 2+ for H 2 S detection.

DFT Calculation.
To gain further insight into the nature of coordination configuration and optical response of sensor NA-LCX toward Cu 2+ , the different coordination structures of NA-LCX-Cu 2+ were examined by density functional theory calculation. All calculations were performed by Gaussian 09 program. The geometries were optimized at the B3LYP/6-31G(d)/SDD level, and the interaction energies were calculated based on the single point energies obtained at the B3LYP/6-31+G(d)/SDD level. As shown in Figure S9, it was obvious that the interaction energy of structure C was higher than structures A and B, which verified the experimental results and presumed the complexation mode of probe NA-LCX with Cu 2+ .

Effect of pH on the Performance of Probe NA-LCX and
Complex NA-LCX-Cu 2+ . To investigate the effect of pH value, fluorescence intensity of probe NA-LCX, complex NA-LCX-Cu 2+ , and complex NA-LCX-Cu 2+ in the presence of S 2was investigated in a wide range of pH values. No significant changes in fluorescence intensity were found at lower pH (pH ≤ 6) ( Figure S10). However, It could be observed that significant fluorescence changes in ligand NA-LCX and NA-LCX-Cu 2+ -Na 2 S at pH > 6, indicating potential of probe NA-LCX-Cu 2+ to detect H 2 S in physiological environments.
3.6. Cell Imaging Experiments. Inspired by the excellent selectivity at physiological pH levels, the cell imaging application of sensor NA-LCX for detection of Cu 2+ and H 2 S was further investigated. Prior to the cell imaging experiment, the MTT cell toxicity assay for probe NA-LCX-Cu 2+ was performed in human liver cancer cells (HepG-2) shown in Figure S11, and no significant cytotoxicity was found in the range of 0~10 μM, even after incubating for 24 h. As shown in Figure 5, significant intracellular green fluorescence in the HepG-2 cells was observed in the presence of probe NA-LCX when excited with blue light (Figure 5(a)), indicating that the sensor NA-LCX was well permeable. However, the complex NA-LCX-Cu 2+ was added to the wells, and the green fluorescence in HepG-2 cells was quenched to a large degree, as expected ( Figure 5(b)). Upon subsequent addition of 2 and 5 equiv. of Na 2 S solution, obvious fluorescence recovery was observed (Figures 5(c) and 5(d)). The fluorescence imaging results suggested the potential of probe NA-LCX-Cu 2+ for in vivo detection of H 2 S.

Conclusion
In this study, a novel two-armed naphthalene derivative probe NA-LCX was synthesized and its spectral performance for sequential recognition of Cu 2+ and H 2 S was studied. The probe NA-LCX showed an obvious "on-off-on" fluorescence response toward Cu 2+ and H 2 S. The probe NA-LCX showed a 1 : 1 binding stoichiometry to Cu 2+ with a complexation constant of 2:6 × 10 4 M −1 . Fluorescence study indicated sen-sitivity and selectivity of the probe NA-LCX-Cu 2+ for H 2 S detection without interference from other amino acids. The detection limit for H 2 S was calculated to be 2.79 μM. The cell imaging results further showed the potential of the probe for Cu 2+ and H 2 S detection in living cells.

Data Availability
The data used to support the findings of this study are included within the article and supplementary information file(s).