Construction and Application of Multipurpose metal-organic frameworks -based Hydrogen Sulfide Probe

Hydrogen sulfide (H2S) is a toxic gas derived from the sulfur industry and trace H2S in the environment can cause serious ecological damage while inhalation can cause serious damage and lead to disease. Therefore, the real-time and accurate detection of trace sulfur ions is of great significance for environmental protection and early disease detection. Considering the shortcoming of current H2S probes in terms of stability and sensitivity, the development of novel probes is necessary. Herein, a novel metal-organic frameworks (MOF)-based material, UiO-66-NH2@BDC, was designed and prepared for the visual detection of H2S with rapid response (< 6 s) and low detection limit of S2− (0.13 µM) by hydrogen bonding. Based on its good optical performance, the UiO-66-NH2@BDC probe can detect S2− in various water environments. More importantly, UiO-66-NH2@BDC probe realize imaging S2− in cells and live zebrafish.


Introduction
With the rapid development of modern industry, pressure on the environment is increasing daily.In the face of increasing environmental safety awareness, it important to quickly detect hazardous substances in the environment in real time.Hydrogen sulfide, derived from the sulfur industry, is a notorious industrial waste gas [1][2][3].As the released form of H 2 S, S 2− is widely distributed in different natural environments, including water and air and trace intake threatens human health and causes various diseases [4][5][6][7].Developing a reliable method that can effectively detect trace S 2− in real time with high sensitivity will contribute to preventing hidden dangers to the environment and human health caused by excessive S 2− .
Recently, fluorescence analysis has gradually replaced traditional analysis methods, including liquid phase, atomic absorption, and plasma emission, owing to its merits of easy operation and rapid response [8][9][10][11][12].To date, reports in living systems and the application of MOFs in biological samples remains limited.The toxicity of trace hydrogen sulfide to environmental and biological systems prompted the development a novel MOF-based material herein (UiO-66-NH 2 @BDC) using UiO-66 as a template via post modification.The UiO-66-NH 2 @BDC probe realized rapid (< 6 s) and super-sensitive detection of S 2− (0.13 µM) with excellent selectivity.In addition, this newly developed probe can be applied for the detection of trace S 2− in different types of water samples, cells, and zebrafish.Fluorescence spectral results were obtained by using F-4700 Fluorescence spectrometer (Hitachi, Japan).Ultraviolet-Visible (UV-vis) absorption was performed with a Cary 50 spectrophotometer (Varian, USA).Infrared spectrum (IR) was performed by ALPHA II (Bruker, Germany).Apreo 2 S ultrahigh resolution scanning electron microscope (Thermo Fisher, USA) was used to research morphology of sample.X-ray photoelectron spectroscopy (XPS) (Thermo Fisher, USA) is further used to study the surface composition of sample.Crystal structure analysis was carried out X-ray diffraction (XRD) (Rigaku, Japan).The Zeta potential was performed by Zetasizer Ultra (Malvern Panalytical, Britain).Fluorescence imaging experiment with Laser confocal microscope (Nikon A1, Japan).

Synthesis of UiO-66-NH 2
UiO-66-NH 2 was obtained by early reported with modifications [27].In this typical synthesis, ZrCl 4 (0.2517 g, 1.08 mmol) was dropwise into the solution containing 10mL DMF and 2mL HCl, reaction for 5 min, and sonicated for 10 min.Mixed DMF (20 mL) containing 2-aminoterephthalic acid (0.2717 g, 1.5 mmol) into the above solution and stirred for 20 min.Then, the mixture was added to a Teflon reactor and heated at 80 O C for 24 h.After cooled to room temperature, the target was gained after centrifugation and cleaned with DMF for three times, and then dried in vacuum of 60 O C for 24 h.

Synthesis of UiO-66-NH 2 @BDC
The synthesis process of UiO-66-NH 2 @BDC was showed in Scheme.1, a mixture of UiO-66-NH 2 (17.53 mg, 0.01 mmol), BDC (36.48 mg, 0.15 mmol) were sonicated in 10 mL ultrapure water.After ultrasonic dissolution, the substance was added to a Teflon reactor and reacted at 80 O C for 12 h.Then, the product is obtained after centrifugation and drying.

Fluorescence Detection of S 2
0.02 g of UiO-66-NH 2 @BDC sample was dispersed into 100mL ultrapure water and the suspension was obtained by ultrasound for 2 min.Then, fluorescence experiments were carried out by gradually adding different concentrations S 2− solution to the suspension of UiO-66-NH 2 @BDC.After both are fully reacted, the suspension was excited at 380 nm.

Detection of S 2− in real Samples
Detection of S 2− in actual samples (tap water, drinking water and industrial wastewater) by standard addition method.The tap water was taken from daily water, drinking water was purchased from supermarkets, and industrial wastewater was taken from Kukdo Chemical Co., Ltd.First, the actual sample was centrifuged and filtered with a 0.22 μm for the next step.Then, S 2− of different concentrations was dropped into the sample for fluorescence test.The actual sample was measured three times in parallel (n = 3) with 380 nm excitation.

Cell Imaging
Hela cells were purchased from the Cell Bank of the Chinese Academy of Sciences.The Hela cells were grown in a humidified incubator containing 10% fetal bovine serum and 5% CO 2 for 24 h.The cultured cells were divided into three groups.Added equal amount of UiO-66-NH 2 @BDC (0.2 mg/mL) to the three groups, then put different concentrations of S 2− (50, 600 µM) in the two groups respectively and cultured at 37 O C for 30 min.Cells were washed five times with phosphate buffered saline for imaging.

Fluorescence Imaging in Zebrafish
The zebrafish were produced by Shanghai FishBio Co., Ltd.Three days old zebrafish were selected for further research.The zebrafish was incubated in the solution containing 0.2 mg/mL UiO-66-NH 2 @BDC for 60 min and washed three times with the culture solution to remove the excess UiO-66-NH 2 @BDC.The group without S 2− as a blank control.For another groups, zebrafish were preconditioned with UiO-66-NH 2 @BDC for 30 min, then incubated with S 2− (50, 600 µM) for 60 min.Then washed three times with PBS.Fluorescence imaging was captured by confocal laser scanning microscope.The excitation wavelength was 405 nm.

Structural Characterization
The morphology of UiO-66-NH 2 and UiO-66-NH 2 @BDC was evaluated by SEM and TEM, as showed in Fig. S1 and Fig. 1, no obvious changes can be observed between UiO-66-NH 2 and UiO-66-NH 2 @BDC, illustrating that the post modification process keep morphology of UiO-66-NH 2 intact.PXRD was further performed to explore crystal structure changes before and after post modification.As illustrated in Fig. 2A, two obvious sharp peaks at 7.4° and 8.5° corresponding to the (111) and (200) crystal planes of UiO-66-NH 2 , respectively, were also observed in UiO-66-NH 2 @BDC.The peak at 16.9° corresponding to BDC was also found in UiO-66-NH 2 @BDC, demonstrating that the small-molecule BDC attached to UiO-66-NH 2 Fig. 2 (A) PXRD patterns of different substances.(B) The XPS spectrum of UiO-66-NH 2 and UiO-66-NH 2 @BDC.
Fig. 1 (A) SEM and (B) TEM of UiO-66-NH 2 @BDC the S 2− concentration and UiO-66-NH 2 @BDC fluorescence.With the concentration of S 2− increased from 0 to 625 µM (Fig. 3A), probe fluorescence was enhanced and showed a good linear relationship between 0 and 200 µM (Fig. 3B).Equation I/I 0 = 1.348 + 0.0031 C with a correlation coefficient of R 2 = 0.9983 was used to express the relationship between S 2− concentration and probe fluorescence ratio.The 0.13 µM ultra-low detection limit can be obtained based on 3 σ/ k.Compared with UiO-66-NH 2 , the UiO-66-NH 2 @BDC probe showed higher sensitivity, indicating that the post modification may endow additional sites for S 2− interaction, thus improving the detection sensitivity.In addition, we systematically compared the sensitivities of different S 2− probes, showing that the novel probes exhibit great advantages in terms of response time, detection limit, and application range [33][34][35][36][37][38] (Table S1).
Specific response is the premise that the probe can be applied to multiple environmental detections.Herein, to assessed the practical applicability of the probe UiO-66-NH 2 @BDC, different potential interfering substances, including Ca 2+ , Na + , Fe , Hcy, Val, Leu, Phe, Met, Tyr, Trp, and Glu were selected for selectivity testing with the UiO-66-NH 2 @BDC probe.As Fig. 4A illustrates, only S 2− induced fluorescence enhancements of the UiO-66-NH 2 @BDC probe in the ion competition experiment (Fig. S9).This experiment demonstrated the UiO-66-NH 2 @BDC probe exhibits excellent selectivity and can be applied for S 2− detection in actual samples.Real time detection capability is another indicator of probe performance and the fluorescence of UiO-66-NH 2 @BDC reached its peak value within 6 s and remained stable demonstrating excellent real-time detection (Fig. 4B).We also investigated the fluorescence stability of UiO-66-NH 2 @BDC, and observed no significant changes after one day, five days, or three weeks (Fig. 4C).In addition, PXRD was used to study the stability of UiO-66-NH 2 @BDC after immersion in water for 3 days and 1 week.The crystal structure remained stable (Fig. 4D), indicating high stability successfully and the crystal configuration of UiO-66-NH 2 remains unchanged in the course of the post modification process [28].FT-IR was also performed and showed in Fig. S2, the characteristic peak of stretching vibration of -NH 2 in UiO-66-NH 2 appear at 3454 and 3356 cm − 1 .After post modification, the amino characteristic peak disappeared, giving rise to the amide characteristic peaks at 3062 and 1290 cm − 1 which further confirmed the PXRD results.Thus, it was confirmed that BDC was connected to UiO-66-NH 2 via acid and amine condensation.XPS was used to determine the composition of the UiO-66-NH 2 and UiO-66-NH 2 @BDC materials (Fig. 2B).UiO-66-NH 2 @BDC exhibited four peaks at 531.1, 398.9, 285.6, and 183.3 eV attributable to O 1s, N 1s, C 1s, and Zr 3d.The high-resolution C 1s spectral peaks at 289.0, 285.2, and 284.6 eV arise from C-C = O, C = O, and C = C groups [29,30], respectively (Fig. S3).For UiO-66-NH 2 @BDC, the high-resolution spectrum of O 1s (Fig. S4) showed peaks at 533.3, 531.9, and 530.6 eV arising from O-C = O, C = O, and Zr-O-Zr [31,32], respectively.Two peaks at 185.3 and 182.9 eV can be found in highresolution Zr spectrogram (Fig. S5) which were considered as Zr 3d 3/2 and Zr 3d 5/2 , respectively [32].The high-resolution N 1s spectra showed a peak at 400.5 eV arising from C-N (Fig. S6).The above results indicate that in the postmodification process, the basic skeleton of UiO-66 has not changed and small molecule BDC was covalently bonded to UiO-66-NH 2 successfully.

Fluorescence
Performance of UiO-66-NH 2 @BDC Fluorescence analysis was performed to explore the optical performance of the MOF probe before and after modification.UiO-66-NH 2 was first used as a sensor for S 2− at concentrations of 0-400 µM where the fluorescence intensity increased gradually (Fig. S7A).The lower detection limit was 4.14 µM by the linear relationship between 25 and 150 µM S 2− (Fig. S7B).Fluorescence titration experiments were performed to illustrate the relationship between Fig. 3 (A) Fluorescence emission spectra and (B) the linear relationship between the fluorescence intensity ration (I/I 0 ) of the UiO-66-NH 2 @BDC with different concentrations of S 2− upon S 2− addition and indicting adsorption of the analyte on UiO-66-NH 2 @BDC (Fig. 5B).The UV/vis absorption spectra (Fig. S10A) showed a slight red shift after S 2− addition from the original peak at 287 nm and the absorption intensity significantly increased.This was ascribed to the incremental electron transfer from the organic linkers to Zr.In addition, the XPS O 1s (Fig. S10B) and N 1s (Fig. S10C) of UiO-66-NH 2 @BDC showed a slight change after S 2− addition due to the difference between S 2− , oxygen, and nitrogen in the hydrogen bonding of UiO-66-NH 2 @BDC [39].The above experimental results illustrated UiO-66-NH 2 @BDC can achieve highly sensitive detection of S 2− via hydrogen bonding.
in water and overall excellent optical performance for the accurate detection of S 2− .

Possible Mechanism
To explore the sensing mechanism between S 2− and UiO-66-NH 2 @BDC, XRD analysis was performed (Fig. 5A).The PXRD peak pattern of UiO-66-NH 2 @BDC remained largely unchanged before and after S 2− addition, indicating that the fluorescence turn-on effect was not caused by structural collapse.The zeta potential was further examined, showing an increase from − 7.241 to -13.78 mV

Cellular Imaging
MOF-based materials have limited applications in biological fields due to their poor aqueous solubility.Post modification of MOFs have gradually solved this problem for applications in biological systems.Because of the excellent optics characteristics of the UiO-66-NH 2 @BDC probe, we further explored its sensing performance for S 2− in cells.HeLa cells were selected and incubated with S 2− (50 and 600 µM) and UiO-66-NH 2 @BDC.The results were showed in Fig. 6, UiO-66-NH 2 @BDC successfully entered the HeLa cells and achieved S 2− imaging.With increasing S 2− concentration, significant fluorescence enhancement was observed, similar to the fluorescence titration.These results indicate that UiO-66-NH 2 @BDC can be used for imaging in cellular environments.

Imaging in Zebrafish
Based on cell imaging research, we further studied the application of the newly developed probes in live zebrafish to provide information for the early diagnosis of disease.Different S 2− concentrations were mixed with UiO-66-NH 2 @BDC and subsequently incubated with zebrafish (Fig. 7).Confocal microscopy revealed that the free probe exhibited no fluorescence, whereas S 2− induced significant blue fluorescence that increased with increasing concentrations.These results were similar to those observed in cells, proving that the UiO-66-NH 2 @BDC probe can be successfully used for S 2− detection in vivo.

Conclusion
A novel post-modification MOF-based material (UiO-66-NH 2 @BDC) was obtained through simple synthesis herein.The UiO-66-NH 2 @BDC probe showed an ultrafast (< 6 s) fluorescence turn-on response to S 2− .A good linear relationship was observed between 0 and 200 µM S 2− and the limit of detection was calculated as 0.13 µM.UiO-66-NH 2 @BDC can also be applied for S 2− detection in different water samples, with S 2− recoveries ranging from 80.36 to 109.33% with a RSD of < 0.26%.In addition, UiO-66-NH 2 @BDC was applied for cellular and zebrafish imaging.The development of MOF-based probes with strong stability and ultra-low detection limits is promising for applied research in environmental and biological systems.

Application
To study the detection ability of the UiO-66-NH 2 @BDC probe for sulfur ions in real water samples, the standard addition method was used.Tap, drinking, and industrial wastewater were used to investigate the detection capability of the newly developed UiO-66-NH 2 @BDC probe (Table 1).The S 2− recovery ranged from 80.36 to 109.33%, while the relative standard deviation (RSD) was < 0.26%.In addition, all interfering substances were added to the solution to comprehensively analyze probe feasibility for practical applications.As shown in Table S2, the recovery ranged between 103.87% and 118.30% and the RSD was < 0.61% in the presence of interferents.

Table 1
Determination of S 2− in water sample (n = 3)