Alizarin red S–zinc(II) fluorescent ensemble for selective detection of hydrogen sulphide and assay with an H₂S donor

Abstract

A new Alizarin Red S based fluorescent probe ARS–Zn(II) has been reported for the detection of H₂S in aqueous buffer solution. In the presence of zinc ions, ARS displayed an increase in fluorescence intensity through the formation of an ARS–Zn(II) ensemble. The addition of H₂S to the ensemble caused the disassembly of the metal and ARS, which restored the fluorescence intensity of ARS. This sensing ensemble exhibited H₂S-selectivity over other biothiols and biologically relevant analytes. The calculated detection limit of ARS–Zn(II) with H₂S was 92 nM. ARS–Zn(II) also detected H₂S in serum under physiological conditions. Moreover, as a practical application, it could be used for the real time monitoring of H₂S released from the H₂S-donor molecule benzoic (methyl carbonic) dithioperoxyanhydride and also applied to live cell imaging. The reported ensemble ARS–Zn(II) has advantages like ready availability, high selectivity with good sensitivity, fast response time, higher excitation (520 nm) and emission (625 nm) wavelengths and real time fluorescence assay with H₂S donors.

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Introduction

Hydrogen sulphide (H₂S) is an unpleasant gas with a characteristic smell of rotten eggs. Endogenously produced H₂S has been recognized as one of the three gasotransmitters along with nitric oxide (NO) and carbon monoxide (CO).¹ Physiological levels of H₂S appear to be involved in various biological functions and it is essential for maintaining human health along with having other roles.² Unusual levels of H₂S in humans has been linked to diseases like Alzheimer’s disease,³ Down’s syndrome⁴ and hypertension.⁵ The physiological and therapeutic importance of H₂S has led to quick progress in research activity involving H₂S.⁶

A number of fluorescent probes for H₂S have been developed in the last decade.^7–12 However, the selectivity over competing biothiols such as glutathione (GSH), cysteine (Cys) and homocysteine (Hcy), and the sensitivity, response time, multistep synthesis and biocompatibility are some of the serious limitations with the existing fluorescent probes.^13–16 These drawbacks show the need for H₂S sensors with specific properties such as selectivity, sensitivity, fast response and ready availability, so that they can be widely used to tackle biological issues and for pharmacological applications. To deal with these demanding issues, we wish to report a displacement approach for the development of practically useful fluorescent probes for H₂S.

The dye displacement approach is a competitive binding supramolecular sensor system,^17–19 where an analyte displaces a dye from a receptor; as a result a colour or fluorescence change is obtained, and can be related to the amount of analyte present.²⁰ In this regard, Alizarin Red-S (ARS) has been successfully employed as an indicator in competitive binding assays for the detection of various analytes such as biological phosphates,^21,22 saccharides,^23,24 and anions.²⁵ ARS is also known for the colorimetric detection of metal ions.^21,26,27 The analyte mediated release of ARS from ARS–copper complex ensembles has been reported for the selective detection of glutathione,²⁸ tiopronin,²⁹ proteins,³⁰ and L-​cysteine.³¹ Similar to copper, ARS is also known to form complexes with the zinc ion with high affinity.³² However ARS–zinc ion based fluorescent ensembles are not well documented for the detection of analytes using the dye displacement method.

Herein, we wish to report a new probe ARS–Zn(II) (Scheme 1) for the selective detection of H₂S under biological conditions. ARS shows a ratiometric absorbance change and fluorescence enhancement upon coordination with zinc to form the ARS–Zn(II) ensemble. In the presence of H₂S, the emission intensity of ARS was immediately restored as it separates from the sensing ensemble with the formation of ZnS. We have also used this ensemble to monitor H₂S released from H₂S donor molecules in aqueous solution.

Scheme 1 Proposed sensing mechanism for the detection of H₂S using the ARS–Zn(II) ensemble.

Experimental section

Materials and reagent

Methoxycarbonylsulfenyl chloride, thiobenzoic acid, and sodium sulfide were purchased from Sigma-Aldrich. Other chemicals were purchased from Loba Chemie or Merck limited unless otherwise mentioned. The solvents used were analytically pure and used without further purification.

Instrumentation methods

¹H NMR was measured on a Bruker Avance 400 MHz. Mass spectra were measured on a Waters Q-Tof mass spectrometer. A Cary Eclipse fluorescence spectrophotometer was used to obtain the fluorescence spectra. UV absorption spectra were recorded on an Evolution 201 UV-Vis spectrophotometer using quartz cells of 1.0 cm pathlength. Live cell images were recorded by using a Nikon Air Laser Scanning Confocal Microscope. Donor studies were performed on a Synergy-HT Multimode Plate reader (BIOTEK). Buffer solutions were prepared by using deionized water and pH studies were done using a HACH sension 2.

General procedures for spectroscopic measurements

Spectral study of ARS with zinc. The absorbance and fluorescence study was performed in a 10 mM MeOH–PBS buffer (3/1 (v/v), pH = 7.4) at RT. For the spectroscopic titrations, 120 μL of the ARS (0.5 mM) stock solution was diluted in 2000 μL by adding buffer. For metal complex formation, the titration was performed with different concentrations of zinc perchlorate (0–30 μM) in buffer. Each spectrum was recorded in a standard quartz cuvette cell of 1 cm in length. The change in absorption was monitored at 530 nm. The emission spectrum was recorded upon excitation at 530 nm with excitation and emission slit widths of 10/10.

Spectral study of ARS–Zn(II) vs. H₂S. For the binding study with H₂S, 120 μL of ARS (0.5 mM) was mixed with 40 μL of zinc perchlorate (1.5 mM) in buffer and diluted to 2000 μL to form an ARS–Zn(II) complex. The concentrations of [ARS] = 30 μM and [Zn(II)] = 30 μM were maintained for all titrations. UV-Vis and fluorescence titrations were done by adding different concentrations of Na₂S (0–44 μM) to this solution. For all the experiments Na₂S was used as an H₂S gas source.

Fluorescence assay with H₂S donor

For the fluorescence assay with H₂S donor molecules, 100 μL of ARS–Zn(II) (60 μM) was prepared in a MeOH : PBS buffer (3/1, (v/v), 10 mM, pH = 7.4, 0.04% THF used as a co-solvent) and diluted by adding 134 μL of buffer to this. 6 μL of glutathione (0.1 M, 100 equivalents) was added and the fluorescence emission was monitored at 645 nm for 30 minutes at 35 °C. The excitation wavelength was 530 nm and emission wavelength was 645/30 nm. The titration was done in 96 well plates using a Synergy-HT multimode micro plate reader.

Results and discussion

Binding studies of ARS with Zn(II)

Alizarin and ARS are known for their complexation behaviour with different metal ions.^32–35 We have studied the complex formation ability of ARS with Zn(II) by using ultraviolet-visible (UV-Vis) and fluorescence spectroscopy in aqueous buffer (MeOH/PBS buffer, 3/1, v/v) at pH 7.4. The UV-Vis spectra resulting from addition of different concentrations of zinc are shown in Fig. 1.

Fig. 1 Absorption spectra of ARS (30 μM) in MeOH/PBS buffer (3 : 1, v/v, pH = 7.4) in the presence of 0–30 μM equivalents of Zn(II).

As shown in Fig. 1, the addition of Zn(II) to ARS induces a ratiometric change with an increase in absorbance at 520 nm, along with a decrease in wavelength at 425 nm and the appearance of an isosbestic point at 465 nm. Based on the UV-Vis spectral change, the binding mode was determined by using a Job’s plot using mixtures of ARS and zinc in buffer solution.

The relative ARS–Zn(II) complex concentration reached a maximum when the molar fraction was 0.33, therefore the Job plot confirmed a 2 : 1 (ARS:Zn(II)) complex formation (SI Fig. 3†), which was similar to the observation of the ARS and Cu²⁺ coordination stoichiometry as reported earlier.³¹ At neutral pH one of the phenolic OH groups of ARS is deprotonated and it forms an ARS–Zn(II) complex as shown in Scheme 1.

The ability of ARS to form complexes with zinc ions was also established by using fluorescence measurements. ARS is a natural dye with weak fluorescence properties in aqueous solution under neutral conditions.³⁶

As depicted in Fig. 2, with increasing concentration of Zn(II) ions, the fluorescence intensity in the orange region at 625 nm increased gradually upon excitation at 530 nm, which was mainly due to a combination of the chelation enhanced fluorescence (CHEF) and internal charge transfer (ICT) mechanisms.

Fig. 2 Fluorescence spectrum of ARS (30 μM) in MeOH/PBS buffer (3 : 1, v/v, pH = 7.4) upon addition of 0–30 μM equivalents of Zn(II).

Moreover, it was observed that Zn(II) ions induced almost a 6 fold increase in the fluorescence intensity of ARS. The fluorescence intensity of ARS reached a maximum at 30 μM of zinc, which makes ARS a more sensitive probe for zinc. But as described earlier, when more than 1 equivalent of Zn(II) was added, the fluorescence intensity dropped.³² The association constant (K_a) was evaluated graphically by plotting 1/ΔF against 1/[Zn(II)] as shown in SI Fig. 4†. The data was linearly fitted according to the Benesi–Hildebrand equation and the apparent binding (K_a) value was obtained from the slope and intercept of the line. The K_a value was found to be 13 826 M⁻¹. Zinc ions induced an increase in the fluorescence intensity and the ratiometric change in the UV-Vis absorbance ensured the determination of H₂S with high sensitivity with the ARS–Zn(II) sensing ensemble.

Effect of H₂S on the optical signal of the ARS–Zn(II) ensemble

H₂S is known to form metal sulphides upon reaction with metal ions. Zinc ions are known to react with sulphides to form stable ZnS species,³⁷ which have a low solubility product constant (K_sp = 1.6 × 10⁻²⁴). So it was expected that the ARS–Zn(II) sensing ensemble would act as a potential sensor for the detection of H₂S through the displacement of zinc thereby recovering the original ARS optical (fluorescence and absorbance) signal. Fig. 3a illustrates the emission spectral changes of the probe with H₂S at pH 7.4 in aqueous buffer solution. ARS–Zn(II) emitted strong fluorescence at 625 nm, and upon addition of different concentrations of H₂S to this solution, the fluorescence intensity decreased. The decrease in fluorescence at 625 nm was achieved immediately (less than a minute) with a fast response time. Real time detection is important for practical applications; consequently the quick response time of the ARS–Zn(II) sensing ensemble is an advantage as compared to that of the DHAQ–Cu²⁺ ensemble reported earlier for H₂S detection, which has a response time of about 4 minutes.³⁸

Fig. 3 (A) Fluorescence spectra of the ARS–Zn(II) ensemble in MeOH/PBS buffer (3 : 1, v/v, pH = 7.4) upon addition of 0–42 μM equivalents of H₂S. (B) Detection limit plot from the fluorescence titration of ARS–Zn(II) with H₂S.

In addition, the original fluorescence intensity of ARS was almost 100% recovered, when the concentration of H₂S was about 42 μM. It confirmed that H₂S disassembles the Zn(II) ion and ARS from the sensing ensemble. These results revealed the applicability of the ARS–Zn(II) system for the detection of H₂S in aqueous medium. The fluorescence recovery could be used to plot the calibration curve to afford quantitative measurement of H₂S in biological samples and H₂S releasing drugs.

Based on the change in emission intensity, the detection limit of H₂S calculated for the sensing ensemble was 92 nM, which was better than those of other probes reported for H₂S, based on the zinc displacement approach.^39–41 The detection limit was calculated by the equation 3σ/slope, where the slope was obtained by plotting the change in fluorescence intensity (ΔF) vs. conc. of H₂S and σ = the standard deviation of the blank signal obtained without H₂S (Fig. 3b).

Furthermore, we have also monitored the UV-Vis absorbance change in the presence of H₂S. As shown in Fig. 4, upon addition of H₂S to ARS–Zn(II) the absorption band at 525 nm decreased gradually and restored the original absorption state of ARS after adding 44 μM of H₂S. It indicates that the dye ARS and Zn(II) have been dissociated from the sensing ensemble in the presence of H₂S.

Fig. 4 Absorption spectra of the ARS–Zn(II) ensemble in MeOH/PBS buffer (3 : 1, v/v, pH = 7.4) upon addition of 0–44 μM of H₂S.

Hence the results obtained from fluorescence and absorbance spectroscopy confirmed the convenience of using the ARS–Zn(II) sensing ensemble for the detection of H₂S in aqueous buffer solution.

Selectivity studies and the effect of interference

The selectivity of the sensing ensemble ARS–Zn(II) for H₂S was examined by fluorescence spectroscopy under the same conditions as discussed above. As shown in Fig. 5a, the probe analysed with different biologically important analytes such as reactive sulphur species (glutathione, cysteine, methionine, S₂O₃²⁻), reactive nitrogen species (NO˙, NO₃⁻ and N₃⁻), biological phosphates (ATP, ADP, AMP, PPi, H₂PO₄⁻ and HPO₄²⁻) and other anions (F⁻, Cl⁻, Br⁻, I⁻, OH⁻, HCO₃⁻ and CH₃COO⁻). Among these biological phosphates (PPi, AMP and H₂PO₄⁻), GSH and cysteine displayed little and other analytes exhibited insignificant changes in the fluorescence spectra of ARS–Zn(II) as shown in Fig. 5a. At a higher concentration of cysteine (1 mM) and glutathione (1 mM), notable changes were obtained, but these changes were small compared to those of H₂S (30 μM) (SI Fig. 11†).

Fig. 5 (a) Fluorescence spectra of the ARS–Zn(II) ensemble in the presence of various analytes in MeOH/PBS buffer (3 : 1, v/v, pH = 7.4) at RT upon excitation at 530 nm. (b) Different analyte responses of ARS–Zn(II) in buffer. [ARS] = 30 μM, [Zn(II)] = 30 μM, [anion] = 300 μM, [H₂S] = 30 μM. The red colour bar indicates only the probe ARS–Zn(II), the dark blue colour indicates ARS–Zn(II) + anions and the sky blue bars indicate ARS–Zn(II) + anions + H₂S.

These results confirm the higher selectivity of H₂S among other physiological and biologically important analytes. The decrease in the fluorescence intensity with H₂S was due to the displacement of zinc and formation of ZnS in aqueous solution. But the non-bonding interaction of glutathione (GSH), cysteine and H₂PO₄⁻ to the Zn(II) center could be responsible for the weak binding and small change in fluorescence intensity with these analytes.

Furthermore, the selectivity of the probe with H₂S was also tested in the presence of interfering species. As shown in Fig. 5b. The competitive experiments revealed minimal interference or no interference with the detection in the coexistence of various species and H₂S. Accordingly, probe ARS–Zn(II) could be applicable for the selective determination of H₂S even in the presence of other biological species like ATP, ADP, AMP, PPi, GSH and cysteine. This result also confirmed the possible use of the probe even when H₂S co-exists with other biological relevant biothiols like glutathione, cysteine, methionine and phosphates.

Effect of pH

The maximum fluorescence of ARS is greatly affected by the pH of the solution.⁴² The influence of the pH on ARS–Zn(II) was studied using fluorescence measurements. The nature of the fluorescence profile shown in Fig. 6 could be a result of the stability of the ARS–Zn(II) sensing ensemble. At pH values less than 6, the competition between the OH-proton and the metal ion could occur. Therefore, the ARS–Zn(II) complex was less stable, which prevented H₂S detection. Therefore, the emission intensity does not change so much with H₂S at pH less than 6. Due to the high concentration of hydroxide ions, at a higher pH (pH > 9.0) it is expected that Zn(II) ions may precipitate and consequently, at higher pH the sensing ensembles would not be stable, therefore it may not be suitable for binding studies. These results described that the detection of H₂S using probe ARS–Zn(II) was pH dependent and the maximum change in emission intensity with H₂S was obtained in the pH range of 6.0–8.0.

Fig. 6 Effect of the pH on the fluorescence intensity of the ARS–Zn(II) ensemble and the ensemble with H₂S. [ARS] = 27 μM, [Zn(II)] = 27 μM, [H₂S] = 27 μM in MeOH/PBS buffer (3 : 1, v/v, pH = 7.4).

Recycling sensing performance

The reusability of any sensor is very important for practical application, which requires that the sensor device should be reversible. Therefore, the recycling sensing performance of ARS–Zn(II) was studied through disassembly and reassembly of ARS by H₂S and zinc.

The reversibility study was carried out by monitoring the change in fluorescence of the ARS–Zn(II) ensemble in a buffered solution. The fluorescence intensity was checked after displacing Zn(II) with H₂S (42 μM) and again, adding Zn(II) (30 μM) to ARS (30 μM). As shown in Fig. 7 even after 5 cycles, the change in fluorescence or decrease in fluorescence for the ARS–Zn(II) solution with H₂S was still relatively good. Thus, switching between the fluorescence OFF–ON states could be repeated more than five times without decomposition of the fluorescence indicator ARS dye. Therefore, these results showing the sensor’s high reversibility and favour real-life applications.

Fig. 7 Fluorescence intensity of the ARS–Zn(II) ensemble upon alternate addition of H₂S and Zn(II) [ARS] = 30 μM, [Zn(II)] = 30 μM, [H₂S] = 42 μM.

Fluorescence study of ARS–Zn(II) with H₂S in serum

After confirming the sensitivity and selectivity of the probe in buffer solution, we have decided to explore the practical application of the probe for the quantization of H₂S, in human serum (HS) and bovine serum albumin (BSA).

As shown in Fig. 8, the background fluorescence of ARS–Zn(II) with blank HS and BSA did not change much. However, upon addition of H₂S spiked (6–30 μM) HS or BSA, the fluorescence intensity of the probe ARS–Zn(II) changed significantly. A linear calibration curve can be found between the change in fluorescence intensity at 620 nm and the concentration of H₂S. The change in fluorescence intensity at 620 nm increased in a linear fashion with a higher percentage of H₂S in the serum solutions (SI Fig. 12†). This calibration curve could be useful for estimating the concentration of H₂S in biological fluids. These results confirmed that probe ARS–Zn(II) can be used to detect H₂S rapidly and quantitatively in biological samples.

Fig. 8 Fluorescence intensity of the ARS–Zn(II) ensemble upon addition of H₂S spiked (6 μM, 12 μM, 18 μM, 24 μM, and 30 μM) buffer, human serum and BSA.

Real time monitoring of H₂S from donor molecules

Recently, the development of novel H₂S donors has become a rapidly growing field, because synthetic H₂S donors are useful research tools as well as potential therapeutic agents.^6,43 These donor molecules release H₂S via different mechanisms with different rates. Therefore it is important to understand the rate of H₂S release from donor molecules. Various methods have been used to determine the concentration of H₂S released by these donor molecules. Fluorescence assaying is one such promising method; in this regard researchers are always looking for better probes that use fluorescence techniques to measure and monitor the release of H₂S from donors. The present probe ARS–Zn(II) has high selectivity, sensitivity and quick response time for the detection of H₂S in biological conditions. Thus, we have decided to make use of this new probe to monitor the H₂S release from a donor molecule (Scheme 2).

Scheme 2 Sensing mechanism for the detection of H₂S released from an H₂S donor molecule.

In this regard, we have prepared the H₂S donor benzoic (methyl carbonic) dithioperoxyanhydride as described in the literature.⁴⁴

Our aim was to examine the feasibility of using ARS–Zn(II) to monitor in situ the production of H₂S from donor molecules. In a typical experimental setup, the sensing ensemble ARS–Zn(II) (1 mM, 100 μL of the stock solution) in phosphate buffer (134 μL) was mixed with GSH (0.1 M, 6 μL of the stock solution). Then, the H₂S donor (1 mM, 60 μL of the stock solution in THF) was added just before starting the fluorescence measurement. The time dependent fluorescence measurement was performed using a Synergy HT micro plate reader at 530/645 nm as the excitation/emission wavelength for a 30 minute period of time.

As shown in Fig. 9, ARS–Zn(II) could determine the H₂S release associated with the decomposition of the donor in the presence of GSH by using a multimode micro plate reader. The amount of H₂S released was expressed as a relative fluorescence unit (F/F₀). The fluorescence assay showed that the spontaneous release of H₂S by donor molecules was easily monitored by the new ARS–Zn(II) ensemble probe based on fluorescence changes.

Fig. 9 H₂S release monitored by the ARS–Zn(II) (2 μM) ensemble with H₂S donor (20 μM) and GSH (200 μM) at 35 °C.

We have also assessed H₂S release assays in a temperature-dependent experiment by varying the assay temperature from 30 to 50 °C (SI Fig. 13†). We found that the rate of the H₂S release from the donor was slightly faster at higher temperatures; it confirmed the stability of the probe at higher temperatures. Hence, the results demonstrated that the H₂S release from donor molecules can be monitored by probe ARS–Zn(II) in real time.

The spectroscopic properties of ARS–Zn(II) and its selectivity for H₂S appeared to be suitable for live cell image studies. Thus we have used cultured C6 cell lines to investigate the potential application of ARS–Zn(II) for the detection of H₂S in biological cells. As shown in Fig. 10, C6 cells images were recorded after incubation with compound ARS for 30 min, and we did not observe any fluorescence signal. But strong fluorescence in the cells was induced after the addition of zinc and the fluorescence disappeared upon treatment with H₂S. This result proves that probe ARS–Zn(II) can be used for the detection of H₂S in cultured cells.

Fig. 10 Fluorescence images of C6 cell lines. (a) DIC image of C6 cells treated with ARS (30 μM) only. (b) Fluorescence image of cells in the red channel treated with ARS (30 μM) for 20 min at 37 °C. (c) DIC image of cells treated with ARS (30 μM) and then treated with 30 μM of Zn(II) ions. (d) Fluorescence image of cells in the red channel upon treatment with 30 μM of ARS and then 30 μM Zn(II) ions for 20 min at 37 °C. (e) DIC image of cells treated with 30 μM of ARS and then treated with 30 μM of Zn(II) ions followed by the addition of Na₂S. (f) Fluorescence images of cells in the red channel upon treatment with 30 μM of ARS and then treated with 30 μM of Zn(II) ions followed by the addition of 44 μM of Na₂S for 20 min at 37 °C. Images were taken at λ_ex = 543 nm with a 40× objective.

Table 1 List of zinc(II) based probes for the detection of H₂S available in the literature^a

Sensor probe	LOD	Medium	Detection time	Reference
a LOD = limit of detection, ND = not determined.
8-Aminoquinoline based Zn-complex	1.2 μM	CH3CN–Tris–HCl	2 min	40
7-Mercapto-4-methylcoumarin based Zn-complex	1 μM	Acetone–HEPES	ND	39
4-Methyl-2,6-diformyl phenol based Zn-complex	μM level	Ethanol–Tris–HCl	ND	41
ARS–Zn(II)	92 nM	Methanol–PBS	<1 min	Present work

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Conclusions

In summary, we have reported an Alizarin Red S based fluorescent sensing ensemble ARS–Zn(II) for the selective detection of H₂S in aqueous solution. The probe displayed good selectivity and remarkable sensitivity. Compared to other reported probes based on zinc complexes for the detection of H₂S (Table 1), we see that the present probe has a number of advantages as listed below: (a) Probe ARS–Zn(II) is commercially available and very cheap, therefore this strategy eliminated the need for multi-step synthesis, purification and characterization by sophisticated analytical instrumentations, (b) longer excitation (λ_exc = 530 nm) and emission (λ_em = 625 nm) wavelengths are more suitable for biological purposes, (c) fast response (<30 seconds) time with H₂S, (d) high detection limit (92 nM), (e) the reversible ON–OFF–ON fluorescence change mode with H₂S and zinc makes ARS–Zn(II) a recyclable probe for H₂S, (f) suitable for real time detection of H₂S released from H₂S donor molecules. In addition, the fluorescence response with H₂S is also applied in live cell imaging. We anticipate that sensing ensemble ARS–Zn(II) based fluorescence assays will be used in future as a powerful tool to monitor the release of H₂S from biological and pharmacological samples.

Acknowledgements

We acknowledge the Director and Head of the Chemistry Department, NIT Kurukshetra, Haryana for the research facilities. The authors also thank Prof. Manoj Kumar for his support in live cell images study. DAJ acknowledges the financial support of the Department of Science and Technology (DST), India, for the SERB-DST project grant SB/FT/CS-195/2013. AG is thankful to the financial support of the DST, India, for the SERB-DST young scientist research project grants (SB/FT/CS-193/2013).

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Footnote

† Electronic supplementary information (ESI) available: Synthesis of the H₂S donor, experimental methods and some UV-Vis and fluorescence spectra are available. See DOI: 10.1039/c5ra11901d

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