Selective Detection of H2S by Copper Complex Embedded in Vesicles

bDepartment of Science & Humanities, Karunya Institute of Technology & Sciences Coimbatore, Tamil Nadu (INDIA). KEYWORDS: Hydrogen sulfide, Metal ...
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Selective Detection of HS by Copper Complex Embedded in Vesicles through Metal Indicator Displacement Approach Rahul Kaushik, Rahul Sakla, Amrita Ghosh, G. Tamil Selvan, P. Mosae Selvakumar, and D. Amilan Jose ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.8b00174 • Publication Date (Web): 01 Jun 2018 Downloaded from http://pubs.acs.org on June 1, 2018

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ACS Sensors

Selective Detection of H2S by Copper Complex Embedded in Vesicles through Metal Indicator Displacement Approach Rahul Kaushik,a Rahul Sakla,a Amrita Ghosh,a G.Tamil Selvan,b P. Mosae Selvakumar,b and D. Amilan Jose*a a

Department of Chemistry, National Institute of Technology (NIT) Kurukshetra, Kurukshetra-136119, Haryana, India

Email: [email protected] b

Department of Science & Humanities, Karunya Institute of Technology & Sciences Coimbatore, Tamil Nadu (INDIA) KEYWORDS: Hydrogen sulfide, Metal Indicator Displacement Approach, Phospholipid Vesicles, Colorimetric, Fluorescent

ABSTRACT: A new approach for the detection of Hydrogen sulfide (H2S) was constructed within vesicles comprising phospholipids and amphiphilic copper complex as receptor. 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) vesicles with embedded metal complex receptor (1.Cu) sites have been prepared. The vesicles selectively respond to H2S in a buffered solution and show colorimetric as well as spectral transformation. Other analytes such as reactive sulphur species, reactive nitrogen species, biological phosphates and other anions failed to induce changes. The H2S detection is established through a metal indicator displacement (MIDA) process, where Eosin-Y (EY) was employed as an indicator. Fluorescence, UV-visible spectroscopy and the naked eye as the signal readout studies confirm the high selectivity, sensitivity and lower detection limit of the vesicular receptor. The application of vesicular receptor for real sample analysis was also confirmed by fluorescence live cell imaging. Hydrogen Sulfide (H2S) has been recognized as one of the three gasotransmitters along with nitric oxide (NO) and carbon monoxide (CO).1-2 The physiological and therapeutic importance of H2S, leading to a quick progress in research activity involving H2S.3-6 Considering the complex biological functions7-8, real-time and selective detection of endogenous H2S is necessary to expand the knowledge about its exact physiological and pathological role. But due to its volatile and reactive nature, the accurate detection of H2S heavily hampered by sample preparation and detection methods. Classical methods such as gas chromatography, atomic absorption spectroscopy (AAS), and electrochemical methods are available for the detection and estimation of H2S. However, due to harsh working conditions and tedious sample preparation, these methods are highly disadvantageous for biological samples. On the other hand, UV-Vis and emission spectroscopy methods are highly reliable due to easy sample preparation, better sensitivity and quick sample analysis time. Therefore, several chemical sensors have been recently reported for the detection H2S using change in fluorescence and Uv/Vis spectral behavior.8-19 Most of the known H2S sensors work mainly based on the H2S specific reactions such as reduction reaction 20-29 and nucleophilic attack.23, 30-34 These strategies have their own limitations such as low water solubility and time-consuming response.35-36 As an alternative approach, we and others have used Metal displacement approach (MDA).18, 37-40 It is promising and overcomes almost all the disadvantages shown by reaction based H2S sensors. MDA is based on the formation of metal sulfide due to a high binding constant of metal sulfides. Similar to MDA, Metal Indicator displacement approach (MIDA) is another approach, in which both metal and indicator get displaced upon binding of H2S with metal centre (Scheme 1). Nevertheless, MIDA based sensor for the detection of H2S in vesicles not known in literature till date.

Scheme 1: Schematic representation of (A) Metal Displacement approach (MDA) and (B) Metal indicator displacement approach (MIDA). Functionalized artificial membranes/bilayers with embedded chemical sensors represent a special class of chemosensors4142 . The artificial bilayers mimic the functions of biomembranes which are known to play a key role in different biological processes associated with the transmitting of signals across cell membranes. The use of functionalized luminescent vesicles for the detection of various biological analytes is known43-45. But in the literature, to the best of our knowledge, the vesicles embedded receptors for the real-time detection of H2S is not known. Vesicles are the perfect platform for the detection of endogenous analytes due to their monodisperse nature and facile preparation. We envisaged that the design of vesicular receptors with MIDA would allow us to replace the multistep synthesis of signalling group attached receptors and also improve bio-compatibility that would certainly broaden their applicability. In this paper, we have reported a different approach by embedding a copper complex functioning as an H2S reporter, along with an indicator dye in the phospholipid vesicles (135 ± 10 nm) for the detection of H2S. The analyte binding at the interface removes the metal in the form of metal sulphide. This results in an expulsion of the indicator and metal from the

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vesicular receptors, thereby triggering an optical response. For the first time, we have introduced the MIDA in the vesicular surface for the selective and sensitive detection of H2S in the pure aqueous medium. Result and discussion To achieve this, we have prepared DPA (Di-(2picolyl)amine) based new amphiphilic copper complex (1.Cu). 1.Cu was embedded into the phospholipids 1,2-distearoyl-snglycero-3-phosphocholine (DSPC) to form vesicular receptor Ves-1.Cu (Chart 1). DPA based Copper complexes are known as prominent receptors to detect H2S due to very low solubility product of CuS (Ksp = 6.36 x10-36).18 Majority of the copper complex based H2S sensors are working in semi-aqueous system that may not be suitable for the detection of H2S in biological system.12 Therefore, we have prepared amphiphilic 1.Cu, that could be incorporated in vesicles surface to work in pure aqueous environment.

indicator. After screening numbers of indicators, EY was selected due to the high water solubility, high quantum yield and ability to bind with copper ions. The characteristic fluorescence emission of EY quenches after binding to copper with the carboxylate group49. Also, EY is not having any binding effect with H2S even after addition of 1000 equivalents (Figure S6-A). Therefore, EY could be an ideal choice as an indicator for the displacement assay in Ves-1.Cu solution.

Chart 1: Structures of 1.Cu, DSPC and Indicator EY. Water-insoluble 1.Cu could be incorporated into the lipids by a simple protocol to give a clear vesicular solution in water at the physiological condition. Lipid bilayers vesicles embedded copper complex (Ves-1.Cu) was prepared by using commercially available synthetic lipid DSPC and amphiphilic complex 1.Cu (25 mol% with respect to used DSPC) in HEPES buffer (pH 7.4, 25 mM) by the well-known filmhydration method.46 The resulting heterogeneous liposomes Ves-1.Cu was homogenized by extrusion method through polycarbonate membranes to yield homogeneous small Ves1.Cu. Homogenized vesicular dispersions were assumed to be free from impurities; therefore, the vesicles solution was not further purified. The particle size distribution of the vesicles Ves-1.Cu was examined by dynamic light scattering (DLS) method. The average size of the receptor incorporated liposomes was found to be 135 ± 10 nm (Figure S4). The dilution of the Ves-1.Cu samples by water did not have much effect on the average size of the Ves-1.Cu as determined by DLS (Figure S5).The prepared vesicle dispersions were stored under dark at 10ºC and used within a week for the sensing study. A convenient approach to detect analytes by using nonfluorescent/colorimetric probes is the indicator displacement approach, which is based on the principle of competitive binding of an indicator and an analyte to the receptor.47-48 Here, we have selected Eosin-Y (EY) as a fluorescent and colorimetric

Figure 1: (A) UV-Vis titration of EY (2.5 ×10⁻6 M) with Ves1.Cu (0-6.4×10⁻6 M) in aqueous medium (phosphate buffer, 20 mM, pH 7.4). (B) Emission titration of EY (2.5 ×10⁻6 M) with Ves-1.Cu (0-6.4×10⁻6 M) in aqueous medium (phosphate buffer, 20 mM, pH 7.4).

Vesicular copper complex and indicator ensemble Ves1.Cu-EY was prepared by mixing Ves-1.Cu and EY in aqueous buffer solution. The formation of sensing ensemble Ves1.Cu-EY was confirmed by UV-Vis and fluorescence titration experiments. EY (2.5 ×10⁻6 M) initially shows absorbance peak at 515 nm (ϵ =82,600 mol-1cm-1) in water (phosphate buffer, 20 mM, pH 7.4). After the successive addition of Ves1.Cu (0-6.4×10⁻6 M) to the EY solution, ratiometric change in absorption spectrum was observed with the decrease in 515 nm peak and increase in peak at 535 nm with about 20 nm red shift (Figure 1A). The color of the EY solution changed from pale pink to dark pink, which could be easily detectable by naked eye. In case of the fluorescence spectrum, EY shows strong emission intensity at 540 nm (λexc = 515 nm). However, the emission intensity is decreased and quenched upon addition of Ves-1.Cu (Figure 1B). The change in emission and UvVis absorption spectrum is due to the interaction between vesicles embedded copper complex and carboxylic acid group of the EY. The binding constant (KsV) for Ves-1.Cu to the EY calculated as 9.09 x 102 M-1 in phosphate buffer (20 mM, pH

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ACS Sensors 7.4). The UV-Vis and the fluorescent study confirm the formation of Ves-1.Cu-EY ensemble in aqueous medium. as CuS, consequently the indicator dye EY get displaced from the system and recovered the original absorption spectrum (scheme 2).

Scheme 2: Schematic representation of H2S sensing using Ves-1.Cu-EY through MIDA approach.

Figure 2: (A) Selectivity studies of Ves-1.Cu-EY with different anions such as reactive sulphur species (glutathione, cysteine, methionine, S2O32¯), reactive nitrogen species (NO, NO2¯ and N3¯), biological phosphates (ATP, ADP, AMP, PPi, H2PO4¯ and HPO42¯) and other anions (F¯, Cl¯, Br¯, I¯, and SO42- ) using UV-Vis spectroscopy in aqueous buffer medium (phosphate buffer, 20 mM, pH 7.4) (B) Absorbance titration of Ves-1.Cu-EY ensemble with H₂S (0- 2.5 × 10⁻5 M) in aqueous medium.

The use of copper complex in IDA (indicator displacement approach) is familiar for the detection of anionic analytes such as PPi and ATP50-51. However, vesicle embedded copper complex for the detection of H2S using IDA is not known. Consequently, we have checked the ability of new ensemble Ves1.Cu-EY for the detection of H2S in aqueous phosphate buffer solution. For all the experiments Na2S has been used as a source for H2S gas. The change in Uv-Vis spectra of Ves1.Cu-EY was monitored in the presence excess amount (10 equivalent) of biologically important analytes such as reactive sulphur species (glutathione, cysteine, methionine, S2O32¯) reactive nitrogen species (NO, NO2¯ and N3¯), biological phosphates (ATP, ADP, AMP, PPi, H2PO4¯ and HPO42¯) and other anions (F¯, Cl¯, Br¯, I¯, and SO42-). As shown in Figure 2A the spectrum drastically changes only with the H2S. Other analytes exhibited an insignificant change in the absorbance spectrum. The Uv-Vis absorption spectrum (λmax = 515 nm) of ensemble Ves-1.Cu-EY with H2S matches perfectly with the absorption spectrum of EY. Copper ions are known to react with sulphide to form stable CuS species,52 which has low solubility products constant (Ksp) = 1.6 x 10-24. So it was expected that, in the presence of H2S, the copper ion removed from the Ves-1.Cu-EY ensemble

The sensitivity of H2S binding was determined via systematic titration by successive addition of H₂S (0- 2.5 × 10⁻5 M) to the Ves-1.Cu-EY ensemble (Figure 2B). Subsequently, the reversibility in the absorption spectrum was observed by restoration of the absorbance peak at 515 nm. As shown in Figure 3, H2S detection could also be monitored by the naked eye, through a prominent color change from dark pink to pale pink. Other analytes failed to induce any color change (Figure 3B). Time dependent study shows that H2S detection was fast and instantaneous (Figure S6-B). Real-time detection is very important for the practical application, consequently, the quick response time of the present sensing ensemble has the advantage over other probes working based on the chemical reactivity of H2S.

Figure 3: (A) Fluorescence change under UV light and (B) naked eye color change; of Ves-1.Cu-EY with different anions (10 equivalents) in phosphate buffer (pH 7.4).

The selectivity of the Ves-1.Cu-EY was also tested in the presence of interfering species such as relevant biothiols and biological phosphates. The competitive experiments revealed that the negligible interference or no interference was observed in the co-existence of various species and H2S (Figure S7). Accordingly, the probe Ves-1.Cu-EY 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 predicts the possible use of the probe even when H2S co-exists with other biological relevant biothiols like glutathione, cysteine, methionine and phosphates.

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Figure 4. (A) Selectivity studies of Ves-1.Cu-EY with different anions such as reactive sulphur species (glutathione, cysteine, methionine, S2O32¯), reactive nitrogen species (NO, NO2¯ and N3¯), biological phosphates (ATP, ADP, AMP, PPi, H2PO4¯ and HPO42¯) and other anions (F¯, Cl¯, Br¯, I¯, and SO42- ) using fluorescence spectroscopy in aqueous buffer medium (phosphate buffer, 20 mM, pH 7.4; λexc = 515 nm) (B) Emission titration of Ves-1.Cu-EY ensemble with H₂S (0- 2.5 × 10⁻5 M) in aqueous medium (phosphate buffer, 20 mM, pH 7.4; λexc = 515 nm).

EY dye is well known for its characteristic fluorescent behaviour; therefore we have recorded the emission spectrum of Ves-1.Cu-EY in the presence and absence of H2S and other analytes. EY has strong emission at 540 nm (λex. = 515 nm) but after binding with Ves-1.Cu it shows very weak emission (Figure 3A). The weak emission peak observed at 540 nm for Ves-1.Cu-EY was increased only in the presence of H2S. Other analytes did not show any change in emission intensity (Figure S8). This is due to the displacement of Cu as CuS and thereby removal of EY from the ensemble. The limit of detection (LOD) for H2S was calculated as 4.06 µM from systematic fluorescent titration (Figure 4A). The observed LOD (Figure 4B) is lower as compared to polymer-based/nanomaterials based probes reported for the detection of H2S.53-59 The LOD is also comparable or better than other small molecule-based probes reported for the H2S detection (table S1). Time-resolved fluorescence studies were carried out to determine the emission decay parameters for EY, Ves-1.CuEY, Ves-1.Cu-EY with H2S (Figure S9). EY exhibited a lifetime τ1=1.17 ns (Quantum yield = 0.236) with single exponential decay. The ensemble probe displayed a lifetime of τ1=1.37 ns and τ2=2.74 ns (Quantum yield = 0.087) with biexponential decay. However, in the presence of H2S (2.5

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×10⁻ 5 M), the lifetime was measured as τ1 =1.04 ns and τ2 = 2.06 ns (Quantum yield = 0.213). The recovery of EY lifetime (τ1) and quantum yield further confirms the displacement of dye from the vesicular surface due to the formation of CuS via MIDA. Detailed UV-Vis and fluorescence studies ensured that the detection of H2S could be done with high sensitivity using Ves-1.Cu-EY sensing ensemble. Further, we have also used Ves-1.Cu-EY to monitor the release of H2S from the donor molecule benzoic (methyl carbonic) dithioperoxyanhydride.39 Time dependent fluorescence measurement was performed at 530/645nm as excitation/emission wavelength for 30 minutes at RT. The kinetic study showed that the spontaneous release of H2S by donor molecules easily monitored by the probe Ves-1.Cu-EY based on fluorescent change (Figure S20). This experiment demonstrated that the use of Ves-1.Cu-EY for the detection and monitoring of released H2S from donor molecules. The morphology of vesicles was investigated under the light microscope. The Ves-1.Cu are spherical in shape and well separated from each other (Figure S10). The ensemble Ves1.Cu-EY alone did not show any fluorescence property in light microscope but upon binding with H2S the vesicles solution show fluorescent behaviour due to the displacement of copper and existence of free EY dye unit (Figure S11). DLS measurement of Ves-1.Cu-EY was also recorded in the absence and presence of H2S. In the presence of H2S, the particle size distribution shows two different size particles in the solution such as 200 nm and about 1050 nm. The higher size particle may be attributed to the CuS precipitation. These results also confirm the MIDA mechanism on the vesicular surface (Figure S4).

Figure 5: (A) Fluorescence microscopic images of C2C12 mouse skeletal muscle fixed cell lines with EY (2.5 µM) only (B) With Ves-1.Cu-EY. (C) After treatment with H2S (37 µM). C2C12 mouse skeletal muscle fixed cell lines with 50−60% confluency using FITC UV filter with a total magnification of 100 X.

H2S is synthesized both enzymatically and nonenzymatically in the human body; therefore, it is highly important to detect H2S in the real biological samples.15, 60-61 To explore the efficiency of the Ves-1.Cu-EY ensemble in real biological sample, live cell imaging study was performed. The cell images were captured using mouse skeletal muscle cells. Fluorescence images of cells show that the cells were highly fluorescent in the presence of EY (2.5×10⁻6 M). Consequently, when the cells were treated with vesicles Ves-1.Cu the decrease in fluorescence intensity was observed due to the formation of non-fluorescent ensemble Ves-1.Cu-EY. Further, when the cells were incubated with H2S, the fluorescence intensity of the cell lines was recovered. This showed that the MIDA mechanism is well supported by the live cell imaging experiment and the Ves-1.Cu-EY ensemble could be used for the detection of H2S in a biological sample at the cellular level (Figure 5).

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ACS Sensors As of now, we have performed the whole H2S binding studies in phosphate buffer. However, inorganic and biological phosphates are known for the binding with copper complex.45, 62 Therefore we have investigated the effect of the buffer by replacing phosphate buffer with HEPES buffer. Uv-Vis and fluorescent results suggest that the probe Ves-1.Cu-EY not very selective for H2S in HEPES buffer, as it is also displayed some changes with PPi, GSH and Cys. The emission spectra showed 15, 8, 4 and 2 fold decrement in emission intensity with H2S, Cys, GSH and PPi respectively (Figure S12). Therefore, it is evident from Uv-vis and emission studies that Ves1.Cu-EY ensemble shows less selectivity (Figure S13-S14). However, the calculated LOD (0.59 µM) suggested that HEPES buffer is useful for sensitive detection of H2S (Figure S15-S18).The LOD calculated by using HEPES buffer is 7 times lesser than the phosphate buffer. The effect of different buffer, lipids and complex loading on H2S binding study is in progress, these results will be published in due course of time. Conclusion In summary, a new strategy for H2S detection was developed through a MIDA process on nano vesicular surface. Where, Cu(II) act as a binding site and EY act as an indicator for detection mechanism. The developed vesicular ensemble showed high selectivity and sensitivity for H2S. The practical application of this vesicular probe was proved by the biological cell imaging study. The Ves-1.Cu- EY ensemble showed a number of advantages over other reported probes. Most of the reported H2S sensors work semi-aqueous environment and lacks applications in biological samples. On the other hand, vesicles as receptor provide a pure aqueous environment at physiological pH with a better sensitivity. The vesicular platform also provides the liberty to modulate the lipid membrane depending upon the cellular environment in the biological sample. However, no such advantage was provided by small molecular probes. Vesicles embedded with copper complex based on MIDA approach opens up the platform to modulate the emission and excitation wavelengths with suitable indicator dyes.

ASSOCIATED CONTENT Supporting Information. Experimental Sections, General procedure for spectroscopic measurements, Uv-Visible absorption, Life time and Fluorescence studies; DLS analyses and color change images are available in the supporting information. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author Email: [email protected]

Author Contributions The manuscript was written through contributions of all authors.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT

The authors acknowledge Department of Chemistry, NITKurukshetra for the lab facilities. We are thankful to Dr. Ashwani Mittal, Biochemistry Department, University College, Kurukshetra University for Fluorescence microscopic images. DAJ acknowledge the financial support for the SERB-DST, New Delhi project grant SB/FT/CS-195/2013 and CSIR grant No.01 (2855)/16/EMR-II. AG thankful to the financial support of DST, India for the SERB-DST young scientist research project grants (SB/FT/CS-193/2013). RS acknowledges DST-Haryana for a HSCST research fellowship.

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