Effect of sulfur doping on graphene oxide towards amplified fluorescence quenching based ultrasensitive detection of hydrogen peroxide

Journal Pre-proofs Full Length Article Effect of sulfur doping on graphene oxide towards amplified fluorescence quenching based ultrasensitive detection of hydrogen peroxide Ayesha Saleem Siddiqui, Akhtar Hayat, Mian Hasnain Nawaz, Muhammad Ashfaq Ahmad, Muhammad Nasir PII: DOI: Reference:

S0169-4332(19)33511-1 https://doi.org/10.1016/j.apsusc.2019.144695 APSUSC 144695

To appear in:

Applied Surface Science

Received Date: Revised Date: Accepted Date:

26 June 2019 22 October 2019 13 November 2019

Please cite this article as: A. Saleem Siddiqui, A. Hayat, M. Hasnain Nawaz, M. Ashfaq Ahmad, M. Nasir, Effect of sulfur doping on graphene oxide towards amplified fluorescence quenching based ultrasensitive detection of hydrogen peroxide, Applied Surface Science (2019), doi: https://doi.org/10.1016/j.apsusc.2019.144695

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Effect of sulfur doping on graphene oxide towards amplified fluorescence quenching based ultrasensitive detection of hydrogen peroxide Ayesha Saleem Siddiquia,b, Akhtar Hayatb, Mian Hasnain Nawazb, Muhammad Ashfaq Ahmada*, Muhammad Nasirb* a Department

of Physics, COMSATS University Islamabad, Lahore Campus, 1.5 KM Defence

Road, Off Raiwand Road, Lahore, Punjab 54000. b Interdisciplinary

Research Centre in Biomedical Materials, COMSATS University Islamabad,

Lahore Campus, 1.5 KM Defence Road, Off Raiwand Road, Lahore, Punjab 54000.

Email of Corresponding Authors: Muhammad Ashfaq Ahmad: [email protected] Muhammad Nasir: [email protected]

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Graphical Scheme Graphical illustration of the fluorescence quenching mechanism catalyzed by sulfur doped graphene oxide in the presence of hydrogen peroxide.

Highlights: 

Graphene oxide was doped with sulfur to tailor its chemical, electronic and optical properties.



Sulfur doped graphene oxide facilitates decomposition of H2O2 by hydroxyl radical generation for robust oxidation of Rhodamine-B.



Sulfur doped graphene oxide catalyzed oxidation of Rhodamine-B results into: ⅰ) amplified fluorescence quenching and ⅱ) ultrasensitive hydrogen peroxide detection.



Enhanced catalytic activity of sulphur doped graphene oxide leads to low limit of detection of 100 pM with the linear range 1 nM–1 μM.

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Keywords: Sulfur doping; graphene oxide; catalytic activity, Rhodamine-B; Hydrogen peroxide; Fluorescence quenching Abstract: Fluorescence quenching of fluorescent dyes by metals, metal oxides or carbon nanomaterials is a trending technique for elemental inspection of nanoscale physical and biochemical processes. Development of graphene oxide-based fluorescence sensors has become a hot spot of research for biomedical applications owing to its profound optical, electrical and sensing properties. Chemical doping of graphene oxide leads to upgradation of its intrinsic features. Hence, it is of great interest to investigate doped counterparts of graphene oxide for credible optical and biosensing. In view of this, we herein report successful sulfur doping of graphene oxide with superb catalytic activity towards enhanced fluorescence quenching and ultrasensitive detection of hydrogen peroxide. Results evidence that sulfur doping via hydrothermal treatment greatly influences the surface chemistry, functionalities and thus the catalytic performance of graphene oxide. The high quenching efficiency of sulfur doped graphene oxide can be attributed to its capabilities of good adsorbability and high electron mobility that substantially catalyzes the decomposition of hydrogen peroxide into -OH radicals, consequently quenching the fluorescence of Rhodamine-B efficiently. Characterization tools such as XRD, SEM, FTIR, UV-Vis, XPS, RAMAN spectroscopy were used to investigate the surface chemistry and morphology and chemical composition of the prepared samples. Different parameters such as concentration of dye, amount of dopant, concentration of particles etc.; were explored for ultrasensitive detection of hydrogen peroxide. Under optimal conditions, the limit of detection (LOD) for hydrogen peroxide was as low as 100 pM with linear detection range from 1 nM to 1 µM. Hence, based on our findings, we propose a highly desirable sensitive and selective H2O2 detection method by virtue of high binding affinity and quenching ability of SGO. 3

1. Introduction Hydrogen peroxide (H2O2) is an important biomolecule, frequently produced as a highly reactive intermediate in chemical/biological reactions [1] and is extensively utilized in cosmetics, disinfectants, detergents, rocketry, mining, textile, food manufacturing [2], oxidizing and bleaching applications [3, 4]. Although generally present in trace amounts in rain and snow, canned food materials, and our own immune system; its higher concentrations or long-term exposure can cause explosions or corrosion to skin and metals [5, 6] and health problems like human hair bleaching, aging, respiratory inflammation, poisoning; or acute diseases like cancer, Alzheimer’s and other neurological disorders [7]. Hence, it is crucial to develop ultrasensitive method for the determination of H2O2. Most of the currently employed methods for H2O2 detection such as high performance liquid chromatography, electrochemistry, chemiluminescence are usually expensive and require sophisticated analytical techniques [5, 8, 9]. On the other hand, the fluorescence spectrophotometry offers selective and sensitive detection of specific analytes using simple preparation methods [8]. Fluorescence quenching, a special type of fluorescence assay, refers to an event in which the observed fluorescence signal is decreased [10] basically due to the nonradiative relaxation of an electronically excited state either by energy or electron transfer [11]. Fluorescence quenching of organic compounds provides useful information about the dynamics of biochemical systems [12]. Graphene oxide is undoubtedly emerging as one of the most promising nano materials [13, 14] exhibiting excellent analytical performance for biomolecular detection [15] owing to its exceptional optical and physiochemical properties [16-22].The oxygenated functional groups on the basal planes and edges of the GO sheets are responsible for the good dispersibility [23, 24]

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high adsorption capacity [25] and providing reactive sites for chemical conjugation or functionalization [8]. Owing to its distinctive optical absorption properties [25], GO exhibits unique fluorescence-quenching capability [17, 26] when organic fluorescent molecules adsorb on its surface by π-π stacking interactions [19, 20]. It acts as an excellent energy acceptor or mediator in energy transfer mechanisms, thereby enhancing the systems optical efficiency [14, 27, 28]. Investigation of GO with respect to quenching of fluorophores is especially interesting, because the photo physical feature of GO makes it a powerful dye quencher [29]. It can act as an active substrate to devise different kinds of fluorescent assays for the detection of proteins, cells, and other biomolecules [26, 30]. The GO–dye composite is therefore proposed for the selective detection of biomolecules [31]. Despite of its worthwhile intrinsic properties, surface modification [30] by heteroatom doping into GO sheets is an effective approach to elevate the electronic structure and chemical properties of GO [18, 19, 25] for various electronic, sensing and biomedical applications [13, 23, 32]. Upon incorporation of heteroatoms, adjacent carbon atoms are activated and cause active edge and structure defects in the lattice network of GO [18, 19] which then augment the catalytic activities and chemical reactions. The surface area, conductivity [28], chemical stability and catalytic properties of GO can be effectively enhanced by the revision [18] of trapping sites for electrons depending on electron-donating [25] or electron-withdrawing groups of dopants [22][33]. Among the different heteroatoms, doping GO with electron-withdrawing sulfur has drawn relentless interests [13, 22, 34, 35]. The S atoms in the hexagonal graphene lattice play crucial roles as the sulfur functional groups improve the electron donating nature of the GO, thereby improving its chemical activity [36]. The potential catalytic centers are the defect sites since sulfur and carbon share similar electronegativity [35, 37]. Sulfur doping of GO is supposed to occur by

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the formation of defect sites and bond rapture [36]. The effective mechanism of sulfur doping may be attributed to the customized polarization and electron density [38], which enhances the catalytic activity of SGO hence making it favorable for surface science and sensing applications [39, 40]. To the best of our knowledge, this is the first report investigating the catalytic activity of sulfur doped graphene oxide towards efficient fluorescence quenching and sensitive detection of H2O2. This work demonstrates the role of the sulfur heteroatom in the surface area and chemical reactivity of GO. SGO can potentially interact with the dye (Rhd-B) and H2O2 owing to its good adsorbing and catalytic abilities. The generation of hydroxyl radicals, upon addition of H2O2, quenches the fluorescence of Rhd-B catalyzed by SGO. Both surface morphology and chemical nature of the SGO contribute to the excellent quenching efficiency and sensitivity of the method. The low cost, facile synthesis and stability of SGO encourages further research on this kind of catalyst material for biomedical applications in future. 2. Experimental Details: 2.1. Reagents and materials. Graphite and Sodium sulfide were received from DAEJUNG. Sulfuric acid 97% and Pluronic F127 were bought from Sigma Aldrich and Hydrochloric acid 98% was from Analar. RhodamineB was purchased from Avonchem. Hydrogen peroxide (29.32% of H2O2) was obtained from Merck. All the routine solutions were prepared in phosphate buffer saline (PBS at pH-7.4); using PBS tablets 10mM, from bio-World, in Deionized water, obtained from Elga PureLab Ultra Water Deionizer. Disposable cuvettes of 20 mM having capacity of volume 4 mL were purchased from Kartell. All the reagents were of scientific analytical category and utilized as received. 2.2. Synthesis of Graphene Oxide.

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Graphene oxide was synthesized by Modified Hummer’s method, followed by the oxidization of graphite powder. In short, graphite powder (2.0 g) was added into concentrated H2SO4 (100 mL), mixture was stirred and allowed to cool to 5 °C in an ice bath. The temperature of the mixture was kept below 5 °C for 30 min. KMnO4 (8.0 g) was then gradually added under constant stirring, to avoid rapid heat evaluation, such that the temperature of the mixture remains less than 10 °C. 100 mL distilled water was added into the mixture under stirring and further diluted up to 300 mL with distilled water after 1 hr. Finally, 30 % H2O2 (20 mL) was added to the mixture to terminate the reaction. The resultant residue was washed several times with 5 % HCl aqueous solution (800 mL) and then with distilled water to remove the residual metal ions till the pH reaches 6. The product dried at 45 °C for 24 h was dissolved in water and sonicated to exfoliate oxidized graphene. 2.3. Synthesis of Sulfur Doped Graphene Oxide. In a typical synthesis of SGO, GO (1 g/20 mL distilled water) and Pluronic F-127 (1 g/20 mL distilled water) were sonicated separately for 30 min and then added together. Sodium sulfide (Na2S) at varying concentrations (3 mM, 5 mm, 10mM, 12mM) was then added to this GO and Pluronic F-127 mixture and diluted up to 150 mL. The mixture was sonicated for another 1 hr. and then transferred to a 200 mL Teflon-lined stainless-steel autoclave in a hydrothermal reactor at 180 °C temperature for 7 hr. After completion of the reaction, the product was collected by centrifugation followed by rigorous washing with deionized water and was kept in vacuum oven for drying at 45 °C for 24 h. Here Na2S has been used as a source of sulfur, with varying concentrations, to study the effect of sulfur doping in graphene oxide. Samples prepared with 3 mM, 5 mM, 10 mM, and 12 mM of Na2S are named as SGO-3, SGO-5, SGO-10 and SGO-12, respectively. Subsequently, the thus obtained sulfur doped graphene oxide (SGO) was used for further characterization and fluorescence-based sensing. 7

2.4. Characterization. A Cary Eclipse fluorescence spectrophotometer (Agilent Technologies) was used to carry out the fluorescence quenching studies. Absorbance measurements were estimated by UVSpectrophotometer Lambda-25 (UV-25, Perkin Singapore) with a bandwidth setting of 1 nm at a scan speed of 960 nm min-1 in the wavelength range of 200–800 nm. The structure and crystalline phase of GO and SGO was explored using powder X-ray diffraction (XRD) analysis, Cu Ka radiation (Panalytical Xpert Powder Diffractometer) and a diffractive beam monochromator operated at 100 mA, 40 kV. Fourier transform Infrared (FTIR) spectra were recorded using Thermo Fisher Scientific (Nicolet 6700, USA) spectrometer with resolution 8 cm-1 and scan range 4000– 600 cm-1 at the Attenuated Total Reflectance (ATR) mode, for the determination of functional groups present in the synthesized samples. The elemental analysis, shape and surface morphology of synthesized SGO was examined using the Scanning Electron Microscope (SEM, Vega 3, LMU, Tescan). Raman spectroscopy a powerful tool for identifying carbon materials was used (In-Via Raman Microscope (Renishaw UK, Raman & PL setup)) to probe the doping effects in our experiment. The chemical state and the surface functional groups were characterized by using xray photoelectron spectroscopy (XPS PHI 5000 VersaProbe). Park Systems AFM XE7 was used in non-contact mode to observe the surface morphology of modified GO. 2.5. Fluorescence assay by SGO. Quenching efficiency of GO and SGO was evaluated before and after addition of the hydrogen peroxide to the fluorescent dye Rhd-B. Briefly, 400 μL of each of the materials GO/SGO (100 mg.L−1), 40 μL of Rhodamine B (100 µg.L−1) and 3560 μL of PBS (pH-7.4) were blended and fluorescent measurements were carried out with excitation wavelength λex=554 nm and emission wavelength λem=577 nm. Quenching measurements were done within 1 min of mixing. Control 8

measurements were performed similarly with same dye and SGO concentration (100 µg.L−1, 100 mg.L−1) and reaction volume (4000μL). 2.6. Detection of H2O2. To perform the analytical measurements, a simple reaction mechanism was employed in which 400 μL of the SGO (100 mg.L−1), 40 μL of Rhd-B (100 µg.L−1) and series concentrations of H2O2 (1 nM – 1 μM) were added and blended in a reaction mixture of 3560 μL of PBS at pH-7.4. The reaction mixture was incubated for a time interval of 5 min at room temperature to complete the interaction between H2O2, SGO and the dye. Finally, the fluorescence response of the solution (excitation 554 nm; emission 577 nm) was recorded by fluorescence spectrophotometer to gauge the emission intensity in the presence of H2O2. The fluorescence response of ⅰ) 40 μL of Rhd-B (100 µg.L−1) alone, ⅱ) 40 μL of Rhd-B (100 µg.L−1) + 20 μL of H2O2 (1 μM), ⅲ) 40 μL of Rhd-B (100 µg.L−1) in the presence of 400 μL of GO and SGO (100 mg.L−1) and ⅳ) 40 μL of Rhd-B (100 µg.L−1) + 400 μL of SGO (100 mg.L−1) + 20 μL of H2O2 (1 μM); in a total buffer volume of 4000 μL was taken to evaluate the fluorescence quenching in the presence of H2O2. The process of the fluorescence quenching of Rhd-B dye by H2O2 with SGO catalyst is shown in the graphical scheme. 2.7. Selectivity of the assay. The selectivity of the assay was explored by implementing the same assay for the determination of isopropyl, glucose, dopamine hydrochloride, uric acid and ascorbic acid (1 μM each) which are the possible interfering compounds. 3. Results & Discussions: Characterization:

9

Raman spectroscopy is one of the most successful tools for examining the electronic and structural properties i.e. thickness, number of layers, degree of order, defects and doping information in GO [36]. The Raman spectral profile shows characteristic D and G bands Fig. 1A. The D band corresponds to the breathing mode of the sp2 ring of the graphene layer, which is related to a series of defects, such as bond angle disorder, bond length disorder, and hybridization, which are caused by heteroatom doping and structural defects. The G band originates from stokes Raman scattering with a single phonon (E2g) emission which is related to the in-plane bond-stretching motion of the pairs of sp2 carbon atoms. In the spectrum of GO, the characteristic bands were found at 1362 and 1592 cm−1 for the D and G bands, respectively. The G band is sensitive to the chemical doping state and under similar conditions, the G peaks of the SGO down-shifted to 1586 cm−1, from 1592 cm−1 in GO [39]. The n-type doping of GO or adsorption of molecules with electron-donating groups leads to downshifts and stiffening of the G band. The ID/IG ratio is used to estimate the defect of the sample and the in-plane crystallite sizes in disordered carbon materials. The intensity ratio of D-band to G-band (ID/ IG) [38, 41, 42] increased from 0.88 to 1.01 in SGO spectrum . The relatively high ID/IG ratio of the SGO relative to GO could be ascribed to a partial restoration of the graphitic framework and a more structural distortion caused by decoration of sulfur on the GO sheets [43, 44]. Moreover, the FWHM (Full Width at Half Maximum) value of D band increased from 125 to 162 for SGO and in case of G band of SGO it rose to 123 from 113.The broadness of D and G bands further evidences the structural defects due to incorporation of Sulfur.

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A

B

D

C

E

Fig. 1. A. Raman spectra B. XRD pattern C. UV absorption spectra D. FTIR spectra for GO and SGO E. SEM images of a) GO and b) SGO

Further structural information of the pristine GO sheets and the resulting SGO sheets can be obtained from the XRD analysis. The XRD patterns of GO and SGO are shown in Fig. 1B. The d002 values were calculated using Bragg’s equation (nλ = 2 d sinθ, where n = 1, λ is the wavelength, 1.5406 Å). After the oxidation of natural graphite by modified Hummers’ method, the (002) reflection peak shifts to 2θ = 23.7°(d-spacing ~3.7) from the 2θ = 26.6° owing to the formation of oxygen-containing functional groups between the layers of the graphite and the presence of intercalated water molecules. Whereas SGO sheets exhibit a well defined peak at 2θ = 25° (dspacing ~3.56) corresponding to the partial restoration of sp2 carbon. The shifting of the diffraction peak towards the higher 2θ can be accredited to the substitution of sulfur atoms [45]. The diameter of a sulfur atom, 2.04 Å is larger than that of a carbon atom 1.54 Å; consequently, the interlayer distance, d002, decreases after the introduction of sulfur in the GO structure. Thus, the XRD results

11

suggest restored conjugated structure of SGO and the sulfur incorporation among GO sheets during the hydrothermal reaction process [36, 37]. Fig. 1C. shows a maximum UV-Vis absorption peak at 233 nm in GO, which is ascribed to the a) b) π-π* transition of aromatic C-C bonds in GO. After GO modification, the absorption peak has broadened and shifted towards the red region, reaching a value of 274 nm. This indicates that the extended π-π conjugated structure of GO has been wrecked during oxidization. However, as the deoxygenation took place, the extensive conjugated sp2-carbon network is rebuilt in the modifying process. The broadening of the absorption peak for SGO refers to the increased transitions to and from the vibrational energy levels available at each electronic energy level which is due to the 2µm

2µm presence of more active sites in case of Sulfur incorporation in carbon lattice. Furthermore, almost

no visible absorption can be seen at 400 nm for SGO, which suggests the incorporation of sulfur into GO [46]. To prove the formation of SGO, FTIR spectra of SGO and GO are recorded and shown in Fig. 1D. FTIR spectra of the pristine GO sheets have proved the presence of different functional groups, such as O–H stretching vibration around 3230 cm-1 (hydroxyl), C–H stretching vibration around 1400 cm-1, C=O stretching mode at 1720 cm-1 (carbonyl), C=C stretching vibration around 1620 cm-1, C–OH stretching vibration at 1220 cm-1 (carboxyl), and C–O stretching mode at 1040 cm-1 (epoxy) [37]. For SGO, it is clearly observed that the relative intensities of O–H, C=O, C–H, C– OH, and C–O are greatly reduced from GO to the resulting SGO sheets, which may be due to the O = S = O asymmetric and symmetric stretching vibrations, implying the efficient restoration of the conjugated structure [37, 45, 47]. SEM images in Fig. 1E. (a) offer a prompt confirmation of the exfoliated GO sheets obtained through the modified Hummers’ and ultrasonic exfoliation process. These synthesized GO sheets 12

have some distinct folds and wrinkling on their surfaces due to the oxygen-containing functional groups on the surface of GO sheet. While Fig. 1E. (b) demonstrates SGO sheets with larger area, more distinct folds and waviness, very different from that of GO sheets in Fig. 1E. (a). The functional groups and the induced contraction of sulfur doping possibly disrupts the original conjugation, resulting in more folds and distortions on the surface of SGO sheets [37]. XPS technique was employed to validate the existence and oxidation state of sulfur in SGO. The full scan XPS spectrum of GO shows the presence of only carbon C1s and oxygen atoms O1s (Fig. 2A) [48, 49]. While the XPS survey spectra of SGO (Fig. 2A) reveals four peaks 532 eV, 284 eV, 232 eV and 169 eV corresponding to oxygen (O1s), carbon (C1s), sulfur(S2s), and sulfur (S2p), respectively [38, 50]. It can be noticed that sulfur doping generates two weak XPS signals from the peaks of S2s and S2p and a decreased O1s peak. The high-resolution C1s spectrum of SGO is convoluted into four peaks (Fig. 2B), featuring the C=C bond at 284.2 eV, the C–C bond at 286.3 eV, the C–O / C–S bond at 288.3 eV, and the C=O bond at 289.7 eV [37, 38]. The high-resolution S2p XPS spectrum (Fig. 2C) of SGO is composed of three peaks centered at 163.1, 164.3 and 166.2 eV, corresponding to S2p3/2 (-C-S-C-), S2p1/2 (-C-S-C-) and oxidized sulfur groups (-CSOx-C-), respectively [48, 49, 51]. Therefore, the above XPS results further suggest that GO reacting with Na2S can incorporate S into the SGO to afford C-S bonded groups by removing most of oxygen containing functional groups [37]. Fig. 2D-E show a 3D AFM topography of GO and SGO. The GO sheets (Fig. 2D) have thickness of about 1.16 nm due to the presence of covalently bonded oxygen and the displacement of the sp3 hybridized carbon atoms [52]. The range of lateral dimension is from several hundred nanometers to several micrometers. The AFM image of SGO (Fig. 2E) shows the lateral dimension is like that of GO, but the apparent thickness is ∼0.6 nm, corresponding to graphene sheet, which is thinner 13

than GO sheets. These results suggest the removal of oxygen-containing functional groups during the hydrothermal treatment of GO to for SGO [53].

A

B

C

D

E

1

1

1

1

1

1

1

1

1

6

6

6

6

6

7

7

7

7

0

2

4

6

8

0

2

4

6

Fig. 2. A. Wide range XPS spectrum for GO and SGO B. High resolution C1s and C. S2p spectrum for SGO D. AFM image of GO E. AFM image of SGO

Optimization of Experimental Conditions: Effect of Dye Concentration: To investigate the effect of concentration of Rhd-B dye on the emission intensity of the system, concentration of dye was varied from 0.01 µg.L-1 to 1000 µg.L-1. It can be seen from Fig. 3. that the fluorescence intensity of system increased gradually with the increase in concentration of RhdB at neutral pH-7.4 of PBS. In order to get a high sensitivity, an intermediate dye amount of 100 µg.L-1 Rhd-B was employed in this assay.

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A

B

Fig. 3. A. Fluorescence emission spectra of Rhd-B at varying concentrations in PBS a) 1000 µg.L-1 b) 750 µg.L-1 c) 500 µg.L-1 d) 250 µg.L-1 e) 100 µg.L-1 f) 50 µg.L-1 g) 10 µg.L-1 h) 0.1 µg.L-1 ⅰ) 0.01 µg.L-1 with λex=554 nm and λem=577 nm B. Calibration plot for varying concentrations of Rhd-B from 0.01 µg.L-1 to 1000 µg.L-1

Effect of Doping Concentration: We studied the effect of sulfur doping on the fluorescence intensity of the system. As displayed in Fig. 4, a gradual decrease of the fluorescence intensity of Rhd-B+SGO was observed with decreasing percentage of sulfur in GO (from 12 to 3%) in a linear fashion. This is attributed to the fact that sulfur in high concentration acts as a scavenger of the highly potent radicals. This is possibly because the excessive amount of sulfur in SGO covers the active sites on the surface and hinders the contact with the Rhd-B molecules. While at lower concentrations of sulfur in the graphene oxide, more electrons are trapped in the structure and greater is the oxidation of the dye due to the availability of superoxide anions or hydroxyl radicals. Similar result is expected with further decrease in sulfur. Therefore, the fluorescence intensity of the Rhd-B+SGO could be modulated by sulfur and GO with minimum sulfur percentage i.e. SGO-3 was selected for the subsequent experiments.

15

Fig. 4. Fluorescence quenching effect of GO and varying concentrations of Sulfur in PBS medium a) Rhd-B dye b) GO c) SGO-12 d) SGO-10 e) SGO-5 f) SGO-3

Effect of SGO Concentration: Fig. 5 show the assay for optimal concentration of SGO-3 that impart better analytical performance in terms of fluorescent quenching. As the concentration of SGO-3 was varied in the system from 1 to 500 mg.L−1; a significant increase in quenching was observed. This could be attributed to the higher number of available electrical charges in the system. Findings indicate that 100 mg.L−1 of SGO-3 imparts sufficient fluorescence quenching (~70%) of the displayed by the dye. Therefore, a concentration of 100 mg.L−1 of SGO-3 was employed for further quenching experiments. A

B

a j

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Fig. 5. A. Effect of change in concentration of SGO while keeping Rhd-B concentration constant in PBS a) Rhd-B, Rhd-B+SGO b) 500 mg.L-1 c) 250 mg.L-1 d) 100 mg.L-1 e) 75 mg.L-1 f) 50 mg.L-1 g) 25 mg.L-1 h) 10 mg.L-1 i) 5 mg.L-1 j)1 mg.L-1 B. Calibration curve for SGO concentration

Detection of Hydrogen peroxide: The present analytical study is based on investigating the molecular interaction between Rhd-B and SGO in the presence of H2O2. The detection of H2O2 was followed by measuring the fluorescence emission of Rhd-B dye and Rhd-B+GO, Rhd-B+SGO at first (Fig. 6A.) and then evaluating the emission intensity after adding hydrogen peroxide to the Rhd-B+GO and RhdB+SGO system (Fig. 6B.). These experiments depict amplified fluorescence quenching with SGO in the presence of H2O2, as compared to GO.

Fig. 6. A. Effect of GO and SGO on fluorescence of Rhd-B B. Effect of GO and SGO on oxidation of Rhd-B by H2O2 : a) Rhd-B b) Rhd-B+GO c) Rhd-B+GO+H2O2 d) Rhd-B+SGO e) Rhd-B+SGO+H2O2

The fluorescence responses of 100 mg.L-1 GO and SGO catalysts towards 100 µg.L-1 RhdB in the presence of H2O2 were investigated as shown in Fig. 6B. Fluorescence is significantly quenched by SGO and upon addition of 1 µM H2O2, the corresponding emission intensity at 577 nm is further decreased. In order to achieve highly sensitive detection of H2O2, the effects of incubation time of Rhd-B+SGO with H2O2 were studied and optimum incubation time of RhdB+SGO system with H2O2 came out to be 5 min. SGO in phosphate buffer solution (pH-7.4) 17

catalyzed H2O2 to form hydroxyl radicals .OH, which can oxidize Rhd-B conjugated molecule to form colorless and non-fluorescence product. This result suggests that the presence of hydrogen peroxide stimulates the interaction of the fluorescent dye with SGO, thereby enhancing the oxidation of the dye by the sample, leading to a fluorescence decrease. In principle, the addition of H2O2 in the system produces hydroxyl radicals, which leads to oxidation of dye and eventually quenches it fluorescence. This is reflected by decreasing of fluorescence intensity signal. The effect of H2O2 concentrations on the fluorescence intensity of the system was investigated. At lower H2O2 concentrations, the process of oxidation of adsorbed dye is slow, hence lesser quenching of fluorescence intensity signals is observed. With the increase in H2O2 concentration, more Rhd-B will be oxidized by more .OH radicals, hence greater quenching of the fluorescence signal is observed (Fig. 7A); mainly due to the saturation of the particle surface and dye adsorption. The fluorescence quenching intensity is found to be linear with the concentration of H2O2 as shown in Fig. 7B. Based on these findings, a new simple and sensitive fluorescence method is proposed for the determination of H2O2.

Fig. 7. A. Fluorescence response of Rhd-B+ SGO system towards different amount of H2O2: a) Rhd-B dye, b) Rhd-B+SGO, Rhd-B+SGO+H2O2 c) 1nM d) 5nM e) 10nM f) 50nM g) 100nM h) 200nM ⅰ) 500nM j) 1µM and B. the related Stern-Volmer plot.

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Calibration Curve for H2O2 Detection: Using the described principle in the optimized conditions, the dye and SGO system was applied for detection of H2O2. Fig. 7. describes the fluorescence intensity response of Rhd-B+SGO upon incubation with a series of different concentrations of H2O2 ranging from of 1 nM–1 µM. It can be seen from the figure that at higher concentrations of H2O2, the resulting fluorescence signals of the dye+SGO are greatly decreased. The resulting calibration curve for H2O2 is shown in Fig. 7B. A good linear relationship (y = -0.14485 x + 78.79141, R2 = 0.999) between the value of emission intensity and H2O2 concentration (1 nM-1 µM) is obtained with a comparably low limit of detection (LOD) of 100 pM (on the basis of 3 times signal-to-background ratio) calculated by following the equation LOD =KS0/S, where K is a numerical factor chosen according to the confidence level desired, S0 is the standard deviation (S.D.) of the blank measurements (n = 9, K= 3) and S is the slope of calibration curve. The lower LOD is attributed to the specificity of RhdB+SGO system to H2O2. The performance of this fluorescent sensor is compared with the previously reported H2O2 sensors (see Table 2) and it is worth noting that our proposed sensor poses quite low limit of detection. Table 1 Analytical performance of the proposed fluorometric sensor for H2O2. Analyte H2O2

Linear Range (µM) 0.001-1

Regression Equation Slope 0.185

Intercept 64.79141

Correction Co-efficient 0.999

LOD (pM) 100

Table 2 A comparison of current work with reported literature regarding the performance (e.g. linear range and LOD) of the H2O2 assay Material

Method

19

Linear Range (µM)

LOD References (nM)

NiS nanocubes Copper oxide-graphite carbon nitride (CuO-g-C3N4 nanocomposites) Pt/rGO-CNT hybrid Pt/MoS2/Ti heterostructures Molybdenum disulfide nanosheets with ortho-phenylenediamine (OPD-MoS2)NS Platinum-Palladium carbon fiber microelectrode (Pt-Pd/CFME) Multifunctional Probe RH-1 nanoporous PtCu flowers (np-PtCu) MoS2 nanosheets conjugated with organic copper nanowires (MoS2–OCu nanohybrids) Iminocoumarin-based probe Catalase conjugated Gold Nanoclusters Mitochondria-based coumarin probe CdTe QDs catalyzed by POM Na10[α-SiW9O34] 4-sulphonatophenyl porphyrin-Fe2+ NPs Iron sulfide (FeSx)/graphene heterostructure Sulfur Doped Graphene Oxide (SGO)

Colorimetric Colorimetric

4-40 2-50

1720 1200

[54] [55]

Electrochemical Electrochemical Colorimetric

1-100 10-160 0.5–200

1000 870 500

[56] [57] [58]

Electrochemical

5–3920 

420

[59]

Fluorescence Electrochemical Electrochemical

5-20 10-1700 85-3800

160 100 76

[60] [61] [62]

Fluorescence Fluorescence

0.0–200.0 0-80

60 25

[63] [64]

Fluorescence

0-80

10

[65]

Fluorescence

0.0078-0.25

3.8

[66]

Fluorescence

0-42

1.24

[67]

Electrochemical

100-1000

0.5

[68]

Fluorescence

0.001-0.05

0.1

This Work

Proposed Quenching Mechanism: Recent developments in the utilization of GO promise its application for detection of trace levels of organic and inorganic analytes. Rich oxygen-containing groups, such as −COOH, −CO, and −OH are known to occupy significant GO surface area. These anchoring sites potentially make GO a more efficient adsorbent as well as a highly surface active functional filler. Due to its active surface structure, it interacts strongly with organic, polymeric, and biological molecules, via 20

noncovalent forces, such as hydrogen bonding, π−π stacking, electrostatic forces, van der Waals forces, and hydrophobic interactions. The biocompatibility and versatile surface modification of GO allows substantial sensing of complex molecules such as bioanalytes [69] . Generally, when doped with a heteroatom, the rigidity of the GO is destroyed, and defects are induced. These modifications and defects are responsible for the enhanced optical and chemical properties of GO. In the present case, doping of GO was done with sulfur atoms via hydrothermal treatment, which results in replacement of some of the C atoms with S atoms, and attachment of few of the S in GO with the oxygenated functional groups, by an electrostatic interaction. These oxygenated functional groups interact with the sulfur atom in the GO and form a C-S bond thereby leading to the formation of defects and destruction of aromaticity of GO [36, 37]. The responsibility for enhanced adsorbability of SGO can be ascribed to the driven noncovalent formation of ᴫ– ᴫ stacking between Rhd-B molecules and aromatic regions of the graphene. The negatively charged SGO interacts electrostatically with the positively charged Rhd-B dye [70]. Photo-induced charge transfer can also occur upon the electronic interaction between SGO and Rhd-B. However, superfluous SGO is detrimental for photon absorption. The results demonstrate that the structure of SGO can effectively increase adsorption of Rhd-B molecules and charge transfer along the SGO sheets [65, 71]. This research work is devoted to gain apprehension into the mechanism responsible for fluorescence quenching of Rhd-B, a well-known fluorescent probe, by as-synthesized SGO in aqueous solution and in the presence of hydrogen peroxide. Literature reveal that GO can effectively quench the fluorescence of Rhd-B in a fashion of combined static quenching and charge transfer [14, 65]. It is possible that ground-state complexes are formed between Rhd-B and SGO during the course of static quenching. In terms of the photo-induced electron transfer quenching,

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the excited state of Rhd-B molecule acts as an electron donor while GO as an electron acceptor [14]. Rhd-B (5.67 eV), after exciting electron, is converted to Rhd-B* (3.08 eV) that injects electrons to the electron acceptor, i.e., SGO (4.8 eV) to become a cationic radical Rhd-B+; which undergoes self-degradation by trapping excited electrons of adsorbed oxygen. [72] . Furthermore, H2O2 generates free radicals such as OH˙, OOH˙, O2- with the help of a catalyst. The decomposition of H2O2 into free radicals [73], in the present study, is induced by the catalyst (SGO), which then oxidizes the Rhd-B molecules. The fluorescence quenching is mainly due to the reaction of these generated ions and radicals. Since hydroxyl radicals and super oxygen anion are very strong oxidizing agents, they can react with the dye molecules to produce intermediates, which subsequently result in the fluorescence quenching of the solution [74]. Based on the above discussion, a possible mechanism for SGO as heterogeneous catalyst for the degradation of Rhd-B dye in the presence of H2O2 is proposed. The quenching efficiency results from the interaction between SGO and H2O2, which could be ascribed to three aspects: ⅰ) the exfoliated GO, after doping with sulphur turns into amorphous SGO and offers more active sites. ii) SGO possesses a large aromatic ring structure that would facilitate the adsorption of Rhd-B, through ᴫ–ᴫ interactions [75]. iii) The increased number of active sites on the catalyst’s surface would facilitate the oxidation of Rhd-B by H2O2 due to the generation of more ions and radicals. Hence, in the presence of the analyte H2O2, Rhd-B is oxidized from the surface of SGO by a stronger interaction, thereby notably quenching the fluorescence signal. The successful quenching capabilities of this Rhd-B-SGO system make it a potential candidate for H2O2 detection in various applications.

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Fig. 8. Mechanism of fluorescence quenching based detection of hydrogen peroxide

Interference Study: Selectivity is a principal factor for evaluating the performance of a newly developed detection method, especially one with potential biomedical applications, for which a high selective response to the target analyte over other possible interference substances is necessary. For probing the detection selectivity of Rhd-B and SGO, the investigation was carried out with H2O2 and various coexistence substrates at a concentration of 1 µM. Fig. 9. depicts that all these substances: dopamine hydrochloride, resorcinol, glucose, isopropyl alcohol, uric acid and ascorbic acid have no obvious interference. This confirm the high specificity and selectivity of the developed fluorometric sensor for hydrogen peroxide determination.

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Fig. 9. Effect of various interfering compounds on fluorescence quenching by SGO

4. Conclusion: In summary, a robust SGO catalyst based fluorescence quenching assay has been designed for the sensitive and selective detection of H2O2. SGO was synthesized by a simple hydrothermal method and its notable features were analyzed by the SEM, XRD, UV–vis, FTIR, XPS, AFM and RAMAN spectra. Significant fluorescence quenching was observed upon the interaction between positively charged Rhd-B dye and negatively charged SGO due to the formation of charge-transfer complex Rhd-B+SGO. Furthermore, H2O2 can be decomposed into ·OH radicals in the presence of SGO. These hydroxyl radicals oxidized Rhd-B dye which was observed in terms of quenching the fluorescence of Rhd-B. Under the optimal reaction conditions, a linear correlation was established between fluorescence intensity and concentration of H2O2 from 1 nM to 1 µM with a low detection limit of 100 pM. The key advance of this Rhd-B+SGO optical sensing framework is attributed to the high catalytic of SGO, which make it an effective candidate for H2O2 detection. We expect that

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the present study would facilitate and provoke new research opportunities for the utilization of SGO catalysts in surface science and biomedical applications. 5. Acknowledgments: The authors gratefully acknowledge the support for this study by Higher Education Commission Pakistan through its NRPU grant 20-4993/R&D/HEC/14 and Pakistan Science Foundation through its PSF-NSFC funded project (Project No. PSF/NSFC-II/Eng/P-COMSATS-Lhr(07).

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