Study on the interactions between graphene quantum dots and Hg(II): Unraveling the origin of photoluminescence quenching of graphene quantum dots by Hg(II)

Journal Pre-proof Study on the interactions between graphene quantum dots and Hg(II): Unraveling the origin of photoluminescence quenching of graphene quantum dots by Hg(II) Jincymol Kappen, Sundararajan Ponkarpagam, S. Abraham John

PII:

S0927-7757(20)30144-8

DOI:

https://doi.org/10.1016/j.colsurfa.2020.124551

Reference:

COLSUA 124551

To appear in:

Colloids and Surfaces A: Physicochemical and Engineering Aspects

Received Date:

15 December 2019

Revised Date:

16 January 2020

Accepted Date:

1 February 2020

Please cite this article as: Kappen J, Ponkarpagam S, John SA, Study on the interactions between graphene quantum dots and Hg(II): Unraveling the origin of photoluminescence quenching of graphene quantum dots by Hg(II), Colloids and Surfaces A: Physicochemical and Engineering Aspects (2020), doi: https://doi.org/10.1016/j.colsurfa.2020.124551

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Study on the interactions between graphene quantum dots and Hg(II): Unraveling the origin of photoluminescence quenching of graphene quantum dots by Hg(II) Jincymol Kappen, Sundararajan Ponkarpagam and S. Abraham John* Centre for Nanoscience and Nanotechnology, Department of Chemistry The Gandhigram Rural Institute, Gandhigram-624 302, Dindigul, Tamilnadu, India Email: [email protected]; [email protected]

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Graphical abstract

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Abstract

The present investigation reveals that when Hg(II) interacts with the colloidal solution of

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graphene quantum dots (GQDs), initially it forms complex with GQDs and then reduces to Hg(0) and Hg(I) resulting the complete quenching of GQDs blue luminescence. This is in contradiction with the earlier reports in which the quenching of GQDs by Hg(II) is attributed to complexation alone. In order to understand the reasons for the quenching of GQDs by Hg(II), a detailed study was undertaken by varying the pH of GQDs solution, incubation time and concentration of Hg(II). The emission studies indicate that formation of Hg(0) is less 1

favorable at pH 7 whereas its formation is more favorable at pH 13. It is assumed that the formation of Hg(OH)2 in alkaline pH facilitates Hg(II) reduction easier than at neutral pH. The SEM and TEM images confirm the presence of spherical Hg(0) particles with different sizes depending upon Hg(II) concentration, pH and incubation time. The results obtained from emission, XPS and differential pulse voltammetry (DPV) studies reveal that Hg(II) was reduced to Hg(0) via Hg(I) on GQDs surface. The differential pulse voltammogram of 40 min incubated Hg(II)-GQDs coated GC electrode shows three oxidation peaks at 0.34, 0.48 and

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0.65 V, corresponding to Hg(I) to Hg(II) and Hg(0) to Hg(II), respectively.

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Keywords

Interaction of GQDs with Hg(II); effect of pH; incubation time; metallic Hg; mechanism for

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1. Introduction

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quenching.

Research on graphene quantum dots (GQDs) is gaining much momentum in recent years

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mainly due to their interesting photoluminescent properties, ease in preparation, low cytotoxicity, excellent solubility, stable photoluminescence and better surface grafting [1-4]. Due to these numerous advantages, they have been exploited for chemosensing, bio-imaging and optoelectronic applications [5-10]. GQDs consist of sp2/sp3 carbons with oxygen rich functional groups like hydroxyl and carboxylic acid, attached on the edges [11]. Both π-π* transitions of aromatic C-C bonds from graphene moiety and n-π* transitions of functional 2

groups on GQDs are responsible for their absorption properties [12]. On the other hand strong photoluminescence (PL) properties of GQDs are associated with quantum confinement, edge effects, structure and size [13]. Further, they show both excitation dependent and independent emission properties depending upon the size distribution and emissive sites [14]. The PL can be tuned by varying the size and number of functional groups on their surfaces by changing the temperature and reaction time [6]. Determination of Hg(II) in water samples is extensively reported in the literature

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based on the quenching of GQDs emission [15-23]. In the reported papers, the quenching is attributed to the strong chelating ability of Hg(II) with oxygen functional groups of GQDs. Recently, two papers have been published in the literature to find the cause for the selective

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complexation of Hg(II) with GQDs [24,25]. These reports suggest that the interaction of Hg(II) with GQDs prevents the radiative recombination of electron-hole pairs which leads to

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quenching of GQDs emission. Jia et al. studied the interaction of GQDs with different metal

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ions including Fe(III) and Hg(II) by spectrofluorimetry [24]. They pointed out that preferential complexation of Fe(III) with GQDs over Hg(II) was expected because the formation constant of the former was greater than the later. However, the spectrofluorimetric

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studies showed that GQDs emission was quenched by Hg(II) even in the presence of high concentration of Fe(III). The authors finally concluded that “certain unknown factors” are

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responsible for the selective quenching of Hg(II) in the presence of Fe(III). Thus, the present

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study aims to unveil the unknown factors for the selective quenching of GQDs by Hg(II) with the aid of spectroscopic and microscopic tools.

In the present study, the GQDs were prepared by pyrolysis of citric acid. Then, a

detailed investigation was carried out to find the interaction of GQDs with different metal ions including Hg(II) by spectrofluorimetry. It was found that the emission of GQDs was completely quenched after the addition of Hg(II) whereas it was unaffected while adding all 3

other metal ions except Fe(III). Further, it was found that the quenching depends on the pH of the GQDs solution, incubation time and concentration of Hg(II). After the quenching of GQDs by Hg(II), the analysis of the GQDs-Hg(II) mixture by SEM, TEM, XPS and differential pulse voltammetry (DPV) reveals the presence of Hg(0) on the surface of GQDs in addition to Hg(I) irrespective of the pH of the solution. The formation of Hg(0) was more favorable at an alkaline pH whereas complexation of Hg(II) with GQDs was more favorable at neutral pH. Thus, the present study disclose for the first time that the pathway for the

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quenching of GQDs is complexation of Hg(II) with GQDs followed by the formation of Hg(0) via Hg(I).

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2. Experimental section 2.1. Materials

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Citric acid, sodium hydroxide, iron(II) sulphate heptahydrate (Fe(SO4).7H2O), cobalt(III) nitrate hexahydrate (Co(NO3)3.6H2O), manganese(II) nitrate tetrahydrate

iron(III)

nitrate

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(Mn(NO3)2.4H2O), zinc nitrate hexahydrate(Zn(NO3)2.6H2O), lead(II) nitrate (Pb(NO3)2), nonahydrate

(Fe(NO3)3.9H2O),

cadmium(II)

nitrate

tetrahydrate

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(Cd(NO3)2.4H2O), cupric(II) nitrate trihydrate (Cu(NO3)2.3H2O), nickel(II) sulphate (NiSO4) and chromium(III) sulphate (Cr2(SO4)3) were purchased from Merck India. Mercury(II)

(Hg2(NO3)2.2H2O)

was

purchased

from

Sigma

Aldrich

and

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dihydrate

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nitrate monohydrate (Hg(NO3)2.H2O) was purchased from Rankem. Mercury (I) nitrate

ethylenediaminetetraacetic acid (C10H14N2O8Na2.2H2O) was purchased from Qualigens. 2.2. Instrumentation Absorption spectra were measured by using JASCO V-750 UV-visible spectrometer. Fluorescence

spectral

measurements

were

carried

out

on

a

JASCO

FP-8500

spectrofluorimeter equipped with a xenon discharge lamp at room temperature. FT-IR 4

measurements were recorded from JASCO FT-IR 460 plus model. FEI-TECNAI-G2 20 TWIN with an accelerating voltage of 200 kV was used to collect high resolution transmission electron microscopy (HR-TEM) images. XPS measurements were done by using PHI 5000 VERSAPROBE scanning ESCA Microprobe. Scanning electron microscopy (SEM) images were taken in VEGA3, TESCAN Czech Republic. Energy dispersive X-ray analysis was done using Bruker Nano, Germany. Electrochemical measurements were carried out using a CHI electrochemical workstation (Model 643B, Austin, TX). Glassy carbon (GC),

electrodes, respectively for electrochemical measurements. 2.3. Synthesis of GQDs

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platinum wire and NaCl saturated Ag/AgCl were used as working, counter and reference

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GQDs were prepared by pyrolysis of citric acid (CA) according to the reported

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protocol [26]. 2 g CA was taken in a round bottom flask and heated to 200 C using a heating mantle. After 5 min, it was melted and the colour of the liquid was changed from yellow to

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orange within 30 min, implying the formation of GQDs. The resultant orange liquid was dissolved in 100 mL of 1% NaOH and purified by dialyzing the solution through dialysis bag

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(1000 Da) and used for further studies. 3. Results and discussion

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3.1. Characterization of GQDs by UV-visible, spectrofluorimetry and FT-IR spectroscopy

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UV-vis spectrum of the as synthesized GQDs is shown in Fig.S1A. It shows an absorption peak at 362 nm, reflecting the presence of n-π* transition. This confirms the presence of surface oxygen functional groups on GQDs [26]. To find the emission maximum, GQDs were excited at different wavelengths from 330 to 370 nm. While varying the excitation wavelength, the emission intensity increases but the emission maximum remains constant at 456 nm. The emission intensity reached maximum at an excitation wavelength of 366 nm and 5

after that it started to decrease at 456 nm (Fig.S1B). Hence, all the emission studies were carried out at an excitation wavelength of 366 nm. The prepared GQDs show blue luminescence under UV light (inset of Fig.S1B). The obtained excitation independent emission confirmed the formation of GQDs with uniform size [5]. It has been already established that GQDs show strong photoluminescence due to the presence of large number of hydroxyl functional groups on their surface [15]. The quantum yield of GQDs was calculated using quinine sulphate as standard and it was found to be 50%. The FT-IR

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spectrum of GQDs shows bands at 1570, 1389 and 3440 cm-1 (Fig.S1D). The band obtained at 1570 cm-1 implies the presence of asymmetric stretching of carboxylic acid. The bending and stretching of -OH groups were observed at 1389 and 3440 cm-1, respectively. The FT-IR

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studies confirmed the presence of both carboxylic and hydroxyl functional groups on the

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surface of GQDs [21,22,26].

3.2. Study on the interaction between metal ions and GQDs by spectrofluorimetry

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The emission titration is carried out by adding 500 μM each metal ions separately into GQDs (1.5 mg/ml) at pH 7. The interaction between GQDs and metal ions was analyzed

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from the changes in GQDs emission intensity at 456 nm. Fig.1A shows the emission spectra of GQDs (pH 7) recorded immediately after the addition of 500 μM each Mn(II), Ni(II),

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Pb(II), Cu(II), Co(III), Zn(II), Cr(III), Fe(II), Fe(III) and Hg(II). No significant change in the GQDs emission intensity was observed after the addition of all metal ions except Fe(III) and

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Hg(II) (curves b-j). The GQDs emission intensity at 456 nm was decreased from 1000 to 300 after the addition of 500 μM Fe(III) (curve k). Interestingly, the emission intensity was completely quenched at 456 nm after the addition of same concentration of Hg(II) to GQDs (curve l). The formation constant of transition and heavy metal ions with carboxylate group of EDTA has been taken from the literature [24]. The formation constants of Fe(II), Mn(II), Cd(II), Pb(II), Cu(II), Hg(II) and Fe(III) are fairly higher than the other metal ions. Among 6

Hg(II) and Fe(III), the formation constant of Fe(III) was higher than Hg(II). Therefore, it is expected that the quenching of GQDs by Fe(III) must be higher than that of Hg(II). But, as shown in Fig.1A, quenching of GQDs by Hg(II) is higher than that of Fe(III). In order to find the exact mechanism for the selective quenching of GQDs emission by Hg(II), a detailed investigation was carried out by varying the GQDs solution pH, incubation time and concentration of Hg(II). 3.3. Effect of GQDs solution pH is

well

known

that

the

PL

properties

of

GQDs

depend

on

the

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protonation/deprotonation of their carboxyl groups [27]. Therefore, emission studies were first carried out by varying the pH of GQDs. Throughout the emission titrations, emission

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intensity of 1000 was maintained irrespective of pH of GQDs solution. As the pH of GQDs

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solution was decreased from 13 to 7, the emission intensity was decreased. Thus, 1, 1.5 and 5 mg/ml of GQDs were used to maintain the intensity of 1000 at pH 13, 7 and 5, respectively.

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First, the emission spectra of GQDs in the presence of 100 μM Hg(II) were studied at pH 5, 7 and 13. As shown in Fig.1B, the emission intensity of GQDs at pH 13 was decreased from

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1000 (curve a) to 600 immediately after the addition of 100 μM Hg(II) (curve b). On the other hand, the emission was completely quenched immediately after the addition of same

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concentration of Hg(II) at both pH 7 and 5 (curves c and d). The obtained results suggest that the interaction of Hg(II) with GQDs differs with respect to pH. It is expected that the

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carboxylic acid groups present on the surface of GQDs were unionized at low pH. As a result, the complexation of Hg(II) with GQDs is less favorable. When the pH increases, the unionized carboxylic acid groups become ionized on the GQDs surface, which in turn favors the complexation of Hg(II) with GQDs. Therefore, the quenching of GQDs by Hg(II) at pH 13 is expected to be higher. However, hydroxide ions interferes the complexation of Hg(II) with carboxylate groups, which resulted incomplete quenching of GQDs at pH 13 (Fig.1B, 7

curve b). According to the previous report, the GQDs are unstable at acidic pH due to aggregation [15]. This was evidenced from their decrease in emission with respect to time at acidic pH (Fig.S2). On the other hand, GQDs are more stable at higher pH due to their enhanced hydrophilicity from their deprotonated acid functionalities [28]. Therefore, detailed studies on the interactions of Hg(II) with GQDs were carried out only at pH 13 and 7. 3.4. Emission titration of GQDs with various concentrations of Hg(II) at pH 13 and 7

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Since the interaction of Hg(II) with GQDs varies with respect to pH, effect of their concentrations on fluorescence was also analyzed with respect to pH. Fig.2A shows the emission spectra of GQDs after the addition of different concentrations of Hg(II) at pH 13.

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For each incremental addition of 10 μM Hg(II) to GQDs, the emission intensity at 456 nm was systematically decreased without affecting the emission maximum. After adding 180 μM

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Hg(II), GQDs emission was completely quenched. The emission intensity of GQDs was also decreased at pH 7 after each addition of 2.5 μM Hg(II) (Fig.2B). Unlike pH 13, complete

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quenching of GQDs was observed with the addition of 3-fold less concentration (60 μM) of Hg(II) at pH 7. These results revealed that complexation between Hg(II) and GQDs is pH

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dependent. As mentioned above, the presence of carboxylate groups on the surface of GQDs will be expected to complex with Hg(II) at pH 13. But, the presence of hydroxide groups in

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solution interferes the complexation. Thus, 3-fold higher concentration of Hg(II) requires for the complete quenching of GQDs at pH 13. On the other hand, the absence of hydroxide

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groups does not interfere the complexation of Hg(II) with GQDs at pH 7. Thus, 3-fold less concentration of Hg(II) is enough for the complete quenching of GQDs when compared to pH 13. 3.5. Effect of incubation time at pH 13 and 7

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To further get insight into the difference in quenching of GQDs with respect to the concentration of Hg(II), the emission spectra were recorded at different incubation time for GQDs in the presence of Hg(II). The emission spectra of GQDs (1mg/ml) were recorded for each 5 min interval in the presence of 10 and 50 μM Hg(II) at pH 13. Fig.3A illustrates the emission spectra of GQDs recorded for each 5 min interval at pH 13 after adding 10 μM Hg(II). When the emission spectrum was recorded immediately after the addition of 10 μM Hg(II), no drastic decrease in emission at 456 nm was observed (curve b). Further, it was

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recorded for each 5 min interval, a slight decrease in the emission intensity was noticed. After 40 min incubation, the emission was decreased from 1000 to 800 (curve c) and no further change in the emission intensity was observed beyond 40 min. The emission intensity-time

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plot (inset of 3A) supports the time consuming reaction kinetics between GQDs and Hg(II). In the reported papers, the quenching of GQDs is attributed to the complexation of Hg(II)

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with GQDs [15,17,19]. Generally, coordination of metal ions with functional groups is a fast

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process [29]. But, as evidenced from Fig.3A, 40 min requires for the mere decrement of 200 emission intensity by Hg(II) under the present experimental conditions.

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Similarly, time dependent emission study was also executed by increasing Hg(II) concentration from 10 to 50 μM under identical conditions and the resultant emission spectra

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are shown in Fig.3B. Here, the quenching was faster than that of 10 μM Hg(II). The emission intensity was decreased to 780 when the spectrum was recorded immediately after

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the addition of 50 μM Hg(II) to GQDs (curves a and b). When the emission spectrum was recorded for every 5 min interval, pronounced quenching was observed and the emission intensity was decreased to 120 after 40 min (curve c) and after that it remains unchanged. The emission intensity-time plot depicts the faster kinetics at the initial stage and slow after 10 min (inset of Fig.3B).

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The time dependent emission study was also carried out at pH 7 under identical conditions as carried out at pH 13. Fig.4A demonstrates the emission spectra of GQDs recorded for each 5 min interval in the presence of 10 μM Hg(II) at pH 7. Unlike pH 13, here the emission intensity of GQDs was quenched from 1000 to 800 immediately after the addition of 10 μM Hg(II) (curve b). This indicates that the complexation of Hg(II) with GQDs was higher at pH 7 when compared to pH 13. Interestingly, the emission intensity of GQDs increases slowly while recording the spectrum for each 5 min interval and saturated at 900 after 40 min (curve

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c). Fig.4B depicts the emission spectra of GQDs recorded for each 5 min interval in the presence of 50 μM Hg(II). The emission was drastically quenched to 150 immediately after the addition of 50 μM Hg(II) (curves a and b). Similar to 10 μM Hg(II), here also the

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emission intensity was started to increase slowly and saturated at 326 after 40 min. The emission intensity-time plot (inset of Fig.4A) indicates the enhancement of emission after the

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initial decrement up to 5 min for 10 μM Hg(II). Similar to pH 13, the quenching was faster

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for 50 μM Hg(II) at pH 7 as evidenced from emission intensity-time plot (inset of Fig.4B). These results reveal that the quenching of GQDs emission not only depends on pH but also the concentration of Hg(II). Besides, the decrease in emission intensity of GQDs at pH 13

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with respect to incubation time indicates that the quenching is not merely due to

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complexation of Hg(II) with GQDs alone but also due to other factors. It has been already demonstrated that quenching of GQDs due to complexation of Hg(II)

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can be recovered by adding EDTA into GQDs-Hg(II) solution [22,24]. Similar experiments were also carried out in the present study by adding 1 mM EDTA into 40 min incubated GQDs solution containing 10 and 50 μM Hg(II) at pH 13. Surprisingly, the emission of GQDs remains unaltered after the addition of 1 mM EDTA into GQDs containing 10 μM Hg(II) (Fig.3A, curve d). On the other hand, the quenched emission was slightly recovered (100) while adding the same concentration of EDTA to GQDs containing 50 μM Hg(II) 10

(Fig.3B, curve d). The increase in emission was attributed to the removal of complexed Hg(II) from GQDs by EDTA since it is a strong chelating agent, which in turn free the surface carboxyl and hydroxyl groups. No significant change in the emission intensity was observed when EDTA concentration was further increased to 5mM into a mixture of GQDsHg(II) solution. In a similar manner, 1 mM EDTA was also added to recover the emission of GQDs containing 10 and 50 μM Hg(II) at pH 7. Interestingly, addition of 1 mM EDTA completely

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recovered the emission of GQDs containing 10 μM Hg(II) (Fig.4A, curve d). But, only slight recovery in the emission intensity (100) was observed for GQDs containing 50 μM Hg(II) (Fig.4B). Nevertheless, the recovery of GQDs emission was higher at pH 7 in contrast to pH

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13. This indicates that complexation of Hg(II) with GQDs is more favorable at pH 7 than pH

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13. But, it also depends on the concentration of Hg(II) because GQDs emission was not completely recovered after the addition of 1 mM EDTA to GQDs containing 50 μM Hg(II) as

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evidenced from Fig.4B, curve d.

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3.6. Evidences for the formation of Hg(0) by SEM Reduction of Ag(I) and Au(III) to corresponding metal nanoparticles by compounds having polyhydroxy functional groups has already been reported [30-32]. For example, the

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hydroxyl end groups present in polyvinyl pyrrolidone (PVP) are utilized for the reduction of

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silver ions into silver nanoplates [30]. When compared to the thermodynamic potentials of transition and heavy metal ions, Hg(II) has more positive potential of +0.851 V (Table S1). Since its reduction potential is close to Ag(I) (0.799 V), the possibility for the reduction of Hg(II) to Hg(0) by the hydroxyl functional groups of GQDs cannot be ruled out. To check the formation of metallic mercury, the incubated GQDs-Hg(II) mixture solution was coated on GC substrate and then SEM studies were carried out. Fig.5A shows the SEM 11

images recorded for 40 min incubated GQDs-10 μM Hg(II) mixture (pH 13) coated on GC substrate. It shows the presence of spherical Hg(0) particles on GQDs surface with a size of 20 nm. On the other hand, the SEM images of 40 min incubated GQDs-50 μM Hg(II) coated on GC substrate shows spherical particles of metallic Hg with a size ranging from 300-400 nm (Fig.5B). The SEM studies revealed that the size of Hg(0) increases when the concentration of Hg(II) in GQDs increases. Interestingly, the SEM images of 40 min incubated GQDs-10 M Hg(II) mixture from pH 7 coated on GC plate shows the absence of

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metallic mercury (Fig.5C). This indicate that the addition of 10 μM Hg(II) to GQDs at pH 7 resulted only complexation. This was evidenced from the complete recovery of GQDs emission by the addition of EDTA (Fig.4A, curve d). On the other hand, the formation of

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spherical Hg(0) particles was noticed for the incubated GQDs-50 μM Hg(II) mixture from pH 7 coated on GC substrate (Fig.5D). This once again indicates that the formation of Hg(0)

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depends on the concentration of Hg(II). The size of the Hg(0) formed was found to be ~100

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nm (Fig.5D). As evidenced from SEM studies, the reduction of Hg(II) to Hg(0) also occurs at pH 7 but the size and number of Hg(0) particles were found to be lesser than that of pH 13

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under identical experimental conditions. In any case, the SEM studies confirmed the formation of Hg(0) both at pH 7 and pH 13.

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The reduction pathway of Hg(II) to Hg(0) can be explained on the basis of thermodynamic potentials of Hg species. The thermodynamic reduction potentials of different forms of Hg

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species are given below.

Hg2+ + 2e-

→ Hg0 (+0.851 V)

…… (1)

2Hg2+ + 2e- → Hg22+ (+0.920 V)

…… (2)

Hg22+ + 2e- → Hg0 (+0.797 V)

…… (3)

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Based on the thermodynamic potentials, the direct one electron reduction of Hg(II) to Hg(I) is more favorable than two electron reduction of Hg(II) to Hg(0). Therefore, the reduction pathway is assumed Hg(II) to Hg(I) followed by Hg(I) to Hg(0). As evidenced from the above studies, EDTA removed the complexed Hg(II) and thereby enhanced the GQDs emission. EDTA can remove both Hg(II) and Hg(I) complexed with GQDs but not Hg(0). Therefore, 1 mM cysteine was added to GQDs-Hg(II) mixture to remove Hg(0) from GQDs surface [33]. The recovery of GQDs emission was noticed after

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the addition of cysteine to the incubated solution of GQDs-50 μM Hg(II) mixture at pH 13 (Fig.3B, curve e). However, no change in the emission was observed when the same concentration of cysteine was added to the incubated solution of GQDs-10 μM Hg(II)

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(Fig.3A, curve e). The SEM studies showed that, the size of the formed Hg(0) from GQDs

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containing 50 μM Hg(II) is much higher than that of Hg(0) formed from 10 μM Hg(II). Therefore addition of cysteine certainly removes Hg(0) in both the cases. But, the removal of

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bigger size Hg(0) from the GQDs surface may free the hydroxyl, carboxyl groups and graphene moiety. Since the emission of GQDs are associated with functional groups, size,

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electron-hole pair interactions in sp2 domain and edge effects, the formation of large sized Hg(0) particles can block the fluorescence. Thus, enhanced emission intensity was observed

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in the case of 50 μM Hg(II) after the addition of cysteine. On the other hand, removal of smaller size Hg(0) from the surface of GQDs does not make any impact on the intensity of

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GQDs. When repeating the similar experiments for GQDs-50 μM Hg(II) mixture at pH 7, the intensity was enhanced after the addition of cysteine indicating the removal of bigger size Hg(0) from the GQDs surface (Fig.4B, curve e). Even though there is an enhancement in the emission after the addition of cysteine, it failed to recover the emission completely at both pH 7 and 13 especially at higher concentration of Hg(II). The reason for the unrecovered intensity was due to the participation 13

of hydroxyl functional groups in the reduction of Hg(II). It was found that the recovery of GQDs emission is less when higher concentration of Hg(II) was used. This is mainly due to the involvement of more hydroxyl functional groups in the reduction of Hg(II) to Hg(I)/Hg(0). From the above results, it is concluded that complexation of Hg(II) with GQDs is entirely different from pH 13 to 7. The pronounced reduction of Hg(II) at alkaline pH was likely to the formation of Hg(OH)2. Reduction potentials of Hg(II) and Hg(OH)2 are 0.851

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and 1.03 V, respectively. Hence, reduction of Hg(OH)2 is more easier than that of Hg(II). At pH 7, the quenching was fast and the recovery of emission through the addition of EDTA was appreciable. Thus, the complexation of Hg(II) with GQDs is more favorable at this pH than

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the tendency to undergo reduction. The enhancement of emission at pH 7 can be explained

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as follows. When Hg(II) was added to GQDs, emission was quenched due to the quick coordination. For the coordination of Hg(II), two coordination sites are needed. After quick

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coordination, electron transfer may start slowly from GQDs to Hg(II). Since Hg(II) has higher electron accepting tendency, it can accept electrons from the GQDs and reduce slowly.

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It is presumed that electron transfer leads to the reduction of Hg(II) to Hg(I) at first. When Hg(II) was reduced to Hg(I), one coordination site (oxygen functional group) becomes free,

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which in turn enhance the emission intensity slowly. Then, Hg(I) undergoes further reduction to form Hg(0). Accordingly, the coordination is the initial step, followed by the electron

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transfer from GQDs to reduce Hg(II) to Hg(I) then subsequently reduction of Hg(I) to Hg(0). 3.7. Effect of concentration of GQDs To understand the effect of concentration of GQDs in the reduction mechanism of Hg(II), two different concentrations of GQDs were chosen and then time dependent emission was recorded. Figs.S3A and S3B illustrate the time dependent emission spectra of 1.5 and 2

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mg/ml GQDs in the presence of 100 μM Hg(II) at pH 7. The emission intensity was 1000 and 1500 for 1.5 and 2 mg/ml GQDs, respectively at pH 7. As shown in Fig.S3A, complexation of Hg(II) with GQDs (1.5 mg/ml) resulted in complete quenching of GQDs. As the incubation time increases, the emission intensity was very slowly increased and saturated at an intensity of 100 after 40 min. On the other hand, same complexation of Hg(II) resulted the quenching of 2 mg/ml GQDs emission intensity from 1500 to 180 but it was saturated at 585 after 40 min incubation. In both the cases, addition of 1 mM EDTA followed by cysteine

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enhanced the emission but not completely. While comparing the unrecovered emission intensity for both the concentrations of GQDs, the unrecovered intensity was higher for 2 mg/ml (620) than that of 1.5 mg/ml (500).

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3.8. Study on the interaction between GQDs and Hg(I)

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According to eqns. (1) and (2), the reduction of Hg(II) to Hg(I) is more favorable than that of direct reduction of Hg(II) to Hg(0). Therefore, the reduction pathway from Hg(II) to

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Hg(0) was assumed to be through Hg(I). Hence, similar time dependent emission studies were repeated for Hg(I) directly at pH 13 and 7. Fig.6A illustrates the time dependent

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emission changes of GQDs with 25 μM Hg(I) at pH 13. The GQDs emission was quenched immediately from 1000 to 600 after the addition of 25 μM Hg(I) (curve b). No drastic

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attenuation was observed after 40 min incubation (curve c). This indicates that the formation of Hg(0) from Hg(I) was faster than that of Hg(II). Similarly, time dependent emission

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studies were also carried out at pH 7. As shown in Fig.6B, addition of 25 μM Hg(I) to GQDs at pH 7 also quenches the emission intensity to 700 immediately (curve b). Then, it was decreased further and saturated at 380 after 40 min (curve c). Because of the direct reduction, no enhancement in emission was observed for Hg(I) in contrast to Hg(II) at pH 7 (Fig.4A and B; curves c). The emission intensity-time plot for Hg(I) incubation at pH 13 and 7 (inset

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of Fig.6B) revealed the immediate emission decay of GQDs at pH 13. All these observations supporting that the reduction of Hg(I) to Hg(0) was faster than Hg(II) to Hg(0). Further, the addition of 1 mM EDTA to GQDs-Hg(I) mixture does not enhance the emission intensity at pH 13 (Fig.6A, curve d). This indicates the absence of coordinated Hg(I). But, addition of EDTA resulted the partial enhancement of emission in the case of pH 7 (Fig.6B, curve d), implying the coordination of Hg(I) with GQDs. Finally, cysteine was added in both cases to recover the emission by removing Hg(0) from the GQDs surface. In

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both cases, addition of cysteine does not enhance the emission intensity (Figs.6A and B, curves e). The reason for the failure in the enhancement of emission intensity after the addition of cysteine was attributed to the formation of smaller Hg(0) particles, which cannot

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mask the functional groups of GQDs. Since the reduction was more favorable than

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could see from the SEM images (Fig.S4).

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complexation for Hg(I), comparatively large number of Hg(0) particles with smaller size

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3.9. Characterization by TEM and EDAX

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Fig.7 shows the TEM images of GQDs before and after the addition of Hg(II) at pH 13. From the TEM images the average particle size of the prepared GQDs from particle size distribution histogram was found to be 1.5 nm (Fig.S5) and SAED pattern supports the crystalline nature of GQDs (Fig.7A; inset). After the addition of 25 μM Hg(II) to GQDs, TEM images were recorded immediately. Interestingly, the formation of Hg(0) was observed and the size was found to be ~ 6 nm (Fig.7B). In the corresponding SAED pattern, crystalline 16

nature of GQDs was disappeared. The formed Hg(0) particles are polycrystalline in nature (Fig.7B; inset). EDAX spectrum was also recorded for Hg(0) produced on the GQDs surface (Fig.S6). The X-ray binding energy corresponds to Lα and Mα were observed at 2.1 and 10 eV, respectively. The absence of characteristic binding energy for oxygen ruled out the possibility for the formation of HgO nanoparticles on GQDs surface. 3.10. Characterization by XPS

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XPS is considered as one of the major tools to find out the chemical state of an element. Fig.8 describes the XPS of prepared GQDs before and after the addition of Hg(II). In the survey spectrum of GQDs, two peaks were observed at 280 and 530 eV due to carbon

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(C1s) and oxygen (O1s) regions, respectively. To find out the nature of elements, wide scan spectrum was deconvoluted and analyzed for C1s and O1s region before and after the

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addition of Hg(II). Fig.8A shows the expanded C1s spectrum for GQDs and GQDs-Hg(II) mixture. The three peaks obtained at 284.8, 285.5 and 288.2 eV in curve a show the presence

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of C=C, C-OH and O-C=O bonding, respectively. The carbon region did not show any shift after the reduction of Hg(II) (curve b). But, in the deconvoluted spectrum of O1s region

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(Fig.8B) the binding energy at 531.2 eV for GQDs was shifted to 531.8 eV for C=O bonding after the introduction of Hg(II) (curve a and b). Similarly, the binding energy for C-O at

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532.8 eV was shifted by 0.4 eV after interacted with Hg(II). The binding energy observed for Hg 4f region at 100 eV in the survey spectrum (Fig.8C) of GQDs-Hg(II) mixture was

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deconvoluted and analyzed. As shown in Fig.8D, three peaks were observed for Hg. The doublet peaks at 100.2 eV for 4f7/2 and 104.5 eV for 4f5/2 indicate the presence of metallic mercury on GQDs surfaces. Along with the Hg(0) peak, another peak at 102.3 eV was also observed. Since it is difficult to differentiate the binding energies of Hg 4f due to the closeness in the binding energy for Hg(I) and Hg(II) in XPS, the binding energy obtained at

17

102.3 eV may be due to the presence of coordinated Hg(I)/Hg(II) with carboxylate groups [34]. 3.11. Characterization by differential pulse voltammetry (DPV) Further, the reduction of Hg(II) to Hg(0) via Hg(I) on GQDs was confirmed by DPV. 10 μL of GQDs (pH 13) with 50 μM Hg(II) after 40 min incubation was drop casted on a well cleaned GC electrode and allowed to dry. Then, DPV was recorded by scanning the potential

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from 0 to +0.8 V. The aim of this experiment was to find out the oxidation states of Hg on GQDs surface. Fig.9 depicts the DPVs obtained for the GQDs-Hg(II) mixture coated from pH 13 and 7 in 0.1 M HClO4. The GQDs-Hg(II) coated from pH 13 showed three oxidation

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peaks (curve a). The oxidation peak at 0.34 V was associated with the oxidation of Hg(I) to Hg(II). To confirm this, Hg(I) was directly added to 0.1 M HClO4 and DPV was recorded

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using GQDs modified GC electrode under identical conditions. It shows the oxidation peak at 0.32 V, which is very close to the dissolution potential assigned for the oxidation of Hg(I) to

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Hg(II) (curve b). The second oxidation peak at 0.48 V was attributed to the oxidation of Hg(0) to Hg(II). Thus, the presence of both Hg(I) and Hg(0) were confirmed from the DPV.

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Along with the association of hydroxyl functional groups, the sp2 domain can also involve in the quenching pathway of GQDs while interacting with metal ions. It is reported

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that the surface state has little effect on the emission from sp2 domain [24]. Even though it is

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less, the involvement of graphene moiety of GQDs on the reduction of Hg(II) cannot be ruled out. The presence of defects sites in GQDs is already established [35]. The reduction of metal ions by materials having defect sites has been reported earlier because the electron transfer is more feasible at these sites [36]. Thus, the Hg(II) reduction was done by hydroxyl functional groups and defect sites on GQDs. The reducing ability of graphene moiety especially the reactive sites on edge graphene is already reported [37,38]. In the second case, initial step is

18

the adsorption of Hg(II) on sp2 domain. According to the previous report, during the interaction of Hg(II)/Hg(I) with GQDs, it would select the on-top site (T) of graphene moiety of GQDs; that is directly on top of a carbon atom (Fig.S7A) [39]. Further, both Hg(II) and Hg(I) should adsorb on T sites and the corresponding binding energies are also higher. Among the heavy metal ions, Hg(II) possesses highest binding energy of 14.563 eV [39]. After the strong adsorption of Hg(II) on graphene domain, electron transfer would occur, resulting the reduction of Hg(II). Since Hg(0) prefers hollow (H) sites on GQDs, that is

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center of the hexagonal ring (Fig.S7B), it is expected that after reduction, Hg(0) would move on to H sites. Therefore, the two dissolution peaks due to Hg(0) to Hg(II) obtained at 0.48 and 0.65 V (Fig.9) can be assigned to the dissolution of Hg(0) formed with the aid of

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hydroxyl functional groups and dissolution of Hg(0) formed with the aid of sp2 domain, respectively. To confirm the presence of unreacted Hg(II), after the dissolution of both Hg(0)

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and Hg(I), the electrode potential was held at -1.0 V for 1 min. The DPV shows a dissolution

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peak at -0.48 V corresponding to the dissolution of Hg(0) to Hg(II) (Fig.S8). This indicates the existence of coordinated Hg(II) on GQDs surface. The overall mechanism for the

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reduction of Hg(II) on the surface of GQDs is schematically shown in Fig.10.

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4. Conclusions

In summary, the systematic investigation on the quenching of GQDs by Hg(II) with

respect to pH, incubation time and concentration of Hg(II) was carried out. It was found that the quenching of GQDs by Hg(II) at pH 7 was faster than pH 13. The complexation of Hg(II) with GQDs was the major contribution for the GQDs quenching at pH 7 whereas the contribution due to Hg(0) was less. On the other hand, emission quenching due to Hg(0) 19

formation was dominant at pH 13 whereas complexation was less. The formation of Hg(0) was confirmed by both SEM and TEM studies. Nevertheless, along with coordination, formation of metallic mercury was observed in both the cases. Because of the formation of Hg(OH)2, the reduction of Hg(II) to Hg(0) was more facile at pH 13. The quenching of GQDs by Hg(II) due to complexation at pH 7 was confirmed from the enhancement of emission by the addition of EDTA especially at low concentration of Hg(II). Among pH 13 and 7, the reduction of Hg(II) to Hg(I) was comparatively slow at pH 7. The reduction pathway of

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Hg(II) to Hg(0) through Hg(I) was confirmed by XPS and DPV. The size of the formed Hg(0) was dependent on the concentration of Hg(II). Further, the reduction of Hg(I) to Hg(0)

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was observed to be faster than Hg(II) to Hg(0) under identical conditions.

Declaration of interests

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The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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Acknowledgments

We thank Department of Biotechnology (DBT), New Delhi, India for the financial support

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(BT/PR20469/BCE/8/1394/2016).

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Figures

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Fig.1. (A) Emission spectra of (a) 1.5 mg/ml GQDs and after the addition of 500 μM each (b) Mn(II), (c) Ni(II),(d) Pb(II), (e) Cu(II), (f) Co(III), (g) Zn(II), (h) Cd(II), (i), Cr(III), (j) Fe(II), (k) Fe(III) and (l) Hg(II) at pH 7.0. (B) Emission spectra recorded

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for (a) GQDs and immediately after the addition of 100 μM Hg(II) to GQDs at pH (b)

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13, (c) 7 and (d) 5.

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Fig.3. (A) Emission spectra recorded for (a) GQDs and GQDs with 10 μM Hg(II) recorded (b) immediately and after (c) 40 min incubation, (d) addition of 1 mM EDTA and (e) the addition of 1 mM cysteine at pH 13 (inset: plot of emission intensity vs. time ). (B) Emission spectra of (a)

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GQDs and GQDs with 50 μM Hg(II) recorded (b) immediately and after (c) 40 min incubation,

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immediately and after (c) 40 min incubation and (d) addition of 1 mM EDTA at pH 7 (inset: Emission intensity vs. time plot). (B) Emission spectra recorded for (a) GQDs and GQDs with 50 μM Hg(II) recorded (b) immediately and after (c) 40 min incubation, (d) addition of 1 mM EDTA

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and (e) 1 mM cysteine at pH 7 (inset: Emission intensity vs. time plot and photograph of GQDs

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under UV light before and after 40 min incubation with 50 μM Hg(II) at pH 7).

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Fig.5. SEM images obtained for GQDs after the addition of (A) 10 and (B) 50 μM Hg

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Fig.6. (A) Emission spectra recorded for (a) GQDs and GQDs with 25 μM Hg(I) recorded (b) immediately, (c) after 40 min incubation, (d) after the addition of 1 mM EDTA and (e) after the addition of 1 mM cysteine at pH 13. (B) (a), (b), (c), (d) and (e) same conditions at

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pH 7 (inset: Emission intensity vs. time plot).

Fig.7. TEM images obtained for GQDs: (A) before and (B) after the addition of 31

Hg(II) at pH 13 (inset of A: particle size histogram and SAED pattern of GQDs and B: SAED pattern after the addition of Hg(II)).

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deconvoluted spectrum of Hg after the incubation of GQDs with Hg(II).

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Fig.9. DPVs obtained for 40 min incubated GQDs-50 μM Hg(II) coated GC electrode at pH (a) 13.0 and (b) 7.0 in 0.1 M HClO4 at a scan rate of 50 mV s-1. 33

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Fig.10. Plausible mechanism for the reduction of Hg(II) by GQDs at pH 7 and 13.

34