Accepted Manuscript

Title: Highly sensitive and selective detection of mercury ions

using N, S-codoped graphene quantum dots and its paper strip
based sensing application in wastewater

Authors: Nguyen Thi Ngoc Anh, Ankan Dutta Chowdhury,

Ruey-an Doong

PII: S0925-4005(17)31391-6
DOI: http://dx.doi.org/doi:10.1016/j.snb.2017.07.177
Reference: SNB 22836

To appear in: Sensors and Actuators B

Received date: 25-5-2017

Revised date: 24-7-2017
Accepted date: 25-7-2017

Please cite this article as: Nguyen Thi Ngoc Anh, Ankan Dutta Chowdhury, Ruey-an
Doong, Highly sensitive and selective detection of mercury ions using N, S-codoped
graphene quantum dots and its paper strip based sensing application in wastewater,
Sensors and Actuators B: Chemicalhttp://dx.doi.org/10.1016/j.snb.2017.07.177

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 Highly sensitive and selective detection of mercury ions

using N, S-codoped graphene quantum dots and its paper

strip based sensing application in wastewater

Nguyen Thi Ngoc Anh1, Ankan Dutta Chowdhury1, Ruey-an Doong1, 2*

1. Institute of Environmental Engineering, National Chiao Tung University, 1001,

University Road, Hsinchu, 30010, Taiwan.

2. Department of Biomedical Engineering and Environmental Sciences, National Tsing

Hua University, 101, Sec. 2, Kuang Fu Road, Hsinchu, 30013, Taiwan.

________________________________

*Corresponding author. Institute of Environmental Engineering, National Chiao Tung

University, Hsinchu, Taiwan; E-mail address: radoong@mx.nthu.edu.tw; Phone number:

+886-3-5726785. Fax number: +886-3-5718649.

1
Highlights

 N, S-GQDs with high quantum yield are fabricated for rapid turn-off detection of Hg2+

ions.

 The dynamic range of Hg2+ by N, S-GQDs are 4 orders of magnitude with LOD of 0.14

nM.

 The N, S-GQDs exhibit excellent selectivity towards Hg2+ detection in real wastewater.

2
Abstract

The development of robust and low-cost sensing materials with superior selectivity and

sensitivity for rapid detection of analytes in a wide variety of samples has attracted much

attention. Herein, the N, S-codoped graphene quantum dots (N, S-GQDs) with high

quantum yield were fabricated by one-pot hydrothermal method for highly sensitive and

selective detection of nanomolar level of mercury ions (Hg2+) in water and wastewater. The

as-prepared N, S-GQDs are uniform in size with mean particle size of 3.5  0.5 nm. The

doping of nitrogen atoms increases the quantum yield to 41.9%, while the introduction of

sulfur atoms enhances the selectivity of Hg2+ via strong coordination interaction. The

fluorescence intensity of N, S-GQDs is quenched proportionally after adding Hg2+ and a

dynamic range of 4 orders of magnitude with limit of detection of 0.14 nM is obtained in

deionized water. The N, S-GQDs nanosensing probes can be successfully applied to the

sewage and dye wastewater samples and a linear range of 0.1 – 15 M with recovery of 96 –

116% is obtained. In addition, the coating of N, S-GQDs onto paper strip provides an

excellently rapid screening and highly selective technique for Hg2+ detection in real

wastewater. Results obtained in this study clearly indicate that the N, S-GQDs are promising

nanomaterials which can open an new avenue to design the GQD based sensing probe for

highly sensitive and selective detection of metal ions and other analytes in environmental

and biological fluid samples.

Keywords: N, S-graphene quantum dots (N, S-GQDs), heteroatom doping, fluorescence

quenching, mercury ions, wastewater.

3
1. Introduction

The contamination of heavy metal ions such as Hg2+, As2+, Pb2+ and Cd2+ has been

becoming one of the most serious problems in the environment because of the increasing

industrial and agricultural activities as well as the improper release of metal ions from

wastewaters and domestic effluents [1-4]. The detection of metal ions in different media by

simple and cost-effective techniques is thus highly needed for environmental safety and

health diagnosis. Among the toxic metal ions, mercury ions (Hg2+) are one of the most

poisonous species to human beings because of the strong interaction of Hg2+ with sulfhydryl

groups of proteins in a wide variety of organs including brain, kidneys, central nervous

system and immune system [5-7]. Although enormous efforts have been carried out to

develop a wide variety of analytical techniques for Hg2+ detection, the most common

method is still dependent on the expensive, time consuming, and hectic instruments like

atomic absorption spectrometry, inductively coupled plasma atomic emission spectrometry

and high-performance liquid chromatography–inductively coupled plasma mass

spectrometry [8-11]. Therefore, there is still an unmet demand to develop new materials for

highly sensitive and selective detection of Hg2+ in real water and wastewater samples.

The optical methods based on nanomaterials have recently been evolved as one of

the promising techniques for detection of metal ions because of their potential advantages of

simple preparation and the sensitive recognition of some special metal ions [12]. Several

studies have used various morphologies of Au-based nanomaterials for the detection of Hg2+

based on the changes in color and absorption [2, 13, 14]. In addition, inorganic metal oxide

quantum dots such as CdSe and CdTe have been developed to detect Hg2+ by fluorescence

quenching methods [15-21]. Yao and Gou have fabricated luminescent core-shell CdTe/CdS

quantum dots for Hg2+ detection and found that the dynamic range of Hg2+ was in the range

of 2 – 500 nM with the limit of detection (LOD) of 1.7 nM [15]. However, the highly toxic

4
effect of inorganic quantum dots on human beings and the environment has hampered the

application of this material to the real environmental application for metal ions detection.

Zero-dimensional carbon-based nanomaterials including carbon dots (CDs) and

graphene quantum dots (GQDs) have recently been emerged as an excellent source of

environmentally friendly fluorescent nanoprobes because of their extra-ordinary properties

such as excellent photoluminescence, tunable surface groups for functionalization, good

stability and easy preparation [22-26]. GQDs have recently ignited the increasing interest in

detection of metal ions and metabolites by fluorescence method using as-prepared [27-30]

and surface modified GQDs [31-33]. Although several advantages can be accounted by

using GQDs as the sensing elements to detect metal ions, the relatively poor quantum yield

of GQDs has limited the detection sensitivity compared with those standard methods.

Doping of GQDs with heteroatoms such as nitrogen (N), boron (B) and sulfur (S) is an

efficient method to improve quantum yield to overcome this drawback [34-37]. Several

studies have depicted that the doping of N atoms on GQDs can enhance the fluorescence

property of GQDs and found that the sensing sensitivity is dependent on the coordination

behavior of graphitic nitrogen atoms [33, 36, 37]. In addition, the N, S-codoped GQDs have

been synthesized for the enhanced visible-light-driven photocatalytic degradation of

rhodamine B [35] or for cyanide detection [38]. However, the development of N, S-GQDs

with high quantum yield for sensitive and selective detection of nanomolar level of Hg2+ has

received less attention. In addition, the development of simple and robust anlaytical

technique for rapid screening of low level of Hg2+ in real wastewater samples based on N, S-

GQDs has not been reported yet.

Herein, we have, for the first time, synthesized the N, S co-doped GQDs with high

quantum yield by one-pot hydrothermal method for highly sensitive and selective detection

of nanomolar level of Hg2+ in wastewater samples. A simple and green fabrication method

5
by hydrothermal treatment of citric acid and thiourea was developed to synthesize N, S-

GQDs. As shown in Scheme 1, the N atoms in N, S-GQDs enhance the fluorescence

quantum yield to 41.9%, while S atoms serve as the active sites for Hg2+ coordination to

increase the selectivity and sensitivity of Hg2+. The interaction of Hg2+ with N, S-GQDs

quenches the fluorescence intensity and a dynamic range of 4 orders of magnitude with

LOD of 0.14 nM is obtained. The N, S-GQDs are then applied for Hg2+ detection in real

sewage and dye wastewaters. The recovery of Hg2+ in different wastewater matrices is in the

range of 96 – 116 %. In addition, the N, S-GQD based paper strip is further prepared by

coating N, S-GQDs onto cellulose filter paper strips for rapid screening of Hg2+ in

wastewater. Results obtained in this study clearly indicate the superiority of as-prepared N,

S-GQDs to detect nanomolar level of Hg2+ in different matrices of water and wastewater.

2. Experimental

2.1 Synthesis of N, S-GQDs

The N, S-GQDs were prepared by a standard hydrothermal method with minor

modification by increasing the weight ratio of thiourea to citric acid [35]. In brief, 0.23 g

citric acid and 0.23 g thiourea (Sigma-Aldrich) were dissolved into 5 mL of deionized water

(18.2 M cm) under stirring conditions to form a clear solution. The solution was then

transferred into a 20-mL of Teflon lined stainless steel autoclave tube and heated up to 160

°C for 4 h. The obtained brown suspension was added into ethanol solution and centrifuged

at 5,000 rpm for 5 min. The final product was re-dispersed into deionized water and stored

at 4 °C for further use. The quantum yield of N, S-GQDs was determined according to our

previous study [28].

2.2 Surface characterization

6
 The morphology as well as particle size of N, S-GQDs was identified by a JEOL

JEM-ARM 200F transmission electron microscope (TEM) and a JEOL JEM-2010 high-

resolution TEM (HR-TEM) both at 200 kV. Atomic force microscopy (AFM) images were

obtained by using an Agilent 5500 scanning probe microscope in tapping mode to elucidate

the topography of N, S-GQDs. The X-ray diffractometry (XRD) was performed by a Bruker

D8 X-ray diffractometer with Ni-filtered Cu Kα radiation (λ = 1.5406 Å). The change in

chemical species of N, S-GQDs was recorded by an ESCA Ulvac-PHI 1600 X-ray

photoelectron spectrometer (XPS) from Physical Electronics using Al Kα radiation photon

energy at 1486.6 ± 0.2 eV. The Fourier transform infrared (FTIR) spectra of N, S-GQDs

were determined by using a Horiba FT720 spectrophotometer. The optical properties of N,

S-GQDs including UV-visible and fluorescence spectra were determined by Hitachi U-4100

UV-visible and F-7000 fluorescence spectrophotometers, respectively. The fluorescence

spectra of N, S-GQDs were recorded by using various excitation wavelengths of 310 – 430

nm. Raman spectra of N, S-GQDs were recorded by Burker Senterra micro-Raman

spectrometer equipped with an Olympus BX 51 microscope and an Andor DU420-OE CCD

camera.

2.3 Detection of Hg2+ by N, S-GQDs

The detection of Hg2+ ions was performed at room temperature. The sensing solution

was prepared by adding 0.16 mg mL-1 N, S-GQD into 0.1 M PBS buffer at pH 7. The

standard solution of Hg2+ was prepared by dissolving 30.75 mg HgCl22H2O into 10 mL of

deionized water. For Hg2+ detection, appropriate volumes of standard Hg2+ solutions were

added into the sensing solution to get the final Hg2+ concentrations of 1 nM – 15 M. The

fluorescence intensity of solution containing N, S-GQDs and Hg2+ was recorded from 350 to

700 nm at excitation wavelength of 340 nm after 2 min of incubation. The calibration curve

7
of Hg2+ was determined by measuring the change in relative fluorescence intensity (I/I0) at

various Hg2+ concentrations ranging from 1 nM to 15 M, where I0 and I are the

fluorescence intensities of sensing solutions in the absence and presence of Hg2+,

respectively. Each experiment was repeated for at least three times for quality control. The

effect of pH on fluorescence intensity was examined by adding 15 µM Hg2+ into the sensing

solutions at pH 5 – 9. The initial pH was adjusted by 0.1 M HCl or NaOH to reach the

desired value.

2.4 Detection of Hg2+ ions in real wastewater samples

To evaluate the applicability of N, S-GQDs to the real wastewaters, 2 effluent

samples including sewage and dye wastewater were collected from different wastewater

treatment plants in Taoyuan City, Taiwan. The physicochemical properties of wastewaters

are listed in Table S1 (see Supplementary data). The wastewater effluents were filtrated by

0.45 µm Millipore filter paper followed by C-18 SPE column to remove suspended solids

and impurities, respectively. After filtration, wastewater samples were spiked with 50 nM –

15 M Hg2+ and then 0.16 mg mL-1 N, S-GQDs were added to the solutions for fluorescence

intensity measurement to evaluate the matrix effect and recovery of Hg2+ in wastewaters.

2.5 Preparation of N, S-GQD-based paper strips for real wastewater detection

The N, S-GQD based paper strips were prepared by cutting cellulose filter paper

(125 mm, Toyo Roshi Kaisha Ltd, Japan) with mean pore size of 25 µm into strips with

dimension of 1 cm (W)  3.5 cm (L). The paper strips were then immersed into 0.16 mg mL-

1
N, S-GQDs solutions for 10 min and then dried in an oven at 60 C for 30 min. For

detection of Hg2+ in wastewaters, the N, S-GQD based paper strips were immersed into the

wastewaters containing 5 – 200 M Hg2+ for 3 sec and then dried in room temperature for

8
another 10 min. The paper strips were then irradiated with 340 nm UV lamp and the change

in fluorescence intensity was recorded by Sony Digital camera for visual detection. In

addition, the selectivity of N, S-GQD based paper filter toward 5 M Hg2+ detection was

evaluated with other metal ions including 100 M Fe2+, Mn2+, Cr3+, Cd2+, Co2+, Pb2+ and

Zn2+.

3. Results and discussion

3.1 Characterization of N, S-GQDs

The surface morphology and particle size distribution of as-prepared N, S-GQDs were

first characterized by (HR)TEM and AFM images. The TEM image of as-prepared N, S-

GQDs shows the homogenous distribution patterns (Fig. 1a) and the particle sizes are in the

range of 2 – 6 nm with an average diameter of 3.5 ± 0.5 nm (Fig. 1b), indicating the

formation of narrow size-distributed N, S-GQDs for sensing applications. Fig. 1c deciphers

the fringes of carbon lattice spacing distances of 2.1 Å, which is similar to the (100) plane of

graphene [28]. In addition, the fast Fourier transform (FFT) pattern (Fig. 1d) also

corroborates that the N, S-GQDs are single crystals with a spacing of 0.21 nm, which is in

good agreement with the result of HRTEM image (Fig. 1e). These results clearly indicate

that the N, S-GQDs are highly nanocrystalline in nature and homogenously distributed. The

AFM image shown in Fig. 1f also shows the uniform distribution of N, S-GQDs. The height

is around 0.5 – 3.0 nm with an average of 1.0 nm, which confirms that the N, S-GQDs only

contain 1 – 5 layers of graphene nanosheets (Fig. 1g).

Fig. 2 shows the XPS survey spectrum and deconvoluted peaks of as-prepared N, S-

GQDs. As illustrated in Fig. 2a, the XPS survey spectrum shows two significant peaks at

284 and 531 eV, which can be assigned as C 1s and O 1s, respectively [39, 40]. Three small

peaks of N 1s (400 eV), S 1s (227 eV) and S 2p (164 eV) are also noticed in the full survey

9
scan of N, S-GQDs, which confirms the successful co-doping of N and S atoms in GQDs. In

addition, the doped amounts of N and S atoms in N, S-GQDs can be estimated from the

XPS full survey spectrum by using CasaXPS software. As shown in Table S2, the doped

amounts of S and N in N, S-GQDs are 1.5 and 8.0 wt%, respectively. A previous study has

used citric acid and rubeanic acid to fabricate N, S-codoped CD and found that the doped

amounts of N and S were 4.0 and 4.7 wt% [39], respectively, which are in a similar range

with our results.

The deconvolution of C 1s region shows peaks of C=C at 284.1 eV, C-N/C-S at

284.3 eV, C–O at 286.2 eV and C=O at 288.3 eV (Fig. 2b). The high contribution of C=C

and C-N/C-S indicates the graphitic structures with strong interaction with N and S atoms,

while C–O and C=O are mainly attributed from the carbonyl and carboxylate groups [41].

Similarly, the deconvoluted peaks of N 1s spectrum show pyrrolic N (C–N–C) and N–H

contributions at 399.7 and 402.1 eV, respectively (Fig. 2c) [42, 43]. The strong pyrrolic

contribution in N 1s peak means that the N atoms with ternary structure are situated higher

than its primary environment (N-H), which is the evidence on the successful N doping onto

the graphitic surface. Moreover, the deconvoluted spectrum of S 2p shows two peaks of

sulfur at 163.8 and 166.8 eV (Fig. 2d), which belong to the spin-orbit coupling of thiophene

2p3/2 and 2p1/2, respectively [44, 45]. The additional peak located at 169.4 eV is the

oxygenated sulfur. In addition, the deconvolution of O 1s spectrum of N, S-GQDs (Fig. S1,

see Supplementary data) displays peaks of C–O, O–H and O–S at 531.1, 532.0 and 532.5

eV, respectively, confirming the successful doping of S atoms into the graphene structures.

Fig. 3a shows the FTIR spectra of N, S-GQDs. A broad peak at 3458 cm-1 is the

stretching vibration of O–H and N–H bonds, which indicates the good hydrophilic nature of

N, S-GQDs because of the amino and hydroxyl groups on the surface. The strong peak at

1639 cm-1 is the C=O in the carboxylic group of graphitic plane. Formation of graphitic

10
layer is confirmed by the C–H and C=C stretching at 1378 and 2096 cm-1, respectively [28].

The C–S and small peak of S–H stretching at 986 and 2340 cm-1, respectively, represent the

sulfur doping [31]. In addition, the broad peak at 662 cm-1 is the weak stretching of C–N

bond, confirming the presence of nitrogen doping on GQDs surface. The Raman spectra

shown in Fig. 3b further confirm the quality of as-prepared N, S-GQDs. The N, S-GQDs

display two distinct D and G bands located at 1378 and 1583 cm−1, respectively, which are

ascribed to the characteristic peaks of graphitic structure [36]. The disordered (D) band at

1378 cm-1 is related to the presence of sp3 defects, while the crystalline (G) band at 1583

cm-1 is attributed from the in-plane vibration of sp2 carbon [28]. The ratio of intensities

(ID/IG) is found to be 0.87 for N, S-GQDs, which means that N, S-GQDs are highly graphitic

materials. In addition, the broad peak at around 24.1° 2 shown in the XRD pattern (Fig. S2,

see Supplementary data) is the (002) plane of graphene, which is in good agreement with

those results obtained from Raman spectra and HRTEM images.

The optical property of as-prepared N, S- GQDs was identified by UV-visible and

fluorescence spectra. Fig. 4a shows the emission fluorescence intensity of N, S-GQDs

excited with various wavelengths ranging from 310 to 430 nm. The N, S-GQDs exhibit an

emission peak at 445 nm and the peak position is independent on the excitation wavelength,

which is perfectly matched with the previously reported results [27, 33, 35]. The

maintenance of emission peak position at 445 nm indicates the sequential absorption of

photons and then results in the emission of fluorescence light at a constant wavelength. It is

also noteworthy that the solution color of as-prepared N, S-GQDs is almost transparent

under visible light irradiation. This means that the as-prepared N, S-GQDs have little up-

conversion and down-conversion properties in the visible light region [39, 46], which is in

good agreement with the emission fluorescence spectra. Fig. 4b shows the UV-visible

absorption spectrum of N, S-GQDs where the strong absorption peak is found at 332 nm,

11
ascribing the n–π* transition of C=O. The absorption bands at 225 and 236 nm are

contributed from the π–π* transition [35]. In addition, the N, S-GQDs emit strong bright

blue fluorescence after the irradiation of UV light at 340 nm (Fig. 4c), indicating the

excellent photoluminescence property for optical sensing. The fluorescence intensity at an

excitation of 340 nm can be maintained for at least 2 months (Fig. S3, see Supplementary

data), clearly depicting the excellent stability of N, S-GQDs for sensing applications.

The emission fluorescence spectra of undoped GQDs was also examined. As shown

in Fig. S4a (see Supplementary data), the emission fluorescence wavelength at 450 nm is

clearly observed when excited at 340 nm. The blue shift in emission wavelength for N, S-

GQDs in comparison with the undoped GQDs is mainly attributed to the doping of electron-

rich N atoms on the surface [47-49], which would result in the high quantum yield. The

quantum yield of N, S-GQDs, determined by using fluorescein (Φ = 0.79) as a reference

[27, 28], is found to be 41.9 %, which is significantly high among the recently reported N-

doped GQDs [27, 30, 33] and N, S-CDs [46]. Our previous study has synthesized the as-

prepared GQDs by the pyrolysis of citric acid and the quantum yield of as-prepared GQDs

was 11.9% [28], clearly showing that the N, S-GQDs have high quantum yield than that of

undoped GQDs. The doping of GQDs with N atoms can provide more electron-enriched

active sites, which facilitate the high yield of radiative recombination as well as the decrease

in non-radiative recombination [42]. It is noteworthy that the doped amount of N atoms in

N, S-GQDs shows that the as-synthesized N, S-GQDs contain higher amount of ternary N

atoms than that of primary NH2 (Fig. 2c). Since the density of ternary N atoms strongly

affect the radiative path of GQDs, the doping of ternary N atoms can make the N, S-GQDs

more fluorescence active compares with the pristine GQDs.

3.2 Detection of Hg2+ using N, S-GQDs

12
 After the successful characterizations, the sensibility of N, S-GQDs toward Hg2+

detection was evaluated by the change in fluorescence intensity before and after the addition

of Hg2+. Fig. 5a shows the change in fluorescence intensity of N, S-GQDs after the addition

of various concentrations of Hg2+ ranging from 1 nM to 15 M. The fluorescence intensity

of N, S-GQDs decreases upon increasing Hg2+ concentration and 90% of the original

fluorescence intensity is quenched at 15 µM Hg2+. It is noteworthy that the quenching of N,

S-GQDs by Hg2+ can be attributed to both static and dynamic quenching. After addition of

Hg2+ into the N, S-GQD solutions, the collision of Hg2+ with N, S-GQDs would produce

dynamic quenching first and then the successful binding of Hg2+ onto the N, S-GQDs

surface produces static quenching. In addition to the carboxylic (−COOH) and hydroxyl (–

OH) groups on the N, S-GQDs surface which can serve as the effective coordination sites

for Hg2+ binding [50], the main reaction mechanism for highly specific binding of Hg2+ is

mainly attributed to the strong coordination reaction between S atoms in the graphitic

carbon structure of GQDs and Hg2+ [39].

Fig. S4b (see Supplementary data) shows the change in fluorescence intensity of

undoped GQDs in the presence of various Hg2+ concentrations ranging from 0 to 50 M. It

is clear that only 3 – 8% decrease in fluorescence intensity at 445 nm is observed when 1 –

50 M Hg2+ are added into the undoped GQD solutions. This sensing performance is much

lower than that of N, S-doped GQDs, clearly showing that doping of N and S atoms can

significantly enhance the sensitivity of GQD based nanomaterials.

Due to the well-known soft acid-soft base interaction between Hg2+ and S atoms,

Hg2+ can be easily attached onto the N, S-GQDs via strong coordination chemistry. This

would lead to the interruption of electronic π-π conjugation of N, S-GQDs to affect the

distribution of excitons obviously, and subsequently results in the hindrance of electron

transfer process as well as the quenching effect of N, S-GQDs. In addition, the pH effect on

13
the sensing efficiency of Hg2+ was further investigated in the presence of 15 µM Hg2+. As

illustrated in Fig. 5b, the fluorescence intensity of N, S-GQDs before the addition of Hg2+

increases with the increase in pH values from 5 to 9 because of the deprotonation of COOH

group at high pHs. However, the fluorescence intensity is quenched obviously at pH 6 – 9

after the addition of 15 M Hg2+, clearly showing that the N, S-GQDs can serve as an

efficient sensing probe for sensitive detection of Hg2+.

Fig. 5c shows the change in fluorescence intensity of N, S-GQDs as a function of

Hg2+ concentration. A two-stage linear relationship between the change in fluorescence

intensity and Hg2+ concentration is observed. It is clear that an excellent linearity in the Hg2+

concentration range of 0.05 – 15 µM with a correlation coefficient of 0.995 is obtained. The

sensing system also obey a linear relationship in the low Hg2+ concentration range of 1 – 50

nM with a correlation coefficient of 0.96 (inset of Fig. 5c). The LOD of Hg2+, which can be

determined by the 3δ/S where δ is the standard deviation of the lowest fluorescence signal

and S is the slope of linear calibration plot [28], is calculated to be 0.14 nM. The superior

sensitivity of N, S-GQDs is mainly attributed to the high quantum yield obtained in this

study. It is noteworthy that the addition of heteroatoms would increase the surface defects of

GQDs, resulting in the increase in isolated sp2-conjugated carbon clusters to enhance the

fluorescence intensity as well as the quantum yield [44]. As shown in Table S2 (see

Supplementary data), the quantum yield of 41.9% for N, S-GQDs is higher than those

prepared by citric acid with rubeanic acid [39] and biomass [46], which can provide a wide

and stable analytical range for sensing, and subsequently enhances the sensitivity of N, S-

GQD toward Hg2+ detection. In this study, the doping of nitr widen the dynamic range and

increase the detection sensitivity of Hg2+ with ultra-low LOD.

Table 1 summarizes the analytical performance of Hg2+ by different inorganic and

organic quantum dots. It is clear that the LOD values for Hg2+ detection by inorganic

14
quantum dots are in the range of 0.47 – 750 nM [15-21]. In addition, the carbon based

materials including carbon dots (CDs) and as-prepared GQDs have been developed for

sensitive detection of Hg2+ and the LODs can be lowered to 0.44 – 180 nM [30, 39, 46, 52,

53]. Wang et al. have recently developed N, S-codoped CDs for detection of Hg2+ and a

dynamic range of 0.1 – 20 M with LOD of 0.18 M was observed [39]. In this study, the

N, S-GQDs are highly sensitive toward Hg2+ detection and the dynamic range can be up to

four orders of magnitude with LOD of 0.14 nM, which is better than those of the reported

data shown in Table 1.

The specificity as well as selectivity is one of the most important parameters for

sensing application. In this study, the selectivity of N, S-GQDs was evaluated by adding 15

M various metal ions including Mn2+, Cr3+, Mg2+, Fe3+, Fe2+, Al3+, Co2+, Cd2+, Cu2+, Ni2+,

Zn2+, Pb2+ and Ca2+ as the interference ions. As shown in Fig. 5d, no obvious decrease in

fluorescence intensity is observed after the addition of interference metal ions. In contrast,

the fluorescence intensity decreases remarkably when Hg2+ is added to the sensing solutions.

When mixing all the metal ions together with Hg2+, the sensor provides almost similar

quenching effect in comparison with the pure 15 µM Hg2+ solution. Several studies have

depicted that the N, S-codoped CDs can effectively detect Hg2+ and Fe3+ [39, 46, 52]. In this

study, the N, S-GQDs show higher selectivity toward Hg2+ detection than that of Fe3+,

which is mainly attributed to the well-known soft acid-soft base coordination interaction

between S atoms in the graphitic carbon structure and Hg2+. It is also noteworthy that N, S-

GQDs prepared by citric acid and cysteine can effectively detect Au3+ in lake and river

waters [27]. In this study, however, the interference of Au3+ is not examined because the Au

concentration is insignificant in wastewater when compares with that of Hg2+.

3.3 Real wastewater analysis

15
 After the demonstration of excellent analytical performances of Hg2+ in deionized

water, the applicability of N, S-GQDs to Hg2+ detection was further examined in the real

wastewater collected from the effluents of sewage treatment plants in Taoyuan city, Taiwan.

Fig. 6a shows the change in fluorescence intensity of N, S-GQDs in sewage effluents in the

presence of various concentrations of Hg2+. The addition of N, S-GQDs to wastewater

sample in the absence of Hg2+ (blank control) still shows good blue fluorescence intensity in

sewage effluent. After spiking with 0.05, 0.5, 1, 10 and 15 μM of Hg2+ ions, however, the

fluorescence intensities of N, S-GQDs decrease dramatically (Fig. 6a). A good linear

relationship between Hg2+ concentration and change in fluorescence intensity with a

correlation coefficient of 0.995 is clearly observed (Fig. 6b), indicating that the matrix effect

of wastewater has less interference on Hg2+ detection by N, S-GQDs.

The recovery of Hg2+ in real wastewater samples was also calculated by spiking

various concentrations of Hg2+ ranging from 50 nM and 15 M into the wastewater samples

collected from the sewage treatment plant and dye wastewater treatment plant. As shown in

Table 2, the recovery of 107  2 % is observed when low concentration of 50 nM Hg2+ is

spiked into the deionized water. Similar to the deionized water, recoveries of 96 – 116 % are

observed when low concentrations of 50 – 100 nM Hg2+ are spiked into the wastewater

samples. In addition, spiking with high concentration of Hg2+ still can get excellent

analytical performance and the recoveries are in the range of 98 – 115 % and 98 – 103 %

when 1 and 15 M Hg2+ are added into different wastewater samples, respectively. The

relative standard deviations for all the spiking analysis are in the range of 2.0 – 6.1 % (n =

3), clearly indicating the excellent analytical performance of N, S-GQDs on Hg2+ detection

in real wastewater samples. It is noteworthy that the COD concentrations for sewage and

dye wastewater effluents are 21.3 and 170 mg/L, respectively (Table S1, see Supplementary

data). However, the recovery of these two wastewater samples has no obvious difference. In

16
addition, the recovery of Hg2+ between the deionized water and real wastewater are quite

similar, clearly depicting that N, S-GQDs are promising nanosensing materials for detection

of Hg2+ in different wastewater samples.

3.4 Rapid screening of Hg2+ by N, S-GQD-based paper strips

The development of potential and rapid screening techniques for wastewater

application is always challenging because of the serious interference of matrix effect and

suitable sensing elements. After the successful detection of Hg2+ by N, S-GQDs in

wastewater effluents, we have also developed an N, S-GQD-based paper strip for rapid

sensing and screening of Hg2+ in wastewaters because of its unique properties of high

selectivity, low detection ability and reliability toward Hg2+ detection. In this study, the low

cost and non-fluorescent cellulose filter papers were selected as the supporting material. Fig.

7a illustrates the images of change in fluorescence intensity of paper strip sensor after

immersion into wastewaters containing various concentrations of Hg2+ ranging from 5 – 200

M. It is clear that the paper strip has no fluorescence property under UV light irradiation.

In contrast, the paper strip coating with N, S-GQDs shows a bright blue fluorescence under

the irradiation of portable UV light and the fluorescence turns into faint when wastewater

contains 5 – 10 μM Hg2+. Moreover, the change in fluorescence intensity becomes distinct

when the Hg2+ concentration increases from 10 to 200 M, clearly showing that the

developed N, S-GQDs paper strips can serve as a platform for rapid and robust detection of

wide concentration range of Hg2+ in wastewaters. In addition, the interference effect of high

concentration of 100 M metal ions including Fe2+, Mn2+, Cr3+, Cd2+, Co2+, Pb2+ and Zn2+

on N, S-GQD based paper strip was evaluated. As illustrated in Fig. 7b, the fluorescence

intensity of paper strip decreases at 5 M Hg2+ in comparison with the as-prepared N, S-

GQD based paper strip. However, the fluorescence intensity of N, S-GQD based paper strip

17
remains almost unchanged when high concentration of interference metal ions are spiked

into the wastewater effluents, clearly indicating the excellent selectivity and sensitivity of N,

S-GQD based paper strip for rapid and robust detection of mercury ions in real wastewaters.

4. Conclusions

In this study, we have developed a highly selective and sensitive N, S-GQD based

sensing technique for rapid and robust detection of nanomolar level of Hg2+ in water and

wastewater. The 2 – 6 nm N, S-GQDs with 1 – 5 layers of graphene nanosheets are

successfully fabricated by using one-pot hydrothermal method. The doping of nitrogen atom

can enhance the quantum yield up to 41.9%, resulting in the high sensitivity and wide

detection range. The introduction of S atoms to N, S-GQDs can selectively detect Hg2+ via

strong coordination based on the soft acid and soft base interaction. The N, S-GQDs exhibit

excellent analytical performance on Hg2+ detection and a dynamic range of 4 orders of

magnitude with LOD of 0.14 nM is obtained. In addition, the N, S-GQDs nanosensing

probes can be successfully applied for sewage and dye wastewater analyses without obvious

matrix interference. A linear range of 0.05 – 15 M with recovery of 96 – 116% is obtained

for Hg2+ detection in different wastewater matrices. In addition, the coating of N, S-GQDs

onto paper strip provides an excellently rapid screening technique for Hg2+ detection in

wastewaters. Results obtained in this study clearly indicate that the N, S-GQDs can

selectively and sensitively detect nanomolar Hg2+ in wastewaters, which can open an avenue

to provide a platform for fabrication of GQD based nanoprobe for highly rapid and robust

detection of metal ions in environmental and biological fluid samples.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in the online version, at doi:

18
Acknowledgements

The authors thank the Ministry of Science and Technology (MOST), Taiwan for

financial support under grant No. 105-2113-M-009-023-MY3.

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Table 1. Analytical performance of Hg2+ by inorganic and organic quantum dot based

sensors.

Dynamic range LOD

Type Probea (nM) References
(M)
Inorganic CdSe@ZnS 36 – 600 180 [16]

CdTe@SiO2 0.002 – 0.014 1.6 [17]

NAC-QDs 5 – 250 750 [18]

CdS 0.04 – 13 8.6 [19]

CdTe@CdS 0.002-0.5 1.7 [15]

0.00047 – 0.05
CdTe/SiO2/CDs 0.47 [54]
0.5 – 21
BSA-CdTe 0.001 – 1 1.0 [21]

Organic GQDs 0.001 – 0.050 0.439 [30]

N, S-CDs 0.1 – 20 180 [39]

N, S-CDs 0.01 – 1.2 10.3 [46]

CDs 0.2 – 15 25 [52]

CNPs 0.25 – 12 42 [53]

N, S-GQDs 0.001 – 15 0.14 This work

a: NAC-QD: N-acetyl-L-cysteine functionalized quantum dots; GQDs: graphene quantum

dots; CDs: carbon dots; CNPs: carbon nanoparticles.

26
 Table 2. Recovery of Hg2+ in real wastewater samples (n = 3).

Spiked Detected Recovery  RSD

Sample
concentration concentration (%)
Deionized Water 50 nM 53 nM 107  2.0

Sewage 50 nM 48 nM 96  3.1

Sewage 100 nM 105 nM 105  3.9

Sewage 1 µM 1.15 µM 115  5.1

Sewage 15 µM 15.5 µM 103  4.5

Dye wastewater 50 nM 58 nM 116  3.8

Dye wastewater 100 nM 96 nM 96  4.7

Dye wastewater 1 µM 1.05 µM 98  5.7

Dye wastewater 15 µM 14.7 µM 98  6.1

27
Figure caption

Scheme 1. Schematic diagram of Hg2+ detection using turn off fluorescence property of N,

S-GQDs and paper based sensor images in real wastewater samples.

Fig. 1. The characterization of N, S-GQDs. (a) TEM image, (b) particle size distribution (n

= 50), (c) HRTEM image of single N, S-GQDs, (d) fast Fourier transform pattern, (e)

lattice spacing of as-prepared N,S-GQDs from HRTEM image, (f) AFM image and (g)

topography of N, S-GQDs from AFM image.

Fig. 2. The XPS (a) survey scan and deconvoluted (b) C 1s, (c) N 1s and (d) S 2p peaks of

as-prepared N, S-GQDs.

Fig. 3. The (a) Raman and (b)FTIR spectra of N, S-GQDs

Fig. 4. (a) The fluorescence spectra of N, S-GQDs excited with various wavelengths ranging

from 310 to 430 nm, (b) UV-Vis spectra of N,S-GQDs and (c) photographs of N, S-

GQDs under daylight (left) and UV light irradiation (right).

Fig. 5. (a) Emission fluorescence spectra of N, S-GQDs in the presence of various

concentrations of Hg2+ ranging from 0 – 15 M, (b) the change in fluorescence

intensity as a function of Hg2+ concentration, (c) the pH effect on the fluorescence

intensity of N, S-GQDs before and after the addition of 15 M Hg2+ at various pHs of

5 – 9 and (d) selectivity of N, S-GQDs in the presence of various metal ions.

Fig. 6. The change in (a) emission fluorescence spectra and (b) fluorescence intensity ratio

as a function of Hg2+ concentration.

Fig. 7. The N, S-GQDs based paper strips for detection of Hg2+ in wastewater effluent. (a)

The change in fluorescence as a function of Hg2 concentration and (b) interference of

100 µM metal ions including Fe2+, Mn2+, Cr3+, Cd2+, Co2+, Pb2+ and Zn2+.

28
Scheme 1.

29
 15

(a) (b) (d) (f)

Particle Distributions
12

0
1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5
Diameter (nm) 0 nm 1 00 nm

(c) (e)200 (g) 3.0

2.5

Height (nm)
150 2.0

Height
1.5
100
1.0

50 0.5

20 nm 0
0.21 nm 0.0

5 nm
-0.5
0.0 0.3 0.6 0.9 1.2 1.5 1.8 0.0 0.2 0.4 0.6 0.8 1.0

Distance (nm) Distance (m)

Fig. 1.

30
 (a) (b)
-O1s

-C1s
C=C

Intensity
Intensity

C-S
-N1s

C=O C-O
-S1s

-S2p

600 500 400 300 200 100 290 288 286 284 282
Binding energy (eV) Binding energy (eV)
(c) C-N (d)
Intensity

S-O
Intensity

thiophene-S

N-H

404 403 402 401 400 399 398 397 176 174 172 170 168 166 164 162 160 158
Binding energy (eV) Binding Energy (eV)

Fig. 2

31
 (a)
Transmittance (%) C-S
C-H S-H
C-C C-H
C-N
C=O / C=N

OH / NH

4000 3500 3000 2500 2000 1500 1000 500

Wavenumber (cm-1)

(b) G
1583
D
Intensity (a.u.)

1378

1200 1300 1400 1500 1600 1700 1800

-1
Raman shift (cm )

Fig. 3

32
 (a) 310 nm (b)
320 nm 0.5
Fluorescence intensity

330 nm
340 nm
350 nm 0.4

Absorbance
360 nm
370 nm
380 nm 0.3
390 nm
400 nm
410 nm
420 nm 0.2
430 nm

0.1

0.0
350 400 450 500 550 600 650 250 300 350 400 450 500

Wavelength (nm) Wavelength (nm)

Fig. 4

33
 (a) 3500 (b) 6000 pH 5
pH 6
2+
Before Hg addition

Intensity (a.u.)
2+
pH 7 After Hg addition

Fluorescecne intensity
3000 5000 pH 8
Hg2+ pH 9
Fl Intensity

2500 0
4000 400 450 500 550 600

2000 15 M Wavelength (nm)

3000
1500

1000 2000

500 1000

0
350 400 450 500 550 600 0
5 6 7 8 9
Wavelength (nm) pH
(c) 1.0 (d)
0.96
1.0
Fluorescence ratio (I/I0)

0.88
I/I0

0.8 0.80
0.8
0.72

y=0.0025x+0.9402
0.64 2
R = 0.96
I/I0

0.6 0 10 20 30 40 50
0.6
Concentration (nM)

0.4 0.4

y = 0.05x+0.909 0.2
2
0.2 R = 0.995
0.0
0 2 4 6 8 10 12 14 16 an
k
II) (II
) ) )
II) (II) (III i(II o(II
)
(II
) ) ) ) )
III (II III (II (I) ix II)
Bl Cu( n( g(
M
g
M Cd Cr N C Zn Al( Fe Fe( Pb Ag M H
Concentration (M)

Fig. 5

34
 (a) 1500
2+
1250 Hg
Fluorescence intensity

50 nM
1000

750
15 M
500

250

400 450 500 550 600

Wavelength (nm)

1.0
(b)
Fluorescence ratio (I/I0)

0.8

0.6

0.4
y = 0.26 log(x) - 1.3
2
0.2 R = 0.9945

0.0
100 1000 10000
Concentration (nM)
Fig. 6

35
Fig. 7.

36
Author Biographies

Nguyen Thi Ngoc Anh received her BS from Can Tho University, Vietnam in

2010. She is now a PhD candidate majoring in Environmental Engineering under the

supervision of Professor Ruey-an Doong at the Institute of Environmental

Engineering, National Chiao Tung University, Taiwan. Her research interest mainly

focused on the fabrication of smart materials for sensing and biosensor applications.

Ankan Dutta Chowdhury received his B.S. degree in 2006 from the Calcutta

University, Kolkata, India and M.S. from G.G.U., Bilaspur, India in Chemistry. He

received his PhD degree in Chemical Sciences Division at Saha Institute of Nuclear

Physics, India in the field of Chemistry. He is currently continuing his research as a

postdoctoral research fellow at National Chiao Tung University, Taiwan on

biosensing, drug delivery and capacitative study using functional nanomaterials. His

research interests lie in the area of biosensors and different nano materials for

biosenssing applications.

Ruey-an Doong is currently a distinguished professor in the Institute of

Environmental Engineering, National Chiao Tung University, Taiwan. His current

research interest includes the fabrication of nanomaterials with novel optical and

electrochemical properties for biosensing of analytes, environmentally benign

nanotechnology for treatment of contaminants, and porous materials for energy

storage and conversion.

37