Author's Accepted Manuscript

A highly sensitive protocol for the determina-tion of Hg2þ in environmental water usingtime-gated mode

Dawei Huang, Chenggang Niu, GuangmingZeng, Xiaoyu Wang, Xiaoxiao Lv

PII: S0039-9140(14)00843-1DOI: http://dx.doi.org/10.1016/j.talanta.2014.10.015Reference: TAL15154

To appear in: Talanta

Received date: 3 August 2014Revised date: 28 September 2014Accepted date: 2 October 2014

Cite this article as: Dawei Huang, Chenggang Niu, Guangming Zeng, XiaoyuWang, Xiaoxiao Lv, A highly sensitive protocol for the determination of Hg2þ

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A highly sensitive protocol for the determination of Hg2+ in environmental water

using time-gated mode

Dawei Huang a,b, Chenggang Niu a,*, Guangming Zeng a, Xiaoyu Wang a, Xiaoxiao Lv a a College of Environmental Science and Engineering, Key Laboratory of Environmental Biology and

Pollution Control (Hunan University), Ministry of Education, Hunan University, Changsha 410082, China b South China Institute of Environmental Sciences, the Ministry of Environmental Protection of PRC,

Guangzhou 510655, China

*Corresponding Author:

E-mail address: cgniu@hnu.edu.cn, cgniu@hotmail.com

Tel: +86-731-88823820, Fax: +86-731-8882282

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ABSTRACT: In this contribution, a sensitive time-gated fluorescent sensing strategy for

mercury ions (Hg2+) monitoring is developed based on Hg2+-mediated thymine (T)-Hg2+-T

structure and the mechanism of fluorescence resonance energy transfer from Mn-doped CdS/ZnS

quantum dots to graphene oxide. The authors employ two T-rich single-stranded DNA (ssDNA)

as the capture probes for Hg2+, and one of them is modified with Mn-doped CdS/ZnS quantum

dots. The addition of Hg2+ makes the two T-rich ssDNA hybrids with each other to form stable

T-Hg2+-T coordination chemistry, which makes Mn-doped CdS/ZnS quantum dots far away from

the surface of grapheme oxide. As a result, the fluorescence signal is increased obviously

compared with that without Hg2+. The time-gated fluorescence intensities are linear with the

concentrations of Hg2+ in the range from 0.20 to 10 nM with a limit of detection of 0.11 nM. The

detection limit is much lower than the U.S. Environmental Protection Agency limit of the

concentration of Hg2+ for drinking water. The time-gated fluorescent sensing strategy is specific

for Hg2+ even with interference by other metal ions based on the results of selectivity experiments.

Importantly, the proposed sensing strategy is applied successfully to the determination of Hg2+ in

environmental water samples.

Keywords: Hg2+; Thymine-Hg2+-thymine; Time-gated fluorescent sensing strategy; Mn:CdS/ZnS

quantum dots; Graphene oxide

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1. Introduction

Hg2+ is a highly toxic metallic pollutant as it has strong bioaccumulation. The cell functions

will be inhibited when Hg2+ is accumulated in the vital organs and tissues, and then causes a

variety of human health problems [1]. The exposure to Hg2+ even at very low concentrations still

has potential risks for human beings [1,2]. The contamination of Hg2+ is a serious environmental

health problem in the world, especially the contamination of drinking water and other

environmental water resources [3]. Therefore, the monitoring of Hg2+ levels with highly

sensitivity and selectivity in aquatic ecosystems is still an important and urgent issue. Traditional

methods including atomic absorption spectroscopy (AAS), atomic fluorescence spectrometry

(AFS), inductively coupled plasma mass spectrometry (ICPMS), and X-ray absorption

spectroscopy have been widely used for the determination of Hg2+ [3�7]. Although these

traditional analytical techniques are very sensitive and selective, the development of new and

efficient Hg2+ detection methods that are cost-effective, rapid, sensitive, selective, and applicable

in routine and in situ is also important and necessary. In previously reported works, it is found

that T-T mismatches could selectively capture Hg2+ to form stable T-Hg2+-T base pairs due to the

intrinsic specific interaction between Hg2+ and thymine [8,9]. Based on the intrinsic specific

interaction between Hg2+ and thymine, a series of fluorescent, colorimetric, and electrochemical

sensors were proposed for Hg2+ quantification [10�29]. As far as fluorescent sensors are

concerned, many of them could be applied to the determination of Hg2+ in aquatic ecosystems

due to the low detection limits which are lower than the toxic level reported by the U.S.

Environmental Protection Agency. However, most fluorescent sensors for Hg2+ are based on

organic dyes or QDs that usually have short lifetime fluorescence. The background noises caused

by the scattering light from solid substrates, the luminescence from the optical components, and

samples themselves might affect the sensitivity of the methods. Hence, it is of interesting to

develop a highly sensitive fluorescence method based on luminophor with long lifetime

fluorescence, which can use time-gated mode to decrease the background noises. It has been

previously reported that Mn-doped QDs have the features of high quantum yield, photostability,

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size-tunable optical properties, and long lifetime fluorescence [30�34]. What’s more, it has been

proved that water soluble Mn-doped CdS/ZnS QDs with long lifetime fluorescence (~4.8 ms) and

excellent stability in aqueous solution exhibited outstanding performances in our previously

reported works [26,27]. The authors thought that the Mn-doped CdS/ZnS QD are good choices

for time-gated fluorescent methods as a fluorescence label.

In recent years, graphene oxide (GO) has emerged and attracted extensive attentions in the

biological and medical applications because of their superior surface properties, unique electronic

properties, and good biocompatibility [35�39]. Especially, GO is an efficient quencher of an

organic fluorophore or inorganic luminescent nanomaterial through either energy transfer or

electron transfer process [40�43]. Without any surface modification, GO can strongly adsorb

unfolded single-stranded DNA (ssDNA) via the �-� stacking interactions while adsorption of

well-folded or double-stranded DNA (dsDNA) is disfavored [44]. Therefore, a series of

GO-based biosensors have been fabricated due to its superior and unique features [45�50].

What’s more, compared with the papers using GNPs as the quencher, there are three main

reasons make us employ graphene oxide (GO) as a new quencher: (1) better quenching efficiency;

(2) stronger adsorption for ssDNA; (3) higher anti-interference ability (such as GO cannot be

affected by salt concentration). Based on these advantages, the authors employ GO as an

effective quencher and want to propose a method with higher sensitivity and selectivity.

Combining advantages of the Mn-doped CdS/ZnS QDs and GO together, a highly sensitive

and selective time-gated fluorescent sensing strategy is designed for Hg2+ detection in aqueous

solution based on the principle of fluorescence resonance energy transfer (FRET). The Mn-doped

CdS/ZnS QDs act as the energy transfer donor while the GO serves as the energy transfer

acceptor. Two T-rich ssDNA, which specifically bind to Hg2+ to form T-Hg2+-T coordination

chemistry, are selected as the Hg2+ recognition probes. It is found that the proposed time-gated

fluorescent method exhibits highly sensitivity and superior selectivity toward Hg2+. In addition,

the determination of Hg2+ in spiked environmental water samples is performed to demonstrate the

applicability of this kind of method.

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2. Experimental section

2.1. Chemicals and apparatus

Two oligonucleotides used in the present study were synthesized and HPLC purified by

Shanghai Sangon Biological Engineering Technology & Services Co., Ltd. (Shanghai, China)

and dissolved in buffer (0.010 M Tris-acetate, pH=7.6), then stocked at –20 �C for future usage.

The sequences of the two oligonucleotides were shown as follows: 5’TTTGTTTGTTGG3’

(ssDNA1); 5’SH-CCTTCTTTCTTA3’ (ssDNA2). 3-Mercaptopropionic acid (MPA, 99+%) was

purchased from Sigma-Aldrich and used as received without further purification.

Tetrabutylammonium chloride was obtained from Tianjin Guangfu Fine Chemical Co., Ltd

(Tianjin, China). Graphite powder was purchased from Tianjin Kemiou Chemical Reagent Co.,

Ltd (Tianjin, China). GO was prepared using a modification of Hummers and Offeman’s method

from environmental graphite powder [51�53]. Mn-doped CdS/ZnS QDs were synthesized

according to the previously reported papers, and the characterization and various performance of

this kind of quantum dots were also presented in previously reported papers [30,31]. All metal

salts used in this work were purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai,

China). All other chemicals and solvents were of analytical reagent grade and obtained from the

commercial sources, then were used without further purification. Ultrapure water was used

throughout the experiments. The time-gated fluorescence intensity was measured and recorded

with a Perkin-Elmer LS-55 spectrofluorimeter (United Kingdom). UV/Vis absorption spectra

were recorded by using Shimadzu UV Spectrophotometer (UV-2550, Kyoto, Japan). Atomic

fluorescence measurements were performed on Atomic Fluorescence Spectrometer (AFS-9700)

(Beijing, China).

2.2. Synthesis of water soluble Mn-doped CdS/ZnS QDs

The Mn-doped CdS/ZnS QDs is hydrophobic. The characteristic of hydrophobic is a

stumbling block for the reaction between QDs and the water soluble alkylthiol-capped

oligonucleotides. Therefore, the ligand-exchange was carried out using hydrophilic

3-mercaptopropionic acid to prepare the water soluble QDs. The details of the ligand-exchange

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experiment are as follows. Firstly, an amount of 2.0~5.0 mg of Mn-doped CdS/ZnS QDs was

dissolved in 1.0 mL of chloroform, and then 1.0 mL MPA solution (1.0 mL of MPA was dissolved

in 1.5 mL water which contained 1.0 g KOH) was dropped to the QDs chloroform solution with

stirring in a glass bottle with a capacity of 10 mL. Stirring was continued for 8.0 h. After that, 2.0

mL of methanol was added to the mixture and blended well by vortex. After standing for 10 min,

a large amount of acetone was added to the mixture, generating a lot of precipitation. To obtain

the precipitate, the suspension was centrifuged at 4000 rpm for 5.0 min. After throwing away the

supernatant, the precipitate was dissolved in 2.0 mL water, and then the solution was centrifuged

at 12600 rpm for 20 min using Centrifugal Filter Devices (Amicon Ultra-0.5) to remove

impurities. Finally, the pure water soluble Mn-doped CdS/ZnS QDs were redispersed into 5.0 mL

of Tris-acetate buffer (0.010 M, pH = 7.6) by vortex and stored at 4 �C for future usage. The

fluorescence excitation and emission of this kind of QDs were 400 nm and 609 nm, respectively.

The absorbance spectrum and fluorescence emission spectrum of the resulting QDs are shown in

Figure 1. The concentration of the resulting QDs was about 5.7 × 10-7 M according to the

absorbance at 400 nm. The resulting water soluble Mn-doped CdS/ZnS QDs have long lifetime

fluorescence (~4.8 ms) and excellent stability in aqueous solution.

Figure 1 should be here

2.3. Preparation of ssDNA2-modified Mn-doped CdS/ZnS QDs

ssDNA2 was conjugated to the Mn-doped CdS/ZnS QDs according to a previously reported

work with minor modifications [54]. Firstly, the Mn-doped CdS/ZnS QDs solution and ssDNA2

solution were mixed together at a mole ratio of 1:5 (1.0 mL, 5.7 × 10-7 M Mn-doped CdS/ZnS

QDs mixed with 30 �L, 0.10 mM ssDNA2). After standing for 12 h, the mixture was brought to

0.15 M NaCl and the particles were aged for an additional 12 h. After that, the NaCl

concentration was then raised to 0.30 M, and the mixture was allowed to stand for a further 40 h.

The mixture was centrifuged using Centrifugal Filter Devices (Amicon® Ultra-0.5) to remove the

unreacted ssDNA2 afterwards. Finally, the ssDNA2-modified Mn-doped CdS/ZnS QDs

(ssDNA2-QDs complex) were redispersed into 5.0 mL Tris-acetate buffer (0.010 M, pH = 7.6) by

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vortex and stored at 4 �C for future usage. The number of ssDNA2 immobilized on the Mn-doped

CdS/ZnS QDs was estimated by measuring the absorbance difference at 260 nm before and after

modification with ssDNA2. The average ssDNA2 loading was about one ssDNA2 per QD. The

final concentration of ssDNA2-modified Mn-doped CdS/ZnS QDs was about 1.0×10-7 M

according to the absorbance at 400 nm. The absorbance and fluorescence emission spectra of the

ssDNA2-modified Mn-doped CdS/ZnS QDs are also shown in Figure 1, and indicated that the

spectra remain unchanged compared to the unmodified QDs.

2.4. General procedures for Hg2+ monitoring

Briefly, 40 �L of 2.0 × 10-7 M ssDNA1, 40 �L of 1.0 × 10-7 M ssDNA2-QDs complex, 40 �L

of different concentrations of Hg2+, and 100 �L of buffer (0.010 M Tris-acetate, 0.10 M NaNO3,

pH=7.6) mixed uniformly by vortex and incubated for 30 min to form T-Hg2+-T coordination

chemistry firstly. Then, 50 �L of 0.30 mg/mL GO was added to the mixture to differentiate the

unfolded and folded ssDNA. The final step is to monitor the time-gated fluorescence intensity of

the mixture above. Similar procedures were used to test the selectivity of the proposed sensing

strategy. The concentrations of Hg2+ used in the sensitivity experiments were 0, 0.20, 0.40, 0.60,

0.80, 1.0, 2.0, 4.0, 6.0, 8.0, 10.0, 50.0, 100.0, and 1000.0 nM, respectively. 1.0 �M of Hg2+ was

used in the optimization experiments. Various other metal ions of 2.0 �M were used in the

selectivity experiments. The other metal ions used in the selectivity experiments are as follows:

Ag+, Co2+, Ni2+, Ca2+, Cd2+, Al3+, Fe3+, Au3+, Cr2+, Mn2+, Pb2+, Cu2+, Mg2+, Zn2+, and Ba2+. The

lake water samples were taken from Taozi Lake in Hunan University. The river water samples

were taken from Xiangjiang River. The spring water samples were taken from the Lush Maple

Spring in Yuelu Mountain. The time-gated fluorescence spectra were measured and recorded by a

Perkin-Elmer LS-55 spectrofluorimeter. The parameters of the spectrofluorimeter were set as �ex=

400 nm, �em= 609 nm, delay time, 0.10 ms, excitation slit, 15 nm, and emission slit, 20 nm.

3. Results and discussion

3.1. Experimental principle

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The proposed sensing strategy was designed based on the principle of FRET from

ssDNA2-QDs complex to GO. The fundamental of the proposed sensing strategy is showed in

Figure 2. In this study, the proposed strategy comprises two ssDNA: one is a 12-mer T-rich

oligonucleotide (ssDNA1), and the other is a 12-mer T-rich oligonucleotide with an alkanethiol

moiety at the 5’-terminus (ssDNA2). Both ssDNA1 and ssDNA2 consist of seven thymine units,

which interact specifically with Hg2+ to form T-Hg2+-T coordination chemistry. ssDNA2 was

functionalized with Mn-doped CdS/ZnS QDs via the alkanethiol moiety at the 5’-terminus. The

ssDNA2-QDs complex exhibited strong and stable fluorescence emission at 609 nm when

excited at 400 nm. ssDNA1 and ssDNA2-QDs complex could not hybridize each other because

of the seven mismatched T-T base pairs in the absence of Hg2+. Upon addition of the Hg2+

solution to the mixture of ssDNA1 and ssDNA2-QDs complex, Hg2+-mediated base pairs

(T-Hg2+-T) induce ssDNA1 and ssDNA2 with seven T-T mismatches hybridize each other. It has

been previously reported that Hg2+ binding to the DNA strand is a synergistic process, and the

binding of one Hg2+ facilitates the binding of another Hg2+ to the same strand [55]. What’s more,

the T-Hg2+-T pair is more stable than the A-T Watson-Crick pair [8]. The hybridization of

ssDNA1 and ssDNA2 formed a stable and rigid structure in aqueous solution. When the

hybridization solution is mixed with GO solution, the well-folded structure after hybridization

could not be adsorbed on the surface of GO which makes the Mn-doped CdS/ZnS QDs far away

from the surface of GO. As a result, the fluorescence intensity of the Mn-doped CdS/ZnS QDs

was observed upon light excitation. The time-gated fluorescence intensity of Mn-doped CdS/ZnS

QDs will be changed upon sequential addition different concentrations of Hg2+ and equivalent

GO to the mixture of ssDNA1 and ssDNA2-QDs complex. Therefore, we can monitor the

concentration of Hg2+ according to the change of time-gated fluorescence intensity of Mn-doped

CdS/ZnS QDs using the proposed sensing strategy. The time-gated fluorescence spectra with and

without the addition of Hg2+ are shown in Figure 3. In addition, a certain amount of Hg2+ was

added to the solution only containing ssDNA2-QDs complex to evaluate the effect of Hg2+ to

Mn-doped CdS/ZnS QDs. The results indicated that the fluorescence performance was not

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sensitive to Hg2+ no matter whether in the presence of different concentrations of Hg2+ or in the

1.0 �M of Hg2+ solution with reaction time increasing.

Figure 2 and Figure 3 should be here

3.2. Optimal conditions for the proposed sensing strategy

The GO concentration was studied firstly because of the intense effect on ssDNA2-QDs

complex. Various concentrations of GO were added to the ssDNA2-QDs complex solution. The

time-gated fluorescence intensity of the mixture above is monitored and recorded in Figure 4(A).

As shows in Figure 4(A), the time-gated fluorescence intensity decreased with an increase in the

GO concentration, and the complete quenching was observed when 0.30 mg/mL or more GO was

added. The reason maybe that all of the ssDNA2-QDs complexes were adsorbed on the surface of

GO because of the strong �-� stacking interactions between ssDNA and GO, which resulted in

the maximized fluorescence quenching of Mn-doped CdS/ZnS QDs. As a result, the authors

chose 0.30 mg/mL as the suitable concentration of GO.

The incubation time after the addition of Hg2+ to the mixture of ssDNA1 and ssDNA2-QDs

complex to form the T-Hg2+-T pairs was investigated afterwards, and the results are shown in

Figure 4(B). The time-gated fluorescence intensity increased with the increase of incubation time

and got constant after 30 min. Hence, the authors chose 30 min as the optimum incubation time.

The adsorption time after the addition of GO to the mixture of ssDNA1, ssDNA2-QDs complex,

and ssDNA1/ssDNA2-QDs was also studied due to the important role in the detection sensitivity.

As indicates in Figure 4(C), the time-gated fluorescence intensity decreased with the increase of

adsorption time and became constant over 35 min. Consequently, the authors chose 35 min as the

optimum adsorption time. In addition, room temperature (28�C–30�C) was selected as the

operational temperature for convenience and the media pH of 7.6 (Tris-acetate buffer) was used

throughout the experiments. Because the PO43-, Cl-, or S2- etc can react with Hg2+ to generate

precipitation, so, it is best to avoid these anions in the buffer used in the system of Hg2+ detection.

Hence, Tris-acetate buffer is selected as the media buffer. Herein, 1.0 �M was selected as the

concentration of Hg2+ in all optimal experiments.

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Figure 4 should be here

3.3. Determination of Hg2+ in buffer using the proposed sensing strategy

Based on the optimum conditions, various concentrations of Hg2+ were introduced to evaluate

the sensitivity of the fluorescent sensing strategy. The fluorescent spectra upon addition of

various concentrations of Hg2+ are recorded in Figure 5(A). As shows in Figure 5(A), it is clearly

that higher concentration of Hg2+ resulted in more fluorescence intensity enhancement.

Importantly, even very low concentration of Hg2+ can induce perceptible change of the

time-gated fluorescence intensity which indicated that the proposed sensing strategy is very

sensitive for Hg2+. The calibration curve of time-gated fluorescence intensity as a function of the

concentration of Hg2+ of the sensing strategy is shown in Figure 5(B). Figure 5(C) shows that the

time-gated fluorescence intensity was linearly dependent on the concentration of Hg2+ in the

range from 0.20 to 10 nM. The equation for the linear calibration curve was y = 4.17 x + 1.89 (x

is the concentration of Hg2+, y is the time-gated fluorescence intensity) with correlation

coefficient of 0.9937. According to the standard deviation of 0.15 for the blank signal with 20

parallel measurements, a limit of detection was estimated to be 0.11 nM based on a 3�/slope

standard (� is the standard deviation of the blank signal). The detection limit is much lower than

the standard of the U.S. Environmental Protection Agency (EPA). The EPA regulates the

maximum allowable level of Hg2+ in drinking water to be 10 nM. The detection limit of the

proposed sensing strategy is much improved in comparison to those previously reported

colorimetric methods and fluorescent methods for Hg2+ [10�12,14,16,18�20,25�29,56]. The

results are summarized in Table 1. The exceptionally low detection limit for Hg2+ of the proposed

sensing strategy may be attributed to four vitally important factors: (1) the super quenching

capability of GO; (2) the strong interaction between ssDNA and GO via �-� stacking interactions;

(3) the specific interaction between Hg2+ and thymine which can form stable T-Hg2+-T

coordination chemistry; (4) the long lifetime fluorescence of Mn-doped CdS/ZnS QDs which can

use time-gated mode to reduce the background noises and increase the signal to noise ratio.

To test the repeatability of this sensor, three kinds of modified QDs and GO were prepared

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with the same procedure and the RSD is about 2.1 % for determination of 100 nM Hg2+ under the

optimum conditions.

Figure 5 should be here

Table 1 should be here

3.4. Selectivity of the proposed sensing strategy for Hg2+ determination

Excellent selectivity is important and necessary for a robust practical method. Herein, two

experiments were conducted to investigate the selectivity of the proposed sensing strategy. First,

every metal ion was studied as an independent sample to investigate the selectivity of the sensing

strategy. The metal ions are Hg2+, Ag+, Co2+, Ni2+, Ca2+, Cd2+, Al3+, Fe3+, Au3+, Cr2+, Mn2+, Pb2+,

Cu2+, Mg2+, Zn2+, and Ba2+. It can be seen from Figure 6 that only the sample of Hg2+ exhibited

significant response. Second, Hg2+ and other metal ions were mixed together to form a mixture

solution as a sample for selectivity testing. As shows in Figure 6, the sample also exhibited

remarkable response. These results above indicated that these metal ions have little effect on the

determination of Hg2+. Hence, the proposed sensing strategy shows excellent anti-jamming

capability and selectivity toward Hg2+ based on the specific interaction between Hg2+ and

thymine. 1.0 �M was selected as the concentration of Hg2+, while the concentrations of other

metal ions were 2.0 �M in the section.

Figure 6 should be here

3.5. Determination of Hg2+ in environmental water samples

The proposed sensing strategy was further tested with environmental water samples to

demonstrate its applicability. The environmental water samples were spring water, river water,

lake water, and tap water samples. These samples were filtered (0.20 �m filter membrane) and

then centrifuged for 15 min at 12000 rpm to remove the visible impurities before use. The

concentrations of total mercury in these samples were 0.019, 0.057, 0.041, and 0.021 nM which

were measured by Atomic Fluorescence Spectrometer (AFS). It was found that the concentration

of Hg2+ in these samples was too low to be detected using the proposed sensing strategy. Hence,

extra Hg2+ was added to the samples to test the feasibility of the sensing strategy. The

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environmental water samples were spiked with different concentrations of Hg2+ (0, 0.80, 4.0, and

8.0 nM), and then tested according to the general procedure with four replicates using the

proposed sensing strategy and AFS. The results are summarized in Table 2 and are in good

agreement with the results obtained by AFS. According to the results, the authors think that the

present sensing strategy can be applied to detect Hg2+ in environmental water samples.

In the experiments of real samples, all the solutions were prepared and diluted with

Tris-acetate buffer except water samples, and when all the solutions were mixed together, the pH

of the mixture was about 7.6 (the same with the pH of Tris-acetate buffer), hence, the effect of

pH is not considered in the paper. If the pH of the environmental water samples is serious acid or

alkali polluted, the pH of the samples should be adjusted to 7.6 using Tris-acetate buffer before

usage.

Table 2 should be here

4. Conclusions

In summary, the authors developed a highly sensitive and selective time-gated fluorescent

sensing strategy for Hg2+ determination. The proposed mtheod is mainly based on the super

quenching capability of GO with strong adsorption function toward ssDNA via �-� stacking

interactions, the specific and selective T-Hg2+-T coordination chemistry between two neighboring

T-rich strands, and the long lifetime fluorescence of Mn-doped CdS/ZnS QDs with unique

photophysical properties. Compared to some other previously reported works for Hg2+, this

fluorescent sensing strategy exhibited an extremely low detection limit. In addition, the present

sensing strategy showed excellent selectivity and a broad linear range from 0.20 to 10 nM.

Particularly, the proposed fluorescent sensing strategy can also work very well in environmental

water samples. Based on the results of the study, the authors believe that the proposed fluorescent

sensing strategy is applicable in routine and in situ Hg2+ monitoring in environmental water

samples.

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Acknowledgments

Special thanks are given to Mr. Y. Charles Cao and his group of the University of Florida, and

Dr. Ou Chen for kind assistance in synthesis of Mn-doped CdS/ZnS core/shell QDs. This work

was financially supported by the National Environmental Science Foundation of China

(20977026), the National 863 High Technology Research Foundation of China (2006AA06Z407),

the Hunan Provincial Environmental Science Foundation of China (14JJ2045), and the Scientific

Research Fund of Hunan Provincial Education Department (13k017).

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Table 1 Sensitivity comparison for Hg2+ between the proposed sensing strategy and some other previously reported methods

Methods No. Linear ranges Detection limits References colorimetric methods

1 2 3 4 5 6 1 2 3 4 5 6 7 8 9

0.1–2.0 �M 100 nM 11 not mentioned 1.0 nM 12 not mentioned 0.5 �M 16

25–500 nM 17 nM 19 not mentioned 1.0 �M 20 0.096–6.4 �M 40 nM 29

fluorescent methods

10–200 nM 5.0 nM 10 2.0–60 nM 2.0 nM 14

not mentioned 2.4 nM 18 3.0–800 nM 3.2 nM 25 1.0–10 nM 0.49 nM 26 1.0–10 nM 0.61 nM 27 0–0.5 �M 20 nM 28

3.6–10000 nM 3.6 nM 56 0.2–10 nM 0.11 nM this work

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Table 2 Determination of Hg2+ in environmental water samples using the proposed sensing strategy and AFS

Samples Added Hg2+ (nM)

Proposed method (meana ± SDb)

AFS (mean ± SD)

Recovery (%)

spring water 1 0 c 0.019 ± 0.001 – spring water 2 0.80 0.77 ± 0.05 0.79 ± 0.02 96.25 % spring water 3 4.0 3.89 ± 0.15 3.97 ± 0.09 97.25 % spring water 4 8.0 7.90 ± 0.17 8.03 ± 0.10 98.75 % river water 1 0 c 0.057 ± 0.006 – river water 2 0.80 0.75 ± 0.07 0.80 ± 0.03 93.75 % river water 3 4.0 4.02 ± 0.15 3.98 ± 0.04 100.5 % river water 4 8.0 8.10 ± 0.21 8.03 ± 0.09 101.25 % lake water 1 0 c 0.041 ± 0.005 – lake water 2 0.80 0.76 ± 0.06 0.79 ± 0.02 95.0 % lake water 3 4.0 3.90 ± 0.15 4.02 ± 0.05 97.5 % lake water 4 8.0 7.87 ± 0.20 8.10 ± 0.11 98.38 % tap water 1 0 c 0.027 ± 0.002 – tap water 2 0.80 0.75 ± 0.05 0.82 ± 0.02 93.75 % tap water 3 4.0 4.10 ± 0.13 4.0 ± 0.06 102.5 % tap water 4 8.0 8.15 ± 0.18 7.95 ± 0.09 101.88 % a Mean of four determinations. b SD, standard deviation. c No Hg2+ concentration could be

detected.

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Highlights:

1. Described a sensing strategy for Hg2+ detection using time-gated manner.

2. Employing long lifetime fluorescence Mn:CdS/ZnS QDs to reduce the background signals.

3. Employing Mn:CdS/ZnS QDs and GO as the energy transfer pairs.

4. Displays the advantages of high sensitivity (LOD: 0.11 nM) and excellent selectivity.

5. Applied successfully to the determination of Hg2+ in environmental water samples.�

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Figure captions

Figure 1. The absorbance and fluorescence emission spectra of water soluble Mn-doped

CdS/ZnS QDs and ssDNA2-modified Mn-doped CdS/ZnS QDs.

Figure 2. Schematic description of the proposed sensing strategy for Hg2+ monitoring.

Figure 3. Time-gated fluorescence emission spectra of the proposed sensing strategy without and

with 1.0 �M Hg2+.

Figure 4. Optimization experiments of the concentration of GO (A), the incubation time (B), and

the adsorption time (C). The error bars are the standard deviation.

Figure 5. (A) Time-gated fluorescence spectra of the proposed sensing strategy after addition of

different concentrations of Hg2+ in buffer. (B) The calibration curve of time-gated fluorescence

intensity as a function of the concentration of Hg2+. (C) Linear region of panel B. The

concentrations of Hg2+ in panel C are 0.2, 0.4, 0.6, 0.8, 1.0, 2.0, 4.0, 6.0, 8.0, and 10 nM. Every

data point is the mean of three measurements. The error bars are the standard deviation.

Figure 6. Selectivity of the proposed sensing strategy towards Hg2+ over other metal ions. The

concentrations of Hg2+ and other metal ions are 1.0 �M and 2.0 �M, respectively. Every data

point is the mean of three measurements. The error bars are the standard deviation.�

Graphical Abstract:

*Graphical Abstract (for review)

Figure 1

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Figure 2

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Figure 3

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Figure 4

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Figure 5

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Figure 6

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