Biosensors and Bioelectronics 55 (2014) 83–90

Contents lists available at ScienceDirect

Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios

Nitrogen-doped carbon quantum dots: Facile synthesis and application as a “turn-off” fluorescent probe for detection of Hg2 þ ions Ruizhong Zhang a,b, Wei Chen a,n a State Key Laboratory of Electroanalytical Chemistry, Changchun institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, Jilin, China b University of Chinese Academy of Sciences, Beijing 100039, China

art ic l e i nf o

a b s t r a c t

Article history: Received 16 September 2013 Received in revised form 7 November 2013 Accepted 28 November 2013 Available online 10 December 2013

A facile, economical and straightforward hydrothermal strategy is used to prepare highly luminescent nitrogen-doped carbon quantum dots (N-CQDs) by using folic acid as both carbon and nitrogen sources. The as-prepared N-CQDs have an average size of 4.5 7 1.0 nm and exhibit excitation wavelengthdependent fluorescence with the maximum emission and excitation at 390 and 470 nm, respectively. Furthermore, due to the effective quenching effect of Hg2 þ ions, such N-CQDs are found to serve as an effective fluorescent sensing platform for lable-free sensitive detection of Hg2 þ ions with a detection limit of 0.23 μM. The selectivity experiments reveal that the fluorescent sensor is specific for Hg2 þ even with interference by high concentrations of other metal ions. Most importantly, the N-CQDs-based Hg2 þ ions sensor can be successfully applied to the determination of Hg2 þ in tap water and real lake water samples. With excellent sensitivity and selectivity, such stable and cheap carbon materials are potentially suitable for monitoring of Hg2 þ in environmental application. & 2013 Elsevier B.V. All rights reserved.

Keywords: Carbon Quantum dots Mercury Sensor Fluorescence Folic acid

1. Introduction Heavy metal ion pollution has become a critical worldwide issue for years due to the severe risks in human health and the environment. As one of the most toxic heavy metals, mercury with the feature of strong toxicity and bioaccumulation can cause serious human health problems even at very low concentration (Nolan and Lippard, 2008; Zahir et al., 2005). The exposure to mercury can cause a number of toxicological effects such as brain damage, kidney failure, and various cognitive and motion disorders (Harris et al., 2003). The solvated Hg2 þ , one of the most stable inorganic forms of mercury, is well-known to be highly toxic due to its good water solubility (Hylander and Goodsite, 2006). Because of the sever poison of Hg2 þ for the environment and health, it is of great necessity to develop rapid and user-friendly Hg2 þ detection methods with cost-efficiency, high sensitivity and selectivity. Traditional methods for Hg2 þ detection include atomic absorption/emission spectroscopy (Cizdziel and Gerstenberger, 2004; Welz and Sperling, 1999), selective cold vapor atomic fluorescence spectrometry (Yamini et al., 1997), X-ray fluorescence spectrometry (Bennun and Gomez, 1997; Greaves et al., 1997), inductively coupled plasma mass spectrometry (ICPMS) (Harrington et al., 2004; Karunasagar et al., 1998; Kenduzler et al., 2012), anodic

n

Corresponding author. Tel.: þ 86 431 85262061. E-mail address: [email protected] (W. Chen).

0956-5663/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bios.2013.11.074

stripping voltammetry (Ugo et al., 1995), and so forth. These methods are very sensitive and selective but they require complicated sample preparation and sophisticated instruments which limit their application in routine Hg2 þ monitoring (Leermakers et al., 2005; Li et al., 2006). Thus, it is still of great challenge to develop a simple method for aqueous Hg2 þ detection. In recent years, novel nanotechnologybased strategies have been proposed for mercury detection. Among them, noble metal-based nanomaterials, such as Au, Ag have been usually used for Hg2 þ detection with electrochemical techniques and fluorescence measurements (Botasini et al., 2013; Leopold et al., 2010; Zhang et al., 2011). However, metal nanoparticle–electrochemical combined strategies require multiple steps, including extensive pre-treatments of the electrode surface (polishing, surface contamination oxidation, etc.), which cannot be easily automated and thus affects their application as ideal systems. Meanwhile, to decrease the cost of the sensors fabricated from noble metals, it is necessary to develop low-cost materials-based biosensors. Because of the high emission quantum yields and size-tunable emission profiles, quantum dots (QDs) have become one of the most extensively optical sensing nanomaterials in the detection of metal ions (Chen and Rosenzweig, 2002). However, these popular QDs have serious toxicity even at relatively low concentrations (Derfus et al., 2004; Kirchner et al., 2005). In addition, their superior photophysical features are usually observed in organic solvents, thus restricting tremendously their analytical potential (Gill et al., 2008). Recently, some methods have been developed to make the QDs water-soluble and biocompatible, such as surface passivation with protective layers and coating

84

R. Zhang, W. Chen / Biosensors and Bioelectronics 55 (2014) 83–90

the QDs with protecting silicon oxide films. Although promising, these protocols are compromised by reducing their photoluminescence efficiency and the time-consuming, complicated, expensive processes (Gerion et al., 2001). Carbon-based materials possess a broad range of properties due to the existence of different allotropes as well as various microstructures. Carbon materials, such as carbon nanotubes, carbon fibers, porous materials, fullerene, and newly discovered graphene, have attracted increasing attention due to their excellent electrical and thermal conductivity, high strength and stability, good flexibility and low weight. Carbon quantum dots (CQDs), a kind of new member of carbon nanomaterials, were first discovered during the purification of single-walled carbon nanotubes in 2004 (Xu et al., 2004). CQDs with size below 10 nm exhibit strong fluorescence with tunable emission depending on the size, surface structure, and excitation wavelength etc. (Baker and Baker, 2010; Liu et al., 2007). Compared to traditional semiconductor quantum dots and organic dyes, photoluminescent CQDs are superior in terms of aqueous solubility, functionalizability, resistance to photobleaching, toxicity, biocompatibility and exhibit broader photoluminescence profiles (Baker and Baker, 2010; Li H.T. et al., 2012; Li et al., 2010). Several strategies have been demonstrated for the synthesis of CQDs with desired possible applications in optoelectronics devices, biological labeling and biomedicines, etc. (Baker and Baker, 2010; Hu et al., 2009; Li H.T. et al., 2012; Sun et al., 2006; Tian et al., 2009; Zhou et al., 2007). In the reported methods, the oxidation of gas soot, carbon soot or activated carbon need a large amount of strong acids like nitric acid which are undesirable and hazardous (Liu et al., 2007; Qiao et al., 2010; Sun et al., 2006; Tian et al., 2009). On the other hand, although carbonization of glucose, sucrose, citric acid, ascorbic acid, etc. has attracted significant attention for the production of fluorescent CQDs, most of these processes need multi-step operations and strong acids as well as post-treatments with surface passivated agents to improve the water solubility and luminescence properties (Puvvada et al., 2012; Zhai et al., 2012; Zhang et al., 2010). Recently, much effort has been made to obtain selfpassivated carbon dots through high temperature or microwaveassisted hydrothermal carbonization of suitable carbon precursors (Hsu and Chang, 2012; Sahu et al., 2012; Yang et al., 2011). However, efficient one-step strategies for the large-scale production of CQDs from renewable precursors with inexpensive and green methods are still challengeable. Current synthetic methods are mainly deficient in accurate control of lateral dimensions and the resulting surface chemistry, as well as in obtaining fluorescent CQDs with high quantum yields and novel applications. Folic acid (FA) is merely the parent structure of the family of vitamin coenzymes. FA and their derivatives have been extensively applied to the field of molecular biology, nutritional biochemistry, and new dynamically functional materials, etc. (Kato et al., 2004; Lucock, 2000). From the structural formula of folic acid molecule, there have several functional groups, such as –NH2 and –COOH, which have been considered as necessary groups to produce high quantum yield of CQDs (Zhu et al., 2013). The nitrous groups and multiple oxygenated groups modified on the surface of CQDs can improve the solubility of the CQDs in water. Meanwhile, the selfpassivated CQDs with nitrous groups and multiple oxygenated groups can enhance their photoluminescence (Sun et al., 2013). Herein, we report for the first time a facile, and economical hydrothermal strategy for the preparation of water-soluble nitrogen-doped carbon quantum dots (N-CQDs) by using folic acid as both carbon and nitrogen sources. The as-prepared N-CQDs have a size around 3–5 nm and exhibit excitation wavelength-dependent photoluminescence (PL) with a high quantum yield of about 15.7% at the strongest emission around 470 nm. Most importantly, due to the presence of oxygenated and nitrous groups on the surface of

carbon quantum dots, significant PL quenching of the N-CQDs occurs upon addition of different concentrations of Hg2 þ ions. These N-CQDs can serve as an effective fluorescent sensing platform for label-free detection of Hg2 þ ions with high sensitivity and selectivity. The N-CQDs can also be successfully applied to the determination of Hg2 þ ions in real water samples.

2. Experimental section 2.1. Chemicals Folic acid (C19H19O6N7, Z97%) and Cys (L-Cysteine) were purchased from Shanghai Huishi Chemical reagent. Quinine sulfate and Glu (L-Glutathione reduced) were obtained from Sigma-Aldrich. Ethylene glycol (C2H6O2, A.R. grade), sulfuric acid (H2SO4, A.R.  95– 98%), hydrochloric acid (HCl, A.R. 36–38%), and nitric acid (HNO3, A.R.  63–70%) were all purchased from Beijing Chemical Reagent. Sodium iodide dehydrate (NaI  2H2O) was obtained from Sinopharm Chemical Reagent Co., Ltd. All other reagents are of analytical reagent grade and used as received. Standard stock (10 mM) solution of Hg2 þ ions was prepared with Hg (NO3)2  1/2H2O in ultrapure water with the same concentration of HNO3. Different concentrations of Hg2 þ ions were obtained by diluting standard stock solutions. Other stock solutions (10 mM) of metal cations were prepared in deionized water from the respective salts of NaCl, KCl, CuCl2  2H2O, ZnCl2, CaCl2, FeCl3  6H2O, AgNO3, MgSO4, Hg(NO3)2  1/2H2O, MnCl2, NiCl2  6H2O, Pb(NO3)2, FeSO4  7H2O, CoCl2  6H2O, Cd(CH3COO)2  2H2O. And all aqueous solutions were prepared with ultrapure water supplied by a Water Purifier Nanopure water system (18.3 MΩ cm). 2.2. Preparation of nitrogen-doped carbon quantum dots (N-CQDs) N-CQDs were prepared as follows: folic acid (0.1 g) and ethylene glycol (5 mL) were mixed with Nanopure water (10 mL) by ultrasonication. The solution was then transferred to a poly (tetrafluoroethylene) (Teflon)-lined autoclave (25 mL) and heated at 180 1C for 12 h. After the reaction, the reactor was cooled down to room temperature naturally. The brown-yellow product was subjected to dialysis in a dialysis bag (retained molecular weight: 8–14 kDa) and the resulting bright-yellow solution was vacuumevaporated in a rotary evaporator at 50 1C and centrifuged at a high speed (15,000 rpm) for 15 min to obtain pure N-CQDs powder on a large scale. Then the N-CQDs were dispersed in ultrapure water as stock solution (0.01 mg mL  1) for further characterization and use. 2.3. Material characterization UV–vis spectra were recorded on a VARIAN CARY 50 UV/vis spectrophotometer. The size and morphology of the product were examined by using a Hitachi H-600 transmission electron microscopy (TEM) operated at 100 kV. The sample was prepared by dropping water dispersion of sample onto carbon-coated copper TEM grids using pipettes and dried under ambient condition. High-resolution TEM (HRTEM) was carried out on a JEM-2010 (HR) microscope operated at 200 kV. X-ray photoelectron spectroscopy (XPS) measurements were performed by using a VG Thermo ESCALAB 250 spectrometer (VG Scientific) operated at 120 W. The binding energy was calibrated against the carbon 1s line. Fouriertransformed infrared spectroscopy (FTIR) study was conducted with a VERTEX 70 FTIR (10 mg catalysts, KBr wafer technique). Fluorescence spectra were measured by using a LS 55 fluorescence spectrometer (Perkin-Elmer), and the concentration of the aqueous dispersions of N-CQDs were controlled to be 0.01 mg mL  1.

R. Zhang, W. Chen / Biosensors and Bioelectronics 55 (2014) 83–90

2.4. Quantum yield measurement The quantum yield (QY) of N-CQDs was measured according to an established procedure. Quinine sulfate in 0.1 M H2SO4 (literature quantum yield 0.54 at 360 nm) was chose as a standard. Absolute values are calculated using the standard reference sample that has a fixed and known fluorescence quantum yield value. To minimize the re-absorption effects, absorbencies in the 10 mm fluorescence cuvette were kept under 0.1 at the excitation wavelength (360 nm) (De and Karak, 2013; Qu et al., 2013). The quantum yield of the N-CQDs was determined at the excitation wavelength of 360 nm by the following equation:   I x Ast ηx 2 Q x ¼ Q st ð1Þ I st Ax ηst where Q is the quantum yield, I is the measured integrated emission intensity, η is the refractive index of the solvent, and A is the optical density. The subscript “st” refers to standard with known QY and “x” for the sample.

85

was confirmed by adding other metal ions stock solutions instead of Hg2 þ ions in a similar way. The fluorescence spectra were recorded under excitation at 360 nm and all experiments were performed at room temperature. 2.6. Detection of Hg2 þ ion in real samples To evaluate the N-CQDs-based sensor for Hg2 þ detection in an artificial system, the performance of the present method for real water sample analysis was examined by lake water samples which obtained from the South Lake of Changchun, Jilin province, China and tap water samples from our lab. The lake water samples were filtered through a 0.20 μm filtered membrane and then centrifuged at 12,000 rpm for 15 min. The resultant water samples were spiked Hg2 þ at different concentration levels and then analyzed with the proposed method. The tap water samples were spiked with the different concentrations of Hg2 þ ions solution directly without any pretreatment.

3. Results and discussion 2.5. Detection of Hg2 þ ion The detection of Hg2 þ ion was performed in ultrapure water solution. In a typical assay, 4 mL aqueous solution of N-CQDs (0.01 mg mL  1) was added in a quartz cuvette, followed by the addition of different concentrations of Hg2 þ ion, and the PL spectra were recorded subsequently. The selectivity for Hg2 þ ion

3.1. Synthesis and characterization of nitrogen-doped carbon quantum dots (N-CQDs) In the present study, a facile hydrothermal synthetic strategy was used to produce water-soluble N-CQDs. The precursor of folic acid combined with ethylene glycol are economic and rich in

Relative Frequency

0.30 0.25 0.20 0.15 0.10 0.05 0.00 1

2

3

4

5

6

7

8

d (nm)

100 nm

C=O

C-C/C=C Intensity (a.u)

C-N

C-N-C N-C

C-O

C-OH/C-O-C

C=O/C=N

280

284

288

292

296

395

400 405 Binding Energy (eV)

410

525

530

535

540

Fig. 1. (A) Low-resolution and (B) high-resolution TEM of the as-synthesized N-doped carbon quantum dots (N-CQDs). The inset of (A) shows the particle size distribution histogram. (C–E) High-resolution XPS spectra of C 1s (C), N 1s (D) and O 1s (E).

86

R. Zhang, W. Chen / Biosensors and Bioelectronics 55 (2014) 83–90

multifunctional groups (–COOH, –OH and –NH2), providing both carbon and nitrogen sources for the formation of fluorescent N-CQDs with high quantum yield. The morphology of the N-CQDs was characterized by TEM and HRTEM. Fig. 1A and B shows the representative TEM and HRTEM images of the N-CQDs dispersed in water. It can be seen that the formed N-CQDs are mostly spherical dots that are uniformly dispersed. One hundred fifty N-CQDs were randomly selected to measure the particle size, and from the particle size distribution histogram shown in Fig. 1A inset, the as-prepared N-CQDs exhibit an average size of around 4.5 71.0 nm. From the HRTEM image, most particles are observed to be amorphous carbon dots without obvious lattice fringes, which is consistent with the previously reported results (Wang et al., 2012; Zhu et al., 2013). The surface chemistry of the N-CQDs was studied by FTIR. The spectrum in Fig. S1 showed that multiple functional groups are present on the surface of N-CQDs, including the characteristic absorption bands from –COO– (ester group) at 1723, 1169 and 1092 cm  1, the absorption of –CONH– group at 3195, 3055, 1651 and 1567 cm  1, IR band of phenyl at 1603 cm  1, and the absorption of –C ¼O (carbonyl) at 1709 cm  1. The composition of the as-prepared N-CQDs was characterized by X-ray photoelectron spectroscopy (XPS). Fig. S2 shows the survey spectrum of the N-CQDs sample. The three predominant peaks at 284.27, 398.80 and 532.04 eV correspond to the binding energies of C1s, N1s and O1s, respectively. This result indicates that the produced quantum dots are mainly composed of three elements of C, O and N. As shown in Fig. S2 inset, the contents of the three elements in the sample were calculated to be 62.54% (C), 19.09% (O) and 18.37% (N), respectively, indicating the high doping concentration of nitrogen in the quantum dots with folic acid as precursor. The high-resolution C1s spectrum is shown in Fig. 1C. According to the previous studies, the spectrum can be

2.5

Ex

Em

400

Abs

1.0

300

1.5 200 1.0 100

0.5 0.0 200

400

500

0.6 0.4 0.2 0.0 350

0 300

340 nm 360 nm 380 nm 400 nm 420 nm

0.8

Intensity

Absorbance

2.0

deconvoluted into four peaks at around 284.3, 285.1, 285.9, and 288.1 eV, which are attributed to C–C/C ¼C, C–N, C–O and C ¼N/ C¼ O groups, respectively (Lu et al., 2012). In the high-resolution N1s spectrum (Fig. 1D), the deconvoluted two peaks at 398.9 and 399.8 eV can be assigned to C–N–C and C–N groups, respectively. From the O1s spectrum shown in Fig. 1E, the two fitted peaks at 531.3 and 532.4 eV are ascribed to C ¼O and C–OH/C–O–C groups, respectively. The characterizations from FTIR and XPS indicated that the surface of the as-synthesized N-CQDs is functionalized by multiple oxygenated and nitrous groups. Fig. 2A shows the UV–vis absorption and fluorescence spectra of the water-soluble N-CQDs. It can be seen that there are three absorbance bands centered at around 235, 285 and 380 nm in the UV–vis absorption spectrum. The peak at 235 nm can be ascribed to the π–π* transitions of the aromatic C ¼C sp2 domains which can not produce observed fluorescence signal (Dong et al., 2013; Eda et al., 2010). The other two absorption peaks at 285 and 380 nm are from the trapping of excited state energy of the surface states, which can lead to strong fluorescence (Anilkumar et al., 2011; Wang et al., 2010). Accordingly, as shown in Fig. 2A, there are two excitation peaks around 275 and 395 nm. When excited at 395 nm, the N-CQDs exhibited strong PL emission centered at 470 nm. From the photographs shown in Fig. 2A inset, the diluted N-CQDs aqueous solution is pale yellow under ambient daylight but exhibits a very intense blue color when irradiated with a UV light (365 nm), further indicating the blue fluorescent property of the N-CQDs. The strong fluorescence of the N-CQDs may result from the emissive traps of the nitrogen-doped surface (Dong et al., 2013; Sun et al., 2006; Wang et al., 2010). Moreover, as observed from other carbon-based quantum dots (De and Karak, 2013; Qu et al., 2013; Tang et al., 2012; Zhu et al., 2013), the fluorescence of the present N-CQDs also changes with the excitation wavelength.

600

400

Wavelength (nm)

0 µM

600

75 µM

200

FL Intensity

800

400

500

550

1000

1000

FL Intensity

FL Intensity

1000

450

600

650

Wavelength (nm)

800 600

R2 =0.996

800 600 400 200 0

5

400

10

15

20

25

[Hg2+]/µM

200

0 400

450

500

Wavelength (nm)

550

0

20

40

60

80

[Hg2+]/µM

Fig. 2. (A) UV–vis absorption, photoluminescence excitation and emission spectra of N-CQDs in aqueous solutions (0.01 mg mL  1), λex ¼ 395 nm and λem ¼ 470 nm. Insets show the photographs of the obtained N-CQDs in aqueous solution under illumination of white (left) and UV (365 nm, right) light. (B) Normalized PL spectra of the N-CQDs at different excitation wavelength. (C) Fluorescence emission spectra of the N-CQDs in aqueous solution upon addition of various concentrations of Hg2 þ (from top to bottom: 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70 and 75 μM), excitation at 360 nm. (D) The dependence of FL intensity on the concentration of Hg2 þ ions within the range of 0–75 μM, inset shows the linear relationship between the FL intensity and Hg2 þ concentration.

R. Zhang, W. Chen / Biosensors and Bioelectronics 55 (2014) 83–90

87

Table 1 Comparison of sensing performance of different fluorescent probes for Hg2 þ detection. Fluorescence probes

Detection limit (μM)

Linear range (μM)

Quantum yield (%)

Ref.

TFIC MNPsa Carbon dots Functionalized Carbon Dots Polymer Sensor Chemosensor RF1 QG compoundb N-CQDs

5.04 1.3 2.69 0.728 0.25 0.5 0.23

4–16 0–2.69 0.1–2.69 1–30 – – 0–25

– – – – 38 – 15.7

Wang et al. (2013) Goncalves H.M.R. et al. (2010) Goncalves H. et al. (2010) Li J.F. et al. (2012) Wu et al. (2008) Ou et al. (2006) This work

a b

TFIC MNPs: TSRh6G-β-cyclodextrin fluorophore/adamantane-modified inclusion complex magnetic nanoparticles. QG compound: molecule containing fluorophore quinoline group and a water-soluble D-glucosamine group.

One can see from Fig. 2B that the maximum PL peak shifts from 462 to 479 nm with the change of excitation wavelength from 340 to 420 nm. Such excitation-dependent PL behavior is related to the different surface states of the N-CQDs (Shang et al., 2012). From FTIR and XPS studies, there are multiple C-, N- and O-containing functional groups on the surface of the produced quantum dots, resulting in various surface states with different energy levels and thus a series of emissive traps. Under different excitation wavelength, the corresponding surface state emissive trap will be dominant, giving excitation-dependent FL. It should be pointed out that the synthesized water-soluble N-CQDs exhibit high stability at room temperature. Even after being stored for three months in air, there is no observation of any floating or precipitated nanoparticles, and no obvious photobleaching loss after irradiation with UV lamp at 365 nm for 3 h, showing their advantages for potential applications. By using quinine sulfate as a reference, the quantum yield (QY) of the N-CQDs was calculated to be 15.7% according to Eq. (1), which is higher than the literature reported values of carbon materials prepared by other methods and the reported highest QY of graphene dots (11.4%) (Tang et al., 2012). 3.2. N-CQDs-based fluorescent chemosensor for sensitive and selective detection of Hg2 þ Because of the serious threat of Hg2 þ ions on environment and human health, it is obviously important to develop efficient Hg2 þ detection method which can monitor Hg2 þ level sensitively and selectively in aqueous systems. From above results, the synthesized N-CQDs meet well the requirements of a fluorescent probe. So we explored the feasibility of using the as-prepared N-CQDs for Hg2 þ detection. 3.2.1. Sensitivity of the chemosensor for Hg2 þ detection Based on above fluorescent properties of N-CQDs, different concentrations of Hg2 þ were added to the aqueous solution of N-CQDs and the fluorescence emission intensity was measured to study the sensitivity of the N-CQDs chemosensor. As shown in Fig. 2C, the FL intensity of the N-CQDs at around 450 nm decreases gradually with Hg2 þ concentration increasing, indicating that addition of Hg2 þ ions can effectively quench the fluorescence of the N-CQDs. The fluorescence quenching may be attributed to the nonradiative electron-transfer from the excited states to the d orbital of Hg2 þ (Xia and Zhu, 2008; Zhou et al., 2012). Meanwhile, the Hg2 þ -induced conversion of the functional group (–CONH–) from spirolactam structure to an opened-ring amide may also make important contribution to the fluorescence quenching (Lu et al., 2011). Fig. 2D presents the PL intensity versus the concentration of Hg2 þ ions. A good linear correlation (R2 ¼ 0.996) is shown under the concentration range from 0 to 25 μM. The limit of detection

(LOD) was estimated to be 0.23 μM based on three times the standard deviation rule (LOD ¼3Sd/s). It should be noted that the obtained limit of detection of the N-CQDs sensor for Hg2 þ detection is much lower than those previously reported (Table 1) with other different fluorescent probes. The results show that our sensing system exhibits some superior sensitivity to the previously reported sensing system and the sensor based on N-CQDs may be useful in environmental applications for mercury detection. The recovery property of the N-CQDs after Hg2 þ detection has also been investigated by adding I  or biothiols into the N-CDQs/ Hg2 þ solution. As observed above, the addition of Hg2 þ ions can quench the fluorescence of the N-CQDs. However, as shown in Fig. S3A, the quenched PL can be recovered immediately upon the addition of I  into the solution of N-CDQs/Hg2 þ , and the PL almost restored to its original intensity with increasing the concentration of I  (Fig. S3B). Such result indicates that the N-CDQs/Hg2 þ sensing system is sensitive to I  , and the “off” state of N-CQDs quenched by Hg2 þ can be turned “on” by adding I  . Except for iodide, the addition of biothiols, such as Cys and Glu, can also lead to the PL recovery (Fig. S3C–F). These results suggest that the PL of N-CDQs/Hg2 þ solution can be recovered due to the strong binding of Hg2 þ to iodide or biothiols, resulting in the removal of Hg2 þ from the surface of the N-CQDs.

3.2.2. Selectivity of the chemosensor for Hg2 þ detection To evaluate the selectivity of the present sensing system, two control experiments were performed. First, 30 μM of various biologically and environmentally relevant metal ions, including K þ , Na þ , Ca2 þ , Mg2 þ , Ni2 þ , Cd2 þ , Cu2 þ , Co2 þ , Pb2 þ , Zn2 þ ,Fe2 þ , Fe3 þ , Ag þ , Mn2 þ and Hg2 þ ions were added into the N-CQDs solution, and the PL responses were then recorded. As shown in Fig. 3A, significant PL quenching effect was observed with the addition of Hg2 þ , while the other metal ions showed only a slight quenching effect and the influence is almost negligible. Fig. 3B shows the photographs of N-CQDs aqueous solution with different metal ions (25 μM) under ambient daylight (top) and UV light (bottom). It can be seen clearly that the fluorescence of N-CQDs was effectively quenched by Hg2 þ ions, while the fluorescence still maintained upon the addition of other metal ions. Such result directly indicates that the N-CQDs sensor has high selectivity for Hg2 þ detection. In another control experiment, 25 μM of Hg2 þ alone (black bars, Fig. 3C) and the mixtures of 25 μM of Hg2 þ and 25 μM of the above-mentioned metal ions (red bars, Fig. 3C) were added into the N-CQDs aqueous solution respectively, then the quenching effects of Hg2 þ and the mixtures of Hg2 þ þMn þ on the fluorescence of the N-CQDs were examined. From the results shown in Fig. 3C, in comparison with the efficient quenching effect of Hg2 þ , the influence from the other coexisting metal ions is negligible. These observations indicate that the N-CQDs sensor is insensitive to other metal ions but selective to Hg2 þ in the mixtures. Besides

88

R. Zhang, W. Chen / Biosensors and Bioelectronics 55 (2014) 83–90

Normalized Intensity

1.0

0.8

0.6

Hg2+ 0.4

0.2

0.0

2.0 1.0 1.5

0.6 0.4

Absorbance

F0-F/F0

0.8

N-CQDs + Hg2+ N-CQDs

1.0

N-CQDs + Mn+

0.5 0.2 0.0

0.0 200

300

400

500

600

700

800

Wavelength (nm) Fig. 3. (A) Normalized fluorescence intensity at λex ¼ 360 nm of aqueous N-CQDs solution (0.01 g mL  1) in the presence of 30 μM of various metal ions. (B) Photographs of the aqueous N-CQDs containing 25 μM of various metal ions under daylight (top) and UV light (bottom, excited at 365 nm). (C) Selective PL response of aqueous N-CQDs solution towards 25 μM Hg2 þ (black bars), and interference of 25 μM of other metal ions with 25 μM Hg2 þ (red bars). (D) UV–vis absorption spectra of aqueous N-CQDs solution and the N-CQDs solution containing different metal ions.

fluorescence, the effects of various metal ions on the UV–vis absorption spectrum of N-CQDs were also studied. Fig. 3D shows the UV–vis spectra of N-CQDs solution before and after addition of different metal ions. Upon addition of 30 μM Hg2 þ , the UV–vis absorption of N-CQDs changed significantly, that is the absorption centered at 235 and 285 nm disappeared, and the absorption at 380 nm became weaker. However, the absorption spectrum almost unchanged after adding the same concentration of other metal ions, which demonstrates the presence of Hg2 þ can influence the surface states of the quantum dots. Above results strongly demonstrate that the present sensor has outstanding selectivity toward Hg2 þ sensing. 3.3. Assay of N-CQDs-based Hg2 þ sensor in environmental water samples The excellent specificity combined with high sensitivity and selectivity of N-CQDs to Hg2 þ ions suggest that the present chemosensor might be directly applied for detecting Hg2 þ in real water samples. Therefore, we further examined the practical application of the assay by testing Hg2 þ from natural water (tap water and lake water). To evaluate the N-CQDs-based Hg2 þ sensor in an artificial system, the performance of the present sensor for real water sample analysis was conducted by lake water samples obtained from the South Lake of Changchun, Jilin province, China. Before evaluating, the lake water samples were filtered through a 0.20 μm membrane and then centrifuged at 12,000 rpm for 15 min. The resultant water samples were spiked with Hg2 þ at different concentration levels and then analyzed with the proposed method. At the same time, the tap water samples obtained

from our lab without any pretreatment were spiked with Hg2 þ at different concentration levels and analyzed with the same way as lake water. Fig. 4 shows the fluorescence response of N-CQDs in presence of tap water (A) and lake water (C) containing different concentrations of Hg2 þ ions, and the corresponding relationship between F0–F/F0 and the concentrations of Hg2 þ of tap water (B) and lake water (D). It can be seen that the PL intensity (excited at 360 nm) gradually decreased with increasing the concentration of Hg2 þ in both tap water and lake water from 5 to 50 μM. The calibration curve for determining Hg2 þ in the tap water and the lake water were obtained by plotting the values of F0–F/F0 versus the concentrations of Hg2 þ . As shown in Fig. 4B and D, good linear correlations have been obtained in the range of 0–30 μM for tap water and in the range of 0–35 μM for the lake water. In spite of the possible presence of minerals and organics in the lake water, this sensing system can still work for the sensitive detection of Hg2 þ . The results indicate that the present N-CQDs-based chemosensor may be a promising sensing platform for the detection of Hg2 þ in real environmental samples.

4. Conclusion In this work, highly photoluminescent N-doped carbon quantum dots (N-CQDs) were synthesized for the first time by a facile one-step hydrothermal treatment of folic acid with a quantum yield of 15.7%. The N-CQDs were found to be highly efficient and sensitive chemosensor for selective Hg2 þ detection with a detection limit of 0.23 μM. The sensitive and selective Hg2 þ detection

R. Zhang, W. Chen / Biosensors and Bioelectronics 55 (2014) 83–90

1000

89

1.0

0 µM 800

0.8

600

0.6

F0-F/F0

FL Intensity

2

400

50 µM

R =0.994

0.4 Experimental data Fitting

0.2

200 0.0 0 400

450

500

550

0

10

20

30

40

50

[Hg2+]/µM

Wavelength (nm)

1.0 1000

0 µM

2

0.8

R =0.995

0.6

F0-F/F0

FL Intensity

800 600

0.4

400

50 µM

Experimental data Fitting

0.2

200 0.0 0 400

450

500

550

0

10

20

30

40

50

[Hg2+]/µM

Wavelength (nm)

Fig. 4. N-CQDs-based Hg2 þ sensor in determination of environmental water samples: PL response of N-CQDs in presence of tap water (A) and lake water (C) containing different concentrations of Hg2 þ (from top to bottom: 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, and 50 μM). Corresponding relationship between F0–F/F0 and the concentrations of Hg2 þ of tap water (B) and lake water (D), where F0 and F refers to the PL intensity of N-CQDs in the absence and presence of Hg2 þ , and all emission spectra were collected at λex ¼ 360 nm.

by the present “turn-off” fluorescent sensor is achieved based on the fluorescence quenching mechanism, which may be attributed to the surface/molecule states in the quantum dots and/or the Hg2 þ -induced conversion of the special functional group (–CONH–) from spirolactam structure to an opened-ring amide. In the present study, the fluorescent N-doped carbon quantum dots can be produced by a simple and green synthesis process with low-cost and commonly available carbon precursor, and without further chemical modification. Moreover, the chemosensor based on N-CQDs eliminates the need to use semiconductor quantum dots, organic dyes and toxic organic solvents, and therefore are environmentally friendly. Most importantly, the N-CQDs exhibit promising application of Hg2 þ detection for real-water samples (tap water and lake water). It is believed that the present strategy may offer a new approach for developing low-cost, high sensitive and selective Hg2 þ sensors in environmental applications.

Acknowledgments This work was supported by the National Natural Science Foundation of China (No. 21275136) and the Natural Science Foundation of Jilin province, China (No. 201215090).

Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2013.11.074.

References Anilkumar, P., Wang, X., Cao, L., Sahu, S., Liu, J.H., Wang, P., Korch, K., Tackett, K.N., Parenzan, A., Sun, Y.P., 2011. Nanoscale 3 (5), 2023–2027. Baker, S.N., Baker, G.A., 2010. Angew. Chem. Int. Ed. 49 (38), 6726–6744. Bennun, L., Gomez, J., 1997. Spectrochim. Acta Part B At. Spectrosc. 52 (8), 1195–1200. Botasini, S., Heijo, G., Mendez, E., 2013. Anal. Chim. Acta 800, 1–11. Chen, Y.F., Rosenzweig, Z., 2002. Anal. Chem. 74 (19), 5132–5138. Cizdziel, J.V., Gerstenberger, S., 2004. Talanta 64 (4), 918–921. De, B., Karak, N., 2013. RSC Adv. 3 (22), 8286–8290. Derfus, A.M., Chan, W.C.W., Bhatia, S.N., 2004. Nano Lett. 4 (1), 11–18. Dong, Y.Q., Pang, H.C., Yang, H.B., Guo, C.X., Shao, J.W., Chi, Y.W., Li, C.M., Yu, T., 2013. Angew. Chem. Int. Ed. 52 (30), 7800–7804. Eda, G., Lin, Y.Y., Mattevi, C., Yamaguchi, H., Chen, H.A., Chen, I.S., Chen, C.W., Chhowalla, M., 2010. Adv. Mater. 22 (4), 505–509. Gerion, D., Pinaud, F., Williams, S.C., Parak, W.J., Zanchet, D., Weiss, S., Alivisatos, A.P., 2001. J. Phys. Chem. B 105 (37), 8861–8871. Gill, R., Zayats, M., Willner, I., 2008. Angew. Chem. Int. Ed. 47 (40), 7602–7625. Goncalves, H., Jorge, P.A.S., Fernandes, J.R.A., da Silva, J.C.G.E., 2010. Sens. Actuators B Chem. 145 (2), 702–707. Goncalves, H.M.R., Duarte, A.J., da Silva, J.C.G.E., 2010. Biosens. Bioelectron. 26 (4), 1302–1306. Greaves, E.D., Sosa, J.A., SajoBohus, L., Alvarez, M., Wobrauschek, P., Streli, C., 1997. Spectrochim. Acta Part B At. Spectrosc. 52 (7), 945–951. Harrington, C.F., Merson, S.A., D'Silva, T.M.D., 2004. Anal. Chim. Acta 505 (2), 247–254. Harris, H.H., Pickering, I.J., George, G.N., 2003. Science 301 (5637), 1203. Hsu, P.C., Chang, H.T., 2012. Chem. Commun. 48 (33), 3984–3986. Hu, S.L., Niu, K.Y., Sun, J., Yang, J., Zhao, N.Q., Du, X.W., 2009. J. Mater. Chem. 19 (4), 484–488. Hylander, L.D., Goodsite, M.E., 2006. Sci. Total Environ. 368 (1), 352–370. Karunasagar, D., Arunachalam, J., Gangadharan, S., 1998. J. Anal. At. Spectrom. 13 (7), 679–682. Kato, T., Matsuoka, T., Nishii, M., Kamikawa, Y., Kanie, K., Nishimura, T., Yashima, E., Ujiie, S., 2004. Angew. Chem. Int. Ed. 43 (15), 1969–1972. Kenduzler, E., Ates, M., Arslan, Z., McHenry, M., Tchounwou, P.B., 2012. Talanta 93, 404–410. Kirchner, C., Liedl, T., Kudera, S., Pellegrino, T., Javier, A.M., Gaub, H.E., Stolzle, S., Fertig, N., Parak, W.J., 2005. Nano Lett. 5 (2), 331–338.

90

R. Zhang, W. Chen / Biosensors and Bioelectronics 55 (2014) 83–90

Leermakers, M., Baeyens, W., Quevauviller, P., Horvat, M., 2005. Trac-Trends Anal. Chem. 24 (5), 383–393. Leopold, K., Foulkes, M., Worsfold, P., 2010. Anal. Chim. Acta 663 (2), 127–138. Li, H.T., Kang, Z.H., Liu, Y., Lee, S.T., 2012. J. Mater. Chem. 22 (46), 24230–24253. Li, J.F., Wu, Y.Z., Song, F.Y., Wei, G., Cheng, Y.X., Zhu, C.J., 2012. J. Mater. Chem. 22 (2), 478–482. Li, Q., Ohulchanskyy, T.Y., Liu, R.L., Koynov, K., Wu, D.Q., Best, A., Kumar, R., Bonoiu, A., Prasad, P.N., 2010. J. Phys. Chem. C 114 (28), 12062–12068. Li, Y.F., Chen, C.Y., Li, B., Sun, J., Wang, J.X., Gao, Y.X., Zhao, Y.L., Chai, Z.F., 2006. J. Anal. At. Spectrom. 21 (1), 94–96. Liu, H.P., Ye, T., Mao, C.D., 2007. Angew. Chem. Int. Ed. 46 (34), 6473–6475. Lu, H., Qi, S.L., Mack, J., Li, Z.F., Lei, J.P., Kobayashi, N., Shen, Z., 2011. J. Mater. Chem. 21 (29), 10878–10882. Lu, Y.Z., Jiang, Y.Y., Wei, W.T., Wu, H.B., Liu, M.M., Niu, L., Chen, W., 2012. J. Mater. Chem. 22 (7), 2929–2934. Lucock, M., 2000. Mol. Genet. Metab. 71 (1-2), 121–138. Nolan, E.M., Lippard, S.J., 2008. Chem. Rev. 108 (9), 3443–3480. Ou, S.J., Lin, Z.H., Duan, C.Y., Zhang, H.T., Bai, Z.P., 2006. Chem. Commun. 42, 4392–4394. Puvvada, N., Kumar, B.N.P., Konar, S., Kalita, H., Mandal, M., Pathak, A., 2012. Sci. Technol. Adv. Mater. 13, 4. Qiao, Z.A., Wang, Y.F., Gao, Y., Li, H.W., Dai, T.Y., Liu, Y.L., Huo, Q.S., 2010. Chem. Commun. 46 (46), 8812–8814. Qu, K.G., Wang, J.S., Ren, J.S., Qu, X.G., 2013. Chem. Eur. J. 19 (22), 7243–7249. Sahu, S., Behera, B., Maiti, T.K., Mohapatra, S., 2012. Chem. Commun. 48 (70), 8835–8837. Shang, J.Z., Ma, L., Li, J.W., Ai, W., Yu, T., Gurzadyan, G.G., 2012. Sci. Rep. 2, 792. Sun, H.J., Gao, N., Wu, L., Ren, J.S., Wei, W.L., Qu, X.G., 2013. Chem. Eur. J. 19 (40), 13362–13368. Sun, Y.P., Zhou, B., Lin, Y., Wang, W., Fernando, K.A.S., Pathak, P., Meziani, M.J., Harruff, B.A., Wang, X., Wang, H.F., Luo, P.J.G., Yang, H., Kose, M.E., Chen, B.L., Veca, L.M., Xie, S.Y., 2006. J. Am. Chem. Soc. 128 (24), 7756–7757. Tang, L.B., Ji, R.B., Cao, X.K., Lin, J.Y., Jiang, H.X., Li, X.M., Teng, K.S., Luk, C.M., Zeng, S.J., Hao, J.H., Lau, S.P., 2012. ACS Nano 6 (6), 5102–5110.

Tian, L., Ghosh, D., Chen, W., Pradhan, S., Chang, X.J., Chen, S.W., 2009. Chem. Mater. 21 (13), 2803–2809. Ugo, P., Moretto, L.M., Mazzocchin, G.A., 1995. Anal. Chim. Acta 305 (1–3), 74–82. Wang, J., Wang, C.F., Chen, S., 2012. Angew. Chem. Int. Ed. 51 (37), 9297–9301. Wang, W., Zhang, Y., Yang, Q.B., Sun, M.D., Fei, X.L., Song, Y., Zhang, Y.M., Li, Y.X., 2013. Nanoscale 5 (11), 4958–4965. Wang, X., Cao, L., Yang, S.T., Lu, F.S., Meziani, M.J., Tian, L.L., Sun, K.W., Bloodgood, M.A., Sun, Y.P., 2010. Angew. Chem. Int. Ed. 49 (31), 5310–5314. Welz, B., Sperling, M., 1999. Atomic Absorption Spectrometry, third ed. Wiley-VCH, Weinheim; Chichester. Wu, D.Y., Huang, W., Lin, Z.H., Duan, C.Y., He, C., Wu, S., Wang, D.H., 2008. Inorg. Chem. 47 (16), 7190–7201. Xia, Y.S., Zhu, C.Q., 2008. Talanta 75 (1), 215–221. Xu, X.Y., Ray, R., Gu, Y.L., Ploehn, H.J., Gearheart, L., Raker, K., Scrivens, W.A., 2004. J. Am. Chem. Soc. 126 (40), 12736–12737. Yamini, Y., Alizadeh, N., Shamsipur, M., 1997. Anal. Chim. Acta 355 (1), 69–74. Yang, Z.C., Wang, M., Yong, A.M., Wong, S.Y., Zhang, X.H., Tan, H., Chang, A.Y., Li, X., Wang, J., 2011. Chem. Commun. 47 (42), 11615–11617. Zahir, F., Rizwi, S.J., Haq, S.K., Khan, R.H., 2005. Environ. Toxicol. Pharmacol. 20 (2), 351–360. Zhai, X.Y., Zhang, P., Liu, C.J., Bai, T., Li, W.C., Dai, L.M., Liu, W.G., 2012. Chem. Commun. 48 (64), 7955–7957. Zhang, F.Q., Zeng, L.Y., Yang, C., Xin, J.W., Wang, H.Y., Wu, A.G., 2011. Analyst 136 (13), 2825–2830. Zhang, J.C., Shen, W.Q., Pan, D.Y., Zhang, Z.W., Fang, Y.G., Wu, M.H., 2010. N. J. Chem. 34 (4), 591–593. Zhou, J.G., Booker, C., Li, R.Y., Zhou, X.T., Sham, T.K., Sun, X.L., Ding, Z.F., 2007. J. Am. Chem. Soc. 129 (4), 744–745. Zhou, L., Lin, Y.H., Huang, Z.Z., Ren, J.S., Qu, X.G., 2012. Chem. Commun. 48 (8), 1147–1149. Zhu, S.J., Meng, Q.N., Wang, L., Zhang, J.H., Song, Y.B., Jin, H., Zhang, K., Sun, H.C., Wang, H.Y., Yang, B., 2013. Angew. Chem. Int. Ed. 52 (14), 3953–3957.