Author’s Accepted Manuscript Graphene quantum dots: Highly active bifunctional nanoprobes for nonenzymatic photoluminescence detection of Hydroquinone Yuezhen He, Jian Sun, Dexiang Feng, Hongqi Chen, Feng Gao, Lun Wang www.elsevier.com/locate/bios
PII: DOI: Reference:
S0956-5663(15)30254-2 http://dx.doi.org/10.1016/j.bios.2015.07.006 BIOS7823
To appear in: Biosensors and Bioelectronic Received date: 10 May 2015 Revised date: 30 June 2015 Accepted date: 3 July 2015 Cite this article as: Yuezhen He, Jian Sun, Dexiang Feng, Hongqi Chen, Feng Gao and Lun Wang, Graphene quantum dots: Highly active bifunctional nanoprobes for nonenzymatic photoluminescence detection of Hydroquinone, Biosensors and Bioelectronic, http://dx.doi.org/10.1016/j.bios.2015.07.006 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Graphene quantum dots: Highly active bifunctional nanoprobes for nonenzymatic photoluminescence detection of hydroquinone Yuezhen He, Jian Sun, Dexiang Feng, Hongqi Chen, Feng Gao and Lun Wang
Anhui Key Laboratory of Chemo-Biosensing; Key Laboratory of Functional Molecular Solids Ministry of Education, Anhui Laboratory of Molecule-based Materials; College of Chemistry and Materials Science; Anhui Normal University, Wuhu 241000, People’s Republic of China
Abstract In this paper, a simple and sensitive photoluminescence method is developed for the hydroquinone quantitation by using graphene quantum dots which simultaneously serve as a peroxidase-mimicking catalyst and a photoluminescence indicator. In the presence of dissolved oxygen, graphene quantum dots with intrinsic peroxidase-mimicking catalytic activity can catalyze the oxidation of hydroquinone to produce p-benzoquinone, an intermediate, which can efficiently quench graphene quantum dots’ photoluminescence. Based on this effect, a novel fluorescent platform is proposed for the sensing of hydroquinone, and the detection limit of 5 nM is found.
Keyword:
Graphene
quantum
dots;
Peroxidase-mimicking
catalytic
Photoluminescence; Hydroquinone.
Corresponding author. Tel: +86 553 5910008; fax: +86 553 5910008.E-mail addresses: [email protected] (L Wang).
activity;
1 Introduction Phenolic compounds are not only frequently used in the chemical industry and agronomic practices, such as plastic, tanning, paint, cosmetics, dyes, rubber, pharmaceuticals, and pesticides, moreover they are also the important side products in oil refineries, steel plants, and pulp mills (Canevari et al., 2013; Yuan et al., 2013). Some of them, such as hydroquinone (H2Q), are extremely harmful effects on humans through oral, dermal, or respiratory tracks, and thus it is among the list of the second kind of environmental pollutants (Wang et al., 2012a). To achieve sensitive and rapid determination of H2Q has attracted considerable research efforts in recent years. In these methods, the peroxidase/H2O2 system has been paid intensive in the areas of the sensing of H2Q, since peroxidase can catalyze the oxidation of H2Q in the presence of H2O2 to produce p-benzoquinone (BQ) intermediates (Caza et al., 1999). For example, Wang et al. utilized a CdTe quantum dots-enzyme hybrid system for the determination of phenolic compounds and hydrogen peroxide (Yuan et al., 2008). Huang et al. used fluorescent conjugated polymer-enzyme hybrid system to detect H2Q and hydrogen peroxide (Huang et al., 2011). However, the practical application of these report methods may suffer from intrinsic limitations of enzyme, such as denaturation, leakage, and high cost of preparation and purification. Thus adopting new designs and novel materials to solve the problems is beneficial for practical application. Graphene quantum dots (GQDs) which are graphene sheets smaller than 100 nm (Ponomarenko et al. 2008), have attracted enormous research because of their excellent performances, such as strong photoluminescence (PL), chemical inertness, water solubility, biocompatibility, and low toxicity (Lin et al., 2014; Shen et al., 2012; Zhang et al., 2012).
These features make GQDs especially useful for optical sensing and imaging (Dong et al., 2012a; Kong et al., 2012; Shi et al., 2015; Yu et al., 2013; Zhang et al., 2015). In addition to the above properties, GQDs consisting of a single-atomic layer of nano-sized carbon nanomaterials, have the excellent performances of graphene. As with grapheme (Song et al., 2010), GQDs have been proved to possess highly-efficient peroxidase-mimicking catalytic activity (Umrao et al., 2015; Zhang et al., 2013), and its activity is much higher than graphene oxide (Zheng et al., 2013). These unique features of GQDs can be highly exploitable in the development of novel optical sensors. In this paper, we described a simple and sensitive sensing system for H2Q by using GQDs which simultaneously functions as a peroxidase-mimicking catalyst and a PL indicator (Scheme 1). In this sensing system, as GQDs have peroxidase-mimicking catalytic activity, when GQDs and H2Q were simply mixed, GQDs could catalyze the oxidation of H2Q to form BQ in the presence of dissolved oxygen. In result, the generated BQ quenched efficiently the PL of GQDs. The coupling of efficient quenching of GQDs’ PL by BQ and a high-efficient catalytic activity of GQDs make this sensor a simple and sensitive method for H2Q detection.
Scheme
2 Experimental sections 2.1 Materials and Instruments. H2Q, BQ and citric acid were purchased from Aladdin chemistry Co. Ltd (Shanghai, China). Other reagents were brought from Sinopharm Chemical Reagent Co. Ltd (Shanghai, China).
All the other reagents were analytical reagent grade and were used without further purification. Doubly distilled water was used throughout the experiments. Stock solution of 0.1 M H2Q and BQ were freshly prepared daily. Transmission electron micrographs (TEM) were carried out by using a Tecnai G220S-TWIN transmission electron microscope (FEI).The fluorescence spectra were performed by using a Hitachi F-4500 fluorescence spectrophotometer (Hitachi, Japan) equipped with a 1 cm quartz cuvette. UV-vis absorption spectra were measured on a UV-3010 recording spectrophotometer (Hitachi, Japan).
2.2 Synthesis of GQDs The GQDs were prepared in aqueous solution by a bottom-up method according to a reported method (Dong et al., 2012b; He et al., 2014). Briefly, 2 g of citric acid was put into a 5 mL beaker and heated to 200 °C for about 15 min, until the citric acid changed to an orange liquid. Then, the obtained orange liquid was added by drop wise into 100 mL of 10 mg mL−1 NaOH solution under vigorous stirring. The obtained pale yellow-green GQDs solution was adjusted to pH 7 with 10 mg mL−1HCl solution. The product was subsequently purified by dialyzing through the molecular porous membrane tubing (Retained molecular weight: 1000 Da).
2.3
Fluorescence Experiments
A 1mg L−1 GQDs suspension was prepared and stored under ambient condition. 10µL of GQDs and various amounts of analytes were placed in a series of 5 mL calibrated test tubes.
The mixtures were diluted to 2 mL with Tris-HCl buffer solution (pH 8.4) and mixed thoroughly. Eight min later, the fluorescence spectra were recorded by operating the fluorescence spectrophotometer at an excitation wavelength of 362 nm. The slot widths of the excitation and emission were both 5.0 nm. The PMT voltage was set at 700V. For real samples detection, tap water (from Lab), lake water (Jinghu Lake, Wuhu), rainwater samples, and wastewater (supplied by Wuhu Sewage Treatment Station) were used and were diluted 10 times with 50 mM Tris-HCl buffer solution (pH 8.4). The diluted water samples were added with different concentrations of H2Q (0.5µM, 1.0µM and 5.0 µM) to prepare the spiked samples. Real samples detection was carried out using the procedure described above.
3 Results and discussion 3.1 Quenching of GQDs PL by BQ The as-prepared GQDs were found to exhibit bright blue PL under excitation of 362 nm UV light (quantum yield ca.13%). The morphology of GQDs is studied by TEM. The result (Fig.1A) indicates that the diameters of the as-prepared GQDs are in the range of 3-10 nm, with an average value of ~8nm in size. The high resolution TEM (HRTEM) result clearly shows high crystallinity of the GQDs, with a lattice parameter of 0.241 nm, (1120) lattice fringes of graphene (Peng et al., 2012).
Fig.1
When BQ was added into the GQDs colloid, the PL of GQDs was remarkably quenched
and 30 µM BQ can produce a quenching extent of 97% (Fig.1B). In this system, BQ is electron-rich and GQDs are electron-deficient, so BQ could absorb on the surface of GQDs by induction interactions (Georgakilas et al., 2012). As a classic electron acceptor, BQ could shuttle the electron from the conduction band to the valence band of the GQDs, resulting in the quenching of GQDs’ PL intensity (Burda et al., 1999). As shown in Fig.S1, intensive quenching of GQDs’ PL intensity by BQ exhibits a good linearity that can be expressed as (F0-F)/F0 = 0.032C + 0.008 (Where F0 and F were the fluorescence intensity of the system at 472 nm in the presence and absence of BQ, and where C was the concentration of BQ, µM) in the BQ concentrations range of 0.005 µM to 30 µM with a correlation coefficient of 0.998.
3.2 The peroxidase-mimicking catalytic activity of GQDs and their PL response to H2Q In the presence of peroxidase and H2O2, H2Q can be transformed to BQ which is efficient PL quencher (Huang et al., 2011; Yuan et al., 2008). As shown in Fig.S2A, when GQDs and H2Q were simply mixed, an inhibition extent of 90% was observed. As GQDs is the only reagent used in this system, we speculated that GQDs could catalyze the oxidation of H2Q by dissolved oxygen to form BQ which can quench the PL of GQDs. To prove our hypothesis, the effect of dissolved oxygen on the quenching GQDs’ PL by H2Q is investigated (Fig.S2A). In N2-saturated GQDs aqueous solution containing H2Q, the PL of GQDs changed little which indicated that H2Q could not quench GQDs without the aid of dissolved oxygen. In the presence of dissolved oxygen, the H2Q can remarkably quench the PL of GQDs. Thus, the dissolved oxygen is one of the most crucial parameter in the sensing system. To investigate the peroxidase-mimicking catalytic activity of the GQDs, the GQDs-catalyzed
oxidation reaction of H2Q with dissolved oxygen was tested. As shown in Fig.S2B, a small color change of H2Q can be observed due to the low reaction rate of H2Q and dissolved oxygen in alkaline medium. With the addition of GQDs to H2Q solution, the color system was increased and a brown color was observed. The color of mixture of H2Q and GQDs becomes darker with the concentration of H2Q increase. The experiment indicates that the reaction of H2Q and dissolved oxygen can be accelerated by the addition of GQDs. The UV-vis absorption spectroscopy of two H2Q solution (1 mM and 5 mM) as well as their mixture with GQDs are depicted in Fig.S2C. The absorption intensity in whole spectrum of the mixture of H2Q and GQDs greatly increases when compared to the absorption intensity of free H2Q, and the absorption intensity increases with the concentration of H2Q increase. These results indicated that GQDs has peroxidase-mimicking catalytic activity. The research of Li et al. (Zheng et al., 2013) found that GQDs possess highly-efficient peroxidase-mimicking catalytic activity and can catalyze H2O2-mediated reaction, which agreed with our experiment well. Therefore, with the catalysis of GQDs, H2Q can be transformed to BQ, a very effective quencher, which can remarkably quench the PL of GQDs.
3.3 Optimization of assay parameters The effect of incubation time on the proposed nanosensor response was investigated. Fig.S3 A and B show the PL quenching effects on GQDs with different incubation times. When 30 µM H2Q was added to 5µg mL−1 GQDs solution in 50 mM Tri-HCl buffer solution (pH 8.4), the PL intensity of GQDs decreased from 898 to 23 with the incubation time changed from 0 to 8 min. When incubation time was longer than 8 min, the quenching effect reached a plain. Therefore,
the incubation time of 8 min was adopted for the subsequent work. The pH of solution is a key factor for this sensing system. As shown in Fig.S3C, the PL activity of GQDs is almost unaffected by altering the pH of solution. The quenching effect of H2Q on the PL intensity of GQDs creases with increasing pH value, and tended to reach a maximum value at 8.4. Therefore, the pH 8.4 was adopted in the subsequent work. Another key factor for the sensing system is the concentration of GQDs. The effect of different amounts of GQDs was investigated (Fig.S3D). The experiment results showed that the PL intensity of the system was increasing gradually with the concentration of GQDs. To find the optimum the concentration of GQDs, the relative PL quenching effect (F0-F)/F0 were compared at different the concentration of GQDs. In order to give attention to both the sensitivity and linear range, 5 µg mL−1 GQDs solution was used for the subsequent FL measurements.
3.4 H2Q detection using GQDs as the catalyst and the PL probe Under the optimized conditions, we evaluated the capability of the sensing system for quantitative detection of H2Q. The response of GQDs to H2Q concentration is shown in Fig.2. Upon stepwise addition of H2Q, the PL intensity of the GQDs gradually decreased. A good linearity between the PL quenching efficiency of the GQDs and the concentration of H2Q was observed from 0.01 µM to 30 µM, and the regression equation is (F0-F)/F = 0.033C + 0.009 (Where F0 and F were the fluorescence intensity of the system at 472 nm in the presence and absence of H2Q, and where C was the concentration of H2Q, µM) with a correlation coefficient of 0.995. The limit of detection for H2Q is 5 nM at 3 times the standard deviation of the blank
response.
Fig.2
For estimating the precision, a series of eleven repetitive measurements of H2Q yielded reproducible signals with a relative standard deviation of 3.56%. Moreover, a comparison between the proposed method and other reported methods for H2Q determination in detection limit and linear range was summed up in Table 1. It was evident that this sensing system reveals impressive sensitivity.
Table 1
3.5 Selectivity of the biosensor To test the selectivity of the sensing system for H2Q before its application in a real sample, we have investigated the interference of ions (Na+, K+, Cd2+, Pb2+, Ba2+, Cu2+, Zn2+, Al3+, Mg2+, Mn2+, Ca2+, Ni2+, Cl−, NO3−, HPO42−, and H2PO4−), reducing organisms (uric acid, glucose, ascorbic acid, caffeine, theocin, cysteine, glycine and benzoic acid), and H2Q-similar species (p-nitrophenol, phenol, aminophenol, catechol, resorcinol, and dopamine, and rutinum). As shown in Fig.3, most of these substances have nearly no interference to the PL response of 25 µM H2Q even at a rather high concentration (Na+, K+, Cl− and NO3− were 5 mM. The others were 0.05 mM). But catechol, dopamine and rutinum significantly interferes with hydroquinone detection. In the present of catechol, the proposed method can determine total hydroquinone
and catechol. Water samples contain little dopamine and rutinum, thus the interference from dopamine and rutinum is not significant in real sample analysis. The results clearly demonstrated that the present method exhibited better selectivity for the detection of H2Q.
Fig.3
3.6 Real samples detection In order to evaluate the feasibility of the sensing system for detection of phenolic compounds in real samples, four kinds of water samples (lake water, rainwater, tap-water, and wastewater) were analyzed in this study. Furthermore, standard addition experiments were performed to detect phenolic compounds. The results were listed in Table. The recovery values are in general from 95% to 110%, which indicated the proposed method has potential in quantitative phenolic compounds in the presence of some coexistent substances.
Table 2
4. Conclusions In summary, we have successfully demonstrated that GQDs both as a peroxidase-mimicking catalyst and a PL indicator can be used as nanosensing platform for H2Q detection. The linear response range of H2Q was from 0.01 to 30 µM. The detection limit was as low as 5 nM. The proposed method was finally applied to detect H2Q in the tap water, lake water, and rainwater samples, indicating that the GQDs-based nanosensor has a potential application for detecting
various analyte which can be translated into quinone. This enzyme-free nanosensor has several advantages: (1) Highly-efficient peroxidase-mimicking graphene quantum dots have several superiorities over natural enzymes, such as low cost, high stability, and ease of preparation. (2) The unique photoluminescence property and high stability of graphene quantum dots give this nanosensor with high sensitivity and selectivity. (3) Graphene quantum dots which are non-toxic and environment-friendly are the only reagents used there, and this method is very green and simple. Therefore, the proposed nanosensor has the great potential to detect hydroquinone in the monitoring and controlling of environmental pollution.
Acknowledgements This work was supported by Natural Science Foundation of China (21275008, 21405004, and 21475001), the Research Culture Funds of Anhui Normal University (2013xmpy11), and PhD Research Startup Funds of Anhui Normal University (2014bsqdjj43).
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Figure captions Scheme 1. Schematic illustration of the PL biosensor by highly active bifunctional GQDs. Fig.1. (A) The TEM image of GQDs, insert of (A) is the HRTEM of GQDs; (B) UV of the obtained GQDs solution, and PL spectrum of the obtained GQDs solution in the absence and presence 30 µM BQ.
Fig.2. (A) PL emission spectra of the sensing system after addition of various amounts of H2Q (a-m: 0, 0.01, 0.1, 1, 3, 5, 7, 9, 10, 15, 20, 25, and 30 µM). (B) The relationship between the PL quenching and the concentration of H2Q. Fig.3 (A) The PL response of the sensing system to various ions. 25 µM H2Q; 5 mM Na+, K+, Cl−, and NO3−, 0.05 mM Cd2+, Pb2+, Ba2+, Cu2+, Zn2+, Al3+, Mg2+, Mn2+, Ca2+, Ni2+, HPO42−, and H2PO4− (B) The PL response of the sensing system to H2Q-similar species and reducing organisms. 25 µM H2Q, 0.05 mM glucose, caffeine, theocin, uric acid, ascorbic acid, cysteine, glycine, benzoic acid, phenol, p-nitrophenol, aminophenol, catechol, resorcinol, dopamine, and rutinum.
Table 1. Comparison of different methods for the detemination of H2Q Table 2. Analysis of H2Q in water samples
Table 1 Linear range
LOD
(µM)
(µM)
Au NPs/PAN nanofibers/Ru(bpy)32+
0.55–37
80
(Wang et al., 2012b)
Electrochemistry
Copper complex
60–2500
30
(de Oliveira et al., 2007)
Electrochemistry
Ionic liquid/carbon paste electrode
10–1500
4
(Zhang and Zheng, 2007)
Electrochemistry
CuS nanocrystals
4.5–4500
1.5
(Zou et al., 2013)
Electrochemistry
Fe3O4–SiO2/laccase
0.1–137.5
0.015
(Zhang et al., 2007)
Fluorometry
Silver NCs/H2O2-peroxidase
0.08–3.2
0.01
(Guo et al., 2013)
Fluorometry
Polymer/H2O2-peroxidase
1–2000
0.5
(Huang et al., 2011)
Fluorometry
CdTe QDs/H2O2-peroxidase
0.5–500
0.5
(Yuan et al., 2008)
Fluorometry
GQDs
0.01-30
0.005
This work
Analytical method
System
Electrochemiluminescence
Ref.
Table 2 H2Q Samples
Tap waterb
Lake waterc
Rain waterd
Wastewatere
a
Recovery (%) a
Added (µM)
Found(µM)
0.5
0.49±0.02
98
1
0.95±0.06
95
5
5.01±0.03
100
0.5
0.48±0.01
96
1
1.02±0.07
102
5
4.99±0.08
100
0.5
0.53±0.03
106
1
1.09±0.05
109
5
5.17±0.11
103
0.5
0.55±0.02
110
1
1.10±0.07
110
5
5.12±0.09
102
Mean of three experiments (±S.D.). bLake water from Jinhu, Wuhu. dRainwater collected during rain in Wuhu city a period from 6 to 8 January 2014, eWastewater sample supplied by Wuhu Sewage Treatment Station.
Highlights Graphene quantum dots (GQDs) are used as sensing platform for hydroquinone detection. GOQs serve as a peroxidase-mimicking catalyst and a photoluminescence indicator. The sensor is very green and simple because GQDs are the only reagents used.
Scheme 1
Fig.1
Fig.2
Fig.3
PII: DOI: Reference:
S0956-5663(15)30254-2 http://dx.doi.org/10.1016/j.bios.2015.07.006 BIOS7823
To appear in: Biosensors and Bioelectronic Received date: 10 May 2015 Revised date: 30 June 2015 Accepted date: 3 July 2015 Cite this article as: Yuezhen He, Jian Sun, Dexiang Feng, Hongqi Chen, Feng Gao and Lun Wang, Graphene quantum dots: Highly active bifunctional nanoprobes for nonenzymatic photoluminescence detection of Hydroquinone, Biosensors and Bioelectronic, http://dx.doi.org/10.1016/j.bios.2015.07.006 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Graphene quantum dots: Highly active bifunctional nanoprobes for nonenzymatic photoluminescence detection of hydroquinone Yuezhen He, Jian Sun, Dexiang Feng, Hongqi Chen, Feng Gao and Lun Wang
Anhui Key Laboratory of Chemo-Biosensing; Key Laboratory of Functional Molecular Solids Ministry of Education, Anhui Laboratory of Molecule-based Materials; College of Chemistry and Materials Science; Anhui Normal University, Wuhu 241000, People’s Republic of China
Abstract In this paper, a simple and sensitive photoluminescence method is developed for the hydroquinone quantitation by using graphene quantum dots which simultaneously serve as a peroxidase-mimicking catalyst and a photoluminescence indicator. In the presence of dissolved oxygen, graphene quantum dots with intrinsic peroxidase-mimicking catalytic activity can catalyze the oxidation of hydroquinone to produce p-benzoquinone, an intermediate, which can efficiently quench graphene quantum dots’ photoluminescence. Based on this effect, a novel fluorescent platform is proposed for the sensing of hydroquinone, and the detection limit of 5 nM is found.
Keyword:
Graphene
quantum
dots;
Peroxidase-mimicking
catalytic
Photoluminescence; Hydroquinone.
Corresponding author. Tel: +86 553 5910008; fax: +86 553 5910008.E-mail addresses: [email protected] (L Wang).
activity;
1 Introduction Phenolic compounds are not only frequently used in the chemical industry and agronomic practices, such as plastic, tanning, paint, cosmetics, dyes, rubber, pharmaceuticals, and pesticides, moreover they are also the important side products in oil refineries, steel plants, and pulp mills (Canevari et al., 2013; Yuan et al., 2013). Some of them, such as hydroquinone (H2Q), are extremely harmful effects on humans through oral, dermal, or respiratory tracks, and thus it is among the list of the second kind of environmental pollutants (Wang et al., 2012a). To achieve sensitive and rapid determination of H2Q has attracted considerable research efforts in recent years. In these methods, the peroxidase/H2O2 system has been paid intensive in the areas of the sensing of H2Q, since peroxidase can catalyze the oxidation of H2Q in the presence of H2O2 to produce p-benzoquinone (BQ) intermediates (Caza et al., 1999). For example, Wang et al. utilized a CdTe quantum dots-enzyme hybrid system for the determination of phenolic compounds and hydrogen peroxide (Yuan et al., 2008). Huang et al. used fluorescent conjugated polymer-enzyme hybrid system to detect H2Q and hydrogen peroxide (Huang et al., 2011). However, the practical application of these report methods may suffer from intrinsic limitations of enzyme, such as denaturation, leakage, and high cost of preparation and purification. Thus adopting new designs and novel materials to solve the problems is beneficial for practical application. Graphene quantum dots (GQDs) which are graphene sheets smaller than 100 nm (Ponomarenko et al. 2008), have attracted enormous research because of their excellent performances, such as strong photoluminescence (PL), chemical inertness, water solubility, biocompatibility, and low toxicity (Lin et al., 2014; Shen et al., 2012; Zhang et al., 2012).
These features make GQDs especially useful for optical sensing and imaging (Dong et al., 2012a; Kong et al., 2012; Shi et al., 2015; Yu et al., 2013; Zhang et al., 2015). In addition to the above properties, GQDs consisting of a single-atomic layer of nano-sized carbon nanomaterials, have the excellent performances of graphene. As with grapheme (Song et al., 2010), GQDs have been proved to possess highly-efficient peroxidase-mimicking catalytic activity (Umrao et al., 2015; Zhang et al., 2013), and its activity is much higher than graphene oxide (Zheng et al., 2013). These unique features of GQDs can be highly exploitable in the development of novel optical sensors. In this paper, we described a simple and sensitive sensing system for H2Q by using GQDs which simultaneously functions as a peroxidase-mimicking catalyst and a PL indicator (Scheme 1). In this sensing system, as GQDs have peroxidase-mimicking catalytic activity, when GQDs and H2Q were simply mixed, GQDs could catalyze the oxidation of H2Q to form BQ in the presence of dissolved oxygen. In result, the generated BQ quenched efficiently the PL of GQDs. The coupling of efficient quenching of GQDs’ PL by BQ and a high-efficient catalytic activity of GQDs make this sensor a simple and sensitive method for H2Q detection.
Scheme
2 Experimental sections 2.1 Materials and Instruments. H2Q, BQ and citric acid were purchased from Aladdin chemistry Co. Ltd (Shanghai, China). Other reagents were brought from Sinopharm Chemical Reagent Co. Ltd (Shanghai, China).
All the other reagents were analytical reagent grade and were used without further purification. Doubly distilled water was used throughout the experiments. Stock solution of 0.1 M H2Q and BQ were freshly prepared daily. Transmission electron micrographs (TEM) were carried out by using a Tecnai G220S-TWIN transmission electron microscope (FEI).The fluorescence spectra were performed by using a Hitachi F-4500 fluorescence spectrophotometer (Hitachi, Japan) equipped with a 1 cm quartz cuvette. UV-vis absorption spectra were measured on a UV-3010 recording spectrophotometer (Hitachi, Japan).
2.2 Synthesis of GQDs The GQDs were prepared in aqueous solution by a bottom-up method according to a reported method (Dong et al., 2012b; He et al., 2014). Briefly, 2 g of citric acid was put into a 5 mL beaker and heated to 200 °C for about 15 min, until the citric acid changed to an orange liquid. Then, the obtained orange liquid was added by drop wise into 100 mL of 10 mg mL−1 NaOH solution under vigorous stirring. The obtained pale yellow-green GQDs solution was adjusted to pH 7 with 10 mg mL−1HCl solution. The product was subsequently purified by dialyzing through the molecular porous membrane tubing (Retained molecular weight: 1000 Da).
2.3
Fluorescence Experiments
A 1mg L−1 GQDs suspension was prepared and stored under ambient condition. 10µL of GQDs and various amounts of analytes were placed in a series of 5 mL calibrated test tubes.
The mixtures were diluted to 2 mL with Tris-HCl buffer solution (pH 8.4) and mixed thoroughly. Eight min later, the fluorescence spectra were recorded by operating the fluorescence spectrophotometer at an excitation wavelength of 362 nm. The slot widths of the excitation and emission were both 5.0 nm. The PMT voltage was set at 700V. For real samples detection, tap water (from Lab), lake water (Jinghu Lake, Wuhu), rainwater samples, and wastewater (supplied by Wuhu Sewage Treatment Station) were used and were diluted 10 times with 50 mM Tris-HCl buffer solution (pH 8.4). The diluted water samples were added with different concentrations of H2Q (0.5µM, 1.0µM and 5.0 µM) to prepare the spiked samples. Real samples detection was carried out using the procedure described above.
3 Results and discussion 3.1 Quenching of GQDs PL by BQ The as-prepared GQDs were found to exhibit bright blue PL under excitation of 362 nm UV light (quantum yield ca.13%). The morphology of GQDs is studied by TEM. The result (Fig.1A) indicates that the diameters of the as-prepared GQDs are in the range of 3-10 nm, with an average value of ~8nm in size. The high resolution TEM (HRTEM) result clearly shows high crystallinity of the GQDs, with a lattice parameter of 0.241 nm, (1120) lattice fringes of graphene (Peng et al., 2012).
Fig.1
When BQ was added into the GQDs colloid, the PL of GQDs was remarkably quenched
and 30 µM BQ can produce a quenching extent of 97% (Fig.1B). In this system, BQ is electron-rich and GQDs are electron-deficient, so BQ could absorb on the surface of GQDs by induction interactions (Georgakilas et al., 2012). As a classic electron acceptor, BQ could shuttle the electron from the conduction band to the valence band of the GQDs, resulting in the quenching of GQDs’ PL intensity (Burda et al., 1999). As shown in Fig.S1, intensive quenching of GQDs’ PL intensity by BQ exhibits a good linearity that can be expressed as (F0-F)/F0 = 0.032C + 0.008 (Where F0 and F were the fluorescence intensity of the system at 472 nm in the presence and absence of BQ, and where C was the concentration of BQ, µM) in the BQ concentrations range of 0.005 µM to 30 µM with a correlation coefficient of 0.998.
3.2 The peroxidase-mimicking catalytic activity of GQDs and their PL response to H2Q In the presence of peroxidase and H2O2, H2Q can be transformed to BQ which is efficient PL quencher (Huang et al., 2011; Yuan et al., 2008). As shown in Fig.S2A, when GQDs and H2Q were simply mixed, an inhibition extent of 90% was observed. As GQDs is the only reagent used in this system, we speculated that GQDs could catalyze the oxidation of H2Q by dissolved oxygen to form BQ which can quench the PL of GQDs. To prove our hypothesis, the effect of dissolved oxygen on the quenching GQDs’ PL by H2Q is investigated (Fig.S2A). In N2-saturated GQDs aqueous solution containing H2Q, the PL of GQDs changed little which indicated that H2Q could not quench GQDs without the aid of dissolved oxygen. In the presence of dissolved oxygen, the H2Q can remarkably quench the PL of GQDs. Thus, the dissolved oxygen is one of the most crucial parameter in the sensing system. To investigate the peroxidase-mimicking catalytic activity of the GQDs, the GQDs-catalyzed
oxidation reaction of H2Q with dissolved oxygen was tested. As shown in Fig.S2B, a small color change of H2Q can be observed due to the low reaction rate of H2Q and dissolved oxygen in alkaline medium. With the addition of GQDs to H2Q solution, the color system was increased and a brown color was observed. The color of mixture of H2Q and GQDs becomes darker with the concentration of H2Q increase. The experiment indicates that the reaction of H2Q and dissolved oxygen can be accelerated by the addition of GQDs. The UV-vis absorption spectroscopy of two H2Q solution (1 mM and 5 mM) as well as their mixture with GQDs are depicted in Fig.S2C. The absorption intensity in whole spectrum of the mixture of H2Q and GQDs greatly increases when compared to the absorption intensity of free H2Q, and the absorption intensity increases with the concentration of H2Q increase. These results indicated that GQDs has peroxidase-mimicking catalytic activity. The research of Li et al. (Zheng et al., 2013) found that GQDs possess highly-efficient peroxidase-mimicking catalytic activity and can catalyze H2O2-mediated reaction, which agreed with our experiment well. Therefore, with the catalysis of GQDs, H2Q can be transformed to BQ, a very effective quencher, which can remarkably quench the PL of GQDs.
3.3 Optimization of assay parameters The effect of incubation time on the proposed nanosensor response was investigated. Fig.S3 A and B show the PL quenching effects on GQDs with different incubation times. When 30 µM H2Q was added to 5µg mL−1 GQDs solution in 50 mM Tri-HCl buffer solution (pH 8.4), the PL intensity of GQDs decreased from 898 to 23 with the incubation time changed from 0 to 8 min. When incubation time was longer than 8 min, the quenching effect reached a plain. Therefore,
the incubation time of 8 min was adopted for the subsequent work. The pH of solution is a key factor for this sensing system. As shown in Fig.S3C, the PL activity of GQDs is almost unaffected by altering the pH of solution. The quenching effect of H2Q on the PL intensity of GQDs creases with increasing pH value, and tended to reach a maximum value at 8.4. Therefore, the pH 8.4 was adopted in the subsequent work. Another key factor for the sensing system is the concentration of GQDs. The effect of different amounts of GQDs was investigated (Fig.S3D). The experiment results showed that the PL intensity of the system was increasing gradually with the concentration of GQDs. To find the optimum the concentration of GQDs, the relative PL quenching effect (F0-F)/F0 were compared at different the concentration of GQDs. In order to give attention to both the sensitivity and linear range, 5 µg mL−1 GQDs solution was used for the subsequent FL measurements.
3.4 H2Q detection using GQDs as the catalyst and the PL probe Under the optimized conditions, we evaluated the capability of the sensing system for quantitative detection of H2Q. The response of GQDs to H2Q concentration is shown in Fig.2. Upon stepwise addition of H2Q, the PL intensity of the GQDs gradually decreased. A good linearity between the PL quenching efficiency of the GQDs and the concentration of H2Q was observed from 0.01 µM to 30 µM, and the regression equation is (F0-F)/F = 0.033C + 0.009 (Where F0 and F were the fluorescence intensity of the system at 472 nm in the presence and absence of H2Q, and where C was the concentration of H2Q, µM) with a correlation coefficient of 0.995. The limit of detection for H2Q is 5 nM at 3 times the standard deviation of the blank
response.
Fig.2
For estimating the precision, a series of eleven repetitive measurements of H2Q yielded reproducible signals with a relative standard deviation of 3.56%. Moreover, a comparison between the proposed method and other reported methods for H2Q determination in detection limit and linear range was summed up in Table 1. It was evident that this sensing system reveals impressive sensitivity.
Table 1
3.5 Selectivity of the biosensor To test the selectivity of the sensing system for H2Q before its application in a real sample, we have investigated the interference of ions (Na+, K+, Cd2+, Pb2+, Ba2+, Cu2+, Zn2+, Al3+, Mg2+, Mn2+, Ca2+, Ni2+, Cl−, NO3−, HPO42−, and H2PO4−), reducing organisms (uric acid, glucose, ascorbic acid, caffeine, theocin, cysteine, glycine and benzoic acid), and H2Q-similar species (p-nitrophenol, phenol, aminophenol, catechol, resorcinol, and dopamine, and rutinum). As shown in Fig.3, most of these substances have nearly no interference to the PL response of 25 µM H2Q even at a rather high concentration (Na+, K+, Cl− and NO3− were 5 mM. The others were 0.05 mM). But catechol, dopamine and rutinum significantly interferes with hydroquinone detection. In the present of catechol, the proposed method can determine total hydroquinone
and catechol. Water samples contain little dopamine and rutinum, thus the interference from dopamine and rutinum is not significant in real sample analysis. The results clearly demonstrated that the present method exhibited better selectivity for the detection of H2Q.
Fig.3
3.6 Real samples detection In order to evaluate the feasibility of the sensing system for detection of phenolic compounds in real samples, four kinds of water samples (lake water, rainwater, tap-water, and wastewater) were analyzed in this study. Furthermore, standard addition experiments were performed to detect phenolic compounds. The results were listed in Table. The recovery values are in general from 95% to 110%, which indicated the proposed method has potential in quantitative phenolic compounds in the presence of some coexistent substances.
Table 2
4. Conclusions In summary, we have successfully demonstrated that GQDs both as a peroxidase-mimicking catalyst and a PL indicator can be used as nanosensing platform for H2Q detection. The linear response range of H2Q was from 0.01 to 30 µM. The detection limit was as low as 5 nM. The proposed method was finally applied to detect H2Q in the tap water, lake water, and rainwater samples, indicating that the GQDs-based nanosensor has a potential application for detecting
various analyte which can be translated into quinone. This enzyme-free nanosensor has several advantages: (1) Highly-efficient peroxidase-mimicking graphene quantum dots have several superiorities over natural enzymes, such as low cost, high stability, and ease of preparation. (2) The unique photoluminescence property and high stability of graphene quantum dots give this nanosensor with high sensitivity and selectivity. (3) Graphene quantum dots which are non-toxic and environment-friendly are the only reagents used there, and this method is very green and simple. Therefore, the proposed nanosensor has the great potential to detect hydroquinone in the monitoring and controlling of environmental pollution.
Acknowledgements This work was supported by Natural Science Foundation of China (21275008, 21405004, and 21475001), the Research Culture Funds of Anhui Normal University (2013xmpy11), and PhD Research Startup Funds of Anhui Normal University (2014bsqdjj43).
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Figure captions Scheme 1. Schematic illustration of the PL biosensor by highly active bifunctional GQDs. Fig.1. (A) The TEM image of GQDs, insert of (A) is the HRTEM of GQDs; (B) UV of the obtained GQDs solution, and PL spectrum of the obtained GQDs solution in the absence and presence 30 µM BQ.
Fig.2. (A) PL emission spectra of the sensing system after addition of various amounts of H2Q (a-m: 0, 0.01, 0.1, 1, 3, 5, 7, 9, 10, 15, 20, 25, and 30 µM). (B) The relationship between the PL quenching and the concentration of H2Q. Fig.3 (A) The PL response of the sensing system to various ions. 25 µM H2Q; 5 mM Na+, K+, Cl−, and NO3−, 0.05 mM Cd2+, Pb2+, Ba2+, Cu2+, Zn2+, Al3+, Mg2+, Mn2+, Ca2+, Ni2+, HPO42−, and H2PO4− (B) The PL response of the sensing system to H2Q-similar species and reducing organisms. 25 µM H2Q, 0.05 mM glucose, caffeine, theocin, uric acid, ascorbic acid, cysteine, glycine, benzoic acid, phenol, p-nitrophenol, aminophenol, catechol, resorcinol, dopamine, and rutinum.
Table 1. Comparison of different methods for the detemination of H2Q Table 2. Analysis of H2Q in water samples
Table 1 Linear range
LOD
(µM)
(µM)
Au NPs/PAN nanofibers/Ru(bpy)32+
0.55–37
80
(Wang et al., 2012b)
Electrochemistry
Copper complex
60–2500
30
(de Oliveira et al., 2007)
Electrochemistry
Ionic liquid/carbon paste electrode
10–1500
4
(Zhang and Zheng, 2007)
Electrochemistry
CuS nanocrystals
4.5–4500
1.5
(Zou et al., 2013)
Electrochemistry
Fe3O4–SiO2/laccase
0.1–137.5
0.015
(Zhang et al., 2007)
Fluorometry
Silver NCs/H2O2-peroxidase
0.08–3.2
0.01
(Guo et al., 2013)
Fluorometry
Polymer/H2O2-peroxidase
1–2000
0.5
(Huang et al., 2011)
Fluorometry
CdTe QDs/H2O2-peroxidase
0.5–500
0.5
(Yuan et al., 2008)
Fluorometry
GQDs
0.01-30
0.005
This work
Analytical method
System
Electrochemiluminescence
Ref.
Table 2 H2Q Samples
Tap waterb
Lake waterc
Rain waterd
Wastewatere
a
Recovery (%) a
Added (µM)
Found(µM)
0.5
0.49±0.02
98
1
0.95±0.06
95
5
5.01±0.03
100
0.5
0.48±0.01
96
1
1.02±0.07
102
5
4.99±0.08
100
0.5
0.53±0.03
106
1
1.09±0.05
109
5
5.17±0.11
103
0.5
0.55±0.02
110
1
1.10±0.07
110
5
5.12±0.09
102
Mean of three experiments (±S.D.). bLake water from Jinhu, Wuhu. dRainwater collected during rain in Wuhu city a period from 6 to 8 January 2014, eWastewater sample supplied by Wuhu Sewage Treatment Station.
Highlights Graphene quantum dots (GQDs) are used as sensing platform for hydroquinone detection. GOQs serve as a peroxidase-mimicking catalyst and a photoluminescence indicator. The sensor is very green and simple because GQDs are the only reagents used.
Scheme 1
Fig.1
Fig.2
Fig.3
