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A novel turn-on fluorescent strategy for sensing ascorbic acid using graphene quantum dots as fluorescent probe
Author’s Accepted Manuscript A novel turn-on fluorescent strategy for sensing ascorbic acid using graphene quantum dots as fluorescent probe Hua Liu, Weidan Na, Ziping Liu, Xueqian Chen, Xingguang Su www.elsevier.com/locate/bios
PII: DOI: Reference:
S0956-5663(17)30074-X http://dx.doi.org/10.1016/j.bios.2017.02.005 BIOS9539
To appear in: Biosensors and Bioelectronic Received date: 24 November 2016 Revised date: 15 January 2017 Accepted date: 3 February 2017 Cite this article as: Hua Liu, Weidan Na, Ziping Liu, Xueqian Chen and Xingguang Su, A novel turn-on fluorescent strategy for sensing ascorbic acid using graphene quantum dots as fluorescent probe, Biosensors and Bioelectronic, http://dx.doi.org/10.1016/j.bios.2017.02.005 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.
A novel turn-on fluorescent strategy for sensing ascorbic acid using graphene quantum dots as fluorescent probe Hua Liu, Weidan Na, Ziping Liu, Xueqian Chen, Xingguang Su* Department of Analytical Chemistry, College of Chemistry, Jilin University, Changchun 130012, China *
Corresponding author. Tel.: +86-431-85168352. [email protected]
Abstract In this paper, a facile and rapid fluorescence turn-on assay for fluorescent detection of ascorbic acid (AA) was developed by using the orange emission graphene quantum dots (GQDs). In the presence of horse radish peroxidase (HRP) and hydrogen peroxide (H2O2), catechol can be oxidized by hydroxyl radicals and converted to o-benzoquinone, which can significantly quench the fluorescence of GQDs. However, when AA present in the system, it can consume part of H2O2 and hydroxyl radicals to inhibit the generation of o-benzoquinone, resulting in fluorescence recovery. Under the optimized experimental conditions, the fluorescence intensity was linearly correlated with the concentration of H2O2 in the range of 3.33-500 µM with a detection limit of 1.2 µM. The linear detection for AA was in the range from 1.11 µM to 300 µM with a detection limit of 0.32 µM. The proposed method was applied to the determination of AA in human serum samples with satisfactory results. Key words: Ascorbic acid, H2O2, Graphene quantum dots (GQDs), Catechol, O-benzoquinone
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1. Introduction In recent decades, graphene quantum dots (GQDs) have gained tremendous attention because of its unique electrical and optical properties, including tunable photoluminescence, molecular size, excellent photostability, chemical inertness, good solubility, high biocompatibility, and ease of functionalization (Wang et al., 2014; Li et al., 2016; Shen et al., 2012). Furthermore, GQDs are considered to be competitive alternatives to organic dyes and heavy metal based quantum dots and they are widely used in biological imaging (Deng et al., 2013; Jiang et al., 2016; Sun et al., 2015; Du et al., 2016) and biomolecule detection (Su et al., 2014; Nirala et al., 2015; Lu et al., 2014; Duan et al., 2014). Jiang’s group reported a novel cast-targeted fluorescent imaging assay for the diagnosis of renal disease using Fe3O4 and GQD nanocomposites (Jiang et al., 2016). Ankan Dutta Chowdhury et al. illustrated a highly sensitive and selective fluorescent sensor to detect Fe3+ in the range of 20 nM to 2 μM based on dopamine functionalized GQDs, and the detection limit is 7.6 nM (Dutta Chowdhury et al., 2016). Wang’s group demonstrated the detection of nuclei acids based on GQDs utilizing cascade amplification by nicking endonuclease and catalytic G-quadruplex DNAzyme (Wang et al., 2016), and the detection limit of nuclei acids is 30 fM. Ascorbic acid (AA), known as vitamin C, plays an extremely important role in human life activities. AA was involved in immune response, disease prevention and oxidative stress reduction and other physiological activities (Liu et al., 2015). Specifically, AA is essential for the synthesis of collagen and neurotransmitters or dissociation of sterols in the body, and it can also promote antibody formation, iron uptake, formation of tetrahydrofolate and maintenance of thiolases activity (Liu et al., 2012; Sharifian et al., 2016; Hillstrom et al., 2003). In addition, some reports showed that it can reduce the risk of cancer (Frajese et al., 2016) and remove free radicals in the
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human body (Pisoschi et al., 2014). The reference ranges of ascorbic acid in the general population are 11.4-76.7 µM (Trezzi et al., 2016; Schleicher et al., 2009). AA deficiency can cause scurvy and hypoimmunity, while too much can cause diarrhea, hyperacidity, coronary heart disease and other diseases (Pournaghi-Azar et al., 2002; Bi et al., 2016; Chen et al., 2003; Massey et al., 2005). Therefore, it is necessary to find a rapid, accurate, sensitive and low-cost determination way of AA. Up to now, many methods for AA detection have been established, including fluorescence methods (Liu et al., 2015; Ma et al., 2013), electrochemical methods (Sharifian et al., 2016; Khairy et al., 2016), colorimetry (Chen et al., 2016; Song et al., 2011), liquid chromatography (Lima et al., 2016), polarography (Dimitrijevic et al., 2016) and etc. Although these methods can meet the requirement of sensitivity, they are usually time-consuming, cumbersome, expensive equipment or the need for complex synthesis or complicated extraction. Compared with the other methods, fluorescence method showed more advantages owing to its operational simplicity, high sensitivity and rapid response (Duan et al., 2014; Li et al., 2014). Liu and co-workers designed a method for detecting AA based on the transformation of Cu2+ and Cu+ at the presence of AA (Liu et al., 2015). Ma and co-workers demonstrated the detection of AA based on the transformation of Fe3+ and Fe2+ at the presence of AA (Ma et al., 2013; Gong et al., 2015). However, these approaches for AA detection are based on the difference abilities of the fluorescence quenching between the different valence states of a metal, and the processes are usually accompanied with heavy metal contamination. In this paper, we developed a novel turn-on fluorescent strategy for sensing AA based on GQDs (Scheme1). Compared with some similar studies on using carbon quantum dots/GQDs for
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detecting AA, the establish method in this work has several mentionable features: The fluorescence probe (GQDs) used in this work has a more ideal fluorescence emission peak (540 nm) than others reported GQDs (less than 500nm); The fluorescence “turn off-on” mode of our sensor possesses the advantages of versatility and high selectivity. Besides, there is no heavy metal introduced in this system for AA detection. It has been reported that substances possessing a benzoquinone structure, known as an electron acceptor, can quench the fluorescence of semiconductor quantum dots (Li et al., 2014). The phenolic hydroxyl of catechol could be oxidized to benzoquinone in the presence of hydroxyl radicals produced by hydrogen peroxide (H2O2) under the catalysis of horse radish peroxidase (HRP), resulting in fluorescence quenching of GQDs. As an effective antioxidant, AA with a g-lactone structure exhibits highly reducibility, which can easily remove H2O2 and free radicals. A part of the H2O2 and hydroxyl radicals could be consumed by AA, which can inhibit the generation of o-benzoquinone and resulting in fluorescence recovery. Thus, a facile and rapid fluorescence turn-on assay for AA detection has been established and the proposed method has potential applications in the detection of AA in pharmaceuticals.
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Scheme 1 Schematic illustration of the process for AA recognition.
2. Experimental section 2.1. Materials All chemicals used were at least of analytical reagent grade and used without further purification. Catechol, HRP, H2O2 (30 %), AA, KMnO4, glucose, proline, tyrosine, glutamic acid, and glycine were obtained from Beijing Dingguo Biotechnology Co. Ltd. Graphite powder (100 mesh), H2SO4 (98 %) were purchased from Huacheng Biological Co., Ltd (Changchun). Sodium dehydrogenized phosphate (NaH2PO4), disodium hydrogen phosphate (Na2HPO4) and sodium phosphate (Na3PO4) were purchased from Beijing Chemical Works. The water used in all experiments had a resistivity higher than 18 MΩ·cm-1. The 10 mM PBS buffered solution (pH 6.5) was used as the medium for detection process. 2.2. Apparatus All fluorescence spectra were measured in a 1 cm path length quartz cuvette with a Shimadzu
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RF-5301 spectrometer (Shimadzu Co., Kyoto, Japan). The schematic diagram of experimental setup is shown in Fig. S1. UV-vis absorption spectra was carried out on a Varian GBC Cintra 10e UV-visible Spectrophotometer. FT-IR spectra were recorded with a Bruker IFS66V FT-IR spectrometer equipped with a DGTS detector (32 scans). All the optical measurements were carried out at room temperature under ambient conditions. All pH measurements were obtained by a PHS-3C pH meter (Tuopu Co., Hangzhou, China). 2.3. Synthesis of GQDs GQDs were prepared from graphite oxide according to a modified Hummer’s method which we had carried out in our previous work (Na et al., 2016). In brief, 1.0 g graphene oxide were added into 40 mL H2SO4 (98%) and 4g KMnO4 were added into the solution in an ice bath after stirred for 10 minutes. Next the mixture was stirred for 60 minutes at 38 ℃. Afterwards, 100 mL water was slowly added dropwise to the solution and the temperature was raised to 90 ℃ for 30 minutes. Subsequently, the mixture of 15 mL H2O2 and 85 mL H2O was slowly added dropwise to the solution. The color of the mixture changed to bright yellow. After cooling down, the mixture was further dialyzed through dialysis membrane with a molecular weight cutoff of 3500Da for 1 day until the pH of the mixture became neutral. GQDs were used in the experiment after centrifuged at 8000 rpm for 8 min, and were stored in 4 ℃. The final concentration of GQDs was 1.5 mg·mL-1. 2.4. Procedure for AA determination For the assay of AA, different amount of AA were added in a series of 2.0 mL calibrated test tubes. Subsequently, H2O2 solution (500 μM), HRP (2 µg·mL-1) and catechol (1000 μM) were added, successively. Then the resulting solution was diluted to 1.4 mL with pH 6.5 PBS (10 mM)
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and mixed thoroughly. The mixtures were shaken thoroughly for 40 min at room temperature followed by the addition of 100 μL GQDs solution before fluorescent measurement. The fluorescence spectra were recorded in the 500-720 nm emission wavelength range at the excitation wavelength of 470 nm, the slit width of emission and excitation were both set at 10 nm. 2.5. Real sample assay The blood samples of healthy persons were supplied by the Hospital of Changchun China, Japan Union Hospital. Some pretreatments to remove impurities are necessary before using in the experiment. Firstly, acetonitrile is added to the blood samples (the volume of acetonitrile and blood was 1.5: 1) and then the product after shaking for 2 minutes was centrifuged at 10000 rpm for 10 min to remove protein. The supernatant was stored in -20℃ until testing. The obtained human serum samples were subjected to a 5-fold dilution and a certain concentration of AA was added to prepare spiked samples which were detected by the method described above. All experiments were performed in compliance with the relevant laws and institutional guidelines, and the writing of informed consent for all samples was obtained from human subjects.
3. Results and discussion 3.1. Characterization of GQDs To explore the properties of GQDs, FT-IR, UV-vis and fluorescence spectrum characterization were carried out. The transmission Electron Microscopy (TEM) image and the size distribution of GQDs was showed in Fig. 1, which can reveal that GQDs was nearly spherical in shape with an average diameter of 7.06 nm and mostly uniform in size. The band gap of GQDs is highly influenced by their size due to the quantum confinement effect, which is particularly significant
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when their diameter is less than 10 nm, and the larger its diameter, the longer wavelength it emits (Jin et al., 2013; Peng et al., 2012). However, the fluorescence properties of GQDs can also be influenced by other factors as well, such as heteroatom doping, edge configuration, attached chemical functionalities, shape and defects (Sk et al., 2014). As shown in Fig. 1 (C), the majority characteristic peaks of GQDs could be clearly found through the characteristic peaks of around 3380 cm-1 from -OH, around 2896 cm-1 from C-H, around 1740 cm-1 from C=O, around 1385 cm-1 from -COO- and around 1110 cm-1 from C-O-O, which is consistent with the previous work (Liu et al., 2015; Li et al., 2014). Fig. 1 (D) showed the UV-vis absorption spectra, the fluorescence excitation and emission spectra of GQDs. It could be seen that the maximum excitation wevelength of GQDs was at 470 nm, and the fluorescence emission peak of GQDs was at 540 nm with the excitation wavelength of 470nm. The UV-vis absorption spectra of GQDs shown in Fig. 1 (D) indicated strong absorbance at 230 nm and a weak shoulder peak at around 300 nm, which maybe result from π–π* transition of aromatic structures and n-π* transition of C=O, respectively (Novoselov et al., 2004; Fang et al., 2012), which is consistent with the previous work.
Fig. 1 (A) The TEM image of GQDs; (B) The size distribution of GQDs; (C) The FTIR spectra of GQDs; (D) The 8
UV-vis absorption spectra, the fluorescence excitation and emission spectra of GQDs.
3.2. The design strategy for sensing H2O2 and AA As illustrated in Scheme 1, catechol became o-benzoquinone in the presence of hydroxyl radicals produced by H2O2 under HRP catalysis, which can quench the fluorescence of GQDs significantly, while the fluorescence was recovered with the addition of AA. In order to prove the feasibility of the strategy, the effect of different materials on the fluorescence intensity of GQDs was studied. As shown in Fig. 2, there is no significant change of the fluorescence intensity of GQDs after mixed with AA, catechol, HRP or H2O2 separately or mixed with both of them. After adding HRP and H2O2, the fluorescence intensity of GQDs decreased greatly with the addition of catechol, while the fluorescence intensity decreased little with the addition of catechol in the presence of HRP, H2O2 and AA. It is consistent with the mechanism illustrated in Scheme 1, and further proves that AA can consume part of the H2O2 and hydroxyl radicals to inhibit the generation of o-benzoquinone.
Fig. 2 The fluorescence spectra of GQDs, GQDs + AA, GQDs + catechol, GQDs + H2O2, GQDs + H2O2 + HRP, GQDs + H2O2 + HRP + catechol, GQDs + H2O2 + HRP + catechol + AA, respectively. Concentration: 1000 µM 9
catechol, 500 μM H2O2, 2 μg·mL-1 HRP, 250 µM AA.
3.3. Optimization for H2O2 and AA detection In order to optimize the detection conditions of AA, we optimized the reaction time, pH, the amount of catechol and HRP. In this work, the reaction time was investigated firstly. According to Fig. S2 (A), it can be observed that the fluorescence intensity of GQDs decreased rapidly in the first 20 min, then kept constant after 39 min. Thus the reaction time of 45 min was adopted in the further experiments. The effect of pH in the range of 6.0-9.0 on the fluorescence intensity of detection system was shown in the Fig. S2 (B). As shown in Fig. S2 (B), the fluorescence intensity of GQDs was stable in the pH range of 6.0-9.0, the fluorescence intensity of the GQDs/H2O2/HRP/catechol system was the lowest at pH 6.5, and the fluorescence intensity of the GQDs/H2O2/HRP/catechol system recovered most obviously at pH 6.5 after adding the same amount of AA. So pH 6.5 was chosen in the further experiment. Then, the effect of catechol and HRP were investigated. As shown in Fig. S2 (C), the fluorescence intensity of GQDs decreased with the increase of the concentration of catechol, and 1000 µM catechol was chosen in the further experiments. Fig. S2 (D) shows the the effect of HRP concentration on the fluorescence intensity of GQDs, it can be seen that the fluorescence intensity of GQDs decreased obviously with the addition of HRP, and kept constant when the concentration of HRP is more than 0.5 µg·mL-1. Thus, we choose 2 µg·mL-1 HRP in the further experiments. 3.4. Detection of H2O2 The effect of H2O2 on the fluorescence intensity of GQDs/H2O2/HRP/catechol system was studied before AA was explored. As shown in Fig. 3, the fluorescence intensity of GQDs was
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gradually decreased with the increase of H2O2 concentration. And Fig. 3 inset showed there was a good linear relationship between I/I0 (I0 and I were the fluorescence intensity of the sensing system in the absence and presence of H2O2, respectively) and the concentration of H2O2 in the range of 3.33-500 µM with a detection limit of 1.2 µM. The linear regression equation was I/I0 = 0.9849 + 0.0011 CH2O2 (µM), with a correlation coefficient R2 = 0.9914. The detection limit was based on the equation LOD=3σ/s, where σ is the standard deviation of the corrected blank signals of the GQDs and s is the slope of the calibration curve. And 500 µM H2O2 was chosen to for AA detection in the further experiment.
Fig. 3 The fluorescence spectra of GQDs/H2O2/HRP/catechol system with different concentrations of H2O2 in the range of 0-1000µM (0, 3.33, 10, 20, 40, 80, 100, 200, 266.7, 300, 400, 500, 700, 900, 1000 µM). The inset showed
the relationship between I/I0 and the concentration of H2O2 in the range of 3.33 to 500 µM. I and I0 were the maximum fluorescence emission intensity of the detection system in the absence and presence of H2O2, respectively. The error bars (RSD) were gained from three parallel test results. Concentration: 1000 µM catechol, 2 μg·mL-1 HRP.
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3.5. Detection of AA Under the optimized experimental conditions, the relationship of the fluorescence intensity and the concentration of AA was investigated. As shown in Fig. 4, the fluorescence intensity of GQDs was restored with the increase of AA concentration. Fig. 4 inset showed there was a good linear relationship between I/I0 and the concentration of AA in the range of 1.11-300 µM. The linear regression equation was I/I0 = 0.9756 + 0.00498 CAA (µM), the correlation coefficient R2 = 0.9929. And the detection limit for AA is 0.32 µM. A comparison of materials, detection limits and linear ranges between this method and some other methods for AA determination reported previously was listed in Table 1. Compared with other methods, the method we established offered a comparable or superior linear range and detection limit.
Fig. 4 The fluorescence spectra of GQDs/H2O2/HRP/catechol/AA system with different concentrations of AA in the range of 0-900 µM (0, 1.11, 6.67, 20, 50, 100, 200, 300, 500, 700, 900 µM). The inset showed the relationship
between I/I0 and the concentration of AA in the range of 1.11 to 300 µM. I and I0 were the maximum fluorescence emission intensity of the detection system in the absence and presence of AA, respectively. The error bars (RSD) were gained from three parallel test results. Concentration: 1000 µM catechol, 500 μM H2O2, 2 μg·mL-1 HRP.
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Table 1 Comparison of methods for AA. Linear range
Detection limit
(µM)
(µM)
PANI/PAA/MWCNTs/SPE
1-1000
0.25
Sharifian et al., 2016
Electrochemical
CuO nanoneedles/SPEs
100-8000
88
Khairy et al., 2016
Colorimetric
ssDNA-AuNPs
1-15
0.3
Song et al., 2011
Colorimetric
PSS-rGO
0.8-70
0.15
Chen et al., 2016
Fluorescence
CdTe QD@SiO2 nanobeads
3.33-400
1.25
Ma et al., 2013
Fluorescence
GQDs
0.3-10
0.094
Liu et al., 2015
Fluorescence
GQDs
1.11-300
0.32
This work
Methods
Materials
Electrochemical
Reference
3.6. Interference study Selectivity is a very important parameter to evaluate the performance of a new fluorescence sensor, which was further evaluated with various common coexisting substances. As shown in Table S1, the interference effect of 500 µM glycine (Gly), 500 µM glutamic acid (Glu), 500 µM proline (Pro), 500 µM tyrosine (Tyr), 2000 µM glucose, 2000 µM Na+, 2000 µM K+, 2000 µM Ca2+ on the determination of AA (150 µM) was demonstrated, respectively. Table S1 showed that the relative error of the interference effect of common metal ions and biomolecules on detecting AA was in the range of -2.37% to 3.17%, which was considered to be tolerable. The results indicated that common metal ions and biomolecules had no obvious interference on the detection of AA. 3.7. Real samples detection In order to further demonstrate the practicality of the experiment, we detected AA in human serum. The results obtained by the standard addition method were shown in Table S2. From Table S2, it was found that the recovery of AA is in the range of 94.1%-105.9%, and the relative
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standard deviation (RSD) was less than 3.0%. These results demonstrated that the proposed method has potential application in practical measurement of AA.
4. Conclusion In summary, a facile and rapid fluorescence method for the detection of AA was developed. Under the optimum condition, a good linear response for AA was found in the range of 1.11-300 µM with a detection limit of 0.32 µM. Moreover, the proposed method was successfully applied to the detection of AA in human serum samples with satisfactory results. In comparison with the previous reports, the proposed method is simple, low cost and has a higher sensitivity and better selectivity.
Acknowledgments This work was finally supported by the National Natural Science Foundation of China (No. 21075050 and No. 21275063), the Science and Technology Development project of Jilin province, China (No. 20150204010GX).
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Highlights
GQDs with a fluorescence emission peak of 540 nm were used to detect H2O2 and AA; The detection for AA is based on a turn-on fluorescent strategy The proposed method was simple, low cost and had a higher sensitivity and better selectivity.
The proposed method was applied to the determination of AA in human serum samples with satisfactory results.
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PII: DOI: Reference:
S0956-5663(17)30074-X http://dx.doi.org/10.1016/j.bios.2017.02.005 BIOS9539
To appear in: Biosensors and Bioelectronic Received date: 24 November 2016 Revised date: 15 January 2017 Accepted date: 3 February 2017 Cite this article as: Hua Liu, Weidan Na, Ziping Liu, Xueqian Chen and Xingguang Su, A novel turn-on fluorescent strategy for sensing ascorbic acid using graphene quantum dots as fluorescent probe, Biosensors and Bioelectronic, http://dx.doi.org/10.1016/j.bios.2017.02.005 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.
A novel turn-on fluorescent strategy for sensing ascorbic acid using graphene quantum dots as fluorescent probe Hua Liu, Weidan Na, Ziping Liu, Xueqian Chen, Xingguang Su* Department of Analytical Chemistry, College of Chemistry, Jilin University, Changchun 130012, China *
Corresponding author. Tel.: +86-431-85168352. [email protected]
Abstract In this paper, a facile and rapid fluorescence turn-on assay for fluorescent detection of ascorbic acid (AA) was developed by using the orange emission graphene quantum dots (GQDs). In the presence of horse radish peroxidase (HRP) and hydrogen peroxide (H2O2), catechol can be oxidized by hydroxyl radicals and converted to o-benzoquinone, which can significantly quench the fluorescence of GQDs. However, when AA present in the system, it can consume part of H2O2 and hydroxyl radicals to inhibit the generation of o-benzoquinone, resulting in fluorescence recovery. Under the optimized experimental conditions, the fluorescence intensity was linearly correlated with the concentration of H2O2 in the range of 3.33-500 µM with a detection limit of 1.2 µM. The linear detection for AA was in the range from 1.11 µM to 300 µM with a detection limit of 0.32 µM. The proposed method was applied to the determination of AA in human serum samples with satisfactory results. Key words: Ascorbic acid, H2O2, Graphene quantum dots (GQDs), Catechol, O-benzoquinone
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1. Introduction In recent decades, graphene quantum dots (GQDs) have gained tremendous attention because of its unique electrical and optical properties, including tunable photoluminescence, molecular size, excellent photostability, chemical inertness, good solubility, high biocompatibility, and ease of functionalization (Wang et al., 2014; Li et al., 2016; Shen et al., 2012). Furthermore, GQDs are considered to be competitive alternatives to organic dyes and heavy metal based quantum dots and they are widely used in biological imaging (Deng et al., 2013; Jiang et al., 2016; Sun et al., 2015; Du et al., 2016) and biomolecule detection (Su et al., 2014; Nirala et al., 2015; Lu et al., 2014; Duan et al., 2014). Jiang’s group reported a novel cast-targeted fluorescent imaging assay for the diagnosis of renal disease using Fe3O4 and GQD nanocomposites (Jiang et al., 2016). Ankan Dutta Chowdhury et al. illustrated a highly sensitive and selective fluorescent sensor to detect Fe3+ in the range of 20 nM to 2 μM based on dopamine functionalized GQDs, and the detection limit is 7.6 nM (Dutta Chowdhury et al., 2016). Wang’s group demonstrated the detection of nuclei acids based on GQDs utilizing cascade amplification by nicking endonuclease and catalytic G-quadruplex DNAzyme (Wang et al., 2016), and the detection limit of nuclei acids is 30 fM. Ascorbic acid (AA), known as vitamin C, plays an extremely important role in human life activities. AA was involved in immune response, disease prevention and oxidative stress reduction and other physiological activities (Liu et al., 2015). Specifically, AA is essential for the synthesis of collagen and neurotransmitters or dissociation of sterols in the body, and it can also promote antibody formation, iron uptake, formation of tetrahydrofolate and maintenance of thiolases activity (Liu et al., 2012; Sharifian et al., 2016; Hillstrom et al., 2003). In addition, some reports showed that it can reduce the risk of cancer (Frajese et al., 2016) and remove free radicals in the
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human body (Pisoschi et al., 2014). The reference ranges of ascorbic acid in the general population are 11.4-76.7 µM (Trezzi et al., 2016; Schleicher et al., 2009). AA deficiency can cause scurvy and hypoimmunity, while too much can cause diarrhea, hyperacidity, coronary heart disease and other diseases (Pournaghi-Azar et al., 2002; Bi et al., 2016; Chen et al., 2003; Massey et al., 2005). Therefore, it is necessary to find a rapid, accurate, sensitive and low-cost determination way of AA. Up to now, many methods for AA detection have been established, including fluorescence methods (Liu et al., 2015; Ma et al., 2013), electrochemical methods (Sharifian et al., 2016; Khairy et al., 2016), colorimetry (Chen et al., 2016; Song et al., 2011), liquid chromatography (Lima et al., 2016), polarography (Dimitrijevic et al., 2016) and etc. Although these methods can meet the requirement of sensitivity, they are usually time-consuming, cumbersome, expensive equipment or the need for complex synthesis or complicated extraction. Compared with the other methods, fluorescence method showed more advantages owing to its operational simplicity, high sensitivity and rapid response (Duan et al., 2014; Li et al., 2014). Liu and co-workers designed a method for detecting AA based on the transformation of Cu2+ and Cu+ at the presence of AA (Liu et al., 2015). Ma and co-workers demonstrated the detection of AA based on the transformation of Fe3+ and Fe2+ at the presence of AA (Ma et al., 2013; Gong et al., 2015). However, these approaches for AA detection are based on the difference abilities of the fluorescence quenching between the different valence states of a metal, and the processes are usually accompanied with heavy metal contamination. In this paper, we developed a novel turn-on fluorescent strategy for sensing AA based on GQDs (Scheme1). Compared with some similar studies on using carbon quantum dots/GQDs for
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detecting AA, the establish method in this work has several mentionable features: The fluorescence probe (GQDs) used in this work has a more ideal fluorescence emission peak (540 nm) than others reported GQDs (less than 500nm); The fluorescence “turn off-on” mode of our sensor possesses the advantages of versatility and high selectivity. Besides, there is no heavy metal introduced in this system for AA detection. It has been reported that substances possessing a benzoquinone structure, known as an electron acceptor, can quench the fluorescence of semiconductor quantum dots (Li et al., 2014). The phenolic hydroxyl of catechol could be oxidized to benzoquinone in the presence of hydroxyl radicals produced by hydrogen peroxide (H2O2) under the catalysis of horse radish peroxidase (HRP), resulting in fluorescence quenching of GQDs. As an effective antioxidant, AA with a g-lactone structure exhibits highly reducibility, which can easily remove H2O2 and free radicals. A part of the H2O2 and hydroxyl radicals could be consumed by AA, which can inhibit the generation of o-benzoquinone and resulting in fluorescence recovery. Thus, a facile and rapid fluorescence turn-on assay for AA detection has been established and the proposed method has potential applications in the detection of AA in pharmaceuticals.
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Scheme 1 Schematic illustration of the process for AA recognition.
2. Experimental section 2.1. Materials All chemicals used were at least of analytical reagent grade and used without further purification. Catechol, HRP, H2O2 (30 %), AA, KMnO4, glucose, proline, tyrosine, glutamic acid, and glycine were obtained from Beijing Dingguo Biotechnology Co. Ltd. Graphite powder (100 mesh), H2SO4 (98 %) were purchased from Huacheng Biological Co., Ltd (Changchun). Sodium dehydrogenized phosphate (NaH2PO4), disodium hydrogen phosphate (Na2HPO4) and sodium phosphate (Na3PO4) were purchased from Beijing Chemical Works. The water used in all experiments had a resistivity higher than 18 MΩ·cm-1. The 10 mM PBS buffered solution (pH 6.5) was used as the medium for detection process. 2.2. Apparatus All fluorescence spectra were measured in a 1 cm path length quartz cuvette with a Shimadzu
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RF-5301 spectrometer (Shimadzu Co., Kyoto, Japan). The schematic diagram of experimental setup is shown in Fig. S1. UV-vis absorption spectra was carried out on a Varian GBC Cintra 10e UV-visible Spectrophotometer. FT-IR spectra were recorded with a Bruker IFS66V FT-IR spectrometer equipped with a DGTS detector (32 scans). All the optical measurements were carried out at room temperature under ambient conditions. All pH measurements were obtained by a PHS-3C pH meter (Tuopu Co., Hangzhou, China). 2.3. Synthesis of GQDs GQDs were prepared from graphite oxide according to a modified Hummer’s method which we had carried out in our previous work (Na et al., 2016). In brief, 1.0 g graphene oxide were added into 40 mL H2SO4 (98%) and 4g KMnO4 were added into the solution in an ice bath after stirred for 10 minutes. Next the mixture was stirred for 60 minutes at 38 ℃. Afterwards, 100 mL water was slowly added dropwise to the solution and the temperature was raised to 90 ℃ for 30 minutes. Subsequently, the mixture of 15 mL H2O2 and 85 mL H2O was slowly added dropwise to the solution. The color of the mixture changed to bright yellow. After cooling down, the mixture was further dialyzed through dialysis membrane with a molecular weight cutoff of 3500Da for 1 day until the pH of the mixture became neutral. GQDs were used in the experiment after centrifuged at 8000 rpm for 8 min, and were stored in 4 ℃. The final concentration of GQDs was 1.5 mg·mL-1. 2.4. Procedure for AA determination For the assay of AA, different amount of AA were added in a series of 2.0 mL calibrated test tubes. Subsequently, H2O2 solution (500 μM), HRP (2 µg·mL-1) and catechol (1000 μM) were added, successively. Then the resulting solution was diluted to 1.4 mL with pH 6.5 PBS (10 mM)
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and mixed thoroughly. The mixtures were shaken thoroughly for 40 min at room temperature followed by the addition of 100 μL GQDs solution before fluorescent measurement. The fluorescence spectra were recorded in the 500-720 nm emission wavelength range at the excitation wavelength of 470 nm, the slit width of emission and excitation were both set at 10 nm. 2.5. Real sample assay The blood samples of healthy persons were supplied by the Hospital of Changchun China, Japan Union Hospital. Some pretreatments to remove impurities are necessary before using in the experiment. Firstly, acetonitrile is added to the blood samples (the volume of acetonitrile and blood was 1.5: 1) and then the product after shaking for 2 minutes was centrifuged at 10000 rpm for 10 min to remove protein. The supernatant was stored in -20℃ until testing. The obtained human serum samples were subjected to a 5-fold dilution and a certain concentration of AA was added to prepare spiked samples which were detected by the method described above. All experiments were performed in compliance with the relevant laws and institutional guidelines, and the writing of informed consent for all samples was obtained from human subjects.
3. Results and discussion 3.1. Characterization of GQDs To explore the properties of GQDs, FT-IR, UV-vis and fluorescence spectrum characterization were carried out. The transmission Electron Microscopy (TEM) image and the size distribution of GQDs was showed in Fig. 1, which can reveal that GQDs was nearly spherical in shape with an average diameter of 7.06 nm and mostly uniform in size. The band gap of GQDs is highly influenced by their size due to the quantum confinement effect, which is particularly significant
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when their diameter is less than 10 nm, and the larger its diameter, the longer wavelength it emits (Jin et al., 2013; Peng et al., 2012). However, the fluorescence properties of GQDs can also be influenced by other factors as well, such as heteroatom doping, edge configuration, attached chemical functionalities, shape and defects (Sk et al., 2014). As shown in Fig. 1 (C), the majority characteristic peaks of GQDs could be clearly found through the characteristic peaks of around 3380 cm-1 from -OH, around 2896 cm-1 from C-H, around 1740 cm-1 from C=O, around 1385 cm-1 from -COO- and around 1110 cm-1 from C-O-O, which is consistent with the previous work (Liu et al., 2015; Li et al., 2014). Fig. 1 (D) showed the UV-vis absorption spectra, the fluorescence excitation and emission spectra of GQDs. It could be seen that the maximum excitation wevelength of GQDs was at 470 nm, and the fluorescence emission peak of GQDs was at 540 nm with the excitation wavelength of 470nm. The UV-vis absorption spectra of GQDs shown in Fig. 1 (D) indicated strong absorbance at 230 nm and a weak shoulder peak at around 300 nm, which maybe result from π–π* transition of aromatic structures and n-π* transition of C=O, respectively (Novoselov et al., 2004; Fang et al., 2012), which is consistent with the previous work.
Fig. 1 (A) The TEM image of GQDs; (B) The size distribution of GQDs; (C) The FTIR spectra of GQDs; (D) The 8
UV-vis absorption spectra, the fluorescence excitation and emission spectra of GQDs.
3.2. The design strategy for sensing H2O2 and AA As illustrated in Scheme 1, catechol became o-benzoquinone in the presence of hydroxyl radicals produced by H2O2 under HRP catalysis, which can quench the fluorescence of GQDs significantly, while the fluorescence was recovered with the addition of AA. In order to prove the feasibility of the strategy, the effect of different materials on the fluorescence intensity of GQDs was studied. As shown in Fig. 2, there is no significant change of the fluorescence intensity of GQDs after mixed with AA, catechol, HRP or H2O2 separately or mixed with both of them. After adding HRP and H2O2, the fluorescence intensity of GQDs decreased greatly with the addition of catechol, while the fluorescence intensity decreased little with the addition of catechol in the presence of HRP, H2O2 and AA. It is consistent with the mechanism illustrated in Scheme 1, and further proves that AA can consume part of the H2O2 and hydroxyl radicals to inhibit the generation of o-benzoquinone.
Fig. 2 The fluorescence spectra of GQDs, GQDs + AA, GQDs + catechol, GQDs + H2O2, GQDs + H2O2 + HRP, GQDs + H2O2 + HRP + catechol, GQDs + H2O2 + HRP + catechol + AA, respectively. Concentration: 1000 µM 9
catechol, 500 μM H2O2, 2 μg·mL-1 HRP, 250 µM AA.
3.3. Optimization for H2O2 and AA detection In order to optimize the detection conditions of AA, we optimized the reaction time, pH, the amount of catechol and HRP. In this work, the reaction time was investigated firstly. According to Fig. S2 (A), it can be observed that the fluorescence intensity of GQDs decreased rapidly in the first 20 min, then kept constant after 39 min. Thus the reaction time of 45 min was adopted in the further experiments. The effect of pH in the range of 6.0-9.0 on the fluorescence intensity of detection system was shown in the Fig. S2 (B). As shown in Fig. S2 (B), the fluorescence intensity of GQDs was stable in the pH range of 6.0-9.0, the fluorescence intensity of the GQDs/H2O2/HRP/catechol system was the lowest at pH 6.5, and the fluorescence intensity of the GQDs/H2O2/HRP/catechol system recovered most obviously at pH 6.5 after adding the same amount of AA. So pH 6.5 was chosen in the further experiment. Then, the effect of catechol and HRP were investigated. As shown in Fig. S2 (C), the fluorescence intensity of GQDs decreased with the increase of the concentration of catechol, and 1000 µM catechol was chosen in the further experiments. Fig. S2 (D) shows the the effect of HRP concentration on the fluorescence intensity of GQDs, it can be seen that the fluorescence intensity of GQDs decreased obviously with the addition of HRP, and kept constant when the concentration of HRP is more than 0.5 µg·mL-1. Thus, we choose 2 µg·mL-1 HRP in the further experiments. 3.4. Detection of H2O2 The effect of H2O2 on the fluorescence intensity of GQDs/H2O2/HRP/catechol system was studied before AA was explored. As shown in Fig. 3, the fluorescence intensity of GQDs was
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gradually decreased with the increase of H2O2 concentration. And Fig. 3 inset showed there was a good linear relationship between I/I0 (I0 and I were the fluorescence intensity of the sensing system in the absence and presence of H2O2, respectively) and the concentration of H2O2 in the range of 3.33-500 µM with a detection limit of 1.2 µM. The linear regression equation was I/I0 = 0.9849 + 0.0011 CH2O2 (µM), with a correlation coefficient R2 = 0.9914. The detection limit was based on the equation LOD=3σ/s, where σ is the standard deviation of the corrected blank signals of the GQDs and s is the slope of the calibration curve. And 500 µM H2O2 was chosen to for AA detection in the further experiment.
Fig. 3 The fluorescence spectra of GQDs/H2O2/HRP/catechol system with different concentrations of H2O2 in the range of 0-1000µM (0, 3.33, 10, 20, 40, 80, 100, 200, 266.7, 300, 400, 500, 700, 900, 1000 µM). The inset showed
the relationship between I/I0 and the concentration of H2O2 in the range of 3.33 to 500 µM. I and I0 were the maximum fluorescence emission intensity of the detection system in the absence and presence of H2O2, respectively. The error bars (RSD) were gained from three parallel test results. Concentration: 1000 µM catechol, 2 μg·mL-1 HRP.
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3.5. Detection of AA Under the optimized experimental conditions, the relationship of the fluorescence intensity and the concentration of AA was investigated. As shown in Fig. 4, the fluorescence intensity of GQDs was restored with the increase of AA concentration. Fig. 4 inset showed there was a good linear relationship between I/I0 and the concentration of AA in the range of 1.11-300 µM. The linear regression equation was I/I0 = 0.9756 + 0.00498 CAA (µM), the correlation coefficient R2 = 0.9929. And the detection limit for AA is 0.32 µM. A comparison of materials, detection limits and linear ranges between this method and some other methods for AA determination reported previously was listed in Table 1. Compared with other methods, the method we established offered a comparable or superior linear range and detection limit.
Fig. 4 The fluorescence spectra of GQDs/H2O2/HRP/catechol/AA system with different concentrations of AA in the range of 0-900 µM (0, 1.11, 6.67, 20, 50, 100, 200, 300, 500, 700, 900 µM). The inset showed the relationship
between I/I0 and the concentration of AA in the range of 1.11 to 300 µM. I and I0 were the maximum fluorescence emission intensity of the detection system in the absence and presence of AA, respectively. The error bars (RSD) were gained from three parallel test results. Concentration: 1000 µM catechol, 500 μM H2O2, 2 μg·mL-1 HRP.
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Table 1 Comparison of methods for AA. Linear range
Detection limit
(µM)
(µM)
PANI/PAA/MWCNTs/SPE
1-1000
0.25
Sharifian et al., 2016
Electrochemical
CuO nanoneedles/SPEs
100-8000
88
Khairy et al., 2016
Colorimetric
ssDNA-AuNPs
1-15
0.3
Song et al., 2011
Colorimetric
PSS-rGO
0.8-70
0.15
Chen et al., 2016
Fluorescence
CdTe QD@SiO2 nanobeads
3.33-400
1.25
Ma et al., 2013
Fluorescence
GQDs
0.3-10
0.094
Liu et al., 2015
Fluorescence
GQDs
1.11-300
0.32
This work
Methods
Materials
Electrochemical
Reference
3.6. Interference study Selectivity is a very important parameter to evaluate the performance of a new fluorescence sensor, which was further evaluated with various common coexisting substances. As shown in Table S1, the interference effect of 500 µM glycine (Gly), 500 µM glutamic acid (Glu), 500 µM proline (Pro), 500 µM tyrosine (Tyr), 2000 µM glucose, 2000 µM Na+, 2000 µM K+, 2000 µM Ca2+ on the determination of AA (150 µM) was demonstrated, respectively. Table S1 showed that the relative error of the interference effect of common metal ions and biomolecules on detecting AA was in the range of -2.37% to 3.17%, which was considered to be tolerable. The results indicated that common metal ions and biomolecules had no obvious interference on the detection of AA. 3.7. Real samples detection In order to further demonstrate the practicality of the experiment, we detected AA in human serum. The results obtained by the standard addition method were shown in Table S2. From Table S2, it was found that the recovery of AA is in the range of 94.1%-105.9%, and the relative
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standard deviation (RSD) was less than 3.0%. These results demonstrated that the proposed method has potential application in practical measurement of AA.
4. Conclusion In summary, a facile and rapid fluorescence method for the detection of AA was developed. Under the optimum condition, a good linear response for AA was found in the range of 1.11-300 µM with a detection limit of 0.32 µM. Moreover, the proposed method was successfully applied to the detection of AA in human serum samples with satisfactory results. In comparison with the previous reports, the proposed method is simple, low cost and has a higher sensitivity and better selectivity.
Acknowledgments This work was finally supported by the National Natural Science Foundation of China (No. 21075050 and No. 21275063), the Science and Technology Development project of Jilin province, China (No. 20150204010GX).
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Highlights
GQDs with a fluorescence emission peak of 540 nm were used to detect H2O2 and AA; The detection for AA is based on a turn-on fluorescent strategy The proposed method was simple, low cost and had a higher sensitivity and better selectivity.
The proposed method was applied to the determination of AA in human serum samples with satisfactory results.
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