Biosensors and Bioelectronics 57 (2014) 239–244
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Mild and novel electrochemical preparation of β-cyclodextrin/ graphene nanocomposite film for super-sensitive sensing of quercetin Zhen Zhang a,b, Shuqing Gu c, Yaping Ding a,n, Mingju Shen a, Lin Jiang a a Department of Chemistry, Shanghai Key Laboratory of Modern Metallurgy & Materials Processing, School of Materials Science and Engineering, Shanghai University, Shanghai 200444, PR China b School of Chemistry & Chemical Engineering, Linyi University, Linyi 276005, PR China c Technical Center for Animal Plant and Food Inspection and Quarantine, Shanghai Entry-Exit Inspection and Quarantine Bureau, Shanghai 200135, China
art ic l e i nf o
a b s t r a c t
Article history: Received 11 November 2013 Received in revised form 5 February 2014 Accepted 6 February 2014 Available online 19 February 2014
A mild and novel preparation tactics based on electrochemical techniques for the fabrication of electrodeposited graphene (E-GR) and polymerized β-cyclodextrin (P-βCD) nanocomposite film were developed. The structure and morphology of GR-based nanocomposite were investigated by scanning electron microscopy (SEM), transmission electron microscopy (TEM) and Raman spectroscopy. Simultaneously, the electrochemical properties of this nanocomposite were characterized by cyclic voltammetry (CV) and differential pulse voltammetry (DPV). Based on the synergistic effect of E-GR and P-βCD, a super-sensitive electrochemical sensor for quercetin was successfully fabricated. Under optimum conditions, the determination range for quercetin was from 0.005 to 20 mM with a low detection limit of 0.001 mM (S/N¼ 3). Moreover, this sensor also displays excellent sensitivity, fine reproducibility and stability. To further study the practical applicability of the proposed sensor, the determination of real samples was carried out with satisfactory results. & 2014 Elsevier B.V. All rights reserved.
Keywords: Electro-deposited grapheme Polymerized β-cyclodextrin Quercetin Nanocomposite film Differential pulse voltammetry
1. Introduction Graphene (GR), a two-dimensional sheet of sp2 conjugated atomic carbon, has attracted fascinating interests across different disciplines because of its ultrahigh specific surface area, unique electronic features (Novoselov et al., 2004; Kim et al., 2008), great mechanical strength (Zhao et al., 2010), and good biocompatibility (Matthew et al., 2010). Benefiting from these advantages, GR has been widely used for immobilizing organic and inorganic molecules (Ohno et al., 2009) and for fabrication of electrochemical biosensors (Shan et al., 2009; Wang et al., 2009; Zhou et al., 2009). β-Cyclodextrin (β-CD), a cyclic oligosaccharide consisting of seven glucose units, presents a toroidal form with a hydrophobic inner cavity and a hydrophilic outer side (Rekharsky and Inoue, 1998). It is well known that β-CD has high molecular selectivity and enantioselectivity. Various inorganic, organic and biological guest molecules can be bound selectively in the inner cavities of β-CD to form stable host–guest inclusion complexes or nanostructure supramolecular assemblies (Freeman et al., 2009; Rekharsky and Inoue, 1998). Therefore, β-CD and its derivatives have been n Corresponding author at: School of Materials Science and Engineering, Iron and Steel Metallurgy Key Laboratory, Shanghai University, Shanghai 200444, PR China. Tel.: þ 86 021 66134734; fax: þ86 21 66132797. E-mail address: [email protected] (Y. Ding).
http://dx.doi.org/10.1016/j.bios.2014.02.014 0956-5663 & 2014 Elsevier B.V. All rights reserved.
extensively used as molecular recognition systems and particularly applied in the field of mimetic enzymes, molecular recognition sensors, discrimination of enantiomers and drug encapsulation (Almirall et al., 2003; Fragoso et al., 2002; Martín et al., 2003; Ng et al., 2002). The nanocomposites based on β-CD can be an ideal substrate material for molecular recognition sensors. Based on the excellent performances of GR and β-CD respectively, a facile effective and efficient preparation strategy of GR–β-CD composite is of considerable significance (Chen et al., 2013; Zhang et al., 2014). Electrochemical technique was as an effective tool for rapid, simple and mild synthesis of various materials. Cyclic voltammetry (CV) (Malitesta et al., 1999; Feng et al., 2004), potentiostatic deposition (Lattach et al., 2012), electrochemical potential pulse technique (Menaker et al., 2009; Choong and Milne, 2010) and the galvanostatic method (Syritskia et al., 2008) have been applied for material preparations. Among them, potentiostatic deposition and CV can ensure similar film thicknesses (Österholma et al., 2012). Until now, there have been no reports concerning electrochemical sensors based on electro-deposited GR (E-GR) and polymer nanocomposite film manufactured by a cooperation of potentiostatic technique and CV. Thus, there is a substantially promising development space in this booming field. Quercetin, as a natural pentahydroxyflavone, is widely distributed in the medicinal plants, flowers, fruits, leaves and variety of beverages as their important constituent (Crozier et al., 1997;
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Hertog et al., 1992). It plays an important role in the science of nutrition and medical science. Quercetin has many biological activities by acting as an effective radical-scavenger against oxidative cell damage, such as antitumor activity (Verma et al., 1998), cardiovascular protection (Scalbert et al., 2005), antiinflammatory, estrogenic and antiallergy activity (Boesch-Saadatmandi et al., 2012). However, an overdose of may lead to kidney cancer (Hsieh et al., 2010). The excess quercetin decreased glutathione S-transferase activity, resulting in DNA damage (Yen et al., 2003). Thus, the low detection of quercetin in the science of nutrition and medical science has attracted an increasing interest. In this work, aiming to explore the unrevealed sensor potential, a mild and novel method has been employed to prepare the electro-deposited graphene/polymerized β-cyclodextrin (E-GR/ P-βCD) nanocomposite film by a cooperation of potentiostatic technique and CV. Quercetin was selected to explore the practical performance of the proposed sensor. The work presented here indicated the effectiveness and feasibility of the proposed strategy and the great potential of E-GR/P-βCD nanocomposite film in electroanalytical fields.
2. Experimental 2.1. Apparatus and reagents All electrochemical experiments were performed on a CHI 660D electrochemical workstation (Shanghai Chenhua Co. Ltd., China). A conventional three-electrode system was employed with a glassy carbon electrode (GCE, 3 mm in diameter) as the working electrode, a platinum electrode as the counter electrode, and a saturated calomel electrode as the reference electrode. The pH value was determined with a pHS-3C acidity meter. Scanning electron micrographs operated on the field emission scanning
electron microscope (Hitachi S4800, Japan) was applied for characterizing the modified materials. Transmission electron microscopy was carried out using a transmission electron microscope (JEOL JEM-200CX working at 160 kV). Graphene was purchased from XFNANO, INC (Nanjing, China). Quercetin and β-cyclodextrin were purchased from Sinopharm Chemical Reagent Co., Ltd., (Shanghai, China). Ascorbic acid and dopamine were purchased from Aladdin Chemical Reagent Co., Ltd. (Shanghai, China). All chemicals employed in this work were analytical grade. All experiments were performed in 0.1 M NaAcHAc buffer solution and double distilled water was used throughout. All electrochemical experiments were carried out at room temperature at about 25 1C.
2.2. Preparation of modified electrodes Prior to use, a bare GCE of 3 mm diameter was firstly polished on the chamois leather with alumina slurries until a mirror-like surface was acquired. Then the GCE was ultrasonicated in HNO3 solution (v/v ¼ 1:1), ethanol and double distilled water for 3 min respectively. The electro-deposited GR/polymerized β-cyclodextrin nanocomposite film (E-GR/P-βCD) modified on GCE was gained via two steps. The first step was the electro-deposition in 10 mL 0.1 M KCl aqueous solution containing 300 μL GR (1 mg/mL dispersed in water) at a deposited voltage of þ1.7 V, then the second step was electro-polymerization in the range from 2.0 to 2.5 V for 6 segments at 100 mV s 1 in 0.1 M phosphate buffer solution (pH 6.0) containing 2.0 mM β-CD. Eventually, the as-prepared electrode was rinsed carefully with double distilled water for further use. At the same time, another two modified electrodes, E-GR/GCE and P-βCD/GCE were also prepared following the same procedure described above for comparison.
Fig. 1. (A) SEM images of graphene; (B) TEM images of graphene; (C) SEM images of E-GR; and (D) Raman spectra of GR (a), E-GR (b).
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Fig. 2. (A) SEM images of ED-GR/P-βCD. The inset is the cross-section image of the nanocomposite. (B) DPVs of bare GCE (a), E-GR/GCE (b), P-βCD/GCE (c) and E-GR/P-βCD/ GCE (d) with 20 μM quercetin in 0.1 M NaAc-HAc buffer solution (pH ¼7.0). (C) CVs of E-GR/P-βCD/GCE with 20 μM quercetin in 0.1 M NaAc-HAc buffer solution (pH ¼ 7.0) at scan rate ranging from 10 to 500 mV s 1. Inset: plots of peak currents vs. scan rates.
3. Results and discussion 3.1. Characterization of the modified electrode The TEM and SEM images of graphene are shown in Fig. 1, from which we can see that graphene was sheet-like, glossy and crimple shape. As shown in Figs. 1C and 2A, the morphologies of E-GR and E-GR/P-βCD on the surface of GCE were investigated by SEM. E-GR film with honeycombed layers acquired from the electro-deposited method can be well modified on the surface of electrode. Compared with GR film on the surface of electrode, specific surface area of E-GR film was increased, which is based on the impetus of the electrochemical power to the changes of Fermi energy level of electrode materials surface (Guo et al., 2009). Raman spectroscopy is a powerful nondestructive tool to distinguish ordered and disordered crystal structures of carbon. The Raman spectra of GR and E-GR are shown in Fig. 1D. As expected, GR (a) displays two prominent peaks at 1345.6 and 1592.9 cm 1, which correspond to the well-documented G and D bands, respectively. By the electro-deposited method, the G and D bands of E-GR (b) shifted to 1352.1 and 1601.1 cm 1. The G band represents the first-order scattering of the E2g vibrational mode while the D band has been attributed to the reduction in size of the in-plane C sp2 atoms. The D/G band intensity ratio expresses the atomic ratio of sp2/sp3 carbons, and is a measure of the extent of disordered graphite. The D/G intensity ratios of GR and E-GR were calculated to be 1.4 and 1.2, respectively. It is anticipated that E-GR film with a distinctive morphology from GR and other reported electrochemical synthesized GR (Zhang et al., 2013; Yang et al., 2012) will support the sensitivity of electrode as an electrochemical sensor. Compared with E-GR film, a relatively flat and smooth surface was obtained after the modification of E-GR/P-βCD nanocomposite film, as showed in Fig. 2A. As seen from the transection images,
β-CD was well implanted in E-GR film with nanosize, implying a favorable distribution with compact multilayer composite-films. These could be the wrinkled E-GR film which offered large surface areas and excellent mechanical durability. It is also expected that the displayed cooperation of β-CD and E-GR will promote the sensitivity and stability of GCE in electrochemical sensing. 3.2. Electrochemical behavior of quercetin on the GCEs The electrochemical oxidations of quercetin on different modified GCEs by differential pulse voltammetry (DPV) were carried out in 0.1 M NaAc-HAc buffer solution (pH 7.0) containing 20 μM quercetin in order to make a comparison of the electrochemical properties, as shown in Fig. 2B. The electrochemical response of E-GR/P-βCD/GCE to quercetin was investigated and the corresponding voltammogram was shown in Fig. 2B, curve d. As a comparison, a similar set of experiments were also performed using the bare GCE (a), E-GR/GCE (b) and P-βCD/GCE (c), shown in Fig. 2B. Compared with the bare GCE, about 20-fold larger current signal was obtained, which demonstrated that the E-GR/P-βCD/ GCE could greatly facilitate the oxidation of quercetin. It is also expected that the displayed cooperation of E-GR and P-βCD will enhance the sensitivity and stability of GCE in electrochemical sensing. E-GR displayed the excellent conductivity and a large surface area which provided more active sites and enhanced the transfer of charge between the electrode and the reagent. Meanwhile, P-βCD improved the uniform distribution, catalytic activity and durability of catalysts. Based on the synergic electrocatalytic effect of E-GR and P-βCD, this E-GR/P-βCD nanocomposite film increased the electrocatalytic active area and promoted the electron transfer between quercetin and the surface of GCE. To investigate the reaction mechanism, CV experiments were carried out at the E-GR/P-βCD/GCE to realize how the scan rate (υ) affected the oxidation of 20 μM quercetin in 0.1 M NaAc-HAc
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buffer solution (pH 7.0). The results are shown in Fig. 2C. The peak current varied linearly with the scan rates from 10 to 500 mV s 1 with a linear regression equation of Ip (μA)¼ 0.3324 c (μM) þ 2.8313 (R¼0.999), indicating a typical adsorption-controlled process. The oxidation potentials (Ep) shifted positively as the increase of scan rate (υ) and showed a linear relationship with log υ, which was further constructed with the equation: Ep(V)¼ 0.055 log v (V s 1)þ 0.236 (V). According to Laviron's theory, Ep can be represented by the following equation (Fei et al., 2005): Ep ¼ F þ ½2:303RT=ð1–αÞnα F log v
ð1Þ
where α is the electron transfer coefficient, and nα is the number of electrons involved in the rate-determining step. R, T and F are the gas constant, temperature and Faraday constant, respectively. The transfer coefficient (α) characterizes the effect of electrochemical potential on the activation energy of an electrochemical reaction, and its reasonable values range from 0.3 to 0.7 for most systems. For our system, the transferred number of electrons (n) during the oxidation of quercetin was calculated to be 2.1 (α¼0.5), suggesting that totally two electrons were involved in the oxidation reaction. This result was in accordance with the reported value (Goyal et al., 2012). The probable mechanism was surmised according to the references (Chen et al., 2012) and shown in Scheme 1. 3.3. Optimization of quercetin sensor performance by DPV 3.3.1. Effect of the applied potential of electro-deposited GR The effect of electro-deposited potential is an important factor affecting the performance of the sensor. The applied potentials ranging from 1.5 V to 1.9 V were investigated for electro-deposited GR. Fig. S1A (Supporting information) shows the current response by different applied potentials after addition of 10 μM quercetin in 0.1 M NaAc-HAc buffer solution (pH 7.0). The maximum peak current towards 10 μM quercetin was obtained on E-GR/GCE with the applied potential at 1.7 V. Therefore, 1.7 V was chosen for subsequent experiments. 3.3.2. Effect of polymerized cycles Electro-polymerized cycle is another important factor affecting the performance of the sensor. Fig. S1B (Supporting information) exhibits the effect of polymerized cycles of β-CD on the current response of 10 μM quercetin. When increasing the electrodeposited cycles from 2 to 10, it was observed that the oxidation current of quercetin increased and reached the maximum at 6 cycles, and then decreased with a higher number of cycles. Hence, 6 cycles were chosen for analytical experiments. 3.3.3. Effect of supporting electrolyte and solution pH To study the influence of pH on the oxidation process of quercetin, 0.1 M NaAc-HAc buffer solution was chosen as the electrolyte to study the influence of pH on the oxidation process of quercetin. An obvious increase of peak current with the pH value in the range of 2.0–7.0 was observed (Fig. S1C, Supporting information). As described in the figure, a negative shift of Ep with the increase of pH value was gained and this indicated that there
OH
O OH
HO
O
OH O
-2H +-2eOH
HO
O
+2H + +2eOH
O
OH
Scheme 1. The reaction mechanism of electrooxidation of quercetin.
O
Fig. 3. DPV of E-GR/P-βCD/GCE at different concentrations of quercetin. Inset: the calibration curve for the determination of quercetin. Each was measured for three repetitive determinations. (RSD o 5%).
was a proton at least involved in the oxidation reaction. Ep and pH were in a good linear relationship described by: Ep ¼0.4336– 0.0278 pH (V) (R¼ 0.998). The current reached a peak value at pH 4.0. However, the oxidation potentials shifted negatively to 0.249 V at pH 7.0, compared with the oxidation potentials (Ep ¼0.316 V) at pH 4.0. The buffer solution of NaAc-HAc at pH 7.0 is close to the physiological level (Xu and Wang, 2005). Accordingly, 0.1 M NaAc-HAc buffer solution at pH 7.0 was chosen for the following experiments.
3.4. Calibration curve and interference DPV is a powerful electrochemical technique that can be applied in both electrokinetic and analytical measurements. In this study, it was used to determine the concentration of quercetin. Fig. 3 shows the DPVs of various concentrations of quercetin at the E-GR/P-βCD/GCE in 0.1 M NaAc-HAc buffer solution (pH 7.0). The current responses are increased linearly with quercetin concentrations in the range of 0.005–20 μM with a current sensitivity of 24.760 μA μM 1 cm 2 (Fig. 3). The corresponding regression equation can be described as follows: Ip (μA) ¼1.74901 c (μM) þ 0.56309 (R¼0.999). The detection limit is 0.001 μM (S/N ¼3). Table 1 displays comparisons of the proposed E-GR/P-βCD/GCE performance with other electrochemical methods reported previously (Chen et al., 2012; Wang et al., 2011; Xu and Kim, 2006; Saber-Tehrani et al., 2013; Gutiérrez et al., 2010; Xiao et al., 2007; Jin et al., 2006). The proposed E-GR/P-βCD/GCE possessed lower detection limit and wider concentration range compared with most reported papers. The excellent performance may be attributed to the synergic electrocatalytic effect of E-GR and P-βCD, this E-GR/P-βCD nanocomposite film increased the electrocatalytic active area and promoted the electron transfer between quercetin and the surface of GCE. Hence, E-GR/P-βCD/GCE in this work is favorable and suitable for practical quercetin detection. Under the optimized experimental conditions described above, the influence of possible interfering substances was investigated with concentration of quercetin fixed at 20 μM. The tolerance limit was taken as the maximum concentration of interfering substances that caused an approximately 75% relative error in the determination of quercetin. Several substances such as glucose, ascorbic acid and uric acid, etc. could possibly co-exist with quercetin in natural samples are studied. The result of interference is shown in Table S1 (Supporting information). Experiment results illustrated that 50-fold of D-glucose, tryptophan, phenylalanine and leucine, 25-fold of ascorbic acid and dopamine, 50-fold of Cu2 þ , Pb2 þ , Zn2 þ , Al3 þ , Ca2 þ and Mg2 þ , 250-fold of Na þ and K þ
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Table 1 Comparison of linear ranges and detection limits at the various modified electrodes for the electrochemical determination of quercetin. Modified electrode
Linear ranges (μM)
Detection limits (μM)
References
Activated silica gel/GCE Flowerlike Co3O4 nanoparticles/GCE Carbon nanotube and Nafion/GCE Graphene nanosheets/GCE MWCNTs dispersed in polyacrylic acid/GCE MWCNTs/GCE MWCNTs/parafin/graphite disc electrode ED-GR/P-β-CD/GCE
0.0165–0.3309 0.5–330 0.02–6.3 0.006–10 and 10–100 0.1–5.0 0.002–0.1 and 0.1–20 0.009–40 0.005–20
0.012 0.1 – 0.0039 0.0075 – 0.0048 0.001
Chen et al. (2012) Wang et al. (2011) Xu and Kim (2006) Saber-Tehrani et al. (2013) Gutiérrez et al. (2010) Xiao et al. (2007) Jin et al. (2006) This work
Table 2 Determination of quercetin in tea and honeysuckle samples (n ¼3). Samples
Detected (μM) Added (μM) Found (μM) Recovery (%) RSD (%)
Tea 6.81 7 0.33 Honeysuckle 7.22 7 0.31
6 6
12.71 13.65
98.6 103.1
4.1 4.5
P-βCD nanocomposite film facilitated the electrocatalytic activity, electron transfer and mass transport. As a result, a super-sensitive electrochemical sensor for detecting quercetin based on E-GR/PβCD nanocomposite film was fabricated with ideal results in real samples. The present strategy provides a way to prepare graphene/ β-cyclodextrin nanocomposite film, thus providing a novel and promising platform for the study of the analytical application of EGR nanocomposite films.
together with 500-fold citric acid hardly caused interference (Table S1, Supporting information). Acknowledgements 3.5. Reproducibility and stability of the sensor The long-term storage and operational stability of the electrode are essential for the continuous monitoring of quercetin. During the test, 10 successive measurements were performed in NaAcHAc (pH 7.0) solution containing 20 μM quercetin by using one E-GR/P-βCD/GCE. The relative standard deviation was 4.05%, confirming that the modified electrode for quercetin sensing was stable. The long-term stability was explored by measuring 10 μM quercetin solutions intermittently. The results show that the catalytic current response maintains 93.6% of its initial value even after storing the modified electrode at room temperature for 2 weeks, reflecting the good stability of the quercetin sensor. 3.6. Real samples analysis To further verify the practical performance of the proposed electrochemical sensor, E-GR/P-βCD/GCE was applied to determine the content of quercetin in tea and honeysuckle samples by the standard addition method. Firstly, tea and honeysuckle were respectively soaked in the double deionized water for about 2 h. Then the solutions were respectively centrifuged for 0.5 h and the supernatants were taken out as the two samples to use for the measurement. The tea and honeysuckle samples were diluted with 0.1 M NaAc-HAc buffer solution, and the final concentration was 1.0–10.0 μM which is in the linear range of the proposed sensor and can be detected sensitively in 10 mL 0.1 M NaAc-HAc buffer solution (pH 7.0). The DPV curve was recorded by using one E-GR/ P-βCD/GCE under optimum conditions, and the results were listed in Table 2. Each sample was determined by three parallel detections, and the RSD was lower than 5%, revealing excellent precision. Moreover, the recovery of quercetin standard was performed to testify the accuracy of this method. The value of recovery ranges from 95.6% to 104.5%, suggesting that this method is effective and reliable.
4. Conclusion A facile and novel E-GR/P-βCD nanocomposite film was successfully synthesized by potentiostatic deposition and cyclic voltammetry techniques. The synergistic effect produced by E-GR and
This research is supported by the National Natural Science Foundation of China (Nos. 21271127 and 61171033), the NanoFoundation of Science and Techniques Commission of Shanghai Municipality (12nm0504200), Leading Academic Discipline Project of Shanghai Municipal Education Commission (J50102).
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Mild and novel electrochemical preparation of β-cyclodextrin/ graphene nanocomposite film for super-sensitive sensing of quercetin Zhen Zhang a,b, Shuqing Gu c, Yaping Ding a,n, Mingju Shen a, Lin Jiang a a Department of Chemistry, Shanghai Key Laboratory of Modern Metallurgy & Materials Processing, School of Materials Science and Engineering, Shanghai University, Shanghai 200444, PR China b School of Chemistry & Chemical Engineering, Linyi University, Linyi 276005, PR China c Technical Center for Animal Plant and Food Inspection and Quarantine, Shanghai Entry-Exit Inspection and Quarantine Bureau, Shanghai 200135, China
art ic l e i nf o
a b s t r a c t
Article history: Received 11 November 2013 Received in revised form 5 February 2014 Accepted 6 February 2014 Available online 19 February 2014
A mild and novel preparation tactics based on electrochemical techniques for the fabrication of electrodeposited graphene (E-GR) and polymerized β-cyclodextrin (P-βCD) nanocomposite film were developed. The structure and morphology of GR-based nanocomposite were investigated by scanning electron microscopy (SEM), transmission electron microscopy (TEM) and Raman spectroscopy. Simultaneously, the electrochemical properties of this nanocomposite were characterized by cyclic voltammetry (CV) and differential pulse voltammetry (DPV). Based on the synergistic effect of E-GR and P-βCD, a super-sensitive electrochemical sensor for quercetin was successfully fabricated. Under optimum conditions, the determination range for quercetin was from 0.005 to 20 mM with a low detection limit of 0.001 mM (S/N¼ 3). Moreover, this sensor also displays excellent sensitivity, fine reproducibility and stability. To further study the practical applicability of the proposed sensor, the determination of real samples was carried out with satisfactory results. & 2014 Elsevier B.V. All rights reserved.
Keywords: Electro-deposited grapheme Polymerized β-cyclodextrin Quercetin Nanocomposite film Differential pulse voltammetry
1. Introduction Graphene (GR), a two-dimensional sheet of sp2 conjugated atomic carbon, has attracted fascinating interests across different disciplines because of its ultrahigh specific surface area, unique electronic features (Novoselov et al., 2004; Kim et al., 2008), great mechanical strength (Zhao et al., 2010), and good biocompatibility (Matthew et al., 2010). Benefiting from these advantages, GR has been widely used for immobilizing organic and inorganic molecules (Ohno et al., 2009) and for fabrication of electrochemical biosensors (Shan et al., 2009; Wang et al., 2009; Zhou et al., 2009). β-Cyclodextrin (β-CD), a cyclic oligosaccharide consisting of seven glucose units, presents a toroidal form with a hydrophobic inner cavity and a hydrophilic outer side (Rekharsky and Inoue, 1998). It is well known that β-CD has high molecular selectivity and enantioselectivity. Various inorganic, organic and biological guest molecules can be bound selectively in the inner cavities of β-CD to form stable host–guest inclusion complexes or nanostructure supramolecular assemblies (Freeman et al., 2009; Rekharsky and Inoue, 1998). Therefore, β-CD and its derivatives have been n Corresponding author at: School of Materials Science and Engineering, Iron and Steel Metallurgy Key Laboratory, Shanghai University, Shanghai 200444, PR China. Tel.: þ 86 021 66134734; fax: þ86 21 66132797. E-mail address: [email protected] (Y. Ding).
http://dx.doi.org/10.1016/j.bios.2014.02.014 0956-5663 & 2014 Elsevier B.V. All rights reserved.
extensively used as molecular recognition systems and particularly applied in the field of mimetic enzymes, molecular recognition sensors, discrimination of enantiomers and drug encapsulation (Almirall et al., 2003; Fragoso et al., 2002; Martín et al., 2003; Ng et al., 2002). The nanocomposites based on β-CD can be an ideal substrate material for molecular recognition sensors. Based on the excellent performances of GR and β-CD respectively, a facile effective and efficient preparation strategy of GR–β-CD composite is of considerable significance (Chen et al., 2013; Zhang et al., 2014). Electrochemical technique was as an effective tool for rapid, simple and mild synthesis of various materials. Cyclic voltammetry (CV) (Malitesta et al., 1999; Feng et al., 2004), potentiostatic deposition (Lattach et al., 2012), electrochemical potential pulse technique (Menaker et al., 2009; Choong and Milne, 2010) and the galvanostatic method (Syritskia et al., 2008) have been applied for material preparations. Among them, potentiostatic deposition and CV can ensure similar film thicknesses (Österholma et al., 2012). Until now, there have been no reports concerning electrochemical sensors based on electro-deposited GR (E-GR) and polymer nanocomposite film manufactured by a cooperation of potentiostatic technique and CV. Thus, there is a substantially promising development space in this booming field. Quercetin, as a natural pentahydroxyflavone, is widely distributed in the medicinal plants, flowers, fruits, leaves and variety of beverages as their important constituent (Crozier et al., 1997;
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Hertog et al., 1992). It plays an important role in the science of nutrition and medical science. Quercetin has many biological activities by acting as an effective radical-scavenger against oxidative cell damage, such as antitumor activity (Verma et al., 1998), cardiovascular protection (Scalbert et al., 2005), antiinflammatory, estrogenic and antiallergy activity (Boesch-Saadatmandi et al., 2012). However, an overdose of may lead to kidney cancer (Hsieh et al., 2010). The excess quercetin decreased glutathione S-transferase activity, resulting in DNA damage (Yen et al., 2003). Thus, the low detection of quercetin in the science of nutrition and medical science has attracted an increasing interest. In this work, aiming to explore the unrevealed sensor potential, a mild and novel method has been employed to prepare the electro-deposited graphene/polymerized β-cyclodextrin (E-GR/ P-βCD) nanocomposite film by a cooperation of potentiostatic technique and CV. Quercetin was selected to explore the practical performance of the proposed sensor. The work presented here indicated the effectiveness and feasibility of the proposed strategy and the great potential of E-GR/P-βCD nanocomposite film in electroanalytical fields.
2. Experimental 2.1. Apparatus and reagents All electrochemical experiments were performed on a CHI 660D electrochemical workstation (Shanghai Chenhua Co. Ltd., China). A conventional three-electrode system was employed with a glassy carbon electrode (GCE, 3 mm in diameter) as the working electrode, a platinum electrode as the counter electrode, and a saturated calomel electrode as the reference electrode. The pH value was determined with a pHS-3C acidity meter. Scanning electron micrographs operated on the field emission scanning
electron microscope (Hitachi S4800, Japan) was applied for characterizing the modified materials. Transmission electron microscopy was carried out using a transmission electron microscope (JEOL JEM-200CX working at 160 kV). Graphene was purchased from XFNANO, INC (Nanjing, China). Quercetin and β-cyclodextrin were purchased from Sinopharm Chemical Reagent Co., Ltd., (Shanghai, China). Ascorbic acid and dopamine were purchased from Aladdin Chemical Reagent Co., Ltd. (Shanghai, China). All chemicals employed in this work were analytical grade. All experiments were performed in 0.1 M NaAcHAc buffer solution and double distilled water was used throughout. All electrochemical experiments were carried out at room temperature at about 25 1C.
2.2. Preparation of modified electrodes Prior to use, a bare GCE of 3 mm diameter was firstly polished on the chamois leather with alumina slurries until a mirror-like surface was acquired. Then the GCE was ultrasonicated in HNO3 solution (v/v ¼ 1:1), ethanol and double distilled water for 3 min respectively. The electro-deposited GR/polymerized β-cyclodextrin nanocomposite film (E-GR/P-βCD) modified on GCE was gained via two steps. The first step was the electro-deposition in 10 mL 0.1 M KCl aqueous solution containing 300 μL GR (1 mg/mL dispersed in water) at a deposited voltage of þ1.7 V, then the second step was electro-polymerization in the range from 2.0 to 2.5 V for 6 segments at 100 mV s 1 in 0.1 M phosphate buffer solution (pH 6.0) containing 2.0 mM β-CD. Eventually, the as-prepared electrode was rinsed carefully with double distilled water for further use. At the same time, another two modified electrodes, E-GR/GCE and P-βCD/GCE were also prepared following the same procedure described above for comparison.
Fig. 1. (A) SEM images of graphene; (B) TEM images of graphene; (C) SEM images of E-GR; and (D) Raman spectra of GR (a), E-GR (b).
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Fig. 2. (A) SEM images of ED-GR/P-βCD. The inset is the cross-section image of the nanocomposite. (B) DPVs of bare GCE (a), E-GR/GCE (b), P-βCD/GCE (c) and E-GR/P-βCD/ GCE (d) with 20 μM quercetin in 0.1 M NaAc-HAc buffer solution (pH ¼7.0). (C) CVs of E-GR/P-βCD/GCE with 20 μM quercetin in 0.1 M NaAc-HAc buffer solution (pH ¼ 7.0) at scan rate ranging from 10 to 500 mV s 1. Inset: plots of peak currents vs. scan rates.
3. Results and discussion 3.1. Characterization of the modified electrode The TEM and SEM images of graphene are shown in Fig. 1, from which we can see that graphene was sheet-like, glossy and crimple shape. As shown in Figs. 1C and 2A, the morphologies of E-GR and E-GR/P-βCD on the surface of GCE were investigated by SEM. E-GR film with honeycombed layers acquired from the electro-deposited method can be well modified on the surface of electrode. Compared with GR film on the surface of electrode, specific surface area of E-GR film was increased, which is based on the impetus of the electrochemical power to the changes of Fermi energy level of electrode materials surface (Guo et al., 2009). Raman spectroscopy is a powerful nondestructive tool to distinguish ordered and disordered crystal structures of carbon. The Raman spectra of GR and E-GR are shown in Fig. 1D. As expected, GR (a) displays two prominent peaks at 1345.6 and 1592.9 cm 1, which correspond to the well-documented G and D bands, respectively. By the electro-deposited method, the G and D bands of E-GR (b) shifted to 1352.1 and 1601.1 cm 1. The G band represents the first-order scattering of the E2g vibrational mode while the D band has been attributed to the reduction in size of the in-plane C sp2 atoms. The D/G band intensity ratio expresses the atomic ratio of sp2/sp3 carbons, and is a measure of the extent of disordered graphite. The D/G intensity ratios of GR and E-GR were calculated to be 1.4 and 1.2, respectively. It is anticipated that E-GR film with a distinctive morphology from GR and other reported electrochemical synthesized GR (Zhang et al., 2013; Yang et al., 2012) will support the sensitivity of electrode as an electrochemical sensor. Compared with E-GR film, a relatively flat and smooth surface was obtained after the modification of E-GR/P-βCD nanocomposite film, as showed in Fig. 2A. As seen from the transection images,
β-CD was well implanted in E-GR film with nanosize, implying a favorable distribution with compact multilayer composite-films. These could be the wrinkled E-GR film which offered large surface areas and excellent mechanical durability. It is also expected that the displayed cooperation of β-CD and E-GR will promote the sensitivity and stability of GCE in electrochemical sensing. 3.2. Electrochemical behavior of quercetin on the GCEs The electrochemical oxidations of quercetin on different modified GCEs by differential pulse voltammetry (DPV) were carried out in 0.1 M NaAc-HAc buffer solution (pH 7.0) containing 20 μM quercetin in order to make a comparison of the electrochemical properties, as shown in Fig. 2B. The electrochemical response of E-GR/P-βCD/GCE to quercetin was investigated and the corresponding voltammogram was shown in Fig. 2B, curve d. As a comparison, a similar set of experiments were also performed using the bare GCE (a), E-GR/GCE (b) and P-βCD/GCE (c), shown in Fig. 2B. Compared with the bare GCE, about 20-fold larger current signal was obtained, which demonstrated that the E-GR/P-βCD/ GCE could greatly facilitate the oxidation of quercetin. It is also expected that the displayed cooperation of E-GR and P-βCD will enhance the sensitivity and stability of GCE in electrochemical sensing. E-GR displayed the excellent conductivity and a large surface area which provided more active sites and enhanced the transfer of charge between the electrode and the reagent. Meanwhile, P-βCD improved the uniform distribution, catalytic activity and durability of catalysts. Based on the synergic electrocatalytic effect of E-GR and P-βCD, this E-GR/P-βCD nanocomposite film increased the electrocatalytic active area and promoted the electron transfer between quercetin and the surface of GCE. To investigate the reaction mechanism, CV experiments were carried out at the E-GR/P-βCD/GCE to realize how the scan rate (υ) affected the oxidation of 20 μM quercetin in 0.1 M NaAc-HAc
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buffer solution (pH 7.0). The results are shown in Fig. 2C. The peak current varied linearly with the scan rates from 10 to 500 mV s 1 with a linear regression equation of Ip (μA)¼ 0.3324 c (μM) þ 2.8313 (R¼0.999), indicating a typical adsorption-controlled process. The oxidation potentials (Ep) shifted positively as the increase of scan rate (υ) and showed a linear relationship with log υ, which was further constructed with the equation: Ep(V)¼ 0.055 log v (V s 1)þ 0.236 (V). According to Laviron's theory, Ep can be represented by the following equation (Fei et al., 2005): Ep ¼ F þ ½2:303RT=ð1–αÞnα F log v
ð1Þ
where α is the electron transfer coefficient, and nα is the number of electrons involved in the rate-determining step. R, T and F are the gas constant, temperature and Faraday constant, respectively. The transfer coefficient (α) characterizes the effect of electrochemical potential on the activation energy of an electrochemical reaction, and its reasonable values range from 0.3 to 0.7 for most systems. For our system, the transferred number of electrons (n) during the oxidation of quercetin was calculated to be 2.1 (α¼0.5), suggesting that totally two electrons were involved in the oxidation reaction. This result was in accordance with the reported value (Goyal et al., 2012). The probable mechanism was surmised according to the references (Chen et al., 2012) and shown in Scheme 1. 3.3. Optimization of quercetin sensor performance by DPV 3.3.1. Effect of the applied potential of electro-deposited GR The effect of electro-deposited potential is an important factor affecting the performance of the sensor. The applied potentials ranging from 1.5 V to 1.9 V were investigated for electro-deposited GR. Fig. S1A (Supporting information) shows the current response by different applied potentials after addition of 10 μM quercetin in 0.1 M NaAc-HAc buffer solution (pH 7.0). The maximum peak current towards 10 μM quercetin was obtained on E-GR/GCE with the applied potential at 1.7 V. Therefore, 1.7 V was chosen for subsequent experiments. 3.3.2. Effect of polymerized cycles Electro-polymerized cycle is another important factor affecting the performance of the sensor. Fig. S1B (Supporting information) exhibits the effect of polymerized cycles of β-CD on the current response of 10 μM quercetin. When increasing the electrodeposited cycles from 2 to 10, it was observed that the oxidation current of quercetin increased and reached the maximum at 6 cycles, and then decreased with a higher number of cycles. Hence, 6 cycles were chosen for analytical experiments. 3.3.3. Effect of supporting electrolyte and solution pH To study the influence of pH on the oxidation process of quercetin, 0.1 M NaAc-HAc buffer solution was chosen as the electrolyte to study the influence of pH on the oxidation process of quercetin. An obvious increase of peak current with the pH value in the range of 2.0–7.0 was observed (Fig. S1C, Supporting information). As described in the figure, a negative shift of Ep with the increase of pH value was gained and this indicated that there
OH
O OH
HO
O
OH O
-2H +-2eOH
HO
O
+2H + +2eOH
O
OH
Scheme 1. The reaction mechanism of electrooxidation of quercetin.
O
Fig. 3. DPV of E-GR/P-βCD/GCE at different concentrations of quercetin. Inset: the calibration curve for the determination of quercetin. Each was measured for three repetitive determinations. (RSD o 5%).
was a proton at least involved in the oxidation reaction. Ep and pH were in a good linear relationship described by: Ep ¼0.4336– 0.0278 pH (V) (R¼ 0.998). The current reached a peak value at pH 4.0. However, the oxidation potentials shifted negatively to 0.249 V at pH 7.0, compared with the oxidation potentials (Ep ¼0.316 V) at pH 4.0. The buffer solution of NaAc-HAc at pH 7.0 is close to the physiological level (Xu and Wang, 2005). Accordingly, 0.1 M NaAc-HAc buffer solution at pH 7.0 was chosen for the following experiments.
3.4. Calibration curve and interference DPV is a powerful electrochemical technique that can be applied in both electrokinetic and analytical measurements. In this study, it was used to determine the concentration of quercetin. Fig. 3 shows the DPVs of various concentrations of quercetin at the E-GR/P-βCD/GCE in 0.1 M NaAc-HAc buffer solution (pH 7.0). The current responses are increased linearly with quercetin concentrations in the range of 0.005–20 μM with a current sensitivity of 24.760 μA μM 1 cm 2 (Fig. 3). The corresponding regression equation can be described as follows: Ip (μA) ¼1.74901 c (μM) þ 0.56309 (R¼0.999). The detection limit is 0.001 μM (S/N ¼3). Table 1 displays comparisons of the proposed E-GR/P-βCD/GCE performance with other electrochemical methods reported previously (Chen et al., 2012; Wang et al., 2011; Xu and Kim, 2006; Saber-Tehrani et al., 2013; Gutiérrez et al., 2010; Xiao et al., 2007; Jin et al., 2006). The proposed E-GR/P-βCD/GCE possessed lower detection limit and wider concentration range compared with most reported papers. The excellent performance may be attributed to the synergic electrocatalytic effect of E-GR and P-βCD, this E-GR/P-βCD nanocomposite film increased the electrocatalytic active area and promoted the electron transfer between quercetin and the surface of GCE. Hence, E-GR/P-βCD/GCE in this work is favorable and suitable for practical quercetin detection. Under the optimized experimental conditions described above, the influence of possible interfering substances was investigated with concentration of quercetin fixed at 20 μM. The tolerance limit was taken as the maximum concentration of interfering substances that caused an approximately 75% relative error in the determination of quercetin. Several substances such as glucose, ascorbic acid and uric acid, etc. could possibly co-exist with quercetin in natural samples are studied. The result of interference is shown in Table S1 (Supporting information). Experiment results illustrated that 50-fold of D-glucose, tryptophan, phenylalanine and leucine, 25-fold of ascorbic acid and dopamine, 50-fold of Cu2 þ , Pb2 þ , Zn2 þ , Al3 þ , Ca2 þ and Mg2 þ , 250-fold of Na þ and K þ
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Table 1 Comparison of linear ranges and detection limits at the various modified electrodes for the electrochemical determination of quercetin. Modified electrode
Linear ranges (μM)
Detection limits (μM)
References
Activated silica gel/GCE Flowerlike Co3O4 nanoparticles/GCE Carbon nanotube and Nafion/GCE Graphene nanosheets/GCE MWCNTs dispersed in polyacrylic acid/GCE MWCNTs/GCE MWCNTs/parafin/graphite disc electrode ED-GR/P-β-CD/GCE
0.0165–0.3309 0.5–330 0.02–6.3 0.006–10 and 10–100 0.1–5.0 0.002–0.1 and 0.1–20 0.009–40 0.005–20
0.012 0.1 – 0.0039 0.0075 – 0.0048 0.001
Chen et al. (2012) Wang et al. (2011) Xu and Kim (2006) Saber-Tehrani et al. (2013) Gutiérrez et al. (2010) Xiao et al. (2007) Jin et al. (2006) This work
Table 2 Determination of quercetin in tea and honeysuckle samples (n ¼3). Samples
Detected (μM) Added (μM) Found (μM) Recovery (%) RSD (%)
Tea 6.81 7 0.33 Honeysuckle 7.22 7 0.31
6 6
12.71 13.65
98.6 103.1
4.1 4.5
P-βCD nanocomposite film facilitated the electrocatalytic activity, electron transfer and mass transport. As a result, a super-sensitive electrochemical sensor for detecting quercetin based on E-GR/PβCD nanocomposite film was fabricated with ideal results in real samples. The present strategy provides a way to prepare graphene/ β-cyclodextrin nanocomposite film, thus providing a novel and promising platform for the study of the analytical application of EGR nanocomposite films.
together with 500-fold citric acid hardly caused interference (Table S1, Supporting information). Acknowledgements 3.5. Reproducibility and stability of the sensor The long-term storage and operational stability of the electrode are essential for the continuous monitoring of quercetin. During the test, 10 successive measurements were performed in NaAcHAc (pH 7.0) solution containing 20 μM quercetin by using one E-GR/P-βCD/GCE. The relative standard deviation was 4.05%, confirming that the modified electrode for quercetin sensing was stable. The long-term stability was explored by measuring 10 μM quercetin solutions intermittently. The results show that the catalytic current response maintains 93.6% of its initial value even after storing the modified electrode at room temperature for 2 weeks, reflecting the good stability of the quercetin sensor. 3.6. Real samples analysis To further verify the practical performance of the proposed electrochemical sensor, E-GR/P-βCD/GCE was applied to determine the content of quercetin in tea and honeysuckle samples by the standard addition method. Firstly, tea and honeysuckle were respectively soaked in the double deionized water for about 2 h. Then the solutions were respectively centrifuged for 0.5 h and the supernatants were taken out as the two samples to use for the measurement. The tea and honeysuckle samples were diluted with 0.1 M NaAc-HAc buffer solution, and the final concentration was 1.0–10.0 μM which is in the linear range of the proposed sensor and can be detected sensitively in 10 mL 0.1 M NaAc-HAc buffer solution (pH 7.0). The DPV curve was recorded by using one E-GR/ P-βCD/GCE under optimum conditions, and the results were listed in Table 2. Each sample was determined by three parallel detections, and the RSD was lower than 5%, revealing excellent precision. Moreover, the recovery of quercetin standard was performed to testify the accuracy of this method. The value of recovery ranges from 95.6% to 104.5%, suggesting that this method is effective and reliable.
4. Conclusion A facile and novel E-GR/P-βCD nanocomposite film was successfully synthesized by potentiostatic deposition and cyclic voltammetry techniques. The synergistic effect produced by E-GR and
This research is supported by the National Natural Science Foundation of China (Nos. 21271127 and 61171033), the NanoFoundation of Science and Techniques Commission of Shanghai Municipality (12nm0504200), Leading Academic Discipline Project of Shanghai Municipal Education Commission (J50102).
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