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2015 © The Japan Society for Analytical Chemistry
Selective Detection toward Quercetin and Kaempferol on NH3-Plasma Treated Carbon Nanotubes Modified Glassy Carbon Electrode Jing-Jing SONG, Yong LU, Si-Wei ZHU, Qin-An HUANG,† and Yan WEI† Department of Chemistry, Wannan Medical College, Wuhu 241002, P. R. China
NH3-plasma treated multi-walled carbon nanotubes (pn-MWCNTs) were prepared based on the plasma technique and developed as sensing materials for detection of quercetin and kaempferol with the differential pulse voltammetry (DPV) and amperometric measurement. Such experimental parameters as pH values, accumulation potential and accumulation time were carefully investigated. The pn-MWCNTs modified electrode (pn-MWCNTs/GCE) was further explored for the analysis of quercetin and kaempferol in diluted blood serum and average recovery rates of 96.91 and 100.5% were obtained, respectively. In addition, the interference and stability measurements were evaluated under the optimized experimental conditions. More importantly, selective detection toward quercetin and kaempferol was achieved, and the proposed electrochemical sensing strategy was available to distinguish substances with similar oxidation potential. Keywords Plasma treatment, multi-walled carbon nanotubes, quercetin, kaempferol, electrochemical detection (Received October 13, 2014; Accepted January 1, 2015; Published March 10, 2015)
Introduction Flavonoids are a vast class of heterogeneous polyphenols that are widely distributed in common foods such as teas, fruits, vegetables, and wine.1 Generally, they are subdivided into seven classes: flavones, flavonols, flavanones, flavanols, flavanonols, isoflavones and anthocyanidins.2,3 Quercetin and kaempferol are of particular importance among flavonols as they are found to be potent antioxidants. Numerous reports have revealed that quercetin and kaempferol possess a wide range of biological activities such as antioxidant ability, anxiolytic effects, antiinflammatory characteristics and the ability to damage in glucose-induced oxidative cells and dysfunction in pancreatic cells.4–7 Due to their popularity as medicinal ingredients, it is of great interest and significance to develop effective and feasible methods for the analysis of quercetin and kaempferol. In the past few years, many conventional methods have been reported on the detection of these two substances, including molecularly imprinted polymers (MIPs),8,9 capillary electrophoresis (CE),10 and high performance liquid chromatography (HPLC).1,11 Although these methods have excellent determination limits and can effectively identify and quantify analytes, they require sophisticated instruments and are time consuming. Alternatively, electrochemical methods have drawn strong interest for the efficient analysis of kaempferol and quercetin because of the advantages of high sensitivity, easy of use, and low cost. And much attention has been paid to the development of sensing J.-J. S. and Y. L. contributed equally to this work. † To whom correspondence should be addressed. E-mail: [email protected] (Y. W.); [email protected] (Q.-A. H.)
materials that are closely reflected in electrochemical Past work reported that such metal performance.12–16 nanomaterials as carbon nanotubes (CNTs) and graphene have been explored as sensing materials and played an extremely important role in electroanalysis. Since first reported by Iijima in 1991,17 CNTs have been a hot topic of research during the past two decades because of their special structural, unique electronic, optical and mechanical properties.18–21 Owing to the enhanced electrode conductivity and large surface area, CNTs have been developed to construct electrochemical sensors. In particular, Wang et al. used β-cyclodextrin-CNTs modified electrodes for the simultaneous determination of adenine and guanine.22 Zeng et al. successfully detected rutin using a single-walled carbon nanotubes modified gold electrode.23 Wu et al. fabricated sensors based on multiwalled carbon nanotubes (MWCNTs) modified electrode for the detection of nitric oxide.24 However, some difficulties were inevitable. Typically, the most common chemical modification with HNO3 and/or H2SO4 may lead to damage bulk properties, decreasing their stability and even cleaving them into shorter pieces; the complicated modification process may result in electrode surface fouling, increasing the instability of the modified electrode.25 In contrast to the common chemical modification method, plasma treatment is a solvent-free, timeefficient, versatile, environment-friendly procedure that does not result in a large amount of structural damage to the CNTs and can also provide a wide range of different functional groups on CNTs.26 To the best of our knowledge, the application of NH3 plasma treated MWCNTs for the detection of quercetin and kaempferol has not been reported yet. In this work, MWCNTs were treated with NH3 plasma and used as sensing materials for the modification of a glassy carbon electrode. After NH3 plasma treatment, amino groups were
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introduced onto the surface of MWCNTs,27–29 which were explored as a selective sorbent for the separation and preconcentration of quercetin and kaempferol. The main purpose of the present work is to achieve the simultaneous detection of quercetin and kaempferol in combination with differential pulse voltammetry (DPV) and amperometric measurements. The optimizing of experimental conditions, such as pH values, accumulation potential, and accumulation time, were carefully investigated. In addition, the interference and stability of an NH3-plasma treated MWCNTs (pn-MWCNTs) modified electrode were also studied.
Experimental Chemical reagents Quercetin and kaempferol were purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). First, 1.0 mM standard solutions of quercetin and kaempferol were prepared by dissolving in ethanol (95%), and then stored in the refrigerator. MWCNTs were purchased from Chengdu Institute of Organic Chemistry of the Chinese Academy of Sciences. The as-received MWCNTs (raw MWCNTs) have a normal hollow structure. The purity was about 95%, with 0.23 and 0.93% of iron and nickel catalysts, respectively. The amorphous carbon content was about 4%. The diameter of raw MWCNTs was about 10 – 30 nm with the length at micron level. All reagents were of analytical grade and used without any further purification. Then, 0.05 M phosphate buffer solutions (PBS) were prepared by mixing 0.05 M NaH2PO4 and 0.05 M Na2HPO4, and then adjusting the pH values with 0.05 M H3PO4 or 0.05 M NaOH. The deionized water (18.2 MΩ cm) used to prepare all solutions was purified with the NANOpureDiamondTM UV water system. Apparatus All electrochemical experiments were recorded using a CHI 660D computer-controlled potentiostat (ChenHua Instruments Co., Shanghai, China). Measurements were carried out in a conventional three-electrode system using the modified or bare glassy carbon electrode (GCE, 3 mm in diameter) as a working electrode, Ag/AgCl as a reference electrode and Pt wire as a counter electrode. Preparation of pn-MWCNTs The pn-MWCNTs were prepared from raw MWCNTs according to the process depicted elsewhere.29 In brief, a plasma generator by radio-frequency inductively coupled plasma was used in this study. Raw MWCNTs were pretreated using Ar plasma at a gas flow rate of 70 sccm and microwave power of 700 W, and then NH3/Ar mixture gases were introduced through mass flow meters. MWCNTs were treated by NH3 plasma under continuous stirring. Then, the MWCNTs after plasma treatment for 30 min were dispersed in ethanol without any further purification. Fabrication of modified electrode Prior to modification, a bare glassy carbon electrode (GCE) was sequentially polished with 1.0, 0.3 and 0.05 μm alumina power slurries to a mirror shiny surface then sonicated with HNO3 solution (v:v 1:1), absolute ethanol and deionized water, respectively. Then, 4 μL of pn-MWCNTs solution was dripped onto the surface of the GCE. The electrode was allowed to air-dry at room temperature. When the modified electrode was not in use, it was stored in 0.05 M PBS (pH 4.0) at 4oC in a
Fig. 1 a) Cyclic voltammograms and Nyquist diagram of electrochemical impedance spectra of raw MWCNTs (black line) and pn-MWCNTs modified GCE (red line) in the solution of 5 mM K3Fe(CN)6 containing 0.1 M KCl. Scanning rate: 100 mV s–1. Frequency: 100000 to 1 Hz.
refrigerator. Electrochemical detection of quercetin and kaempferol Quercetin and kaempferol were preconcentrated in 0.05 M PBS (pH 4.0) for 120 s under stirring condition. Differential pulse voltammetry was performed in the potential range of 0 to 0.8 V at the following parameters: frequency, 15 Hz; amplitude, 25 mV; increment potential, 4 mV; pulse width, 0.2 s; sampling width, 0.02 s. Quantitative detection of quercetin and kaempferol was performed using an amperometric technique by adding substances every 50 s and setting the applied potential at 0.375 and 0.45 V, respectively. Cyclic voltammograms (CV) and electrochemical impedance spectra (EIS) were performed in mixing solution of 5 mM K3[Fe(CN)6] with 0.1 M KCl and the scanning rate was 100 mV s–1.
Results and Discussion Electrochemical characterization of NH3-plasma treated MWCNTs The cyclic voltammetric response of raw MWCNTs and pn-MWCNTs modified GCE has been characterized in the solution of 5 mM Fe(CN)63–/4– containing 0.1 M KCl. Figure 1a shows the CV responses of raw MWCNTs and pn-MWCNTs
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modified electrodes. As compared with the raw MWCNTs modified electrode, the anodic and cathodic peak currents of pn-MWCNTs modified electrode increased. We suggested that the effective surface area of pn-MWCNTs, increased by the plasma treatment, was responsible for the increased peak current. The electrochemical impedance spectrum (EIS) was employed to characterize the interface properties of the pn-MWCNTs modified electrode. It is well-known that a typical EIS consists of two parts, a semicircle part and a linear part. The semicircle portion corresponds to the electron-transfer resistance (Ret) at a higher frequency range, while a linear part at a lower frequency range represents the diffusion process. As presented in Fig. 1b, Ret for raw MWCNTs modified electrode was about 100 Ω. In contrast, the EIS for pn-MWCNTs modified electrode was nearly a straight line, indicating the promotion of the electron transfer process on the modified electrode surface. The results of electrochemical impedance spectra were in good accordance with that from CV. Optimization of experimental conditions In order to obtain the high sensitive electrochemical performance with pn-MWCNTs modified GCE for detecting quercetin and kaempferol, the experimental parameters of pH value, accumulation time and accumulation potential were optimized. The effect of pH value is very significant in the experimental environment. DPV responses of 1 μM quercetin and 1 μM kaempferol were assessed on the pn-MWCNTs modified GCE in 0.05 M PBS by varying the pH value from 3.0 to 8.0 (Fig. 2a). Apparently, the highest responses were observed at pH 4.0 for both 1 μM quercetin (black line) and 1 μM kaempferol (red line). It was suggested that due to the acidic hydroxyl groups of quercetin and kaempferol, the pKa value is about 4 and slightly less than 7, which is in agreement with a previous report.30 At pH 4.0, it may bring favorable possibility for the chemical reactions of hydroxyl in quercetin and kaempferol, such as hydroxylation, dimerization or the intramolecular of reaction. Thus, as the optimized pH value, 4.0 was employed. However, the reasonable mechanism on the effect of pH needs to be further verified in detail. Figure 2b depicts the DPV responses of 1 μM quercetin and 1 μM kaempferol with accumulation potential ranging from –0.3 to 0.2 V. As shown, the peak currents of quercetin (black line) increased from –0.3 to –0.2 V and then decreased significantly. In respect to kaempferol (red line), there was no obvious change in peak currents from –0.3 to 0.1 V and an apparent decrease was only observed at 0.2 V. Considering sensitivity for these two substances, the optimized accumulation potential was selected as –0.2 V. Different accumulation times of 0, 30, 60, 90, 120, and 150 s were examined using DPV in an analysis of 1 μM quercetin and 1 μM kaempferol at –0.2 V in 0.05 M PBS (pH 4.0). As seen in Fig. 2c, the peak currents increased with the increase of the accumulation time ranging from 0 to 120 s, and then started to level off at 120 s. Therefore, to achieve a lower detection limit and wider response range, an optimized accumulation time of 120 s was used throughout. Electrochemical detection of quercetin and kaempferol on pnMWCNTs modified electrode The electrochemical performance of pn-MWCNTs modified electrode toward quercetin and kaempferol was firstly evaluated with the DPV technique. Figure 3 presents the DPV analytical characteristics of 1.0 μM quercetin and kaemferol on the raw MWCNTs and pn-MWCNTs modified electrodes in 0.05 M
Fig. 2 Effect of a) pH values, b) accumulation potential and c) accumulation time on DPV responses of 1 μM quercetin (Que) and 1 μM kaempferol (Kae), respectively.
PBS (pH 4.0). In the case of the raw MWCNTs modified electrode, there were only weak responses for 1.0 μM quercetin (red line in Fig. 3a) and kaempferol (red line in Fig. 3b) at 0.38 and 0.42 V. Whereas, the strong and well-defined responses of quercetin and kaempferol (pink lines) can be clearly observed at the same concentrations without potential shift, which were much higher than that on raw MWCNTs/GCE. The significantly enhanced responses on pn-MWCNTs/GCE were due to the efficiency of MWCNTs after NH3 plasma treatment, resulting
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Fig. 4 DPV responses of mixture of quercetin and kaempferol ranging from 0.01 to 4.0 μM on pn-MWCNTs modified GCE in the solution of 0.05 M PBS (pH 4.0). Inset: the DPV response of mixture of quercetin and kaempferol ranging from 0.5 to 2.5 μM on raw MWCNTs modified GCE. DPV conditions are identical to those in Fig. 3.
Fig. 3 DPV responses of a) 1.0 μM quercetin (Que) and b) 1.0 μM kaempferol (Kae) on raw MWCNTs (red line) and pn-MWCNTs modified GCE (pink line) in the solution of 0.05 M PBS (pH 4.0) with accumulation time of 120 s and desorption time of 150 s. Frequency, 15 Hz; amplitude, 25 mV; increment potential, 4 mV; pulse width, 0.2 s; sampling width, 0.02 s. Dotted line: corresponding DPV responses in the absence of quercetin or kaempferol.
from the promotion of electron transfer. Simultaneous determination of quercetin and kaempferol Due to a similarity in the oxidation potentials for quercetin and kaempferol and possible appearance of the overlapping peaks, it was recognized as an important objective to distinguish quercetin and kaempferol when they co-existed. Figure 4 shows the simultaneous determination of quercetin and kaempferol with different concentrations (molar ratio, 1:1) under the optimized experimental conditions. As depicted, two separated peaks for quercetin and kaempferol on pn-MWCNTs/GCE were observed over the concentration range of 0.01 – 4.0 μM. And there was an obvious potential of 60 mV between the two peaks. We suggested that in comparison with the individual determination of quercetin and kaempferol, their interaction would have an effect on the peak potential and broaden the potentials in the simultaneous determination. In contrast, only a single and broad oxidation peak for quercetin and kaempferol on raw MWCNTs/GCE was obtained even at high concentrations (inset in Fig. 4). The results demonstrated the efficiency of pnMWCNTs. We suggested that MWCNTs after plasma treatment can improve the electron transfer, thus being of benefit for the simultaneous determination of quercetin and kaempferol.
Amperometric determination of quercetin and kaempferol As for the quantitative analysis of quercetin and kaempferol, the amperometric measurement at a certain applied potential (Eapp) was performed by successive addition of different qualities of quercetin and kaempferol under constant stirring. Figures 5a and 5b show the typical amperometric responses on pnMWCNTs/GCE after successive additions of quercetin (Eapp: 0.375 V) and kaempferol (Eapp: 0.45 V) into the 0.05 M PBS (pH 4.0). Herein Eapp selected refers to the peak potential in DPV measurement. That is, 0.375 and 0.45 V are similar to or slightly positive than the oxidation potential of Que and Kae, respectively. Well-defined responses proportional to the concentration of quercetin and kaempferol were observed. It was also found that the current responses of a steady state were reached within 5 s after each addition, indicating the fast response for the sensor. Calibration plots were obtained from the amperometric responses in the range of 0.05 – 4.55 μM (see the insets) with linear equations, i/μA = 0.001 + 0.02 c/μM (R2 = 0.999) for quercetin and i/μA = 0.005 + 0.0196 c/μM (R2 = 0.99) for kaempferol, respectively. The limits of detection (LODs) calculated in linear equations for quercetin and kaempferol were 0.05 μM. Moreover, with a applied potential at 0.375 V, the successful determination of quercetin was achieved without the interference from kaempferol (Fig. 5a). Figure 5c illustrates the current responses for alternate additions of two substances each 50 s at 0.45 V, and the corresponding current responses continuously increased on the basis of the former. Therefore, it can be confirmed the feasibility analysis of individual or simultaneous determination of kaempferol or the two substances on the pn-MWCNTs modified electrode. Moreover, no obvious interference was observed in the detection of the two substances. Stability and reproducibility study The stability was examined to check out the long-term performance of pn-MWCNTs modified electrode in 0.05 M PBS (pH 4.0) using DPV. As shown in Fig. 6, with 12 repetitive measurements, we found that the DPV responses of the modified electrode was nearly constant. The relative standard deviations (RSD) were 0.62 and 0.43% for quercetin and kaempferol, respectively. The results indicated that the modified electrode
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Fig. 6 Stability measurement of DPV responses on pn-MWCNTs modified GCE in 0.05 M PBS (pH 4.0) containing 1.5 μM quercetin (Que, black dot) or 0.5 μM kaempferol (Kae, red dot). DPV conditions are identical to those in Fig. 3.
Table 1 Determination of quercetin in blood serum samples and recovery study n cQue/μM 1 2 3 4 5
0.05 0.15 0.30 0.50 0.75
iMin/μA
iMax/μA
iAve/μA
Deviation, %
1.02 × 10–3 3.96 × 10–3 10.0 × 10–3 20.3 × 10–3 34.4 × 10–3
1.09 × 10–3 4.10 × 10–3 10.5 × 10–3 21.5 × 10–3 35.6 × 10–3
1.05 × 10–3 4.03 × 10–3 10.3 × 10–3 20.8 × 10–3 34.9 × 10–3
6.7 3.5 5.0 5.4 3.5
Table 2 Determination of kaempferol in blood serum samples and recovery study n cKae/μM
Fig. 5 Amperometric responses on the pn-MWCNTs/GCE in 0.05 M PBS (pH 4.0) for detection of quercetin or kaempferol. a) applied potential: Eapp = 0.375 V; successive addition of quercetin over the concentration of 0.05, 0.15, 0.30, 0.50, 0.75, 1.05, 1.40, 1.80, 2.25, 2.75, 3.30, 3.90, 4.55 μM (a → m), followed by a final addition of kaempferol; b) applied potential: Eapp = 0.45 V, successive addition of kaempferol over the same concentrations as in panel a (a → m). Insets in panels a) and b) were the corresponding calibration plots. c) Alternate addition of two substances with the concentration 0.05 μM, interval time = 50 s, Eapp =0.45 V.
can be reused without any obvious loss in response and robust stability was therefore obtained. The reproducibility of the modified electrode was also tested after one day and one week. The current response decreased by about 0.18 and 0.36% after one day, 10.16 and 13.97% after one week for the detection of quercetin and kaempferol, respectively, indicating the favorable reproducibility of the modified electrode. Analytical applications The pn-MWCNTs/GCE was further developed for the analysis of quercetin and kaempferol in diluted blood serum based on the amperometric technique. Prior to determination, the blood serum was pretreated by adding 0.1 M hydrochloric acid to
1 2 3 4 5
0.05 0.15 0.30 0.50 0.75
iMin/μA
iMax/μA
iAve/μA
Deviation, %
2.26 × 10–3 6.77 × 10–3 14.0 × 10–3 21.1 × 10–3 34.2 × 10–3
2.3 × 10–3 7.12 × 10–3 15.0 × 10–3 21.6 × 10–3 35.2 × 10–3
2.29 × 10–3 6.67 × 10–3 14.4 × 10–3 21.3 × 10–3 34.6 × 10–3
1.7 5.2 7.1 2.3 2.7
remove uric acid.31 After filtration by membrane (f 0.22 μM), the solution was mixed with ethanol in a volume ratio of 1:1, where 0.1 mM quercetin and 0.1 mM kaempferol were contained. And then mixed blood serum of different amounts was added into 0.05 M PBS (pH 4.0) solution. The concentrations of the analytes (Que and Kae) were determined using standard addition method and named as CQue and CKae. After three independent measurements, the currents were recorded and imax and imin denoted the maximum value and minimum value from the measurements. The results are summarized in Tables 1 and 2. The recovery values were calculated to be 96.91% for quercetin and 100.5% for kaempferol, suggesting the efficiency and reliability of pn-MWCNTs modified electrode for the determination of quercetin and kaempferol in practical applications. Interferences An interference study was performed in the presence of quercetin and kaempferol with the addition of other possible interferents. With individual additions of 500 μM glycine,
230 400 μM L(+)-tartaric acid, 600 μM glucose, 200 μM ascorbic acid into 0.05 M PBS (pH 4.0), no obvious interference (RSD < 5%) on the current responses of 1 μM quercetin and 1 μM kaempferol (data not shown) was observed. This indicated that these compounds did not affect the redox reaction of quercetin and kaempferol even at a very high concentration.
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8. 9. 10.
Conclusions In summary, we have demonstrated the highly selective detection toward quercetin and kaempferol based on pn-MWCNTs modified GCE. Simultaneous determination of quercetin and kaempferol in solution and actual samples were successfully achieved based on DPV and amperometric measurement. Moreover, the pn-MWCNTs modified electrode offered excellent stability and high sensitivity in the electrochemical determination of quercetin and kaempferol. This finding provides a simple, sensitive and reliable method to evaluate quercetin and kaempferol.
11. 12. 13. 14. 15. 16. 17. 18.
Acknowledgements
19.
This work was supported by the National Natural Science Foundation of China (21105073), 2014 Program for Excellent Young Talents at the University of Anhui Province and the State Key Laboratory of Electroanalytical Chemistry (SKLEAC201307). Q.-A. H. acknowledges the Project of Educational Committee of Anhui Province (KJ2010B240).
20.
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Selective Detection toward Quercetin and Kaempferol on NH3-Plasma Treated Carbon Nanotubes Modified Glassy Carbon Electrode Jing-Jing SONG, Yong LU, Si-Wei ZHU, Qin-An HUANG,† and Yan WEI† Department of Chemistry, Wannan Medical College, Wuhu 241002, P. R. China
NH3-plasma treated multi-walled carbon nanotubes (pn-MWCNTs) were prepared based on the plasma technique and developed as sensing materials for detection of quercetin and kaempferol with the differential pulse voltammetry (DPV) and amperometric measurement. Such experimental parameters as pH values, accumulation potential and accumulation time were carefully investigated. The pn-MWCNTs modified electrode (pn-MWCNTs/GCE) was further explored for the analysis of quercetin and kaempferol in diluted blood serum and average recovery rates of 96.91 and 100.5% were obtained, respectively. In addition, the interference and stability measurements were evaluated under the optimized experimental conditions. More importantly, selective detection toward quercetin and kaempferol was achieved, and the proposed electrochemical sensing strategy was available to distinguish substances with similar oxidation potential. Keywords Plasma treatment, multi-walled carbon nanotubes, quercetin, kaempferol, electrochemical detection (Received October 13, 2014; Accepted January 1, 2015; Published March 10, 2015)
Introduction Flavonoids are a vast class of heterogeneous polyphenols that are widely distributed in common foods such as teas, fruits, vegetables, and wine.1 Generally, they are subdivided into seven classes: flavones, flavonols, flavanones, flavanols, flavanonols, isoflavones and anthocyanidins.2,3 Quercetin and kaempferol are of particular importance among flavonols as they are found to be potent antioxidants. Numerous reports have revealed that quercetin and kaempferol possess a wide range of biological activities such as antioxidant ability, anxiolytic effects, antiinflammatory characteristics and the ability to damage in glucose-induced oxidative cells and dysfunction in pancreatic cells.4–7 Due to their popularity as medicinal ingredients, it is of great interest and significance to develop effective and feasible methods for the analysis of quercetin and kaempferol. In the past few years, many conventional methods have been reported on the detection of these two substances, including molecularly imprinted polymers (MIPs),8,9 capillary electrophoresis (CE),10 and high performance liquid chromatography (HPLC).1,11 Although these methods have excellent determination limits and can effectively identify and quantify analytes, they require sophisticated instruments and are time consuming. Alternatively, electrochemical methods have drawn strong interest for the efficient analysis of kaempferol and quercetin because of the advantages of high sensitivity, easy of use, and low cost. And much attention has been paid to the development of sensing J.-J. S. and Y. L. contributed equally to this work. † To whom correspondence should be addressed. E-mail: [email protected] (Y. W.); [email protected] (Q.-A. H.)
materials that are closely reflected in electrochemical Past work reported that such metal performance.12–16 nanomaterials as carbon nanotubes (CNTs) and graphene have been explored as sensing materials and played an extremely important role in electroanalysis. Since first reported by Iijima in 1991,17 CNTs have been a hot topic of research during the past two decades because of their special structural, unique electronic, optical and mechanical properties.18–21 Owing to the enhanced electrode conductivity and large surface area, CNTs have been developed to construct electrochemical sensors. In particular, Wang et al. used β-cyclodextrin-CNTs modified electrodes for the simultaneous determination of adenine and guanine.22 Zeng et al. successfully detected rutin using a single-walled carbon nanotubes modified gold electrode.23 Wu et al. fabricated sensors based on multiwalled carbon nanotubes (MWCNTs) modified electrode for the detection of nitric oxide.24 However, some difficulties were inevitable. Typically, the most common chemical modification with HNO3 and/or H2SO4 may lead to damage bulk properties, decreasing their stability and even cleaving them into shorter pieces; the complicated modification process may result in electrode surface fouling, increasing the instability of the modified electrode.25 In contrast to the common chemical modification method, plasma treatment is a solvent-free, timeefficient, versatile, environment-friendly procedure that does not result in a large amount of structural damage to the CNTs and can also provide a wide range of different functional groups on CNTs.26 To the best of our knowledge, the application of NH3 plasma treated MWCNTs for the detection of quercetin and kaempferol has not been reported yet. In this work, MWCNTs were treated with NH3 plasma and used as sensing materials for the modification of a glassy carbon electrode. After NH3 plasma treatment, amino groups were
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introduced onto the surface of MWCNTs,27–29 which were explored as a selective sorbent for the separation and preconcentration of quercetin and kaempferol. The main purpose of the present work is to achieve the simultaneous detection of quercetin and kaempferol in combination with differential pulse voltammetry (DPV) and amperometric measurements. The optimizing of experimental conditions, such as pH values, accumulation potential, and accumulation time, were carefully investigated. In addition, the interference and stability of an NH3-plasma treated MWCNTs (pn-MWCNTs) modified electrode were also studied.
Experimental Chemical reagents Quercetin and kaempferol were purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). First, 1.0 mM standard solutions of quercetin and kaempferol were prepared by dissolving in ethanol (95%), and then stored in the refrigerator. MWCNTs were purchased from Chengdu Institute of Organic Chemistry of the Chinese Academy of Sciences. The as-received MWCNTs (raw MWCNTs) have a normal hollow structure. The purity was about 95%, with 0.23 and 0.93% of iron and nickel catalysts, respectively. The amorphous carbon content was about 4%. The diameter of raw MWCNTs was about 10 – 30 nm with the length at micron level. All reagents were of analytical grade and used without any further purification. Then, 0.05 M phosphate buffer solutions (PBS) were prepared by mixing 0.05 M NaH2PO4 and 0.05 M Na2HPO4, and then adjusting the pH values with 0.05 M H3PO4 or 0.05 M NaOH. The deionized water (18.2 MΩ cm) used to prepare all solutions was purified with the NANOpureDiamondTM UV water system. Apparatus All electrochemical experiments were recorded using a CHI 660D computer-controlled potentiostat (ChenHua Instruments Co., Shanghai, China). Measurements were carried out in a conventional three-electrode system using the modified or bare glassy carbon electrode (GCE, 3 mm in diameter) as a working electrode, Ag/AgCl as a reference electrode and Pt wire as a counter electrode. Preparation of pn-MWCNTs The pn-MWCNTs were prepared from raw MWCNTs according to the process depicted elsewhere.29 In brief, a plasma generator by radio-frequency inductively coupled plasma was used in this study. Raw MWCNTs were pretreated using Ar plasma at a gas flow rate of 70 sccm and microwave power of 700 W, and then NH3/Ar mixture gases were introduced through mass flow meters. MWCNTs were treated by NH3 plasma under continuous stirring. Then, the MWCNTs after plasma treatment for 30 min were dispersed in ethanol without any further purification. Fabrication of modified electrode Prior to modification, a bare glassy carbon electrode (GCE) was sequentially polished with 1.0, 0.3 and 0.05 μm alumina power slurries to a mirror shiny surface then sonicated with HNO3 solution (v:v 1:1), absolute ethanol and deionized water, respectively. Then, 4 μL of pn-MWCNTs solution was dripped onto the surface of the GCE. The electrode was allowed to air-dry at room temperature. When the modified electrode was not in use, it was stored in 0.05 M PBS (pH 4.0) at 4oC in a
Fig. 1 a) Cyclic voltammograms and Nyquist diagram of electrochemical impedance spectra of raw MWCNTs (black line) and pn-MWCNTs modified GCE (red line) in the solution of 5 mM K3Fe(CN)6 containing 0.1 M KCl. Scanning rate: 100 mV s–1. Frequency: 100000 to 1 Hz.
refrigerator. Electrochemical detection of quercetin and kaempferol Quercetin and kaempferol were preconcentrated in 0.05 M PBS (pH 4.0) for 120 s under stirring condition. Differential pulse voltammetry was performed in the potential range of 0 to 0.8 V at the following parameters: frequency, 15 Hz; amplitude, 25 mV; increment potential, 4 mV; pulse width, 0.2 s; sampling width, 0.02 s. Quantitative detection of quercetin and kaempferol was performed using an amperometric technique by adding substances every 50 s and setting the applied potential at 0.375 and 0.45 V, respectively. Cyclic voltammograms (CV) and electrochemical impedance spectra (EIS) were performed in mixing solution of 5 mM K3[Fe(CN)6] with 0.1 M KCl and the scanning rate was 100 mV s–1.
Results and Discussion Electrochemical characterization of NH3-plasma treated MWCNTs The cyclic voltammetric response of raw MWCNTs and pn-MWCNTs modified GCE has been characterized in the solution of 5 mM Fe(CN)63–/4– containing 0.1 M KCl. Figure 1a shows the CV responses of raw MWCNTs and pn-MWCNTs
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modified electrodes. As compared with the raw MWCNTs modified electrode, the anodic and cathodic peak currents of pn-MWCNTs modified electrode increased. We suggested that the effective surface area of pn-MWCNTs, increased by the plasma treatment, was responsible for the increased peak current. The electrochemical impedance spectrum (EIS) was employed to characterize the interface properties of the pn-MWCNTs modified electrode. It is well-known that a typical EIS consists of two parts, a semicircle part and a linear part. The semicircle portion corresponds to the electron-transfer resistance (Ret) at a higher frequency range, while a linear part at a lower frequency range represents the diffusion process. As presented in Fig. 1b, Ret for raw MWCNTs modified electrode was about 100 Ω. In contrast, the EIS for pn-MWCNTs modified electrode was nearly a straight line, indicating the promotion of the electron transfer process on the modified electrode surface. The results of electrochemical impedance spectra were in good accordance with that from CV. Optimization of experimental conditions In order to obtain the high sensitive electrochemical performance with pn-MWCNTs modified GCE for detecting quercetin and kaempferol, the experimental parameters of pH value, accumulation time and accumulation potential were optimized. The effect of pH value is very significant in the experimental environment. DPV responses of 1 μM quercetin and 1 μM kaempferol were assessed on the pn-MWCNTs modified GCE in 0.05 M PBS by varying the pH value from 3.0 to 8.0 (Fig. 2a). Apparently, the highest responses were observed at pH 4.0 for both 1 μM quercetin (black line) and 1 μM kaempferol (red line). It was suggested that due to the acidic hydroxyl groups of quercetin and kaempferol, the pKa value is about 4 and slightly less than 7, which is in agreement with a previous report.30 At pH 4.0, it may bring favorable possibility for the chemical reactions of hydroxyl in quercetin and kaempferol, such as hydroxylation, dimerization or the intramolecular of reaction. Thus, as the optimized pH value, 4.0 was employed. However, the reasonable mechanism on the effect of pH needs to be further verified in detail. Figure 2b depicts the DPV responses of 1 μM quercetin and 1 μM kaempferol with accumulation potential ranging from –0.3 to 0.2 V. As shown, the peak currents of quercetin (black line) increased from –0.3 to –0.2 V and then decreased significantly. In respect to kaempferol (red line), there was no obvious change in peak currents from –0.3 to 0.1 V and an apparent decrease was only observed at 0.2 V. Considering sensitivity for these two substances, the optimized accumulation potential was selected as –0.2 V. Different accumulation times of 0, 30, 60, 90, 120, and 150 s were examined using DPV in an analysis of 1 μM quercetin and 1 μM kaempferol at –0.2 V in 0.05 M PBS (pH 4.0). As seen in Fig. 2c, the peak currents increased with the increase of the accumulation time ranging from 0 to 120 s, and then started to level off at 120 s. Therefore, to achieve a lower detection limit and wider response range, an optimized accumulation time of 120 s was used throughout. Electrochemical detection of quercetin and kaempferol on pnMWCNTs modified electrode The electrochemical performance of pn-MWCNTs modified electrode toward quercetin and kaempferol was firstly evaluated with the DPV technique. Figure 3 presents the DPV analytical characteristics of 1.0 μM quercetin and kaemferol on the raw MWCNTs and pn-MWCNTs modified electrodes in 0.05 M
Fig. 2 Effect of a) pH values, b) accumulation potential and c) accumulation time on DPV responses of 1 μM quercetin (Que) and 1 μM kaempferol (Kae), respectively.
PBS (pH 4.0). In the case of the raw MWCNTs modified electrode, there were only weak responses for 1.0 μM quercetin (red line in Fig. 3a) and kaempferol (red line in Fig. 3b) at 0.38 and 0.42 V. Whereas, the strong and well-defined responses of quercetin and kaempferol (pink lines) can be clearly observed at the same concentrations without potential shift, which were much higher than that on raw MWCNTs/GCE. The significantly enhanced responses on pn-MWCNTs/GCE were due to the efficiency of MWCNTs after NH3 plasma treatment, resulting
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Fig. 4 DPV responses of mixture of quercetin and kaempferol ranging from 0.01 to 4.0 μM on pn-MWCNTs modified GCE in the solution of 0.05 M PBS (pH 4.0). Inset: the DPV response of mixture of quercetin and kaempferol ranging from 0.5 to 2.5 μM on raw MWCNTs modified GCE. DPV conditions are identical to those in Fig. 3.
Fig. 3 DPV responses of a) 1.0 μM quercetin (Que) and b) 1.0 μM kaempferol (Kae) on raw MWCNTs (red line) and pn-MWCNTs modified GCE (pink line) in the solution of 0.05 M PBS (pH 4.0) with accumulation time of 120 s and desorption time of 150 s. Frequency, 15 Hz; amplitude, 25 mV; increment potential, 4 mV; pulse width, 0.2 s; sampling width, 0.02 s. Dotted line: corresponding DPV responses in the absence of quercetin or kaempferol.
from the promotion of electron transfer. Simultaneous determination of quercetin and kaempferol Due to a similarity in the oxidation potentials for quercetin and kaempferol and possible appearance of the overlapping peaks, it was recognized as an important objective to distinguish quercetin and kaempferol when they co-existed. Figure 4 shows the simultaneous determination of quercetin and kaempferol with different concentrations (molar ratio, 1:1) under the optimized experimental conditions. As depicted, two separated peaks for quercetin and kaempferol on pn-MWCNTs/GCE were observed over the concentration range of 0.01 – 4.0 μM. And there was an obvious potential of 60 mV between the two peaks. We suggested that in comparison with the individual determination of quercetin and kaempferol, their interaction would have an effect on the peak potential and broaden the potentials in the simultaneous determination. In contrast, only a single and broad oxidation peak for quercetin and kaempferol on raw MWCNTs/GCE was obtained even at high concentrations (inset in Fig. 4). The results demonstrated the efficiency of pnMWCNTs. We suggested that MWCNTs after plasma treatment can improve the electron transfer, thus being of benefit for the simultaneous determination of quercetin and kaempferol.
Amperometric determination of quercetin and kaempferol As for the quantitative analysis of quercetin and kaempferol, the amperometric measurement at a certain applied potential (Eapp) was performed by successive addition of different qualities of quercetin and kaempferol under constant stirring. Figures 5a and 5b show the typical amperometric responses on pnMWCNTs/GCE after successive additions of quercetin (Eapp: 0.375 V) and kaempferol (Eapp: 0.45 V) into the 0.05 M PBS (pH 4.0). Herein Eapp selected refers to the peak potential in DPV measurement. That is, 0.375 and 0.45 V are similar to or slightly positive than the oxidation potential of Que and Kae, respectively. Well-defined responses proportional to the concentration of quercetin and kaempferol were observed. It was also found that the current responses of a steady state were reached within 5 s after each addition, indicating the fast response for the sensor. Calibration plots were obtained from the amperometric responses in the range of 0.05 – 4.55 μM (see the insets) with linear equations, i/μA = 0.001 + 0.02 c/μM (R2 = 0.999) for quercetin and i/μA = 0.005 + 0.0196 c/μM (R2 = 0.99) for kaempferol, respectively. The limits of detection (LODs) calculated in linear equations for quercetin and kaempferol were 0.05 μM. Moreover, with a applied potential at 0.375 V, the successful determination of quercetin was achieved without the interference from kaempferol (Fig. 5a). Figure 5c illustrates the current responses for alternate additions of two substances each 50 s at 0.45 V, and the corresponding current responses continuously increased on the basis of the former. Therefore, it can be confirmed the feasibility analysis of individual or simultaneous determination of kaempferol or the two substances on the pn-MWCNTs modified electrode. Moreover, no obvious interference was observed in the detection of the two substances. Stability and reproducibility study The stability was examined to check out the long-term performance of pn-MWCNTs modified electrode in 0.05 M PBS (pH 4.0) using DPV. As shown in Fig. 6, with 12 repetitive measurements, we found that the DPV responses of the modified electrode was nearly constant. The relative standard deviations (RSD) were 0.62 and 0.43% for quercetin and kaempferol, respectively. The results indicated that the modified electrode
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Fig. 6 Stability measurement of DPV responses on pn-MWCNTs modified GCE in 0.05 M PBS (pH 4.0) containing 1.5 μM quercetin (Que, black dot) or 0.5 μM kaempferol (Kae, red dot). DPV conditions are identical to those in Fig. 3.
Table 1 Determination of quercetin in blood serum samples and recovery study n cQue/μM 1 2 3 4 5
0.05 0.15 0.30 0.50 0.75
iMin/μA
iMax/μA
iAve/μA
Deviation, %
1.02 × 10–3 3.96 × 10–3 10.0 × 10–3 20.3 × 10–3 34.4 × 10–3
1.09 × 10–3 4.10 × 10–3 10.5 × 10–3 21.5 × 10–3 35.6 × 10–3
1.05 × 10–3 4.03 × 10–3 10.3 × 10–3 20.8 × 10–3 34.9 × 10–3
6.7 3.5 5.0 5.4 3.5
Table 2 Determination of kaempferol in blood serum samples and recovery study n cKae/μM
Fig. 5 Amperometric responses on the pn-MWCNTs/GCE in 0.05 M PBS (pH 4.0) for detection of quercetin or kaempferol. a) applied potential: Eapp = 0.375 V; successive addition of quercetin over the concentration of 0.05, 0.15, 0.30, 0.50, 0.75, 1.05, 1.40, 1.80, 2.25, 2.75, 3.30, 3.90, 4.55 μM (a → m), followed by a final addition of kaempferol; b) applied potential: Eapp = 0.45 V, successive addition of kaempferol over the same concentrations as in panel a (a → m). Insets in panels a) and b) were the corresponding calibration plots. c) Alternate addition of two substances with the concentration 0.05 μM, interval time = 50 s, Eapp =0.45 V.
can be reused without any obvious loss in response and robust stability was therefore obtained. The reproducibility of the modified electrode was also tested after one day and one week. The current response decreased by about 0.18 and 0.36% after one day, 10.16 and 13.97% after one week for the detection of quercetin and kaempferol, respectively, indicating the favorable reproducibility of the modified electrode. Analytical applications The pn-MWCNTs/GCE was further developed for the analysis of quercetin and kaempferol in diluted blood serum based on the amperometric technique. Prior to determination, the blood serum was pretreated by adding 0.1 M hydrochloric acid to
1 2 3 4 5
0.05 0.15 0.30 0.50 0.75
iMin/μA
iMax/μA
iAve/μA
Deviation, %
2.26 × 10–3 6.77 × 10–3 14.0 × 10–3 21.1 × 10–3 34.2 × 10–3
2.3 × 10–3 7.12 × 10–3 15.0 × 10–3 21.6 × 10–3 35.2 × 10–3
2.29 × 10–3 6.67 × 10–3 14.4 × 10–3 21.3 × 10–3 34.6 × 10–3
1.7 5.2 7.1 2.3 2.7
remove uric acid.31 After filtration by membrane (f 0.22 μM), the solution was mixed with ethanol in a volume ratio of 1:1, where 0.1 mM quercetin and 0.1 mM kaempferol were contained. And then mixed blood serum of different amounts was added into 0.05 M PBS (pH 4.0) solution. The concentrations of the analytes (Que and Kae) were determined using standard addition method and named as CQue and CKae. After three independent measurements, the currents were recorded and imax and imin denoted the maximum value and minimum value from the measurements. The results are summarized in Tables 1 and 2. The recovery values were calculated to be 96.91% for quercetin and 100.5% for kaempferol, suggesting the efficiency and reliability of pn-MWCNTs modified electrode for the determination of quercetin and kaempferol in practical applications. Interferences An interference study was performed in the presence of quercetin and kaempferol with the addition of other possible interferents. With individual additions of 500 μM glycine,
230 400 μM L(+)-tartaric acid, 600 μM glucose, 200 μM ascorbic acid into 0.05 M PBS (pH 4.0), no obvious interference (RSD < 5%) on the current responses of 1 μM quercetin and 1 μM kaempferol (data not shown) was observed. This indicated that these compounds did not affect the redox reaction of quercetin and kaempferol even at a very high concentration.
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8. 9. 10.
Conclusions In summary, we have demonstrated the highly selective detection toward quercetin and kaempferol based on pn-MWCNTs modified GCE. Simultaneous determination of quercetin and kaempferol in solution and actual samples were successfully achieved based on DPV and amperometric measurement. Moreover, the pn-MWCNTs modified electrode offered excellent stability and high sensitivity in the electrochemical determination of quercetin and kaempferol. This finding provides a simple, sensitive and reliable method to evaluate quercetin and kaempferol.
11. 12. 13. 14. 15. 16. 17. 18.
Acknowledgements
19.
This work was supported by the National Natural Science Foundation of China (21105073), 2014 Program for Excellent Young Talents at the University of Anhui Province and the State Key Laboratory of Electroanalytical Chemistry (SKLEAC201307). Q.-A. H. acknowledges the Project of Educational Committee of Anhui Province (KJ2010B240).
20.
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