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Highly sensitive determination of hyperin on poly(diallyldimethylammonium chloride)-functionalized graphene modified electrode
Journal of Electroanalytical Chemistry 776 (2016) 105–113
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Journal of Electroanalytical Chemistry journal homepage: www.elsevier.com/locate/jelechem
Highly sensitive determination of hyperin on poly(diallyldimethylammonium chloride)-functionalized graphene modified electrode Huichao Li, Kai Sheng, Zhengkun Xie, Lina Zou ⁎, Baoxian Ye ⁎ College of Chemistry and Molecular Engineering, Zhengzhou University, Zhengzhou 450001, PR China
a r t i c l e
i n f o
Article history: Received 10 May 2016 Received in revised form 16 June 2016 Accepted 29 June 2016 Available online 01 July 2016 Keywords: Hyperin Functionalized graphene Poly(diallyldimethylammonium chloride) Voltammetric sensor
a b s t r a c t A simple electrochemical method for the highly sensitive determination of hyperin was established based on poly(diallyldimethylammonium chloride)-functionalized graphene modified electrode (PDDA-Gr/GCE). The nanocomposite was characterized by X-ray diffraction (XRD), ultraviolet/visible spectra (UV–vis), Fourier Transform infrared spectroscopy (FT-IR) and transmission electron microscopy (TEM). The electrochemical characteristics of hyperin were studied in detail via cyclic voltammetry (CV), chronocoulometry (CC). Dynamics parameters of electrode process were also calculated to discuss the reaction mechanism. Under the optimized conditions, a lower detection limit of 5 × 10−9 mol L−1 (S/N = 3) and a wide linear detection range from 7 × 10−9 to 7 × 10−7 mol L−1 were achieved by the differential pulse voltammetry (DPV). Additionally, this simple, sensitive sensor was proved to be suitable for the determination of hyperin in Chinese herb Hypericum Perforatum with satisfactory results. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Hyperin (Fig. 1) is a natural flavonol glycoside compound present in many plants. It has many pharmacological activities, including anti-inflammatory [1,2], antioxidants [3,4], anti-cancer [5], liver protection [6], anti-depressant [7,8], anti-tumor [9]. Results of clinical application reveal that it has significant curative effect for treating coronary heart [10], canker sore [11,12] and other diseases. Based on the pharmacological activities and clinical application, it was necessary to develop a sensitive and convenient method for the determination of hyperin. The most common analytical methods for hyperin include high performance liquid chromatography (HPLC) [13–18], reverse phase high performance liquid chromatography (RP-HPLC) [19,20], capillary electrophoresis [21–23]. These methods offer excellent accuracy and precision, but suffer from expensive devices. Comparing with these methods, electrochemical methods have some advantages in analysis of electroactive molecules, such as sensitivity, time-saving, lower cost easy handling and so on. Moreover, the electrochemical techniques provide more information including redox mechanism and pharmacological action. To the best of our knowledge, one literature about determination of hyperin by voltammetric sensor was reported, the hyperin was detected by carboxylic multiwalled carbon nanotube modified composite pencil graphite electrode (c-MWCNT/CPGE) with the detection limit of 1 × 10−7 mol L−1 [24], but no information of redox mechanism was ⁎ Corresponding authors. E-mail addresses: [email protected] (L. Zou), [email protected] (B. Ye).
http://dx.doi.org/10.1016/j.jelechem.2016.06.044 1572-6657/© 2016 Elsevier B.V. All rights reserved.
given, in addition, the preparation process of the modified materials was complicated. To improve the sensitivity and explore the mechanism of hyperin, new voltammetric sensor should be constructed. Graphene (Gr), a two-dimensional, single-layer sheet of sp2 hybridized carbon atoms, has attracted tremendous attention, owing to its exceptional physical properties, such as high electronic conductivity, good thermal stability and excellent mechanical strength [25]. These properties of Gr make them extremely attractive in the field of electrochemical sensor [26,27]. Chemical reduction of exfoliated graphene oxide (GO) was an efficient approach to production of Gr. However, the Gr sheets tend to form irreversible agglomerates through π-π stacking and Van Der Waals interaction in the process of reduction [28]. To avoid this problem, some functionalized reagents were introduced to the Gr surface, such as poly(diallyldimethylammonium chloride) (PDDA) [29]. PDDA, a widely used cationic polyelectrolyte, can effectively enhance the dispersion of Gr and preserve the structural properties of Gr [30, 31]. PDDA functionalized graphene (PDDA-Gr) possess good stability, high conductivity, efficient electron transfer ability, biocompatibility and good electrocatalytic activity [32]. Therefore, the PDDA-Gr can serve as a sort of favorable material for modified electrode. Some methods for preparing the PDDA-Gr were reported with their own advantages, such as cost-effective and eco-friendly in microwave method [33,34] and simplification in wet chemical method, which only need common chemical regents [35,36]. In this work, PDDA-Gr was prepared via a simple wet chemical method. The characterization of the composite was carried out using transmission electron microscope (TEM), UV–visible spectrophotometry (UV–vis),
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2.2. Preparation of PDDA-Gr Graphene oxide (GO) was prepared from natural graphite flakes by the Hummers method [37]. GO (100 mg) was dispersed in 100 mL water with ultrasonication for 5 h, PDDA (5 mL) was added to the yellow-brown dispersion with stirred vigorously for 1 h at room temperature. After that, hydrazine (1 mL) was added to the mixture. And then, the mixture was heated to 90 °C for 3 h. Subsequently, the mixture was centrifugated and washed with water for three times. Finally, the obtained PDDA-Gr was dried at 60 °C for 12 h. The Gr was synthesized in the same condition without PDDA. 2.3. Preparation of modified electrode
Fig. 1. The chemical structure of hyperin.
Fourier Transform infrared spectroscopy (FT-IR) and X-ray diffraction analysis (XRD). PDDA-Gr was used to fabricate a voltammetric sensor for determination of hyperin. Moreover, electrochemical behavior and the redox mechanism of hyperin at poly(diallyldimethylammonium chloride)-functionalized graphene modified electrode (PDDA-Gr/GCE) were investigated. Different linear relationships between peak current and hyperin concentration were obtained via differential pulse voltammetry (DPV) with different accumulation times. The detection limit reached 5 × 10−9 mol L−1 under the accumulation of 150 s. In addition, this voltammetric sensor was also successfully employed for detection of hyperin in pharmaceutical samples with satisfactory results.
PDDA-Gr was dispersed in water and sonicated for 1 h to obtain a uniformly black suspension (0.5 mg mL−1). Prior to the modification, the bare GCE was mechanically polished to a mirror-like with 0.3 μm alumina slurry, and subsequently sonicated with water, absolutely ethanol and dried in air naturally. The PDDA-Gr/GCE was established by depositing PDDA-Gr (5 μL) on a fresh GCE surface using a micro-injector, and then dried under an infrared lamp. For comparison, Gr/GCE was fabricated with the similar procedures. 2.4. Analytical procedure Before the experiment, the prepared PDDA-Gr/GCE was scanned 5 cycles between 0.0 V and 0.8 V with the scan rate of 0.1 V s−1 in 0.1 mol L−1 (pH 2.0) by cyclic voltammetry (CV). And then, a certain amount of standard stock solution of hyperin was added to 0.1 mol L−1 (pH 2.0), after that, accumulation of 150 s was performed under an open circuit. Because of the adsorptive character of hyperin on the electrode, it was necessary to refresh the electrode surface. After each measurement, the PDDA-Gr/GCE was scanned in 0.1 mol L−1 (pH 8.0) for 2 cycles between 0.0 V and 0.8 V using CV to renew the electrode surface.
2. Experimental 2.5. Sample solution preparation 2.1. Apparatus and reagents All electrochemical experiments were carried out using an CHI610A electrochemical system (Chenhua Instrument Company, Shanghai, China) with a three-electrode system, a bare GCE (d = 3 mm) or modified GCE as working electrode, a saturated calomel electrode (SCE) and a platinum wire served as reference and counter electrode, respectively. The type of pH meter was pHS-3C (Shanghai Techcomp Jingke Scientific Instruments, Shanghai, China). High performance liquid chromatography was performed on 1260 Infinity Quaternary LC System (Agilent Technologies Inc., Santa Clara, California, USA). Characterization of morphology and crystalline structure was performed by transmission electron microscopy (TEM, JEOL-2100 Hiroshima, Japan), x-ray powder diffraction (XRD, Rigaku D/max-IIIA, Tokyo, Japan), Infrared spectrogram was recorded on Infrared Spectrometer (Thermo Nicolet Corporation, Santa Clara, California, USA) and UV–vis spectra was obtained from a Lambda 35 UV–vis spectrometer (Pgeneral General Instrument, Beijing, China). Hyperin (≥ 98%) and PDDA (MW b 1,000,000, 35% in water) were purchased from Aladdin (http://www.aladdin-e.com). Natural graphite flakes with the average diameter of 200 mesh were obtained from the Sigma-Aldrich (Sigma-Aldrich, St. Louis, Missouri, USA). Hypericum perforatum was purchased from local drugstore. The standard stock solution of hyperin was prepared with methanol and kept in darkness at 4 °C. The supporting electrolyte of 0.1 mol L−1 PBS were prepared by mixing 0.1 mol L−1 NaH2PO4 with Na2HPO4. The lower pH value of PBS was adjusted with 0.1 mol L−1 H3PO4. All the other reagents used in experiments were analytical grade and used without further purification and all experiments were performed at room temperature.
A certain amount of Chinese herb Hypericum Perforatum (100 mg) were put in volumetric flask and dissolved with 10 mL methanol. Hyperin was extracted by ultrasonic for 3 h, then the suspension was centrifuged for 10 min at 5000 rpm, this step was repeated three times for ensuring extraction completely. All solution were put together and concentrated to 10 mL in fume hood for analysis. Before each measurement, a certain amount of 20 μL was diluted with supporting electrolyte. 3. Results and discussion 3.1. Characterization of PDDA-Gr Fig. 2A showed the UV–vis spectrum of GO (curve a) and PDDA-Gr (curve b). GO had a characteristic absorption peak at 230 nm (the π-π* transitions of C_C bonds) [38], while the PDDA-Gr exhibited a strong absorption peak at 270 nm (n → π* transitions of C_O bonds) [39], indicating that the GO was reduced [40]. Fig. 2B showed the IR spectra for GO (curve a), Gr (curve b) and PDDA-Gr (curve c). As expected, the IR spectrum for GO exhibited characteristic peaks at approximately 1724 cm−1 which represent the skeletal vibration of C_O, indicating the existence of oxygen functionalities at GO surface [41]. The other characteristic peaks were also showed in the spectra: O\\H (3434 cm−1), C_C (1625 cm−1), C\\O\\C (1216 cm−1) and C\\O (1050 cm−1). The IR of Gr and PDDA-Gr were different from the GO. The characteristic absorption bands of the oxygen functional group (C_O, C\\O\\C) decreased dramatically, which indicated that GO had been reduced to
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Fig. 2. (A) UV–vis absorption spectra of GO (a) and PDDA-Gr (b). (B) The FT-IR spectra of GO (a), Gr (b) and PDDA-Gr (c). (C) XRD patterns of GO (a) and PDDA-Gr (b).
Gr. In addition, some new bands were appearance in the PDDA-Gr: 2923 cm−1 (CHn) and 1113 cm−1 (C\\N), which consist with the characteristic bands of PDDA [42]. The results indicated the functionalization of Gr with PDDA. Fig. 2C showed the XRD images of GO (curve a) and PDDA-Gr (curve b). As expected, GO had characteristic peak at 2θ = 10° due to the introduction of oxygenated functional groups onto the carbon sheets corresponding to the (002) plane. The diffration peak at 2θ = 43° was associated with the hexagonal structure of carbon ( (100) plane). For PDDA-Gr, it was still keeping the hexagonal structure with a diffration peak at 2θ = 43°. However, the (002) plane shifted to greater angle (about 22°), which indicated the removal of oxygen-containing
functional groups. From this, we can conclude that the GO has been reduced to Gr completely [43]. The structure and morphology of GO, Gr and PDDA-Gr were analyzed using TEM. Fig. 3A showed the TEM of GO, which exhibited the typical wrinkle morphology with well dispersibility on the substrate without any aggregation. After chemical reduction process, Gr (Fig. 3B) exhibited obvious re-stacking effect due to strong π-π stacking interaction and Van Der Waals forces. The re-stacking effect could highly reduce the specific surface area of the material. In contrast, PDDA-Gr (Fig. 3C) remained a well dispersibility without clear re-stacking effect due to the surface protection of PDDA. The presence of PDDA not only could prevent the natural force of Gr layers stacking together, but also
Fig. 3. (A) The TEM of GO. (B) The TEM of Gr. (C) The TEM of PDDA-Gr.
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of Gr. However, in the case of PDDA-Gr/GCE (curve d), the redox peak currents of K3Fe(CN)6 increased dramatically with ΔEp of 60 mV. Much higher peak currents and relatively smaller ΔEp obtained on PDDA-Gr/GCE were mainly ascribed to its large surface area and positive surface charge, which provide more active site and electrostatic attraction for Fe(CN)36 −. Chronocoulometry was used to evaluate electrochemically effective area of the electrodes [44]. The electrochemically effective area of the modified electrodes were measured by single potential step chronocoulometry, the potential was stepped from −0.2 V to 0.6 V with the step time of 5 s. Fig. 5A was the chronocoulometry curves of K3Fe(CN)6 (1 × 10−4 mol L−1, containing 1 mol L−1 KCl) obtained at bare GCE (curve a), GO/GCE (curve b), Gr/GCE (curve c) and PDDA-Gr/GCE (curve d). The corresponding Q-t1/2 curves were shown in Fig. 5B. According to the formula of Anson theory [45]. Q¼
Fig. 4. Cyclic voltammograms of 1.0 × 10−3 mol L−1 K3[Fe(CN)6] containing 0.1 mol L−1 KCl at bare GCE (a), GO/GCE (b), Gr/GCE (c), PDDA-Gr/GCE (d).
provide repulsion between the sheets by its surface positive charges, which was favorable for forming a stable suspension. The observation of PDDA-Gr showed a general flake-like structure of Gr, indicating the surface functionalization did not change the morphology of the Gr. The UV–vis, IR, XRD and TEM data provide evidence that the PDDAGr were successfully synthesized. 3.2. The electrochemical characterization of PDDA-Gr/GCE Fig. 4 displayed cyclic voltammograms of K3Fe(CN)6 (1 × 10−3 mol L−1 containing 0.1 mol L−1 KCl) at different electrodes. A pair of redox peaks were observed on the GCE (curve a) with peak difference (ΔEp) of 79 mV. When GO was immobilized on the GCE surface (curve b), the redox peak currents decreased obviously with ΔEp of 100 mV. The reason of ΔEp increment can attribute to a large number of oxygen containing function groups on GO surface, which reduced the redox rate of K3Fe(CN)6. After modified with Gr (curve c), both the cathodic and anodic peak currents were increased with the ΔEp of 75 mV, which may contributed to the excellent electrical conductivity
2nFAcðDtÞ1=2 þ Q dl þ Q ads π1=2
where A is the area of electrode, D is diffusion coefficient, c is the concentration of the species, t is the potential pulse width, Qdl is the double-layer charge, Qads is the Faradaic charge. For 1 × 10− 4 mol L−1 K3Fe(CN)6, n = 1, D = 7.6 × 10−6 cm2 s−1. According to the slope of Q-t1/2 from Fig. 5B: Q (10− 4 C) = 0.023 t1/2 (s1/2) + 0.080 (R = 0.999), Q (10− 4 C) = 0.078 t1/2 (s1/2) + 0.20 (R = 0.999), Q (10−4 C) = 0.12 t1/2 (s1/2) + 0.70 (R = 0.998) and Q (10−4 C) = 0.13 t1/2 (s1/2) + 1.10 (R = 0.999), the A of GCE, GO/GCE, Gr/GCE and PDDAGr/GCE were calculated to be 0.080 cm2, 0.260 cm2, 0.399 cm2 and 0.433 cm2, respectively. The results further confirmed the enlarged effect of PDDA-Gr on the electrode surface area. 3.3. Electrochemical behavior of hyperin at the PDDA-Gr/GCE Fig. 6A illustrated the CV of hyperin (3 × 10−6 mol L−1) at bare GCE (b), Gr/GCE (c) and PDDA-Gr/GCE (e) in 0.1 mol L− 1 PBS (2.0). The curve a and curve d were the bare GCE and PDDA-Gr/GCE in blank solution. Fig. 6B was the enlarged curve a and curve b. For the blank solution of bare GCE (curve a) and PDDA-Gr/GCE (curve d), no redox peak was observed, meaning that there was no redox reaction of the PDDA-Gr membrane in a potential window of 0.0–0.8 V. From bare GCE (curve b), a very weak response was obtained at Epa = 0.527 V, Epc = 0.500 V. On the Gr/GCE, a pair of well-defined redox peaks were obtained with the Epa = 0.559 V and Epc = 0.500 V, the redox peak currents are significantly increased, which were about 10 times higher than at the bare GCE, which might be attributed to the excellent conductivity of Gr. Comparing with the GCE and Gr/GCE, the PDDA-Gr/GCE show the
Fig. 5. (A) Chronocoulometric curves of the 1 × 10−4 mol L−1 K3[Fe(CN)6] containing 1 mol L−1 KCl at the bare GCE (a), GO/GCE (b), Gr/GCE (c), PDDA-Gr/GCE (d). (B) The corresponding Q-t1/2 plots. The potential was stepped from −0.2 V to 0.6 V, step time: 5 s.
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Fig. 6. (A) Cyclic voltammograms of hyperin (3 × 10−6 mol L−1) at bare GCE (b), Gr/GCE (c) and PDDA-Gr/GCE (e) in 0.1 mol L−1 PBS 2.0, the bare GCE (a) and PDDA-Gr/GCE (d) in blank solution. (B) The enlarged curve a and curve b, scan rate 0.1 V s−1, accumulation time: 150 s.
best sensitivity response which were 20 times higher than at bare GCE with the Epa = 0.577 V and Epc = 0.510 V. This could be reasonably ascribed to the enlarged surface area, the extraordinary electron-transfer
properties of PDDA-Gr and the strong adsorption between hyperin and PDDA-Gr. So PDDA-Gr/GCE was selected as the voltammetric sensor in the following experiment.
Fig. 7. (A) Cyclic voltammograms of hyperin (3.0 × 10−6 mol L−1) with different supporting electrolytes at PDDA-Gr/GCE including 0.1 mol L−1 phosphate buffer (pH 3.0 curve a), acetate buffer (pH 3.6 curve b), Britton-Robinson (pH 3.78 curve c) and sulfuric acid (pH 3.03 curve d). (B) Cyclic voltammograms of hyperin (3.0 × 10−6 mol L−1) with different pH (a–g: 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0). (C) The relationship between the peak potentials and pH, scan rate 0.1 V s−1, accumulation time: 150 s.
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3.4. Optimization conditions 3.4.1. The influence of supporting electrolyte and pH To get the best response of hyperin (3 × 10−6 mol L−1) at PDDA-Gr/ GCE, the influence of different supporting electrolytes were investigated by CV (Fig. 7A), including 0.1 mol L−1 phosphate buffer (pH 3.0 curve a), acetate buffer (pH 3.6 curve b), Britton-Robinson (pH 3.78 curve c) and sulfuric acid (pH 3.03 curve d). In consideration of the peak shape and sensitivity, 0.1 mol L− 1 PBS was chosen as supporting electrolyte in the following experiments. The influence pH value of supporting electrolytes on the response of hyperin at PDDA-Gr/GCE was also investigated by CV. As shown in Fig. 7B, the redox currents changed with the increase of pH from 2.0 to 8.0. The redox peak potentials shifted negatively with the increase of pH from 2.0 to 8.0. It indicated that proton take part in the electrode reaction process [46]. From the relation of Ep and pH (Fig. 7C): Epa = 0.721–0.0586 pH (R = 0.992), Epc = 0.649–0.0581 pH (R = 0.997). According to the formula: dEp/dpH = 2.303mRT/nF (m is the number of proton, n is the number of electron), m/n was calculated to be 1 for the hyperin redox process. This result indicates that protons and electrons in a ratio of 1:1 in the electrode reaction process of hyperin. The other information obtained from this group of experiments was that the reductive peak almost disappeared when the pH value was equal to or higher than 7.0. These results demonstrate that the acidic condition leads to a more reversible electrochemical reaction of hyperin on the proposed electrode. It showed that the highest response of peak
current was obtained at pH 2.0, So 0.1 mol L−1 (pH 2.0) was selected as supporting electrolyte for the determination of hyperin. 3.4.2. Influence of scan rate To further explore the electrode reaction mechanism of hyperin, the influence of different scan rates were investigated by CV with scan rate changed from 0.03 to 0.35 V s−1 (Fig. 8A). It was found that the anodic current and cathodic currents were increased with the increase of scan rates. The linear relationship between ip and v were obtained (Fig. 8B): ipa (μA) = 168.08 + 7.667 v (V s−1) (R = 0.996), ipc (μA) = −127.78– 5.4151 v (V s−1) (R = 0.997), suggesting that the electrode reaction of hyperin was a adsorption-controlled process. Moreover, with the increase of scan rate, the anodic peak potential shifted positively, while the cathodic peak potential shifted negatively, as depicted in Fig. 8C, the anodic (Epa) and cathodic (Epc) peak potentials have a linear relationship with the Napierian logarithm of scan rate (lnv): Epa (V) = 0.0255 lnv (V s− 1) + 0.6386 (R = 0.994), Epc (V) = − 0.0208 lnv (V s−1) + 0.4657 (R = 0.992). According to the Formulas (1) and (2) [47], n was estimated to be 2, α = 0.55. In addition, the electron transfer rate constant ks = 1.16 s−1 was obtained from Formula (3) [47]. 0
RT ln v αnF
ð1Þ
0
RT ln v ð1−αÞnF
ð2Þ
Epc ¼ E0 − Epa ¼ E0 −
Fig. 8. (A) The voltammograms of hyperin (3.0 × 10−6 mol L−1) with different scan rates at PDDA-Gr/GCE (a–k: 0.03, 0.04, 0.06, 0.08, 0.1, 0.12, 0.15, 0.2, 0.25, 0.30, 0.35 V s−1) in 0.1 mol L−1 PBS 2.0, accumulation time: 150 s. (B) The relationship between ip and v. (C) Ep and lnv.
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Fig. 9. The proposed redox mechanism of hyperin at the PDDA-Gr/GCE.
logks ¼ α logð1−αÞ þ ð1−αÞ logα− log
RT nFΔEp −αð1−αÞ 2:3RT nFv
ð3Þ
According to the results, two electrons and two protons were involved in the redox reaction of hyperin. Therefore, a possible electrochemical reaction mechanism of hyperin at PDDA-Gr/GCE could be described as following: (See Fig. 9) 3.5. Chronocoulometry studies Considering of the adsorptive character of hyperin at the PDDA-Gr/ GCE, it is necessary to calculate the saturated adsorption capacity of hyperin at PDDA-Gr/GCE surface. The saturated adsorption capacity could be calculated according to the Laviron's theory of Qads = nFAΓ* [47]. The Qads of hyperin on PDDA-Gr/GCE was measured in 0.1 mol L−1 (pH 2.0) by single potential step chronocoulometry. The potential was stepped from 0.0 V to 0.8 V, step time 5 s. As shown in the Fig. 10A, the Q-t curves were recorded in the absence (curve a) and presence (curve b) hyperin solution (1 × 10−5 mol L−1) after stirring for 10 min to achieve saturated adsorption. The corresponding plots of Q against t1/2 were shown in Fig. 10B for oxidation process. The two straight lines were almost parallel, further implying the oxidation process of hyperin on the PDDA-Gr/GCE was driven by adsorption. According to the intercept, the value of Qads was calculated to be 1.730 × 10−4 C for the oxidation process. The saturated adsorption capacity Γ* of hyperin was calculated to 2.692 × 10− 9 mol cm− 1 for the oxidation process.
3.6. Analytical applications and methods validation 3.6.1. Influence of accumulation time For an adsorptive driven electrode process, a step of accumulation has a significant effect on the detection sensitivity. The relationship was recorded between accumulation time (tacc) from 30 s to 210 s and peak currents in a hyperin solution (3 × 10−6 mol L−1) via CV. Fig. 11 showed that with the increase of accumulation time, the responding anodic peak currents increased gradually up to 150 s, and then enhanced slowly. Considering the detection sensitivity and linear range, an accumulation time of 150 s was chosen to set up the calibration curve in following experiments. 3.6.2. Calibration curve, detection limit, repeatability and stability Under the optimum conditions described above, DPV was performed to investigate the relationship between the peak currents and the concentration of hyperin with the accumulation time of 150 s at the proposed electrochemical sensor. Oxidation peak current was recorded and used as the detected signal (Fig. 12A). As the Fig. 12B shown, a good linear relationship could be obtained between peak currents (ipa) and hyperin concentrations (C) in the range of 7 × 10−9 mol L−1–7 × 10−7 mol L−1. The linear regression equation was ipa (μA) = 44.04 C (μM) + 2.305 (R = 0.997) with a detection limit of 5 × 10−9 mol L−1 (S/N = 3). Table 1 lists the data for comparison of the present methods for the determination of hyperin. In this part, in order to further evaluate the influence of tacc on the determination, we chose a shorter time 30 s as tacc. As a result, the linear range was 2 × 10−8 mol L−1 to 6 × 10−6 mol L−1, which is wider than
Fig. 10. (A) Chronocoulometric responses curves of absence (a) and presence (b) of 1.0 × 10−5 mol L−1 hyperin in 0.1 mol L−1 PBS 2.0. (B) The corresponding Q-t1/2 plots of anodic peaks. The potential was stepped from 0.0 V to 0.8 V, step time: 5 s.
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The repeatability and stability of PDDA-Gr/GCE were evaluated by DPV in hyperin solution (3 × 10−6 mol L−1). A relative standard deviation (RSD) of the peak current from five electrodes was obtained of 2.9%, while the RSD of 3.0% was estimated with one electrode for 10 successive measurements. These results showed that the sensors exhibited repeatability and reproducibility. The stability of the sensor was evaluated by examining its response current after storage in dark two weeks, the current of hyperin reduced 4.9%, indicating the excellent stability.
Fig. 11. The relation between the anodic currents of hyperin (3 × 10−6 mol L−1) and the accumulation time (30 s, 60 s, 90 s, 120 s, 150 s, 180 s, 210 s.). Supporting electrolyte: 0.1 mol L−1 PBS 2.0, scan rate: 0.1 V s−1.
that of 150 s tacc. The linear regression equation was ipa (μA) = 7.40 C (μM) + 1.965 (R = 0.996) with a detection limit of 1 × 10−8 mol L−1 (S/N = 3). The results indicated that with the accumulation time, the sensibility can be increased, however, the liner range was narrow.
3.6.3. Interference studies Potential interferences of some possible inorganic and organic compounds in real samples were explored in 3 × 10−7 mol L−1 hyperin by DPV (Fig. 13). The results indicated that the inorganic ions: Ca2+, Cu2+, − Al3+, Mg2+, Zn2+, SO2− 4 , NO3 in 100 folds of hyperin had no interfering with the determination of hyperin. Moreover some organic compounds: ascorbic acid, glucose, dopamine in 20-fold of hyperin, it showed that b5.0% of the peak current variation for the detection of hyperin. Considering in natural products, there are some other flavonol compounds coexisting with hyperin. So, the electrochemical behaviors of other flavonol compounds were investigated. The results showed that the hesperetin and diosmin in 20 folds of hyperin were no interfering. The rutin and quercetin in 6-folds of hyperin were no interfering. All these results indicated the excellent anti-interference of the sensor. 3.6.4. Real sample analysis In order to validate the practical application of this proposed method, the fabricated PDDA-Gr/GCE was employed to determine the concentration of hyperin in an actual sample (Chinese Herba Hypericum perforatum). The sample treatment processes were described in the
Fig. 12. (A) The DPV curves of different concentrations of hyperin at PDDA-Gr/GCE under optimum conditions, accumulation time: 150 s. Hyperin concentrations: 7.0 × 10−9, 1.0 × 10−8, 4.0 × 10−8, 6.0 × 10−8, 8.0 × 10−8, 1.0 × 10−7, 3.0 × 10−7, 5.0 × 10−7 and 7 × 10−7 mol L−1. (B) The relationship of peak current with hyperin concentration.
Table 1 Comparison of different methods for determination of hyperin (the concentration unit in the references cited had been converted to mol L−1). Method
Sample pre-treatment
Linear range (10−6 mol L−1)
Detection limit (10−6 mol L−1)
Reference
HPLC HPLC HPLC HPLC HPLC RPHPLC Capillary electrophoresis Capillary electrophoresis Capillary electrophoresis c-MWCNT/CPGE PDDA-Gr/GCE
Extraction and separation Extraction and separation Extraction and separation Extraction and separation Extraction and separation Extraction and separation Extraction and separation Extraction and separation Extraction and separation Extraction Extraction
2.15–193.82 9.39–1174.60 1.08–21.53 21.53–215.34 10.76–215.34 2.15–129.20 21.535–107.67 2.76–1119.79 14.45–1076.73 0.22–30.15 0.0070–0.070
0.71 9.39 1.08 0.15 1.14 0.0082 0.13 2.76 6.82 0.10 0.0050
13 15 16 17 18 20 21 22 23 24 This work
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Acknowledgements The authors are very grateful for the financial support from the National Natural Science Foundation of China (Grant no. 21575130; 21275132; U1504216). References
Fig. 13. Column chart of the oxidation peak current of 3 × 10−7 mol L−1 hyperin in 0.1 mol L−1 PBS 2.0, the PBS solution containing metal ions and some organic compounds and other flavonol compounds.
experimental section. After determination of the hyperin content in sample solution, the recovery tests were carried out using the standard addition method and the results were listed in Table 2. And the average content was calculated to be 2.0816 mg/g with RSD of 3.4% for three repeated measurements. For testing the accuracy of the proposed method, the same samples were analyzed using HPLC method and the results showed that there was no significant difference between them.
4. Conclusion In summary, PDDA-Gr composite was synthesized via a simple wet chemical method and applied in the fabrication of electrochemical sensor for the determination of hyperin. The high specific surface area, excellent conductivity and the strong adsorption of PDDA-Gr for hyperin affords excellent sensitivity. The electrochemical behaviors of hyperin on the PDDA-Gr/GCE were studied in detail. Otherwise the sensor exhibited a low detection limit of 5 × 10−9 mol L−1 (S/N = 3) and a wide linear detection range of from 7 × 10−9 to 7 × 10−7 mol L−1 by DPV with excellent reproducibility, repeatability and long-term stability. This study demonstrated that PDDA-Gr/GCE would be a promising sensor and useful for the hyperin analysis in biological samples.
Table 2 Determination results of hyperin in Chinese Herba Hypericum perforatum sample by DPV and HPLC. DPV
HPLC
Sample Addeda (μL) (10−7 mol
20
a
Founda (10−7 mol
L−1)a
L−1)
0 2 4
0.8965 2.882 4.901
Recovery RSD (%) (%)
Founda (10−7 mol
RSD (%)
L−1) 99.16 100.5
Average value of three replicate measurements.
3.4 2.0 2.1
0.8750
0.69
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Highly sensitive determination of hyperin on poly(diallyldimethylammonium chloride)-functionalized graphene modified electrode Huichao Li, Kai Sheng, Zhengkun Xie, Lina Zou ⁎, Baoxian Ye ⁎ College of Chemistry and Molecular Engineering, Zhengzhou University, Zhengzhou 450001, PR China
a r t i c l e
i n f o
Article history: Received 10 May 2016 Received in revised form 16 June 2016 Accepted 29 June 2016 Available online 01 July 2016 Keywords: Hyperin Functionalized graphene Poly(diallyldimethylammonium chloride) Voltammetric sensor
a b s t r a c t A simple electrochemical method for the highly sensitive determination of hyperin was established based on poly(diallyldimethylammonium chloride)-functionalized graphene modified electrode (PDDA-Gr/GCE). The nanocomposite was characterized by X-ray diffraction (XRD), ultraviolet/visible spectra (UV–vis), Fourier Transform infrared spectroscopy (FT-IR) and transmission electron microscopy (TEM). The electrochemical characteristics of hyperin were studied in detail via cyclic voltammetry (CV), chronocoulometry (CC). Dynamics parameters of electrode process were also calculated to discuss the reaction mechanism. Under the optimized conditions, a lower detection limit of 5 × 10−9 mol L−1 (S/N = 3) and a wide linear detection range from 7 × 10−9 to 7 × 10−7 mol L−1 were achieved by the differential pulse voltammetry (DPV). Additionally, this simple, sensitive sensor was proved to be suitable for the determination of hyperin in Chinese herb Hypericum Perforatum with satisfactory results. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Hyperin (Fig. 1) is a natural flavonol glycoside compound present in many plants. It has many pharmacological activities, including anti-inflammatory [1,2], antioxidants [3,4], anti-cancer [5], liver protection [6], anti-depressant [7,8], anti-tumor [9]. Results of clinical application reveal that it has significant curative effect for treating coronary heart [10], canker sore [11,12] and other diseases. Based on the pharmacological activities and clinical application, it was necessary to develop a sensitive and convenient method for the determination of hyperin. The most common analytical methods for hyperin include high performance liquid chromatography (HPLC) [13–18], reverse phase high performance liquid chromatography (RP-HPLC) [19,20], capillary electrophoresis [21–23]. These methods offer excellent accuracy and precision, but suffer from expensive devices. Comparing with these methods, electrochemical methods have some advantages in analysis of electroactive molecules, such as sensitivity, time-saving, lower cost easy handling and so on. Moreover, the electrochemical techniques provide more information including redox mechanism and pharmacological action. To the best of our knowledge, one literature about determination of hyperin by voltammetric sensor was reported, the hyperin was detected by carboxylic multiwalled carbon nanotube modified composite pencil graphite electrode (c-MWCNT/CPGE) with the detection limit of 1 × 10−7 mol L−1 [24], but no information of redox mechanism was ⁎ Corresponding authors. E-mail addresses: [email protected] (L. Zou), [email protected] (B. Ye).
http://dx.doi.org/10.1016/j.jelechem.2016.06.044 1572-6657/© 2016 Elsevier B.V. All rights reserved.
given, in addition, the preparation process of the modified materials was complicated. To improve the sensitivity and explore the mechanism of hyperin, new voltammetric sensor should be constructed. Graphene (Gr), a two-dimensional, single-layer sheet of sp2 hybridized carbon atoms, has attracted tremendous attention, owing to its exceptional physical properties, such as high electronic conductivity, good thermal stability and excellent mechanical strength [25]. These properties of Gr make them extremely attractive in the field of electrochemical sensor [26,27]. Chemical reduction of exfoliated graphene oxide (GO) was an efficient approach to production of Gr. However, the Gr sheets tend to form irreversible agglomerates through π-π stacking and Van Der Waals interaction in the process of reduction [28]. To avoid this problem, some functionalized reagents were introduced to the Gr surface, such as poly(diallyldimethylammonium chloride) (PDDA) [29]. PDDA, a widely used cationic polyelectrolyte, can effectively enhance the dispersion of Gr and preserve the structural properties of Gr [30, 31]. PDDA functionalized graphene (PDDA-Gr) possess good stability, high conductivity, efficient electron transfer ability, biocompatibility and good electrocatalytic activity [32]. Therefore, the PDDA-Gr can serve as a sort of favorable material for modified electrode. Some methods for preparing the PDDA-Gr were reported with their own advantages, such as cost-effective and eco-friendly in microwave method [33,34] and simplification in wet chemical method, which only need common chemical regents [35,36]. In this work, PDDA-Gr was prepared via a simple wet chemical method. The characterization of the composite was carried out using transmission electron microscope (TEM), UV–visible spectrophotometry (UV–vis),
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2.2. Preparation of PDDA-Gr Graphene oxide (GO) was prepared from natural graphite flakes by the Hummers method [37]. GO (100 mg) was dispersed in 100 mL water with ultrasonication for 5 h, PDDA (5 mL) was added to the yellow-brown dispersion with stirred vigorously for 1 h at room temperature. After that, hydrazine (1 mL) was added to the mixture. And then, the mixture was heated to 90 °C for 3 h. Subsequently, the mixture was centrifugated and washed with water for three times. Finally, the obtained PDDA-Gr was dried at 60 °C for 12 h. The Gr was synthesized in the same condition without PDDA. 2.3. Preparation of modified electrode
Fig. 1. The chemical structure of hyperin.
Fourier Transform infrared spectroscopy (FT-IR) and X-ray diffraction analysis (XRD). PDDA-Gr was used to fabricate a voltammetric sensor for determination of hyperin. Moreover, electrochemical behavior and the redox mechanism of hyperin at poly(diallyldimethylammonium chloride)-functionalized graphene modified electrode (PDDA-Gr/GCE) were investigated. Different linear relationships between peak current and hyperin concentration were obtained via differential pulse voltammetry (DPV) with different accumulation times. The detection limit reached 5 × 10−9 mol L−1 under the accumulation of 150 s. In addition, this voltammetric sensor was also successfully employed for detection of hyperin in pharmaceutical samples with satisfactory results.
PDDA-Gr was dispersed in water and sonicated for 1 h to obtain a uniformly black suspension (0.5 mg mL−1). Prior to the modification, the bare GCE was mechanically polished to a mirror-like with 0.3 μm alumina slurry, and subsequently sonicated with water, absolutely ethanol and dried in air naturally. The PDDA-Gr/GCE was established by depositing PDDA-Gr (5 μL) on a fresh GCE surface using a micro-injector, and then dried under an infrared lamp. For comparison, Gr/GCE was fabricated with the similar procedures. 2.4. Analytical procedure Before the experiment, the prepared PDDA-Gr/GCE was scanned 5 cycles between 0.0 V and 0.8 V with the scan rate of 0.1 V s−1 in 0.1 mol L−1 (pH 2.0) by cyclic voltammetry (CV). And then, a certain amount of standard stock solution of hyperin was added to 0.1 mol L−1 (pH 2.0), after that, accumulation of 150 s was performed under an open circuit. Because of the adsorptive character of hyperin on the electrode, it was necessary to refresh the electrode surface. After each measurement, the PDDA-Gr/GCE was scanned in 0.1 mol L−1 (pH 8.0) for 2 cycles between 0.0 V and 0.8 V using CV to renew the electrode surface.
2. Experimental 2.5. Sample solution preparation 2.1. Apparatus and reagents All electrochemical experiments were carried out using an CHI610A electrochemical system (Chenhua Instrument Company, Shanghai, China) with a three-electrode system, a bare GCE (d = 3 mm) or modified GCE as working electrode, a saturated calomel electrode (SCE) and a platinum wire served as reference and counter electrode, respectively. The type of pH meter was pHS-3C (Shanghai Techcomp Jingke Scientific Instruments, Shanghai, China). High performance liquid chromatography was performed on 1260 Infinity Quaternary LC System (Agilent Technologies Inc., Santa Clara, California, USA). Characterization of morphology and crystalline structure was performed by transmission electron microscopy (TEM, JEOL-2100 Hiroshima, Japan), x-ray powder diffraction (XRD, Rigaku D/max-IIIA, Tokyo, Japan), Infrared spectrogram was recorded on Infrared Spectrometer (Thermo Nicolet Corporation, Santa Clara, California, USA) and UV–vis spectra was obtained from a Lambda 35 UV–vis spectrometer (Pgeneral General Instrument, Beijing, China). Hyperin (≥ 98%) and PDDA (MW b 1,000,000, 35% in water) were purchased from Aladdin (http://www.aladdin-e.com). Natural graphite flakes with the average diameter of 200 mesh were obtained from the Sigma-Aldrich (Sigma-Aldrich, St. Louis, Missouri, USA). Hypericum perforatum was purchased from local drugstore. The standard stock solution of hyperin was prepared with methanol and kept in darkness at 4 °C. The supporting electrolyte of 0.1 mol L−1 PBS were prepared by mixing 0.1 mol L−1 NaH2PO4 with Na2HPO4. The lower pH value of PBS was adjusted with 0.1 mol L−1 H3PO4. All the other reagents used in experiments were analytical grade and used without further purification and all experiments were performed at room temperature.
A certain amount of Chinese herb Hypericum Perforatum (100 mg) were put in volumetric flask and dissolved with 10 mL methanol. Hyperin was extracted by ultrasonic for 3 h, then the suspension was centrifuged for 10 min at 5000 rpm, this step was repeated three times for ensuring extraction completely. All solution were put together and concentrated to 10 mL in fume hood for analysis. Before each measurement, a certain amount of 20 μL was diluted with supporting electrolyte. 3. Results and discussion 3.1. Characterization of PDDA-Gr Fig. 2A showed the UV–vis spectrum of GO (curve a) and PDDA-Gr (curve b). GO had a characteristic absorption peak at 230 nm (the π-π* transitions of C_C bonds) [38], while the PDDA-Gr exhibited a strong absorption peak at 270 nm (n → π* transitions of C_O bonds) [39], indicating that the GO was reduced [40]. Fig. 2B showed the IR spectra for GO (curve a), Gr (curve b) and PDDA-Gr (curve c). As expected, the IR spectrum for GO exhibited characteristic peaks at approximately 1724 cm−1 which represent the skeletal vibration of C_O, indicating the existence of oxygen functionalities at GO surface [41]. The other characteristic peaks were also showed in the spectra: O\\H (3434 cm−1), C_C (1625 cm−1), C\\O\\C (1216 cm−1) and C\\O (1050 cm−1). The IR of Gr and PDDA-Gr were different from the GO. The characteristic absorption bands of the oxygen functional group (C_O, C\\O\\C) decreased dramatically, which indicated that GO had been reduced to
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Fig. 2. (A) UV–vis absorption spectra of GO (a) and PDDA-Gr (b). (B) The FT-IR spectra of GO (a), Gr (b) and PDDA-Gr (c). (C) XRD patterns of GO (a) and PDDA-Gr (b).
Gr. In addition, some new bands were appearance in the PDDA-Gr: 2923 cm−1 (CHn) and 1113 cm−1 (C\\N), which consist with the characteristic bands of PDDA [42]. The results indicated the functionalization of Gr with PDDA. Fig. 2C showed the XRD images of GO (curve a) and PDDA-Gr (curve b). As expected, GO had characteristic peak at 2θ = 10° due to the introduction of oxygenated functional groups onto the carbon sheets corresponding to the (002) plane. The diffration peak at 2θ = 43° was associated with the hexagonal structure of carbon ( (100) plane). For PDDA-Gr, it was still keeping the hexagonal structure with a diffration peak at 2θ = 43°. However, the (002) plane shifted to greater angle (about 22°), which indicated the removal of oxygen-containing
functional groups. From this, we can conclude that the GO has been reduced to Gr completely [43]. The structure and morphology of GO, Gr and PDDA-Gr were analyzed using TEM. Fig. 3A showed the TEM of GO, which exhibited the typical wrinkle morphology with well dispersibility on the substrate without any aggregation. After chemical reduction process, Gr (Fig. 3B) exhibited obvious re-stacking effect due to strong π-π stacking interaction and Van Der Waals forces. The re-stacking effect could highly reduce the specific surface area of the material. In contrast, PDDA-Gr (Fig. 3C) remained a well dispersibility without clear re-stacking effect due to the surface protection of PDDA. The presence of PDDA not only could prevent the natural force of Gr layers stacking together, but also
Fig. 3. (A) The TEM of GO. (B) The TEM of Gr. (C) The TEM of PDDA-Gr.
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of Gr. However, in the case of PDDA-Gr/GCE (curve d), the redox peak currents of K3Fe(CN)6 increased dramatically with ΔEp of 60 mV. Much higher peak currents and relatively smaller ΔEp obtained on PDDA-Gr/GCE were mainly ascribed to its large surface area and positive surface charge, which provide more active site and electrostatic attraction for Fe(CN)36 −. Chronocoulometry was used to evaluate electrochemically effective area of the electrodes [44]. The electrochemically effective area of the modified electrodes were measured by single potential step chronocoulometry, the potential was stepped from −0.2 V to 0.6 V with the step time of 5 s. Fig. 5A was the chronocoulometry curves of K3Fe(CN)6 (1 × 10−4 mol L−1, containing 1 mol L−1 KCl) obtained at bare GCE (curve a), GO/GCE (curve b), Gr/GCE (curve c) and PDDA-Gr/GCE (curve d). The corresponding Q-t1/2 curves were shown in Fig. 5B. According to the formula of Anson theory [45]. Q¼
Fig. 4. Cyclic voltammograms of 1.0 × 10−3 mol L−1 K3[Fe(CN)6] containing 0.1 mol L−1 KCl at bare GCE (a), GO/GCE (b), Gr/GCE (c), PDDA-Gr/GCE (d).
provide repulsion between the sheets by its surface positive charges, which was favorable for forming a stable suspension. The observation of PDDA-Gr showed a general flake-like structure of Gr, indicating the surface functionalization did not change the morphology of the Gr. The UV–vis, IR, XRD and TEM data provide evidence that the PDDAGr were successfully synthesized. 3.2. The electrochemical characterization of PDDA-Gr/GCE Fig. 4 displayed cyclic voltammograms of K3Fe(CN)6 (1 × 10−3 mol L−1 containing 0.1 mol L−1 KCl) at different electrodes. A pair of redox peaks were observed on the GCE (curve a) with peak difference (ΔEp) of 79 mV. When GO was immobilized on the GCE surface (curve b), the redox peak currents decreased obviously with ΔEp of 100 mV. The reason of ΔEp increment can attribute to a large number of oxygen containing function groups on GO surface, which reduced the redox rate of K3Fe(CN)6. After modified with Gr (curve c), both the cathodic and anodic peak currents were increased with the ΔEp of 75 mV, which may contributed to the excellent electrical conductivity
2nFAcðDtÞ1=2 þ Q dl þ Q ads π1=2
where A is the area of electrode, D is diffusion coefficient, c is the concentration of the species, t is the potential pulse width, Qdl is the double-layer charge, Qads is the Faradaic charge. For 1 × 10− 4 mol L−1 K3Fe(CN)6, n = 1, D = 7.6 × 10−6 cm2 s−1. According to the slope of Q-t1/2 from Fig. 5B: Q (10− 4 C) = 0.023 t1/2 (s1/2) + 0.080 (R = 0.999), Q (10− 4 C) = 0.078 t1/2 (s1/2) + 0.20 (R = 0.999), Q (10−4 C) = 0.12 t1/2 (s1/2) + 0.70 (R = 0.998) and Q (10−4 C) = 0.13 t1/2 (s1/2) + 1.10 (R = 0.999), the A of GCE, GO/GCE, Gr/GCE and PDDAGr/GCE were calculated to be 0.080 cm2, 0.260 cm2, 0.399 cm2 and 0.433 cm2, respectively. The results further confirmed the enlarged effect of PDDA-Gr on the electrode surface area. 3.3. Electrochemical behavior of hyperin at the PDDA-Gr/GCE Fig. 6A illustrated the CV of hyperin (3 × 10−6 mol L−1) at bare GCE (b), Gr/GCE (c) and PDDA-Gr/GCE (e) in 0.1 mol L− 1 PBS (2.0). The curve a and curve d were the bare GCE and PDDA-Gr/GCE in blank solution. Fig. 6B was the enlarged curve a and curve b. For the blank solution of bare GCE (curve a) and PDDA-Gr/GCE (curve d), no redox peak was observed, meaning that there was no redox reaction of the PDDA-Gr membrane in a potential window of 0.0–0.8 V. From bare GCE (curve b), a very weak response was obtained at Epa = 0.527 V, Epc = 0.500 V. On the Gr/GCE, a pair of well-defined redox peaks were obtained with the Epa = 0.559 V and Epc = 0.500 V, the redox peak currents are significantly increased, which were about 10 times higher than at the bare GCE, which might be attributed to the excellent conductivity of Gr. Comparing with the GCE and Gr/GCE, the PDDA-Gr/GCE show the
Fig. 5. (A) Chronocoulometric curves of the 1 × 10−4 mol L−1 K3[Fe(CN)6] containing 1 mol L−1 KCl at the bare GCE (a), GO/GCE (b), Gr/GCE (c), PDDA-Gr/GCE (d). (B) The corresponding Q-t1/2 plots. The potential was stepped from −0.2 V to 0.6 V, step time: 5 s.
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Fig. 6. (A) Cyclic voltammograms of hyperin (3 × 10−6 mol L−1) at bare GCE (b), Gr/GCE (c) and PDDA-Gr/GCE (e) in 0.1 mol L−1 PBS 2.0, the bare GCE (a) and PDDA-Gr/GCE (d) in blank solution. (B) The enlarged curve a and curve b, scan rate 0.1 V s−1, accumulation time: 150 s.
best sensitivity response which were 20 times higher than at bare GCE with the Epa = 0.577 V and Epc = 0.510 V. This could be reasonably ascribed to the enlarged surface area, the extraordinary electron-transfer
properties of PDDA-Gr and the strong adsorption between hyperin and PDDA-Gr. So PDDA-Gr/GCE was selected as the voltammetric sensor in the following experiment.
Fig. 7. (A) Cyclic voltammograms of hyperin (3.0 × 10−6 mol L−1) with different supporting electrolytes at PDDA-Gr/GCE including 0.1 mol L−1 phosphate buffer (pH 3.0 curve a), acetate buffer (pH 3.6 curve b), Britton-Robinson (pH 3.78 curve c) and sulfuric acid (pH 3.03 curve d). (B) Cyclic voltammograms of hyperin (3.0 × 10−6 mol L−1) with different pH (a–g: 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0). (C) The relationship between the peak potentials and pH, scan rate 0.1 V s−1, accumulation time: 150 s.
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3.4. Optimization conditions 3.4.1. The influence of supporting electrolyte and pH To get the best response of hyperin (3 × 10−6 mol L−1) at PDDA-Gr/ GCE, the influence of different supporting electrolytes were investigated by CV (Fig. 7A), including 0.1 mol L−1 phosphate buffer (pH 3.0 curve a), acetate buffer (pH 3.6 curve b), Britton-Robinson (pH 3.78 curve c) and sulfuric acid (pH 3.03 curve d). In consideration of the peak shape and sensitivity, 0.1 mol L− 1 PBS was chosen as supporting electrolyte in the following experiments. The influence pH value of supporting electrolytes on the response of hyperin at PDDA-Gr/GCE was also investigated by CV. As shown in Fig. 7B, the redox currents changed with the increase of pH from 2.0 to 8.0. The redox peak potentials shifted negatively with the increase of pH from 2.0 to 8.0. It indicated that proton take part in the electrode reaction process [46]. From the relation of Ep and pH (Fig. 7C): Epa = 0.721–0.0586 pH (R = 0.992), Epc = 0.649–0.0581 pH (R = 0.997). According to the formula: dEp/dpH = 2.303mRT/nF (m is the number of proton, n is the number of electron), m/n was calculated to be 1 for the hyperin redox process. This result indicates that protons and electrons in a ratio of 1:1 in the electrode reaction process of hyperin. The other information obtained from this group of experiments was that the reductive peak almost disappeared when the pH value was equal to or higher than 7.0. These results demonstrate that the acidic condition leads to a more reversible electrochemical reaction of hyperin on the proposed electrode. It showed that the highest response of peak
current was obtained at pH 2.0, So 0.1 mol L−1 (pH 2.0) was selected as supporting electrolyte for the determination of hyperin. 3.4.2. Influence of scan rate To further explore the electrode reaction mechanism of hyperin, the influence of different scan rates were investigated by CV with scan rate changed from 0.03 to 0.35 V s−1 (Fig. 8A). It was found that the anodic current and cathodic currents were increased with the increase of scan rates. The linear relationship between ip and v were obtained (Fig. 8B): ipa (μA) = 168.08 + 7.667 v (V s−1) (R = 0.996), ipc (μA) = −127.78– 5.4151 v (V s−1) (R = 0.997), suggesting that the electrode reaction of hyperin was a adsorption-controlled process. Moreover, with the increase of scan rate, the anodic peak potential shifted positively, while the cathodic peak potential shifted negatively, as depicted in Fig. 8C, the anodic (Epa) and cathodic (Epc) peak potentials have a linear relationship with the Napierian logarithm of scan rate (lnv): Epa (V) = 0.0255 lnv (V s− 1) + 0.6386 (R = 0.994), Epc (V) = − 0.0208 lnv (V s−1) + 0.4657 (R = 0.992). According to the Formulas (1) and (2) [47], n was estimated to be 2, α = 0.55. In addition, the electron transfer rate constant ks = 1.16 s−1 was obtained from Formula (3) [47]. 0
RT ln v αnF
ð1Þ
0
RT ln v ð1−αÞnF
ð2Þ
Epc ¼ E0 − Epa ¼ E0 −
Fig. 8. (A) The voltammograms of hyperin (3.0 × 10−6 mol L−1) with different scan rates at PDDA-Gr/GCE (a–k: 0.03, 0.04, 0.06, 0.08, 0.1, 0.12, 0.15, 0.2, 0.25, 0.30, 0.35 V s−1) in 0.1 mol L−1 PBS 2.0, accumulation time: 150 s. (B) The relationship between ip and v. (C) Ep and lnv.
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Fig. 9. The proposed redox mechanism of hyperin at the PDDA-Gr/GCE.
logks ¼ α logð1−αÞ þ ð1−αÞ logα− log
RT nFΔEp −αð1−αÞ 2:3RT nFv
ð3Þ
According to the results, two electrons and two protons were involved in the redox reaction of hyperin. Therefore, a possible electrochemical reaction mechanism of hyperin at PDDA-Gr/GCE could be described as following: (See Fig. 9) 3.5. Chronocoulometry studies Considering of the adsorptive character of hyperin at the PDDA-Gr/ GCE, it is necessary to calculate the saturated adsorption capacity of hyperin at PDDA-Gr/GCE surface. The saturated adsorption capacity could be calculated according to the Laviron's theory of Qads = nFAΓ* [47]. The Qads of hyperin on PDDA-Gr/GCE was measured in 0.1 mol L−1 (pH 2.0) by single potential step chronocoulometry. The potential was stepped from 0.0 V to 0.8 V, step time 5 s. As shown in the Fig. 10A, the Q-t curves were recorded in the absence (curve a) and presence (curve b) hyperin solution (1 × 10−5 mol L−1) after stirring for 10 min to achieve saturated adsorption. The corresponding plots of Q against t1/2 were shown in Fig. 10B for oxidation process. The two straight lines were almost parallel, further implying the oxidation process of hyperin on the PDDA-Gr/GCE was driven by adsorption. According to the intercept, the value of Qads was calculated to be 1.730 × 10−4 C for the oxidation process. The saturated adsorption capacity Γ* of hyperin was calculated to 2.692 × 10− 9 mol cm− 1 for the oxidation process.
3.6. Analytical applications and methods validation 3.6.1. Influence of accumulation time For an adsorptive driven electrode process, a step of accumulation has a significant effect on the detection sensitivity. The relationship was recorded between accumulation time (tacc) from 30 s to 210 s and peak currents in a hyperin solution (3 × 10−6 mol L−1) via CV. Fig. 11 showed that with the increase of accumulation time, the responding anodic peak currents increased gradually up to 150 s, and then enhanced slowly. Considering the detection sensitivity and linear range, an accumulation time of 150 s was chosen to set up the calibration curve in following experiments. 3.6.2. Calibration curve, detection limit, repeatability and stability Under the optimum conditions described above, DPV was performed to investigate the relationship between the peak currents and the concentration of hyperin with the accumulation time of 150 s at the proposed electrochemical sensor. Oxidation peak current was recorded and used as the detected signal (Fig. 12A). As the Fig. 12B shown, a good linear relationship could be obtained between peak currents (ipa) and hyperin concentrations (C) in the range of 7 × 10−9 mol L−1–7 × 10−7 mol L−1. The linear regression equation was ipa (μA) = 44.04 C (μM) + 2.305 (R = 0.997) with a detection limit of 5 × 10−9 mol L−1 (S/N = 3). Table 1 lists the data for comparison of the present methods for the determination of hyperin. In this part, in order to further evaluate the influence of tacc on the determination, we chose a shorter time 30 s as tacc. As a result, the linear range was 2 × 10−8 mol L−1 to 6 × 10−6 mol L−1, which is wider than
Fig. 10. (A) Chronocoulometric responses curves of absence (a) and presence (b) of 1.0 × 10−5 mol L−1 hyperin in 0.1 mol L−1 PBS 2.0. (B) The corresponding Q-t1/2 plots of anodic peaks. The potential was stepped from 0.0 V to 0.8 V, step time: 5 s.
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The repeatability and stability of PDDA-Gr/GCE were evaluated by DPV in hyperin solution (3 × 10−6 mol L−1). A relative standard deviation (RSD) of the peak current from five electrodes was obtained of 2.9%, while the RSD of 3.0% was estimated with one electrode for 10 successive measurements. These results showed that the sensors exhibited repeatability and reproducibility. The stability of the sensor was evaluated by examining its response current after storage in dark two weeks, the current of hyperin reduced 4.9%, indicating the excellent stability.
Fig. 11. The relation between the anodic currents of hyperin (3 × 10−6 mol L−1) and the accumulation time (30 s, 60 s, 90 s, 120 s, 150 s, 180 s, 210 s.). Supporting electrolyte: 0.1 mol L−1 PBS 2.0, scan rate: 0.1 V s−1.
that of 150 s tacc. The linear regression equation was ipa (μA) = 7.40 C (μM) + 1.965 (R = 0.996) with a detection limit of 1 × 10−8 mol L−1 (S/N = 3). The results indicated that with the accumulation time, the sensibility can be increased, however, the liner range was narrow.
3.6.3. Interference studies Potential interferences of some possible inorganic and organic compounds in real samples were explored in 3 × 10−7 mol L−1 hyperin by DPV (Fig. 13). The results indicated that the inorganic ions: Ca2+, Cu2+, − Al3+, Mg2+, Zn2+, SO2− 4 , NO3 in 100 folds of hyperin had no interfering with the determination of hyperin. Moreover some organic compounds: ascorbic acid, glucose, dopamine in 20-fold of hyperin, it showed that b5.0% of the peak current variation for the detection of hyperin. Considering in natural products, there are some other flavonol compounds coexisting with hyperin. So, the electrochemical behaviors of other flavonol compounds were investigated. The results showed that the hesperetin and diosmin in 20 folds of hyperin were no interfering. The rutin and quercetin in 6-folds of hyperin were no interfering. All these results indicated the excellent anti-interference of the sensor. 3.6.4. Real sample analysis In order to validate the practical application of this proposed method, the fabricated PDDA-Gr/GCE was employed to determine the concentration of hyperin in an actual sample (Chinese Herba Hypericum perforatum). The sample treatment processes were described in the
Fig. 12. (A) The DPV curves of different concentrations of hyperin at PDDA-Gr/GCE under optimum conditions, accumulation time: 150 s. Hyperin concentrations: 7.0 × 10−9, 1.0 × 10−8, 4.0 × 10−8, 6.0 × 10−8, 8.0 × 10−8, 1.0 × 10−7, 3.0 × 10−7, 5.0 × 10−7 and 7 × 10−7 mol L−1. (B) The relationship of peak current with hyperin concentration.
Table 1 Comparison of different methods for determination of hyperin (the concentration unit in the references cited had been converted to mol L−1). Method
Sample pre-treatment
Linear range (10−6 mol L−1)
Detection limit (10−6 mol L−1)
Reference
HPLC HPLC HPLC HPLC HPLC RPHPLC Capillary electrophoresis Capillary electrophoresis Capillary electrophoresis c-MWCNT/CPGE PDDA-Gr/GCE
Extraction and separation Extraction and separation Extraction and separation Extraction and separation Extraction and separation Extraction and separation Extraction and separation Extraction and separation Extraction and separation Extraction Extraction
2.15–193.82 9.39–1174.60 1.08–21.53 21.53–215.34 10.76–215.34 2.15–129.20 21.535–107.67 2.76–1119.79 14.45–1076.73 0.22–30.15 0.0070–0.070
0.71 9.39 1.08 0.15 1.14 0.0082 0.13 2.76 6.82 0.10 0.0050
13 15 16 17 18 20 21 22 23 24 This work
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Acknowledgements The authors are very grateful for the financial support from the National Natural Science Foundation of China (Grant no. 21575130; 21275132; U1504216). References
Fig. 13. Column chart of the oxidation peak current of 3 × 10−7 mol L−1 hyperin in 0.1 mol L−1 PBS 2.0, the PBS solution containing metal ions and some organic compounds and other flavonol compounds.
experimental section. After determination of the hyperin content in sample solution, the recovery tests were carried out using the standard addition method and the results were listed in Table 2. And the average content was calculated to be 2.0816 mg/g with RSD of 3.4% for three repeated measurements. For testing the accuracy of the proposed method, the same samples were analyzed using HPLC method and the results showed that there was no significant difference between them.
4. Conclusion In summary, PDDA-Gr composite was synthesized via a simple wet chemical method and applied in the fabrication of electrochemical sensor for the determination of hyperin. The high specific surface area, excellent conductivity and the strong adsorption of PDDA-Gr for hyperin affords excellent sensitivity. The electrochemical behaviors of hyperin on the PDDA-Gr/GCE were studied in detail. Otherwise the sensor exhibited a low detection limit of 5 × 10−9 mol L−1 (S/N = 3) and a wide linear detection range of from 7 × 10−9 to 7 × 10−7 mol L−1 by DPV with excellent reproducibility, repeatability and long-term stability. This study demonstrated that PDDA-Gr/GCE would be a promising sensor and useful for the hyperin analysis in biological samples.
Table 2 Determination results of hyperin in Chinese Herba Hypericum perforatum sample by DPV and HPLC. DPV
HPLC
Sample Addeda (μL) (10−7 mol
20
a
Founda (10−7 mol
L−1)a
L−1)
0 2 4
0.8965 2.882 4.901
Recovery RSD (%) (%)
Founda (10−7 mol
RSD (%)
L−1) 99.16 100.5
Average value of three replicate measurements.
3.4 2.0 2.1
0.8750
0.69
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