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Fabrication of Electrochemical Sensor Modified with Porous Graphene for Determination of Trace Calycosin
CHINESE JOURNAL OF ANALYTICAL CHEMISTRY Volume 47, Issue 2, February 2019 Online English edition of the Chinese language journal
Cite this article as: Chinese J. Anal. Chem., 2019, 47(2): 271–280
RESEARCH PAPER
Fabrication of Electrochemical Sensor Modified with Porous Graphene for Determination of Trace Calycosin SUN Bo-Lu1, CAI Jin-Ying1, LI Dai1, GOU Xiao-Dan2, GOU Yu-Qiang3, LI Wen1,*, HU Fang-Di1,* 1
School of Pharmacy, Lanzhou University, Lanzhou 730000, China School of Chemistry and Chemical Engineering, Nanjing University 210046, China 3 Lanzhou Military Command Center for Disease Prevention and Control, Lanzhou 730000, China 2
Abstract:
In this study, the porous graphene (PG) with excellent structure was successfully prepared by a simple pyrolysis process,
and applied to construct electrochemical sensor (PG@GCE) for detection of calycosin (CYS). PG was characterized by Raman spectroscopy, Fourier transform infrared spectroscopy and scanning electron microscopy. Also, the electrochemical properties of the proposed sensors were characterized by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS), and the results indicated that sensors had relatively large specific surface area and higher electron transport rate. Based on all those characteristics, CYS, a traditional Chinese medicine (TCM) active ingredient, displayed a great response on the surface of PC@GCE. Under the optimized conditions, the sensors displayed a good linear relationship between the peak current and the CYS concentration in the range of 1.8 × 10–7–4.4 × 10–5 M with the detection limit of 5.8 × 10–8 M (S/N = 3). This study provided a novel analytical method for detection of CYS, rapid identification of Radix Hedysari and Radix Astragali, and gave another way for the trace analysis of CYS in biological samples. Furthermore, it would also deepen the application of PG in the field of pharmaceutical analysis. Key Words:
1
Porous graphene; Calycosin; Electrochemical sensor; Trace analysis
Introduction
Calycosin (CYS), as the index component used to assess the quality of Radix Hedysari and Radix Astragali, could bind to the estrogen receptor on the cell membrane, and modulate mitogen activated protein kinase signaling pathway[1]. CYS has important significance for cancer treatment[2], inflammation reduction[3], improvement of diabetic cognitive impairment[4] and the remission of cardiovascular disease[5]. Therefore, it is of great significance to establish a simple, fast and precise method for analysis of CYS content in herbal materials, herbal preparations and human blood with the minimum blood concentration to realize the quality control of Chinese medicine and the diagnosis of diseases. CYS, also called 3′,7-dihydroxy-4′-methoxyisoflavone, is a
typical isoflavone compounds. It has a p-π conjugation system between B ring and C2=C3, the 4-carbonyl group and the A ring, which effectively promotes the electron delocalization and results in rising of the activity of 3’-OH[6], so that CYS is prone to be involved in oxidation and reduction reaction. Therefore, the electrochemical method can be used to detect the electrochemical signal and rapidly analyze CYS in the samples. Currently, the most common methods for detection of CYS include chromatography[7–9] and chromatographymass spectrometry[10,11]. However, these methods are not conducive for the popularization due to time-consuming and complex preprocessing, even requirement of professional technicians. While, as a novel analytical technique with high selectivity, low cost, convenient operation, miniaturized equipment, simple pretreatment and high automation[12–14],
________________________ Received 5 September 2018; accepted 28 November 2018 *Corresponding author. Email: [email protected]; [email protected] This work was supported by the Major Science and Technology Project of Gansu Provincial Science and Technology Department, China (No. 17ZD2FA009), the Patent Conversion Project of Lanzhou Science and Technology Bureau, China (No. 2017-4-119), the Achievement Transformation Base Project of Lanzhou, China (No. 2016-2-80), the National Key Research and Development Program of China (No. 2018YFC1706300), and the National Traditional Chinese Medicine Standardization Project (No. ZYBZH-Y-GS-10). Copyright © 2019, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences. Published by Elsevier Limited. All rights reserved. DOI: 10.1016/S1872-2040(19)61141-2
SUN Bo-Lu et al. / Chinese Journal of Analytical Chemistry, 2019, 47(2): 271–280
electroanalytical technique has been used in the field of water pollution monitoring, food safety and quality evaluation of herbal medicinal products[15–18]. However, there are few reports about other electrochemical analysis for the detection of CYS so far. Thus, a simple electrochemical analytical technique for real-time, efficient determination of CYS is highly desirable. Porous graphene (PG), as a new type of graphene derivatives, has excellent dispersibility in aqueous phase because it overcomes the issues of accumulation and selfaggregation, which is caused by the strong π-π stacking of graphene[19,20]. Therefore, PG has a large specific surface area and mechanical stability compared to graphene. Furthermore, the pore structure in PG facilitates rapid movement of charge carrier, which makes this material have excellent electrical conductivity[20,21]. According to reports in the literature, PG has been widely used in the field of sensors and electronic devices[22–24]. Yang et al[25] constructed a glucose sensor based on CuCo2O4 polyhedron/porous reduced graphene oxide (PrGO) composite with hollow structure with a detection limit of 0.5 μM (S/N = 3) for glucose. Dong et al[26] proposed a novel electrochemical sensor for the detection of dopamine, which was fabricated by PG using chemical vapor deposition, and this sensor eliminated the interference of uric acid, a common interferent to dopamine detection. However, as we know, there has been no report about the sensor fabricated by PG used to detect traditional Chinese medicine (TCM) with electrical activity in complex systems. In this study, PG was prepared by high-temperature synthesis and water cracking. In addition, glassy carbon electrode (GCE)
Fig.1
was successfully modified with PG to fabricate PG@GCE, which was used to investigate electrochemical behavior of CYS. Finally, determination of trace CYS in real samples was achieved with satisfactory results (Fig.1).
2
Experimental
2.1
Instruments and reagents
Electrochemical measurements were performed on a CHI 6041E workstation (Shanghai Chenhua Company, China) with a conventional three-electrode system. A bare or modified GCE was used as working electrode. A saturated calomel electrode (SCE) and a platinum wire electrode were used as reference electrode and auxiliary electrode, respectively. pHs-3B digital pH-meter (Shanghai Precision Scientific Instrument, China), KQ-50 Ultrasonic Cleaners (Kun Shan Ultrasonic Instruments Co., Ltd), JSM-6701F Cold field emission type sweeping mirror (Japan electronics optics corporation), Finder One Micro-zone laser Raman spectrometer (Beijing Zolix Instrument, China) and Nicolet IS5 Fourier transform infrared spectroscopy (FTIR) (Thermo, US) were used in this study. CYS was purchased from Baoji Chenguang Biotechnology Co., Ltd. All chemical reagents were of analytical pure grade, and all solutions were prepared with distilled water. 2.2 2.2.1
Experimental method Preparation of PG
Schematic diagram of the synthesis of PG and the fabrication process of the PG@GCE for determination of CYS
SUN Bo-Lu et al. / Chinese Journal of Analytical Chemistry, 2019, 47(2): 271–280
PG was prepared by pyrolysis method[27]. 2 g of Na and 5 mL ethanol (the molar ratio is 1:1) were added into stainless steel reactor. After evenly mixing, the reactor was heated under 220ºC for 48 h. The mixture naturally cooled at room temperature, and a white product was obtained. The crude product PG (black) was obtained by pyrolysis with deionized water. The generated product was washed with deionized water several times, then a lyophilization process was carried out to get the final product, which was dissolved in isopropyl alcohol to get 2 mg mL–1 PG solution for subsequent experiments. 2.2.2
Pretreatment of Radix Astragali and Hedysari Radix The mixture of Radix Astragali powder (4 g) and methanol (40 mL) was added into a 250-mL round-bottom flask for extraction at 80 ºC under reflux for 1 h twice, followed by filtering, merging of filtrates, and concentration of filtrates to 10 mL. Radix Hedysari was treated with the same method. Pretreatment of biosample 5 mL blood was sampled from rat carotid artery, and kept it the heparin anticoagulant tubes at 4 ºC. Then the obtained 5 mL anticoagulant plasma (added with a certain amount of CYS) was centrifuged at 4000 rpm for 15 min. The isolated supernatant was measured by the proposed sensors.
Preparation of modified electrode
3 The bare GCE was carefully polished with 0.3 and 0.05 μm Gamma alumina powder (γ-Al2O3) on chamois leather, and then ultrasonically washed with water and ethanol several times. The electrode was dried by natural drying. Then, 5 μL PG solution (2 mg mL–1) was dripped on the bare GCE, the PG@GCE electrode was obtained. The resulting PG@GCE was washed with water and stored at 4 ºC until use. 2.2.3 Electrochemical measurements of modified electrode Cyclic voltammetry (CVs) with potential scanning rang from –0.2 V to 0.8 V and a sweep rate of 100 mV s–1 as well as electrochemical impedance spectroscopy (EIS) with amplitude of 0.005 V, voltage of 0.2 V and frequency range from 0.1 Hz to 105 Hz were applied in solutions containing 5.0 mM K3[Fe(CN)6]/K4[Fe(CN)6] and 0.1 M KCl to investigate electrochemical characters of different modified electrodes. 2.2.4
Pretreatment of samples
3.1
Results and discussion Characterization of PG@GCE
The morphology of the graphene oxides (GO) and PG were characterized by SEM. As shown in Fig.2A, GO were partially packed with their basal planes, intensely crumpled, and folded into a typical wrinkled structure. The surface of PG (Fig.2B) and its interior (Fig.2C) exhibited hollow network structure, and a large number of pores served as the support of PG structure, effectively preventing aggregation of the graphene nanosheet layer[28]. Therefore, the specific surface area of the PG was greatly increased, and more active sites were provided for enrichment of small molecule electroactive compounds and adsorption of biomolecules. In addition, the porous network structure of PG provided the possibility of rapid movement of charge carriers in their continuous and interconnected pore sizes, and played an important role in increasing the sensitivity of electrochemical sensor detection[27].
Fig.2 SEM images of GO (A) and PG (B, C); (D) FT-IR spectra of GO and PG; (E) Raman spectra of GO and PG
SUN Bo-Lu et al. / Chinese Journal of Analytical Chemistry, 2019, 47(2): 271–280
GO and PG were characterized by FTIR. As shown in Fig.2D, the infrared absorption of PG (Curve b) at 3000–3700 cm–1 was significantly lower than that of GO (Curve a), which was due to the small number of oxygen-containing functional groups carried on the surface of the PG. In addition, the absorption peaks of GO at 1400 and 1048 cm–1 correspond to the characteristic absorption of O‒H bond and C-O bond, respectively[29], while the characteristic absorption peak of PG here was obviously weakened or even disappeared, indicating that PG prepared in this study had a lower degree of oxidation. As shown in Fig.2E, two distinct peaks of GO (Curve a) at 1346 cm–1 and PG (Curve b) at 1580 cm–1 are observed, corresponding to D and G bands, respectively. The G band is the first order Raman scattering of the E2g optical mode, which corresponds to the ordered sp2 structure. The D band is the A1g phonon mode of the K-point of the Brillouin zone, corresponding to the degree of disorder and the local defects of the edge. The signal intensity ratio of D peak to G peak (r = ID/IG) can be used to measure the degree of defects and chaos of the material. The D band is caused by the conversion of sp2 hybridization to sp3, which affects the ordering[30]. By calculating the intensity ratios of D and G peaks of GO and PG (rGO = 0.780, rPG = 1.05), it indicated that the prepared PG had relatively more defect structures, which were closely related to the hollow network structure of PG. This result was consistent with the SEM characterization results. And the results further proved that PG was successfully synthesized.
coefficient of 0.5 mM K3[Fe(CN)6] solution containing 1.0 M KCl is 1.39 × 10−4 cm2 s–1), n is the electron transfer number, Qdl is the electric charge of the electric double layer, Qads is the induced electric charge, A is the effective surface area of the electrode, and F is the Faraday constant. As can be seen from Fig.3B, Q and t1/2 have a good linear relationship. According to the slope of the linear equation, the effective surface area of GCE, GO@GCE and PG@GCE were calculated as 0.16, 0.23 and 0.47 cm2, respectively. Attributing to the increased effective surface area of PG@GCE, the amount of CYS accumulated on the electrode surface was larger, which enhanced the electrochemical response signal of CYS.
3.2
3.4
Calculation of electrochemical effective surface area
Using K3[Fe(CN)6] as a probe molecule, the effective surface areas of GCE, GO@GCE and PG@GCE electrodes were studied by chronocoulometry with the electrochemical experiment parameters set as follows: starting potential, 0.5 V; termination potential, –0.2 V; pulse width, 0.25 s; and sampling interval of 2.5 × 10−4 s. According to Anson equation[31]: Q(t) = 2nFAc(Dt/π)1/2 + Qdl + Qads (1) where, c represents the concentration of the substrate in the electrolyte, D is the diffusion coefficient (the diffusion
3.3
Electrochemical characterization of the sensor
The fabrication process of the sensor was characterized by EIS and CV. As shown in Fig.4A, the background current of PG@GCE is larger than that of GCE, indicating that PG@GCE had excellent electrical conductivity and larger effective surface area. This phenomenon should be ascribed to PG with interconnected multilayer networks structure, which increased the surface layer and improved the electron exchange efficiency. The construction process of the sensor was also characterized by EIS, as shown in Fig.4B. Compared with GCE, PG@GCE showed a lower electron-transfer resistance (Ret), indicating that PG increased the electron exchange efficiency. This was probably due to the outstanding electric conductivity of PG. Electrochemical response of CYS
The effect of the buffer solution on the sensor response was tested in three different buffer solutions (NaAc-HAc buffer solution, phosphate buffered solution and citrate buffer solution) with the concentration of 0.1 M. A stable baseline and high sensitivity were achieved in NaAc-HAc buffer. Therefore, NaAc-HAc buffer solution was chosen as the supporting solution. Figure 4C shows CVs of GCE and PG@GCE in NaAc-HAc buffer solution (0.1 M, pH 7.0) with 28 μM CYS or without CYS at scan rate of 100 mV s–1. No redox peak on GCE
Fig.3 (A) Q-t curves of three electrodes in 0.5 mM K3[Fe(CN)6]/1.0 M KCl solution; (B) Q-t1/2 linear plot of the electrode
SUN Bo-Lu et al. / Chinese Journal of Analytical Chemistry, 2019, 47(2): 271–280
Fig.4
(A) The cyclic voltammetry (CV) curves of GCE and PG@GCE in 0.5 mM [Fe(CN)6]3−/4− (containing 0.1 M KCl) solution, Scan rate: 100 mV s–1; (B) Electrochemical impedance spectroscopy (EIS) of the GCE and PG@GCE in 0.5 M [Fe(CN)6]3−/4− (containing 0.1 M KCl); (C) CVs of GCE and PG@GCEin NaAc-HAc buffer solutionand NaAc-HAc buffer solution containing 2.8 × 10−5 M CYS; (D) CVs of GCE in NaAc-HAc buffer solution and NaAc-HAc buffer solution containing 2.8 × 10−5 M CYS
and PG@GCE was observed in the NaAc-HAc buffer solution without CYS. After CYS was added, the CV curve of bare GCE presented a relatively weak oxidation and reduction peaks of CYS, while the oxidation and reduction peaks of PG@GCE were significantly enhanced at Epa = 0.230 V and Epc = 0.412 V, ∆Ep ＝ 182 mV, demonstrating that CYS had a good electrochemical response on PG@GCE. The voltammetric response of PG@GCE to CYS was much more sensitive than that of GCE under the same conditions. These results might be ascribed to the network structure and electrical conductivity of PG, which effectively increased the specific surface area and significantly promoted the rapid propagation of electrons on the electrode surface. 3.5 3.5.1
Optimization of experimental conditions Effect of immobilized amount of PG
The immobilized amount of PG on the surface of sensor influenced oxidation and reduction behaviors of CYS. As shown in Fig.5A, the oxidation peak current of CYS increased with the amount increasing from 0 to 5.0 μL, and then decreased with further increasing, indicating that if the modified film was too thick, it would increase the resistance of interface electron transfer and affect the sensitivity of sensors. Thus, 5 μL of PG dispersion was chosen in further experiments.
3.5.2
Effect of pH value
The peak potentials and the peak currents were closely linked to the pH value of NaAc-HAc buffer solution. Figure 5B shows the electrochemical response of CYS on PG@GCE in the buffer solution with different pH values (3.0–8.0). With the increase of pH value, the redox peaks negatively shifted. In addition, the oxidation peak current conversely decreased when pH further increased. This result indicated the electrochemical process including some protons transfer. A good linear relationship between Epa and pH was expressed as Epa (V) = 0.4343 – 0.04464pH, R2 = 0.9903 (Fig.5D). According to Nernst equation[32] Epa = Eθ – [(2.303mRT)/(nF)] pH, the ratio of m/n was calculated as 0.924. This result indicated that protons and transferred electrons were identical during the electrode reaction. Moreover, a maximum of the oxidation peak current was achieved at pH 4.0 (Fig.5C). Thus, pH 4.0 was chosen for subsequent experiments. 3.5.3 Effect of scan rate The CVs behavior of CYS on PG@GCE at different scan rates was investigated (Fig.5E). The peak currents of CYS increased with the increasing of scan rates. Moreover, the oxidation peak currents (Ipa) for CYS increased linearly with the square root of scan rate from 10 mV s–1 to 300 mV s–1 (Fig.5F), and the linear relationship was expressed as Ipa (μA) = 3.4492υ1/2 (mV s–1)1/2 – 7.25897 with R2 = 0.9974. The results
SUN Bo-Lu et al. / Chinese Journal of Analytical Chemistry, 2019, 47(2): 271–280
indicated that the electrochemical reaction of CYS on the sensor was a diffusion-controlled process. At high scan rate (200‒300 mV s–1), a linear relationship was found between the redox potential and the logarithm of scan rate (Fig.5G), the regression equations was Epa (V) = 0.2003lgυ + 0.5787 (Epa: V, υ: V s–1), R2 = 0.9968; Epc (V) = 0.0050 − 0.2567lgυ (Epc: V, υ: V s–1), R2 = 0.9925. According to Laviron’s equations[33,34]: Epa = Eθ′ + 2.303RT/[(1 ‒ α)nF]lgv (2) Epc = Eθ′ － 2.303RT/(αnF)lgv (3) lgks = αlg(1 ‒ α) + (1 ‒ α)lgα ‒ lg(RT/nFv) ‒ α(1 ‒ α)nFΔEp/2.303RT (4) Hence, the electron-transfer coefficient (α), electrontransfer number (n) and electrode reaction rate constant (ks) were calculated as 0.43, 0.53 and 0.67 s–1, respectively, indicating that CYS had a rapid electron transfer process on PG@GCE surface. This was because the large specific surface area of PG could increase the adsorbing capacity of CYS on the electrode. In addition, the superior conductivity of PG effectively promoted the electron transfer on the electrode surface, thereby enhancing the electrochemical response of CYS. Also, PG provided a microenvironment for electron transport of CYS, which promoted the electrochemical
reaction of CYS. Based on those reasons, the proposed sensor realized the sensitive determination of CYS. A possible reaction mechanism of CYS in PG@GCE is shown in Fig.1. 3.5.4
Effect of accumulation time
The proposed sensor was placed in NaAc-HAc buffer solution containing 28 μM CYS. CYS was enriched on the surface of the sensor using open circuit potential every 30 s, and the CV curves of CYS at PG@GCE were obtained under different accumulation time. As shown in Fig.5H, the oxidation peak current increased rapidly before 300 s, whereafter, the peak current increased slowly with accumulation time prolonging. Therefore, accumulation for 300 s was adopted. 3.6
Calibration curve and detection limit
Under the optimal conditions, differential pulse voltammetry (DPV) was adopted to study the anodic peak current response of CYS on PG@GCE. As shown in Fig.6, the oxidation peak current had a good linear relationship with CYS
Fig.5 (A) Plots of anodic peak currents against volume of modification solutions; (B) CV curves of CYS at different pH values; (C) relationship between the formal redox potentials and the pH values; (D) relationship between anodic peak currents and pH values; (E) CV curves of CYS at different scan rates; (F) relationship between peak currents and square root of scan rates (ν1/2); (G) plots of anodic and cathodic potentials against logarithm of scan rates; (H) relationship between anodic peak currents and the accumulation time
SUN Bo-Lu et al. / Chinese Journal of Analytical Chemistry, 2019, 47(2): 271–280
Fig.6 (A) Differential pulse voltammograms of different concentrations of CYS (a to f: 0.176, 9.560, 18.421, 27.280, 36.133 and 44.012 μM, respectively) on PG@GCE; (B) calibration curve between the anodic peak currents and the concentration of CYS
concentration in the range of 1.8 × 10–7 – 4.4 × 10–5 M, and the linear regression equation was Ipa (μA) = 0.2609C (μM) + 0.1187 (R2 = 0.9928). The detection limit for CYS was calculated to be 5.8 × 10–8 M based on a signal-to-noise ratio of 3. Table 1 shows comparison of analytical performance of this CYS sensor with other methods reported previously, and it can be seen that the PG@GCE was comparable or superior to some reported methods. Therefore, the PG@GCE sensor could serve as a good platform for determination of CYS. 3.7
Reproducibility, stability and selectivity of the sensor
Five PG/GCE electrodes were prepared in parallel, and the reproducibility was investigated by measuring the oxidation peak current in the 10 mL NaAc-HAc buffer (pH 4.0) containing 28 μM CYS. The relative standard deviation (RSD)
was 4.4%, indicating that the proposed sensor had a good reproducibility. The stability was also investigated by measuring the oxidation peak current at interval of one week. During the investigation period, the sensor was stored in a refrigerator at 4 °C. The sensor retained 95.7% of the original response. A series of solutions added with various inorganic ions and molecules as the interferents into 10 mL NaAc-HAc buffer solution (pH 4.0) containing 28 µM CYS was used to investigate the selectivity of the sensor. It was found that 2.8 × 10–4 M quercetin, formononetin, 1.4 × 10–3 M glycine, glutamic acid, L-rhamnose monohydrate, sucrose and 2.8 × 10–3 M inorganic ions of Fe3+, Cu2+, Mg2+, Ca2+, Cl– and SO42– had no influence on the CV signals of CYS. As shown in Fig.7, the performance of the sensor was not obviously affected by those interferents.
Table 1 Comparison of different analytical methods for detection of CYS Detection methods
Linear range (M)
Detection limit (M)
References
HPLC
1.09 × 10–5–8.70 × 10−4
—
6
LC-MS/MS
1.24 × 10–8–7.96 × 10−7
2.2 × 10–9
9
LC-DAD-TOF/MS
7.04 × 10−7–1.83 × 10–4
—
10
DPV (gold electrode/GCE)
3.5 × 10–7–7.04 × 10−6 7.04 × 10–6–7.04 × 10−5
—
35
–6
−4
HPLC
5.6 × 10 –7.0 × 10
DPV (Porous carbon@GCE)
1.76 × 10–7–4.4 × 10−5
—
Present
5.78 × 10–8
Present
Fig.7 Interference of different species to DPV determination of CYS with the proposed sensor (1: Quercetin, 2: Formononetin, 3: Glycine, 4: Glutamic acid, 5: L-rhamnose, 6: Sucrose, 7: Trace element (Fe3+, Cu2+, Mg2+, Ca2+, Cl– and SO42–), 8: CYS, 9: Quercetin + CYS, 10: Formononetin + CYS, 11: Glycine + CYS, 12: Glutamic acid + CYS, 13: L-rhamnose + CYS, 14: Sucrose + CYS, 15: Trace element + CYS)
SUN Bo-Lu et al. / Chinese Journal of Analytical Chemistry, 2019, 47(2): 271–280
Table 2 Detection of CYS in Astragali Radix and Hedysari Radix using PG@GCE Found (mg)
Added (mg)
Total found (mg)
Recovery (%)
RSD (%, n = 3)
1
0.00505
0.0375
0.04345
102.4
1.7
2
0.01009
0.0375
0.04885
103.4
0.5
3
0.01514
0.0375
0.05123
96.2
1.5
1
0.00153
0.0375
0.03888
99.6
1.2
2
0.00306
0.0375
0.04080
100.6
1.7
3
0.00459
0.0375
0.04247
101.0
0.6
Sample Radix Astragali
Radix Hedysari
The above results showed that PG@GCE had good selectivity, stability and reproducibility, and could be applied to the determination of CYS in the complex sample. 3.8
Table 3 Detection of CYS in plasma samples using PG@GCE Biological sample
Added (mg)
Measured (mg)
Recovery (%)
RSD (%, n = 3)
1 2 3
0.0510 0.0306 0.0102
0.0524 0.0291 0.0101
102.7 95.0 99.2
0.2 0.6 1.1
Analytical applications
3.8.1
Determination of CYS in Radix Astragali and Radi Hedysari
The standard addition experiments were then carried out by adding successive concentrations of CYS. As shown in Table 2, 100 μL of the test samples (Radix Astragali or Radi Hedysari) was added into 10 mL NaAc-HAc buffer solution (0.1 M, pH = 4.0), and the concentration of CYS was adjusted in the linear range of the PG@GCE. The concentration of the test samples (Radix Astragali or Radi Hedysari) was also tested by HPLC method. The recovery for the determination of CYS was in the range of 96.2%–103.4%, which indicated that the proposed CYS sensor was suitable for determination of CYS in complex samples.
enrichment. Also, the porous structure effectively accelerated the electrons transfer rate on the surface of the sensor. The electrochemical response of CYS at the surface of PG@GCE increased significantly. Under the optimal conditions, PG@GCE displayed satisfactory analytical performance with wide linear range, low detection limit, superior selectivity and high stability for detection of CYS. Furthermore, the application of PG@GCE to detect CYS in Radix Astragali, Radix Hedysari and plasma sample exhibited satisfactory results. This study provided a novel analytical method for rapid evaluation of the quality of Radix Astragali, Radix Hedysari and real-time analysis of trace CYS in biological samples. It also demonstrated that PG could be extensively applied in designing high performance electrochemical sensors for drug analysis.
3.8.2
References
Determination of CYS in biological samples
The PG@GCE was also used to measure CYS concentration in plasma samples. 5 mL pretreated plasma samples solution with CYS was diluted to 10 mL with NaAc-HAc buffer (pH 4.0), then detected by DPV with standard addition method. The results are shown in Table 3. The sensor expressed a sensitive, simple and fast detection for CYS in biological samples with satisfactory recoveries from 95.0% to 102.7%, clarifying that the proposed method could be used to measure CYS in plasma samples.
4
Conclusions
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Cite this article as: Chinese J. Anal. Chem., 2019, 47(2): 271–280
RESEARCH PAPER
Fabrication of Electrochemical Sensor Modified with Porous Graphene for Determination of Trace Calycosin SUN Bo-Lu1, CAI Jin-Ying1, LI Dai1, GOU Xiao-Dan2, GOU Yu-Qiang3, LI Wen1,*, HU Fang-Di1,* 1
School of Pharmacy, Lanzhou University, Lanzhou 730000, China School of Chemistry and Chemical Engineering, Nanjing University 210046, China 3 Lanzhou Military Command Center for Disease Prevention and Control, Lanzhou 730000, China 2
Abstract:
In this study, the porous graphene (PG) with excellent structure was successfully prepared by a simple pyrolysis process,
and applied to construct electrochemical sensor (PG@GCE) for detection of calycosin (CYS). PG was characterized by Raman spectroscopy, Fourier transform infrared spectroscopy and scanning electron microscopy. Also, the electrochemical properties of the proposed sensors were characterized by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS), and the results indicated that sensors had relatively large specific surface area and higher electron transport rate. Based on all those characteristics, CYS, a traditional Chinese medicine (TCM) active ingredient, displayed a great response on the surface of PC@GCE. Under the optimized conditions, the sensors displayed a good linear relationship between the peak current and the CYS concentration in the range of 1.8 × 10–7–4.4 × 10–5 M with the detection limit of 5.8 × 10–8 M (S/N = 3). This study provided a novel analytical method for detection of CYS, rapid identification of Radix Hedysari and Radix Astragali, and gave another way for the trace analysis of CYS in biological samples. Furthermore, it would also deepen the application of PG in the field of pharmaceutical analysis. Key Words:
1
Porous graphene; Calycosin; Electrochemical sensor; Trace analysis
Introduction
Calycosin (CYS), as the index component used to assess the quality of Radix Hedysari and Radix Astragali, could bind to the estrogen receptor on the cell membrane, and modulate mitogen activated protein kinase signaling pathway[1]. CYS has important significance for cancer treatment[2], inflammation reduction[3], improvement of diabetic cognitive impairment[4] and the remission of cardiovascular disease[5]. Therefore, it is of great significance to establish a simple, fast and precise method for analysis of CYS content in herbal materials, herbal preparations and human blood with the minimum blood concentration to realize the quality control of Chinese medicine and the diagnosis of diseases. CYS, also called 3′,7-dihydroxy-4′-methoxyisoflavone, is a
typical isoflavone compounds. It has a p-π conjugation system between B ring and C2=C3, the 4-carbonyl group and the A ring, which effectively promotes the electron delocalization and results in rising of the activity of 3’-OH[6], so that CYS is prone to be involved in oxidation and reduction reaction. Therefore, the electrochemical method can be used to detect the electrochemical signal and rapidly analyze CYS in the samples. Currently, the most common methods for detection of CYS include chromatography[7–9] and chromatographymass spectrometry[10,11]. However, these methods are not conducive for the popularization due to time-consuming and complex preprocessing, even requirement of professional technicians. While, as a novel analytical technique with high selectivity, low cost, convenient operation, miniaturized equipment, simple pretreatment and high automation[12–14],
________________________ Received 5 September 2018; accepted 28 November 2018 *Corresponding author. Email: [email protected]; [email protected] This work was supported by the Major Science and Technology Project of Gansu Provincial Science and Technology Department, China (No. 17ZD2FA009), the Patent Conversion Project of Lanzhou Science and Technology Bureau, China (No. 2017-4-119), the Achievement Transformation Base Project of Lanzhou, China (No. 2016-2-80), the National Key Research and Development Program of China (No. 2018YFC1706300), and the National Traditional Chinese Medicine Standardization Project (No. ZYBZH-Y-GS-10). Copyright © 2019, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences. Published by Elsevier Limited. All rights reserved. DOI: 10.1016/S1872-2040(19)61141-2
SUN Bo-Lu et al. / Chinese Journal of Analytical Chemistry, 2019, 47(2): 271–280
electroanalytical technique has been used in the field of water pollution monitoring, food safety and quality evaluation of herbal medicinal products[15–18]. However, there are few reports about other electrochemical analysis for the detection of CYS so far. Thus, a simple electrochemical analytical technique for real-time, efficient determination of CYS is highly desirable. Porous graphene (PG), as a new type of graphene derivatives, has excellent dispersibility in aqueous phase because it overcomes the issues of accumulation and selfaggregation, which is caused by the strong π-π stacking of graphene[19,20]. Therefore, PG has a large specific surface area and mechanical stability compared to graphene. Furthermore, the pore structure in PG facilitates rapid movement of charge carrier, which makes this material have excellent electrical conductivity[20,21]. According to reports in the literature, PG has been widely used in the field of sensors and electronic devices[22–24]. Yang et al[25] constructed a glucose sensor based on CuCo2O4 polyhedron/porous reduced graphene oxide (PrGO) composite with hollow structure with a detection limit of 0.5 μM (S/N = 3) for glucose. Dong et al[26] proposed a novel electrochemical sensor for the detection of dopamine, which was fabricated by PG using chemical vapor deposition, and this sensor eliminated the interference of uric acid, a common interferent to dopamine detection. However, as we know, there has been no report about the sensor fabricated by PG used to detect traditional Chinese medicine (TCM) with electrical activity in complex systems. In this study, PG was prepared by high-temperature synthesis and water cracking. In addition, glassy carbon electrode (GCE)
Fig.1
was successfully modified with PG to fabricate PG@GCE, which was used to investigate electrochemical behavior of CYS. Finally, determination of trace CYS in real samples was achieved with satisfactory results (Fig.1).
2
Experimental
2.1
Instruments and reagents
Electrochemical measurements were performed on a CHI 6041E workstation (Shanghai Chenhua Company, China) with a conventional three-electrode system. A bare or modified GCE was used as working electrode. A saturated calomel electrode (SCE) and a platinum wire electrode were used as reference electrode and auxiliary electrode, respectively. pHs-3B digital pH-meter (Shanghai Precision Scientific Instrument, China), KQ-50 Ultrasonic Cleaners (Kun Shan Ultrasonic Instruments Co., Ltd), JSM-6701F Cold field emission type sweeping mirror (Japan electronics optics corporation), Finder One Micro-zone laser Raman spectrometer (Beijing Zolix Instrument, China) and Nicolet IS5 Fourier transform infrared spectroscopy (FTIR) (Thermo, US) were used in this study. CYS was purchased from Baoji Chenguang Biotechnology Co., Ltd. All chemical reagents were of analytical pure grade, and all solutions were prepared with distilled water. 2.2 2.2.1
Experimental method Preparation of PG
Schematic diagram of the synthesis of PG and the fabrication process of the PG@GCE for determination of CYS
SUN Bo-Lu et al. / Chinese Journal of Analytical Chemistry, 2019, 47(2): 271–280
PG was prepared by pyrolysis method[27]. 2 g of Na and 5 mL ethanol (the molar ratio is 1:1) were added into stainless steel reactor. After evenly mixing, the reactor was heated under 220ºC for 48 h. The mixture naturally cooled at room temperature, and a white product was obtained. The crude product PG (black) was obtained by pyrolysis with deionized water. The generated product was washed with deionized water several times, then a lyophilization process was carried out to get the final product, which was dissolved in isopropyl alcohol to get 2 mg mL–1 PG solution for subsequent experiments. 2.2.2
Pretreatment of Radix Astragali and Hedysari Radix The mixture of Radix Astragali powder (4 g) and methanol (40 mL) was added into a 250-mL round-bottom flask for extraction at 80 ºC under reflux for 1 h twice, followed by filtering, merging of filtrates, and concentration of filtrates to 10 mL. Radix Hedysari was treated with the same method. Pretreatment of biosample 5 mL blood was sampled from rat carotid artery, and kept it the heparin anticoagulant tubes at 4 ºC. Then the obtained 5 mL anticoagulant plasma (added with a certain amount of CYS) was centrifuged at 4000 rpm for 15 min. The isolated supernatant was measured by the proposed sensors.
Preparation of modified electrode
3 The bare GCE was carefully polished with 0.3 and 0.05 μm Gamma alumina powder (γ-Al2O3) on chamois leather, and then ultrasonically washed with water and ethanol several times. The electrode was dried by natural drying. Then, 5 μL PG solution (2 mg mL–1) was dripped on the bare GCE, the PG@GCE electrode was obtained. The resulting PG@GCE was washed with water and stored at 4 ºC until use. 2.2.3 Electrochemical measurements of modified electrode Cyclic voltammetry (CVs) with potential scanning rang from –0.2 V to 0.8 V and a sweep rate of 100 mV s–1 as well as electrochemical impedance spectroscopy (EIS) with amplitude of 0.005 V, voltage of 0.2 V and frequency range from 0.1 Hz to 105 Hz were applied in solutions containing 5.0 mM K3[Fe(CN)6]/K4[Fe(CN)6] and 0.1 M KCl to investigate electrochemical characters of different modified electrodes. 2.2.4
Pretreatment of samples
3.1
Results and discussion Characterization of PG@GCE
The morphology of the graphene oxides (GO) and PG were characterized by SEM. As shown in Fig.2A, GO were partially packed with their basal planes, intensely crumpled, and folded into a typical wrinkled structure. The surface of PG (Fig.2B) and its interior (Fig.2C) exhibited hollow network structure, and a large number of pores served as the support of PG structure, effectively preventing aggregation of the graphene nanosheet layer[28]. Therefore, the specific surface area of the PG was greatly increased, and more active sites were provided for enrichment of small molecule electroactive compounds and adsorption of biomolecules. In addition, the porous network structure of PG provided the possibility of rapid movement of charge carriers in their continuous and interconnected pore sizes, and played an important role in increasing the sensitivity of electrochemical sensor detection[27].
Fig.2 SEM images of GO (A) and PG (B, C); (D) FT-IR spectra of GO and PG; (E) Raman spectra of GO and PG
SUN Bo-Lu et al. / Chinese Journal of Analytical Chemistry, 2019, 47(2): 271–280
GO and PG were characterized by FTIR. As shown in Fig.2D, the infrared absorption of PG (Curve b) at 3000–3700 cm–1 was significantly lower than that of GO (Curve a), which was due to the small number of oxygen-containing functional groups carried on the surface of the PG. In addition, the absorption peaks of GO at 1400 and 1048 cm–1 correspond to the characteristic absorption of O‒H bond and C-O bond, respectively[29], while the characteristic absorption peak of PG here was obviously weakened or even disappeared, indicating that PG prepared in this study had a lower degree of oxidation. As shown in Fig.2E, two distinct peaks of GO (Curve a) at 1346 cm–1 and PG (Curve b) at 1580 cm–1 are observed, corresponding to D and G bands, respectively. The G band is the first order Raman scattering of the E2g optical mode, which corresponds to the ordered sp2 structure. The D band is the A1g phonon mode of the K-point of the Brillouin zone, corresponding to the degree of disorder and the local defects of the edge. The signal intensity ratio of D peak to G peak (r = ID/IG) can be used to measure the degree of defects and chaos of the material. The D band is caused by the conversion of sp2 hybridization to sp3, which affects the ordering[30]. By calculating the intensity ratios of D and G peaks of GO and PG (rGO = 0.780, rPG = 1.05), it indicated that the prepared PG had relatively more defect structures, which were closely related to the hollow network structure of PG. This result was consistent with the SEM characterization results. And the results further proved that PG was successfully synthesized.
coefficient of 0.5 mM K3[Fe(CN)6] solution containing 1.0 M KCl is 1.39 × 10−4 cm2 s–1), n is the electron transfer number, Qdl is the electric charge of the electric double layer, Qads is the induced electric charge, A is the effective surface area of the electrode, and F is the Faraday constant. As can be seen from Fig.3B, Q and t1/2 have a good linear relationship. According to the slope of the linear equation, the effective surface area of GCE, GO@GCE and PG@GCE were calculated as 0.16, 0.23 and 0.47 cm2, respectively. Attributing to the increased effective surface area of PG@GCE, the amount of CYS accumulated on the electrode surface was larger, which enhanced the electrochemical response signal of CYS.
3.2
3.4
Calculation of electrochemical effective surface area
Using K3[Fe(CN)6] as a probe molecule, the effective surface areas of GCE, GO@GCE and PG@GCE electrodes were studied by chronocoulometry with the electrochemical experiment parameters set as follows: starting potential, 0.5 V; termination potential, –0.2 V; pulse width, 0.25 s; and sampling interval of 2.5 × 10−4 s. According to Anson equation[31]: Q(t) = 2nFAc(Dt/π)1/2 + Qdl + Qads (1) where, c represents the concentration of the substrate in the electrolyte, D is the diffusion coefficient (the diffusion
3.3
Electrochemical characterization of the sensor
The fabrication process of the sensor was characterized by EIS and CV. As shown in Fig.4A, the background current of PG@GCE is larger than that of GCE, indicating that PG@GCE had excellent electrical conductivity and larger effective surface area. This phenomenon should be ascribed to PG with interconnected multilayer networks structure, which increased the surface layer and improved the electron exchange efficiency. The construction process of the sensor was also characterized by EIS, as shown in Fig.4B. Compared with GCE, PG@GCE showed a lower electron-transfer resistance (Ret), indicating that PG increased the electron exchange efficiency. This was probably due to the outstanding electric conductivity of PG. Electrochemical response of CYS
The effect of the buffer solution on the sensor response was tested in three different buffer solutions (NaAc-HAc buffer solution, phosphate buffered solution and citrate buffer solution) with the concentration of 0.1 M. A stable baseline and high sensitivity were achieved in NaAc-HAc buffer. Therefore, NaAc-HAc buffer solution was chosen as the supporting solution. Figure 4C shows CVs of GCE and PG@GCE in NaAc-HAc buffer solution (0.1 M, pH 7.0) with 28 μM CYS or without CYS at scan rate of 100 mV s–1. No redox peak on GCE
Fig.3 (A) Q-t curves of three electrodes in 0.5 mM K3[Fe(CN)6]/1.0 M KCl solution; (B) Q-t1/2 linear plot of the electrode
SUN Bo-Lu et al. / Chinese Journal of Analytical Chemistry, 2019, 47(2): 271–280
Fig.4
(A) The cyclic voltammetry (CV) curves of GCE and PG@GCE in 0.5 mM [Fe(CN)6]3−/4− (containing 0.1 M KCl) solution, Scan rate: 100 mV s–1; (B) Electrochemical impedance spectroscopy (EIS) of the GCE and PG@GCE in 0.5 M [Fe(CN)6]3−/4− (containing 0.1 M KCl); (C) CVs of GCE and PG@GCEin NaAc-HAc buffer solutionand NaAc-HAc buffer solution containing 2.8 × 10−5 M CYS; (D) CVs of GCE in NaAc-HAc buffer solution and NaAc-HAc buffer solution containing 2.8 × 10−5 M CYS
and PG@GCE was observed in the NaAc-HAc buffer solution without CYS. After CYS was added, the CV curve of bare GCE presented a relatively weak oxidation and reduction peaks of CYS, while the oxidation and reduction peaks of PG@GCE were significantly enhanced at Epa = 0.230 V and Epc = 0.412 V, ∆Ep ＝ 182 mV, demonstrating that CYS had a good electrochemical response on PG@GCE. The voltammetric response of PG@GCE to CYS was much more sensitive than that of GCE under the same conditions. These results might be ascribed to the network structure and electrical conductivity of PG, which effectively increased the specific surface area and significantly promoted the rapid propagation of electrons on the electrode surface. 3.5 3.5.1
Optimization of experimental conditions Effect of immobilized amount of PG
The immobilized amount of PG on the surface of sensor influenced oxidation and reduction behaviors of CYS. As shown in Fig.5A, the oxidation peak current of CYS increased with the amount increasing from 0 to 5.0 μL, and then decreased with further increasing, indicating that if the modified film was too thick, it would increase the resistance of interface electron transfer and affect the sensitivity of sensors. Thus, 5 μL of PG dispersion was chosen in further experiments.
3.5.2
Effect of pH value
The peak potentials and the peak currents were closely linked to the pH value of NaAc-HAc buffer solution. Figure 5B shows the electrochemical response of CYS on PG@GCE in the buffer solution with different pH values (3.0–8.0). With the increase of pH value, the redox peaks negatively shifted. In addition, the oxidation peak current conversely decreased when pH further increased. This result indicated the electrochemical process including some protons transfer. A good linear relationship between Epa and pH was expressed as Epa (V) = 0.4343 – 0.04464pH, R2 = 0.9903 (Fig.5D). According to Nernst equation[32] Epa = Eθ – [(2.303mRT)/(nF)] pH, the ratio of m/n was calculated as 0.924. This result indicated that protons and transferred electrons were identical during the electrode reaction. Moreover, a maximum of the oxidation peak current was achieved at pH 4.0 (Fig.5C). Thus, pH 4.0 was chosen for subsequent experiments. 3.5.3 Effect of scan rate The CVs behavior of CYS on PG@GCE at different scan rates was investigated (Fig.5E). The peak currents of CYS increased with the increasing of scan rates. Moreover, the oxidation peak currents (Ipa) for CYS increased linearly with the square root of scan rate from 10 mV s–1 to 300 mV s–1 (Fig.5F), and the linear relationship was expressed as Ipa (μA) = 3.4492υ1/2 (mV s–1)1/2 – 7.25897 with R2 = 0.9974. The results
SUN Bo-Lu et al. / Chinese Journal of Analytical Chemistry, 2019, 47(2): 271–280
indicated that the electrochemical reaction of CYS on the sensor was a diffusion-controlled process. At high scan rate (200‒300 mV s–1), a linear relationship was found between the redox potential and the logarithm of scan rate (Fig.5G), the regression equations was Epa (V) = 0.2003lgυ + 0.5787 (Epa: V, υ: V s–1), R2 = 0.9968; Epc (V) = 0.0050 − 0.2567lgυ (Epc: V, υ: V s–1), R2 = 0.9925. According to Laviron’s equations[33,34]: Epa = Eθ′ + 2.303RT/[(1 ‒ α)nF]lgv (2) Epc = Eθ′ － 2.303RT/(αnF)lgv (3) lgks = αlg(1 ‒ α) + (1 ‒ α)lgα ‒ lg(RT/nFv) ‒ α(1 ‒ α)nFΔEp/2.303RT (4) Hence, the electron-transfer coefficient (α), electrontransfer number (n) and electrode reaction rate constant (ks) were calculated as 0.43, 0.53 and 0.67 s–1, respectively, indicating that CYS had a rapid electron transfer process on PG@GCE surface. This was because the large specific surface area of PG could increase the adsorbing capacity of CYS on the electrode. In addition, the superior conductivity of PG effectively promoted the electron transfer on the electrode surface, thereby enhancing the electrochemical response of CYS. Also, PG provided a microenvironment for electron transport of CYS, which promoted the electrochemical
reaction of CYS. Based on those reasons, the proposed sensor realized the sensitive determination of CYS. A possible reaction mechanism of CYS in PG@GCE is shown in Fig.1. 3.5.4
Effect of accumulation time
The proposed sensor was placed in NaAc-HAc buffer solution containing 28 μM CYS. CYS was enriched on the surface of the sensor using open circuit potential every 30 s, and the CV curves of CYS at PG@GCE were obtained under different accumulation time. As shown in Fig.5H, the oxidation peak current increased rapidly before 300 s, whereafter, the peak current increased slowly with accumulation time prolonging. Therefore, accumulation for 300 s was adopted. 3.6
Calibration curve and detection limit
Under the optimal conditions, differential pulse voltammetry (DPV) was adopted to study the anodic peak current response of CYS on PG@GCE. As shown in Fig.6, the oxidation peak current had a good linear relationship with CYS
Fig.5 (A) Plots of anodic peak currents against volume of modification solutions; (B) CV curves of CYS at different pH values; (C) relationship between the formal redox potentials and the pH values; (D) relationship between anodic peak currents and pH values; (E) CV curves of CYS at different scan rates; (F) relationship between peak currents and square root of scan rates (ν1/2); (G) plots of anodic and cathodic potentials against logarithm of scan rates; (H) relationship between anodic peak currents and the accumulation time
SUN Bo-Lu et al. / Chinese Journal of Analytical Chemistry, 2019, 47(2): 271–280
Fig.6 (A) Differential pulse voltammograms of different concentrations of CYS (a to f: 0.176, 9.560, 18.421, 27.280, 36.133 and 44.012 μM, respectively) on PG@GCE; (B) calibration curve between the anodic peak currents and the concentration of CYS
concentration in the range of 1.8 × 10–7 – 4.4 × 10–5 M, and the linear regression equation was Ipa (μA) = 0.2609C (μM) + 0.1187 (R2 = 0.9928). The detection limit for CYS was calculated to be 5.8 × 10–8 M based on a signal-to-noise ratio of 3. Table 1 shows comparison of analytical performance of this CYS sensor with other methods reported previously, and it can be seen that the PG@GCE was comparable or superior to some reported methods. Therefore, the PG@GCE sensor could serve as a good platform for determination of CYS. 3.7
Reproducibility, stability and selectivity of the sensor
Five PG/GCE electrodes were prepared in parallel, and the reproducibility was investigated by measuring the oxidation peak current in the 10 mL NaAc-HAc buffer (pH 4.0) containing 28 μM CYS. The relative standard deviation (RSD)
was 4.4%, indicating that the proposed sensor had a good reproducibility. The stability was also investigated by measuring the oxidation peak current at interval of one week. During the investigation period, the sensor was stored in a refrigerator at 4 °C. The sensor retained 95.7% of the original response. A series of solutions added with various inorganic ions and molecules as the interferents into 10 mL NaAc-HAc buffer solution (pH 4.0) containing 28 µM CYS was used to investigate the selectivity of the sensor. It was found that 2.8 × 10–4 M quercetin, formononetin, 1.4 × 10–3 M glycine, glutamic acid, L-rhamnose monohydrate, sucrose and 2.8 × 10–3 M inorganic ions of Fe3+, Cu2+, Mg2+, Ca2+, Cl– and SO42– had no influence on the CV signals of CYS. As shown in Fig.7, the performance of the sensor was not obviously affected by those interferents.
Table 1 Comparison of different analytical methods for detection of CYS Detection methods
Linear range (M)
Detection limit (M)
References
HPLC
1.09 × 10–5–8.70 × 10−4
—
6
LC-MS/MS
1.24 × 10–8–7.96 × 10−7
2.2 × 10–9
9
LC-DAD-TOF/MS
7.04 × 10−7–1.83 × 10–4
—
10
DPV (gold electrode/GCE)
3.5 × 10–7–7.04 × 10−6 7.04 × 10–6–7.04 × 10−5
—
35
–6
−4
HPLC
5.6 × 10 –7.0 × 10
DPV (Porous carbon@GCE)
1.76 × 10–7–4.4 × 10−5
—
Present
5.78 × 10–8
Present
Fig.7 Interference of different species to DPV determination of CYS with the proposed sensor (1: Quercetin, 2: Formononetin, 3: Glycine, 4: Glutamic acid, 5: L-rhamnose, 6: Sucrose, 7: Trace element (Fe3+, Cu2+, Mg2+, Ca2+, Cl– and SO42–), 8: CYS, 9: Quercetin + CYS, 10: Formononetin + CYS, 11: Glycine + CYS, 12: Glutamic acid + CYS, 13: L-rhamnose + CYS, 14: Sucrose + CYS, 15: Trace element + CYS)
SUN Bo-Lu et al. / Chinese Journal of Analytical Chemistry, 2019, 47(2): 271–280
Table 2 Detection of CYS in Astragali Radix and Hedysari Radix using PG@GCE Found (mg)
Added (mg)
Total found (mg)
Recovery (%)
RSD (%, n = 3)
1
0.00505
0.0375
0.04345
102.4
1.7
2
0.01009
0.0375
0.04885
103.4
0.5
3
0.01514
0.0375
0.05123
96.2
1.5
1
0.00153
0.0375
0.03888
99.6
1.2
2
0.00306
0.0375
0.04080
100.6
1.7
3
0.00459
0.0375
0.04247
101.0
0.6
Sample Radix Astragali
Radix Hedysari
The above results showed that PG@GCE had good selectivity, stability and reproducibility, and could be applied to the determination of CYS in the complex sample. 3.8
Table 3 Detection of CYS in plasma samples using PG@GCE Biological sample
Added (mg)
Measured (mg)
Recovery (%)
RSD (%, n = 3)
1 2 3
0.0510 0.0306 0.0102
0.0524 0.0291 0.0101
102.7 95.0 99.2
0.2 0.6 1.1
Analytical applications
3.8.1
Determination of CYS in Radix Astragali and Radi Hedysari
The standard addition experiments were then carried out by adding successive concentrations of CYS. As shown in Table 2, 100 μL of the test samples (Radix Astragali or Radi Hedysari) was added into 10 mL NaAc-HAc buffer solution (0.1 M, pH = 4.0), and the concentration of CYS was adjusted in the linear range of the PG@GCE. The concentration of the test samples (Radix Astragali or Radi Hedysari) was also tested by HPLC method. The recovery for the determination of CYS was in the range of 96.2%–103.4%, which indicated that the proposed CYS sensor was suitable for determination of CYS in complex samples.
enrichment. Also, the porous structure effectively accelerated the electrons transfer rate on the surface of the sensor. The electrochemical response of CYS at the surface of PG@GCE increased significantly. Under the optimal conditions, PG@GCE displayed satisfactory analytical performance with wide linear range, low detection limit, superior selectivity and high stability for detection of CYS. Furthermore, the application of PG@GCE to detect CYS in Radix Astragali, Radix Hedysari and plasma sample exhibited satisfactory results. This study provided a novel analytical method for rapid evaluation of the quality of Radix Astragali, Radix Hedysari and real-time analysis of trace CYS in biological samples. It also demonstrated that PG could be extensively applied in designing high performance electrochemical sensors for drug analysis.
3.8.2
References
Determination of CYS in biological samples
The PG@GCE was also used to measure CYS concentration in plasma samples. 5 mL pretreated plasma samples solution with CYS was diluted to 10 mL with NaAc-HAc buffer (pH 4.0), then detected by DPV with standard addition method. The results are shown in Table 3. The sensor expressed a sensitive, simple and fast detection for CYS in biological samples with satisfactory recoveries from 95.0% to 102.7%, clarifying that the proposed method could be used to measure CYS in plasma samples.
4
Conclusions
[1]
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