Carboxylated Multiwalled Carbon Nanotubes Nanocomposite

CHINESE JOURNAL OF ANALYTICAL CHEMISTRY Volume 47, Issue 8, August 2019 Online English edition of the Chinese language journal

Cite this article as: Chinese J. Anal. Chem., 2019, 47(8): e19095–e19103

RESEARCH PAPER

A Novel Genistein Electrochemical Sensor Based on Molecularly Imprinted Polycarbazole/Carboxylated Multiwalled Carbon Nanotubes Nanocomposite NIU Li-Ting1,2, LI Gen-Gen1,2, LI Hai-Feng2, CUI Fan2, ZHANG Jing2, HUANG Ying2, CHEN Kai2, ZHANG Jian-Fu1,3,4,*, LI Wen-Fei1,3,*, LIU Wei-Lu2 1

School of Chemistry and Environmental Engineering, Changchun University of Science and Technology, Changchun 130022, China School of Pharmacy, Shenyang Pharmaceutical University, Shenyang 110016, China 3 Jilin Provincial Science and Technology Innovation Center of Optical Materials and Chemistry, Changchun 130022, China 4 State Key Laboratory of Polymer Physics and Chemistry, Changchun Institute of Applied Chemistry Chinese Academy of Sciences, Changchun 130022, China 2

Abstract:

Genistein is an important isoflavone that has been widely used to prevent blood disease and cancer. In this work, a novel

genistein electrochemical sensor was developed based on the composite of molecularly imprinted polymer (MIP) and carboxylated multiwalled carbon nanotubes (cMWCNTs). The MIP layer was electropolymerizated on the cMWCNTs modified electrode using carbazole as functional monomer and genistein as template molecule. The morphology and electrochemical performance of MIP/cMWCNTs were characterized by scanning electron microscopy (SEM) and cyclic voltammetry (CV), respectively. A series of experimental conditions were optimized, including the pH value of supporting electrolyte, electropolymerization potential range, molar ratio of functional monomers to template molecules, numbers of cycle, accumulation potential and accumulation time. Under the optimal conditions, the resulting electrochemical sensor (MIP/cMWCNTs/GCE) showed high performance, such as high sensitivity and selectivity towards genistein, a wide linear range (0.02–7.00 M) and a low limit detection of 0.006 M (S/N = 3). The electrochemical sensor was applied to determination of genistein in tablets and human urine samples with satisfactory recoveries (97.9%–102.8%), and the accuracy of the sensor was demonstrated with the HPLC method. Key Words:

Genistein; Electrochemical sensor; Molecularly imprinted polymer; Multiwalled carbon nanotube

1 Introduction Genistein (4,5,7-trihydroxy isoflavones) is a typical phytoestrogen and an important member of the isoflavones family[1]. As a nature product produced from leguminous plants, genistein exhibits multitudinous biological properties and pharmacological effects. Previous researchers found that it could modulate various hormone-mediated pathways in thyroid hormone receptors regulating their target genes for

controlling organ development and functional maintenance [2]. Besides, it is able to act as an inhibitor of protein tyrosine kinases, NF-kB and AKT[3] (an important prosurvival factors), as well as DNA topoisomerase II[4], and all of them have vital impact in inhibiting growth factor and cytokine-stimulated proliferation of both normal and transformed cancer cells[5–7]. In addition, genistein exhibits effects of cancer-preventive[8], anti-osteoporosis[9] and regulating energy metabolism for menopause female[10]. However, concerns have been raised

________________________ Received 8 January 2019; accepted 4 June 2019 *Corresponding author. Email: [email protected]; [email protected] This work was supported by the National Natural Science Foundation of China (Nos. 81503037, 21504008), the Doctoral Research Funding of Liaoning Province, China (No. 201601141), the Basic Research Projects of Liaoning Provincial Department of Education, China (No. 2017LQN06), the Career Development Support Plan for Young and Middle-aged Teachers in Shenyang Pharmaceutical University, China (No. ZQN2015029), and the Scientific Research Fund of Liaoning Provincial Education Department of China (No. L2014385). Copyright © 2019, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences. Published by Elsevier Limited. All rights reserved. DOI: 10.1016/S1872-2040(19)61176-X

NIU Li-Ting et al. / Chinese Journal of Analytical Chemistry, 2019, 47(8): e19095–e19103

because low concentration of genistein in MCF-7 cells usually leads to proliferation while high concentration genistein can attenuate cell growth and shows anticancer property[11]. Therefore, a facile, fast and sensitive measurement technique for detecting genistein would be useful in the field of pharmaceutical analysis. Compared with the traditional method for detecting genistein, such as high-performance liquid chromatography (HPLC)[12], gas chromatography (GC)[13], capillary electrophoresis (CE)[14] etc, electrochemical method is less time-consuming, more sensitive and convenient. Daniela et al[5] prepared electrochemical sensor by using a supporting electrolyte consisted of phosphate buffer–methanol solution and cationic surfactant cetyltrimetylammonium bromide to analyze genistein in soy flours and soy based supplements and obtained accurate and precise results. Surinya et al[15] developed an in-house screen-printed electrochemical sensor decorated with cobalt (II) phthalocyanine for genistein determination in Derris scandens with high sensitivity and rapid operation. These reports indicated the applicability of electrochemical sensor in genistein determination. The selectivity and sensitivity are crucial issues in the development of electrochemical sensors. Molecularly imprinted polymers (MIPs), as artificial receptors, offer a specific cavity for target molecule recognition, which can be used as a recognition element of the electrochemical sensor. Compared with natural recognition elements such as enzymes, DNA and aptamers, MIPs exhibit many advantages such as good stability, low cost, convenience for storage and preparation[16]. Nevertheless, it is worth noting that the sensitivity is a crucial aspect in constructing high-performance MIP sensor. The MIPs prepared by the bulk polymerization are often nonconductive and lack of catalytic centers, resulting in slow mass transfer ability and low eletrocatalytical activity. To improve response and conductivity of electrochemical sensor, efforts have been made to fabricate the thin film of MIPs on nanomaterials surface. According to recent reports, several nanomaterials have been utilized to enhance the detection performance of MIP electrochemical sensor, such as silver nanodendrites[17], TiO2 nanotube[18], reduced graphene oxide-Pt core-shell microspheres[19], g-C3N4-AuNPs[20], and SiO2 nanoparticles[21]. MWCNT is a promising substrate material due to its remarkable properties in structure, chemistry, electrical conductivity, and mechanical behavior. The high conductivity and large surface area of MWCNT can produce a highly sensitive electrochemical sensor. In this work, a novel electrochemical sensor based on MIP/cMWCNTs composite was prepared. cMWCNTs were utilized to enhance the sensing performance due to its high surface specific area and good conductivity, which resulted in a high sensitivity in genistein detection. MIP was uniformly electro-polymerized onto the surface of cMWCNTs modified electrode, which possessed high selectivity to gensitein over the structure analogs by forming certain shaped cavities that

just perfectly matched with genistein molecules. A three dimensional network of MIP/cMWCNTs was observed on the electrode surface by scanning electron microscope (SEM). The preparation conditions of the MIP/cMWCNTs composite were optimized. Finally, the MIP/cMWCNTs composite was applied to detect gensitein in tables and urine samples with satisfactory results.

2 Experimental 2.1

Materials and apparatus

MWCNTs were purchased from Chengdu Institute of Organic Chemistry, Chinese Academy of Sciences. Genistein, formononetin, rutin, quercetin, luteolin, dopamine, ascorbic acid, uric acid, glucose, reduced glutathione, tyrosine, K3Fe(CN)6, K4Fe(CN)6 and carbazole were purchased from Aladdin reagent China. All other reagents were of analytical grade and obtained from Sinopharm chemical reagent. The electrode material was characterized using SEM (Hitachi-SU8010). Electrochemical properties of electrode materials were executed by CV using a redox probe of 10 mM [Fe(CN)6]3−/4− and 0.1 M KCl solution. HPLC data was obtained on a Shimadzu LC-20A with UV detection. HPLC conditions are described as follows: ODS-C18 (200 mm × 4.6 mm, 5 μm) column; mobile phase, methanol and water (25:75, V/V) solution; flow rate 0.8 mL min‒1; detection wavelength 260 nm. 2.2

Carboxylation of MWCNTs

cMWCNTs were prepared according to the literature [22] method with minor modification. Briefly, 150 mg of MWCNTs was dissolved in 20 mL of HNO3 (mass fraction 65%) and sonication for 0.5 h until attached to homodisperse, then the mixture was refluxed in 90 ºC for 10 h to obtain cMWCNTs. After being cooled to room temperature, the resulting mixture was centrifuged and then the precipitate was collected and washed with ultrapure water several times until the pH value of the solution was within the range of 6.5–7.0. Finally, the cMWCNTs were dispersed in 10 mL of ethanol for further use. 2.3

Fabrication of MIP/cMWCNTs composite

The preparation procedure of the sensor is shown in the top of Fig.1. Prior to the modification, the glassy carbon electrode (GCE) was polished to a mirror-like surface with 10, 3 and 0.3 µm Al2O3 slurry on chamois leather, respectively, followed by successive sonication with aqueous nitrate solution (1:1, V/V), ethanol and ultrapure water for 3 min to remove the residual alumina powder. After drying in a nitrogen stream, 5 μL of cMWCNTs dispersion (0.1 mg mL‒1) was dropped on the GCE.

NIU Li-Ting et al. / Chinese Journal of Analytical Chemistry, 2019, 47(8): e19095–e19103

excipients contained 80% starch and 20% sodium carboxymethylcellulose. Ten pieces genistein tablets were grinded into powder with an agate mortar and 20 mg of powder were accurately weighed and dissolved in 10 mL of ethanol. Subsequently, the obtained extraction solution was centrifuged at 10000 rpm for 10 min, and then the extraction solution was collected into the 25-mL volumetric flask after repeated the extraction procedure again. Finally, the solution was diluted to suitable concentration with ethanol for real sample testing and the recovery experiment.

3 Results and discussion Fig.1

Synthetic route of MIP/cMWCNTs composite and genistein oxidation mechanism on MIP/cMWCNTs

The modified electrode (cMWCNTs/GCE) was activated in H2SO4 (0.1 M) with CV method. Then, the MIP layer was electrodeposited on the surface of cMWCNTs modified electrode using CV method in a potential range from 0 V to 1.5 V for 40 scanning cycles at a scan rate of 0.05 V s–1. The electrodeposition solution was a mixed solution of acetate buffer solution (ABS, 0.1 M, pH 5.0) containing carbazole (1.5 mM) as the monomer and genistein (0.1 mM) as the template. All the electrodeposition solutions were bubbled with nitrogen to remove dissolved oxygen. Finally, the genistein template molecules were removed from the polymer film by washing in a mixed solution of 0.1 M NaOH aqueous solution/ethanol (3:7, V/V) at 35 ºC for 30 min. 2.4

Electrochemical measurements

Electrochemical measurements were conducted using CHI830d electrochemical workstation (Chenhua Instruments, China) that equipped with a three electrode cell. The MIP/cMWCNTs modified GCE, platinum wire, Ag/AgCl electrode (KCl saturated) were employed as the working electrode, counter electrode and reference electrode, respectively. All the potential values in this work were obtained reference to the Ag/AgCl electrode. 2.5

3.1

Fabrication and characterization of the MIP/cMWCNTs composite

The cyclic voltammograms (CVs) of polymerization for carbazole (1.5 mM) in the presence of genistein (0.1 mM) are shown in Fig.2. There was an oxidation peak of carbazole at the potential of 1.2 V, and the oxidation peak gradually decreased with the increase of cycles, indicating that a non-conductive polycarbazole film was formed on the surface of cMWCNT/GCE. Morphologies of the cMWCNTs and MIP/cMWCNTs were characterized by SEM. Figure 3A shows a spaghetti shaped structure with smooth walls on the surface of cMWCNTs modified GCE. After the MIP was deposited, a network-like structure with rough interface could be observed in Fig.3B, which indicated that the MIP layer was successfully wrapped on the cMWCNTs. Owing to the combination of MIP layer and cMWCNTs, the electrode with large surface area provided more binding sites for genistein detection. The redox probe of K3Fe(CN)6/K4Fe(CN)6 was used to characterize the electrochemical property and recognition ability of MIP/cMWCNTs composite (Fig.3C). The redox probe of K3Fe(CN)6/K4Fe(CN)6 was used to characterize the recognition ability of the MIP/cMWCNTs composite (Fig.3C). Before the template was removed from the MIP/cMWCNTs composite, no electrochemical response was observed (curve a), owing to that

Sample preparation

The urine sample from a healthy male volunteer in our lab was pretreated by precipitation to remove the interfering proteins. Urine sample (120 μL) was treated with acetonitrile (300 L) and centrifuged at 10000 rpm for 10 min. The supernatant was collected and dried with a low speed flow of nitrogen stream at 37 ºC. Finally, the sample was reconstituted in 6 mL of phosphate buffer solution (PBS, 0.1 M, pH 5.0) for analysis. Genistein tablets were provided by Pharmaceutics Laboratory, Shenyang Pharmaceutical University. The ingredients consisted of 3% genistein and 97% excipients, and

Fig.2

Cyclic voltammograms for electropolymerization of polycarbazole (1.5 mM) in 0.1 M ABS (pH 5.0)/ethanol (6:4, V/V) containing genistein (0.1 mM). The scan rate is 50 mV s‒1

NIU Li-Ting et al. / Chinese Journal of Analytical Chemistry, 2019, 47(8): e19095–e19103

Fig.3

SEM images of (A) cMWCNTs modified electrode surface and (B) MIP/cMWCNTs modified electrode surface; (C) Cyclic voltammograms of K3Fe(CN)6/K4Fe(CN)6 (1 mM) obtained from MIP/cMWCNTs modified electrode before (curve a) and after (curve b) removal of template, and after recognition of 70 μM genistein (curve c)

the redox reaction of K3Fe(CN)6/K4Fe(CN)6 was obstructed by the nonconductive MIP layer. After removal of template from the MIP layer, a pair of reversible redox peaks appeared (curve b) because the access to the electrode surface was regenerated, which allowed the redox probe of the electron to transfer through the MIP/cMWCNTs composite. After genistein was recognized on the MIP/cMWCNTs composite, the access for the electron transfer was blocked again, which resulted in the decrease of the peak current of the redox probe (curve c). The change of the response of the redox probe indicated the well-established recognition ability of the MIP/cMWCNTs towards the template genistein and the genistein molecule could be specifically recognized on the imprinted binding sites (curve b). Considering the structure of genistein and the MIP film, the interaction between templates and MIPs could be hydrogen bonding, electrostatic interaction and π-π stacking. 3.2

Electrochemical behavior of genistein at different modified electrodes

The CVs of genistein (40 μM) obtained at different electrodes in PBS solution (0.1 M, pH 5.0) is displayed in Fig.4. The unmodified GCE exhibited a tiny oxidation peak with the peak current of 0.70 A and the peak potential of 0.807 V (curve a). At the MIP/cMWCNTs modified electrode, the peak potential was negatively shifted to 0.768 V and the peak peak current was 2.1 times than that of the bare GCE (curve b), because the imprinted binding cites on the polymer matrix could specifically adsorb the target genistein molecules. At the cMWCNTs modified electrode (curve c), the peak potential was shifted to more cathodic value of 0.726 V and higher peak current of 4.6 µA, which contributed to the catalytic property and specific adsorption of cMWCNTs. At the MIP/cMWCNTs modified electrode (curve d), the peak potential was negatively shifted to 0.745 V and the peak current was 5.2 times of that of the bare GCE, which was attributed to a synergistic effect of MIP and cMWCNTs. 3.3

Optimization of preparation conditions of MIP/cMWCNTs composite

To obtain high-performance MIP electrochemical sensor, the electropolymerization conditions of MIPs were discussed and optimized. The detection sensitivity obtained from the slop of the calibration curve was used to evaluate the sensing performance of the MIP/cMWCNTs composite. The pH value of polyelectrolyte solution determines the existence form of template molecule and functional monomer, which is crucial to the formation of hydrogen bonds between template molecule and MIP layer. The influences of polyelectrolyte solution were investigated by performing DPV tests in ABS with different pH values. Figure 5A exhibits that the detection sensitivity of genistein increases as the pH value increases from 2.5 to 5.0, reaching a maximum at pH 5.0. Further increase in the pH would lead to the decreased detection sensitivity because of the destruction of hydrogen bond. Therefore, pH 5.0 was selected for further experiments. The influence of potential range of electropolymerization on MIP/cMWCNTs/GCE was evaluated. The results demonstrated a maximal detection sensitivity in 0–1.5 V, as shown in Fig.5B. To investigate the effects of different monomer/ template ratio, different DPV responses were obtained by changing the monomer concentration and fixing the concentration of template (0.1 mM) constant. The highest detection sensitivity was obtained at a monomer/template ratio of 15:1 (Fig.5C). It

Fig.4 Cyclic voltammograms of genistein (40 μM) obtained at bare GCE (curve a), MIP modified GCE (curve b), cMWCNTs modified GCE (curve c), and MIP/cMWCNTs modified GCE (curve d) in PBS solution (pH 5.0). The scan rate is 100 mV s‒1

NIU Li-Ting et al. / Chinese Journal of Analytical Chemistry, 2019, 47(8): e19095–e19103

Fig.5

Effects of pH value of polyelectrolyte solution (A), electrochemical polymerization range (B), molar ratio of monomer to template (C) and numbers of cycle (D) on detection sensitivity (slop of the calibration curve)

is supposed that the excess amount of monomer will result in high polymerization degree which reduces the conductivity. However, inadequate amount of monomer may lead to inadequate polymerization and difficulty in forming effective imprinted binding sites. The numbers of the cycle in the polymerization process is another key factor. As shown in Fig.5D, MIP/cMWCNTs modified GCE possessed the highest sensitivity at 40 scanning cycles. This could be ascribed to the proper thickness of MIP layer. When the scanning cycles were over 40, a relatively thicker film could be obtained, which resulted in the high mass-transfer resistance and difficulty for target molecules to attach the active sites. On the other hand, a thinner MIP film obtained at less scanning cycles was easily broken while removing template molecule. 3.4

Effect of pH values and scan rate

The pH value of buffer solution is a significant factor influencing the oxide peak current and potential of genistein. MIP/cMWCNTs/GCE was immersed in PBS with different pH (from 3.0 to 9.0). Figure 6A shows the CVs of genistein oxidation obtained at 0.1 M PBS with different pH values. Inset 1 indicated that the oxide peak current of genistein increased as the pH value increased from 3.0 to 5.0, and the maximum peak current was obtained at pH 5.0. When the pH value of buffer solution was over 5, the peak current decreased. Thus, PBS solution (0.1 M, pH 5.0) was selected as the buffer solution for the following genistein determination. As shown

in inset 2, the oxide peak potential was negatively shifted as the pH value increased, which indicated that protons participated in the redox reaction on the electrode surface. The relationships between Epa of genistein and pH could be expressed as follows: Epa = –0.0592pH + 0.9918 (R = 0.995), this slop value was equal to the Nernstian value of 0.0592 V pH−1, demonstrating that equal numbers of protons and electrons were transferred during genistein oxidation. To further study the kinetics of the electrode reactions happening at the MIP/cMWCNTs, the relation of scan rate vs. peak current of genistein was also investigated by CV. Figure 6B displays the superimposed CVs of genistein with scan rate in the range from 0.03 V s−1 to 0.3 V s−1. As shown in inset 1, the peak currents increased with the increasing scan rate gradually, indicating that the electrochemical oxidation of genistein was an adsorption-controlled electrode process. Moreover, the peak potential positively shifted with the increase of scan rate. A good linear relationship between peak potential (Ep) and logarithm of scan rate (υ) could be described as Ep = –6.655lgυ + 7.026 (R = 0.996) (inset 2). In view of Laviron’s method[23,24], the number of electrons (n) transferred in the electrochemical oxidation reaction could be determined using the following equation: Ep = E0 + (2.303RT/anF)[lg(RTk0/anF) + lgυ] (1) where, E0 is the formal standard potential, υ is the scan rate, k0 is the standard rate constant of the reaction, α is the chargetransfer coefficient. In irreversible electrode process, α is generally assumed as 0.5, the other symbols represent their usual meanings. The number of the electron involved in the

NIU Li-Ting et al. / Chinese Journal of Analytical Chemistry, 2019, 47(8): e19095–e19103

electrode reaction was found to be 1.2 according to the slope value of Ep-lgυ plot. According to the above results, the mechanism of genistein electrochemical oxidation at the MIP/cMWCNTs modified electrode was a one-electron and one-proton process. The mechanism of genistein oxidation was described in the bottom of Fig.1. 3.5

Effect of accumulation time and accumulation potential

To enhance the sensitivity of the fabricated sensor, we examined the effects of the accumulation time and accumulation potential on the oxidation peak current of genistein. As presented in Fig.7A, the peak current of genistein continuously increases within the increasing 80 s accumulation time and the peak current maintaines stable over 80 s. As shown in Fig.7B, the peak current of genistein reaches maximum at 0 V as the accumulation potential increases from –0.6 V to 0.4 V. Thus, the accumulation time of 80 s and accumulation potential of 0 V were used as the optimized conditions. 3.6

Calibration curve

In this work, the DPV response of the genistein oxidation

reaction at the MIP/cMWCNTs modified electrode was recorded to establish calibration curve for detecting genistein. Under optimized conditions, the oxidation peak currents were proportional to genistein concentrations in the range of 0.02‒7.00 µM. Figure 8A shows the DPV oxidation peaks of different concentrations of genistein. Figure 8B shows the linear dependence of the DPV peak currents on the genistein concentrations. The regression equation was described as I p (µA) = 0.1594C (µM) + 0.4766 (R = 0.991) and the detection limit (LOD) was calculated as 0.006 μM (S/N = 3). As shown in Table 1, a comparison study of different methods was conducted based on the analytical parameters for genistein. The data showed that the proposed method in this study had a broader linear range and a lower detection limit compared with other methods. In summary, MIP/cMWCNTs was a good platform for genistein sensing. 3.7

Selectivity

The selectivity of the MIP/cMWCNTs modified electrode was evaluated by testing its responses to genistein in the presence of structural analogs and other coexisting ions. Figure 8A (curve a) shows the DPV response of genistein (5 μM) in the absence of interfering substance. Figure 8A (curve b to curve e)

Fig.6 (A) Cyclic voltammograms of genistein (40 μM) obtained at MIP/cMWCNTs modified electrode in PBS solution (0.1 M) with different pH values (3.0, 4.0, 5.0, 6.0, 7.0, 8.0 and 9.0). The scan rate is 100 mV s‒1. Inset (1) is plot of peak currents (Ip) vs pH, inset (2) is plot of peak potential (Ep) vs pH values. (B) Cyclic voltammograms of genistein (40 μM) obtained at MIP/cMWCNTs modified electrode in PBS solution (0.1 M, pH 5.0) with different scan rates (from a to h: 0.03, 0.06, 0.09, 0.12, 0.15, 0.18, 0.21, 0.24, 0.27 and 0.30 V s‒1, respectively). Inset (1) is plot of peak current (Ip) vs scan rate (υ), inset (2) is plot of peak potential (Ep) vs lgυ

Fig.7 Effect of accumulation time (A) and accumulation potential (B) on oxidation peak current of genistein (7 μM) in PBS solution (0.1 M, pH 5.0)

NIU Li-Ting et al. / Chinese Journal of Analytical Chemistry, 2019, 47(8): e19095–e19103

Fig.8

(A) DPV responses of genistein oxidation at MIP/cMWCNTs composite in PBS solution (pH 5.0). The concentration of genistein is, from bottom to top, 0.02, 0.07, 0.2, 0.7, 2.0 and 7.0 µM, respectively. (B) Plot of DPV oxidation peak current (Ip) vs concentration of genistein Table 1

Methods SWV SWV ILd-CEZe CEZ HPLC-DADf DPV

Comparison of different methods for determination of genistein

Electrode material

Linear range (μM)

LOD (μM)

References

HMDEa CPb/SPCEc — — — cMWCNTs/MIP/GCE

0.114‒1.09 2.5‒150 31.6‒1018.5 1.85‒185 0.14‒5.56 0.02‒7

0.034 1.5 7.96 0.31 0.014 0.006

[5] [15] [25] [26] [27] This work

HMDEa: Hanging mercury drop electrode; CPb: Cobalt(II) phthalocyanine; SPCEc: Screen-printed electrode; ILd: Ionic liquid; CEZe : Capillary zone electrophoresis; HPLC-DADf: High-performance liquid chromatography-diode array detection.

shows the DPV response of genistein (5 μM) in the presence of 2 μM formononetin, luteolin, rutin and quercetin, respectively. The result indicated that the peak current of genistein barely changed in the presence of interfering substance. Figure 9B shows the ratio of peak current of genistein in the presence and absence of interfering substances. The ratio was kept within the range of 98.1%–103.6%, indicating the structural analogs had negligible effect on the detection of genistein. The effect of some common interfering substances on the detection of genistein was also evaluated. It was observed that 50-fold dopamine, ascorbic acid, uric acid, glucose, reduced glutathione and tyrosine, 100-fold K+, Na+, Ca2+, Mg2+, SO42‒ and CO32‒ did not interfere with the

determination of 5 μM genistein (signal change < 6.0%). Herein, the target genistein could be specifically recognized on the imprinted binding sites, which resulted in the good selectivity to genistein over the interferents. 3.8

Reproducibility, repeatability and stability

To investigate the reproducibility of the fabricated electrodes, five different MIP/cMWCNTs/GCEs were prepared for detection of genistein (7 μM) under the same conditions. A satisfactory reproducibility with a relative standard deviation (RSD) value of 3.5% was obtained. Moreover, eleven repetitive DPV tests were conducted in different buffer solutions

Fig.9 (A) DPV responses of genistein (5.0 μM) in the absence (curve a) and presence of 2.0 μM interfering substance (curve b to curve e: formononetin, luteolin, rutin and quercetin). (B) Ratio of peak current of genistein in the presence and absence of interfering substance (n = 3)

NIU Li-Ting et al. / Chinese Journal of Analytical Chemistry, 2019, 47(8): e19095–e19103

(pH 5.0), such as phosphate buffer solution (PBS), acetate buffer solution (ABS) and Britton-Robinson buffer solution (B-R), and gave RSDs of 2.3%, 2.8%, 4.1%, respectively. This indicated that the sensor possessed acceptable repeatability. To investigate the influence of work voltage on the stability of the sensor, experiments were conducted by changing the work voltage to higher values (0–1.2 V, 0–1.5 V, 0–1.8 V and 0–2.0 V, respectively), and then detecting genistein (7 μM) at normal work voltage (0–1.0 V). The DPV responses remained 95.3%, 90.6%, 85.8% and 70.4%, respectively of its initial response. This indicated that high working voltage would destroy the stability of the membrane over 1.8 V. The stability of the sensor was also tested after the sensor was stored four weeks at 4 °C, and the response current retained 92.3% of its initial response. These results reflected the good stability of the sensor. 3.9

HPLC method. The calculated t values were smaller than the tabulated values. Therefore, there was no significant difference between results of MIP/cMWCNTs/GCE sensor and the HPLC method at the 95% confidence level. The results of standard addition experiment and t-test revealed that the proposed sensor was accurate and reliable in detection of genistein in real samples.

4

In this work, MIP/cMWCNTs composite was employed to construct a novel genistein electrochemical sensor. The MIP/cMWCNTs modified electrochemical sensor exhibited high sensitivity and good selectivity, which could be attributed to its large specific surface area, high electrocatalysis activity and loads of binding sites for genistein recognition. It was found that the genistein underwent a one-electron and oneproton oxidation process on MIP/cMWCNTs surface. Under the optimized conditions, the proposed method exhibited low detection limit, good stability and reproducibility in detection of genistein. Moreover, this electrochemical sensor showed satisfied results in determination of genistein in genistein tablets and human urine samples. The analysis results were in accordance with the results of HPLC method. Thus, the proposed method could provide a reliable reference for the genistein analysis in real samples.

Real sample analysis

To explore the feasibility of MIP/cMWCNTs/GCE for practical analysis, the modified electrode was used for determination of genistein in human urine sample and tablets. As listed in Table 2, the recoveries for the added standard were in the range of 97.9%–102.8% with RSD value of less than 4.1%. The determination results of genistein concentrations by this sensor were compared with those by Table 2 Samples

Conclusions

Determination of genistein in human urine and tablet (n = 3)

Detected by MIP/cMWCNTs sensor Found (M)

Urine Urine Tablet Tablet

— — 3.0 3.1

Added (M)

Total found (M)

2.0 4.0 1.0 1.0

0.197 4.114 4.013 4.079

Recovery (%) 98.5 102.8 101.3 97.9

RSD (%)

Detected by HPLC (M)

t-Test*

1.8 4.1 3.5 2.1

0.196 4.115 4.012 4.083

2.078 (2.776) 3.312 (2.776) 2.273 (2.776) 2.176 (2.776)

*Values in parenthesis are tabulated t values at p = 0.05.

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