High Sensitivity Detection of Capsaicin in Red Pepper Oil Based on Reduced Graphene Oxide Enhanced by β-Cyclodextrin

Red pepper oil is a kind of condiment used in food factories, and its spicy degree can be reflected by the content of capsaicin. However, the classification method of red pepper oil is too subjective to be applied in food factories. In our work, a sensitivity electrochemical sensor for detecting capsaicin was constructed based on the glassy carbon electrode (GCE) modified with β-cyclodextrin (β-CD) assisted reduced graphene oxide (rGO). The results showed that the introduction of β-CD made rGO more dispersed, increasing the electrochemical active surface area of β-CD/rGO, thus improving the charge transfer rate. Furthermore, polycyclodextrin with selective recognition ability was dispersed on the surface of rGO, providing the possibility of capsaicin enrichment on the surface of the sensor. Based on these reasons, the sensor had a lower detection limit (0.05 μg/mL), and acceptable stability and anti-interference. Most importantly, β-CD/rGO/GCE displayed a satisfactory recovery rate (94.83 ~ 115.75%) in the detection of red pepper oil, and there was no statistical significance difference between this method and the LC–MS method.


Introduction
Red pepper oil, also known as chili oil, is made by hot maceration of dried chilies in cooking oil (Rui et al. 2020;Xza et al. 2020). As a flavoring oil, red pepper oil is often used to process various snack foods to give them a spicy taste (Ornelas-Paz et al. 2010;Zhang et al. 2021). Depending on the intensity of spiciness, red pepper oil is usually classified as slightly spicy, medium spicy, and extra spicy. However, due to regional and individual differences, consumers have different cognition and acceptance of spicy flavor, leading to this classification method being too subjective. This makes its application in food more based on experience, which is not conducive to the standardization of food production. Therefore, it is necessary to establish a more scientific classification method for grading the spiciness of red pepper oil. In fact, the main source of spiciness in red pepper oil is capsaicin, the content of which is directly related to spiciness of food and is commonly used to evaluate spiciness of food. Therefore, the establishment of determination method for capsaicin content is of great significance to the classification of red pepper oil Liu et al. 2022).
A variety of methods for the analysis of capsaicin have been reported and generally recognized, such as spectrophotometry (Perucka and Oleszek 2000), ELISA , high-performance liquid chromatography , liquid chromatography-mass spectrometry (Kozukue et al. 2005), gas chromatography (Cisneros-Pineda et al. 2006;Peña-Alvarez et al. 2008), and TLC-ESI-MS (Santos et al. 2021). Spectrophotometry is simple and inexpensive, but it lacks specificity and sensitivity, and cannot achieve the purpose of trace detection (Perucka and Oleszek 2000). ELISA has higher specificity, but due to the difficulty in screening specific antibodies, it cannot be widely used in the detection of capsaicin . Chromatography and mass spectrometry methods have the characteristics of high precision and good accuracy, but they need long detection period, frequent operation, and expensive equipment and labor costs (Kaale et al. 2002;Peña-Alvarez et al. 2008;Wu et al. 2018).
Compared to usual tests, electrochemical analysis has significant advantages in terms of economy, timeliness, accuracy, miniaturization, and sensitivity (Sheela et al. 2011;Da Silva Antonio et al. 2019;Supchocksoonthorn et al. 2020). Up to now, there have been reports on the detection of capsaicin by electrochemical sensors based on metals and their oxides (Wang et al. 2016), carbon nanomaterials (Salimi et al. 2008;Yardm and entürk 2013), and other semiconductor substances (Yu et al. 2012;Søpstad et al. 2018). Among them, rGO is considered a good modification material for detecting capsaicin because it not only has excellent electrical conductivity, but also can produce a large number of topological defects and continuous changes of carbon vacancies, thus enhancing the adsorption, embedding, and activation properties of target molecules (Zhou et al. 2010;Zhong et al. 2019). However, rGO sheets tend to accumulate and lose the advantage of nanomaterials because of their van der Waals forces (Wang et al. 2013;Mutyala and Mathiyarasu 2016). β-CD is an oligosaccharide consisting of seven glucose units, which can effectively disperse various nanomaterials to enhance their electrochemical detection performance. At the same time, β-CD has a toroidal structure with a hydrophobic inner cavity and a hydrophilic outer side, enabling them to selectively combine with some organic molecules (Przybyla et al. 2020). This interesting property will undoubtedly enrich the organic molecules on the electrode surface, thus improving the sensitivity of electrochemical detection (Díaz et al. 2018;Gu et al. 2021). Therefore, we speculate that β-CD can further enhance the detection performance of capsaicin by electrochemical sensors based on rGO.
In our work, a sensitive electrochemical sensor for detecting capsaicin was constructed by introducing β-CD into the polymerization of rGO. Firstly, the mixed solution of β-CD and graphene oxide (GO) was scanned by cyclic voltammetry (CV) to deposit β-CD/rGO composite material on the surface of GCE. Secondly, the electrochemical reaction mechanism of capsaicin on the sensor we prepared (β-CD/rGO/GCE) and the optimal deposition parameters of β-CD/rGO composite material were further investigated. Finally, the methodological evaluation and the application potential of β-CD/rGO/ GCE for the detection of capsaicin were studied.

Reagents and Materials
Graphene oxide (GO) dispersion was purchased from Suzhou Tanfeng Graphene Technology Co. Ltd (Suzhou, China). β-CD, monopotassium phosphate, dipotassium phosphate, hydrochloric acid (HCl), sucrose, ascorbic acid, sodium glutamate, potassium chloride, potassium ferrocyanide, and potassium ferricyanide were purchased from Shanghai Sinopharm Chemical Reagents Co. Ltd (Shanghai, China). Capsaicin standard, anhydrous ethanol, and methanol were purchased from Shanghai Aladdin biochemical Technology Co. Ltd (Shanghai, China). The ultrapure water was purified with a water purification system, and its resistivity was 18.2 MΩ·cm (Sichuan Youpu Ultrapure Science and Technology Co. Ltd, Chengdu, China). Red pepper oil was obtained by Three Squirrels Inc (Wuhu, China). All electrodes were purchased from Shanghai Xianren Instrument Co. Ltd (Shanghai, China). All reagents except capsaicin used were of analytical grade.

Synthesis of β-CD/rGO
Preparation of β-CD/GO Dispersion β-CD dispersion was obtained by dissolving 1 mg β-CD into 3.7 mL phosphate buffers (0.1 mol/L, pH 5) and then ultrasonic treatment (40 kHz, 75 W) for 10 min. Afterward, 1.3 mL GO (2 mg/mL) dispersion was added to the mixture, which was performed by ultrasonic treatment for 10 min to obtain β-CD/GO dispersion.

Preparation of β-CD/rGO/GCE
The GCE was polished using 0.05 μm of alumina slurry and ultrasonically washed for 10 min in ethanol and ultrapure water, respectively. To obtain β-CD/rGO/GCE, bare GCE was treated in β-CD/GO dispersion by cyclic voltammetry (CV) scanning of 25 segments between − 1.4 and + 0.4 V with the scan rate of 25 mV/s. Next, β-CD/rGO/GCE was further reduced in PBS solution (0.1 mol/L, pH 5) by CV scanning. The preparation method of rGO/GCE was the same as that of β-CD/rGO/GCE, except that there was no β-CD in the dispersion. The preparation process of β-CD/ rGO/GCE was shown in Scheme 1.

Characterization Methods of β-CD/rGO/GCE
The characterization methods referred to previous researches with a slight modification ).
The microscopic images of GCE, rGO/GCE, and β-CD/ rGO/GCE were obtained by a SU8010 scanning electron microscope (Hitachi, Tokyo, Japan). The Raman spectra of different samples were analyzed by a Raman spectrometer (Horiba scientific, Kyoto, Japan) using a 532nm laser in the range of 500 ~ 2500 cm −1 . The charge transfer rate and electrochemical impedance spectroscopy of different modified electrodes were studied in Fe[(CN) 6 ] 3−/4− solution (5 mmol/L).

Application of Real Samples Detection
The β-CD/rGO/GCE sensor was applied to detect the content of capsaicin in red pepper oil. The preparation method of real samples was as follows: 4 g of sample was placed into a centrifuge tube after homogenizing the red pepper oil with a tissue masher. Afterwards, 15 mL of methanol solution was added into the tube. Ultrasonic treatment was carried out (40 kHz, 150 W) at 60℃ for 15 min. To collect the supernatant, the sample was centrifuged at 8000 g for 5 min. The centrifugal extraction process was repeated twice. Finally, the solution was adjusted to 50 mL using methanol solution, and then it was filtered using a 0.22-μm filter membrane before detection. To obtain the recovery rate of our method, different concentrations (5, 10, and 20 μg/L) of capsaicin standard solution were added to the filtrate. At the same time, the LC-MS method was compared with our method. LC-MS experiments were carried out according to the Chinese national standard of GB/T 40,348-2021 ) by a TRIPLE QUAD 5500 LC-MS (Waters Corporation, Connecticut, USA).

Electrochemical Experiments
All the electrochemical experiments were performed on a LK2010 electrochemical workstation (Tianjin Lanlike Chemical Electronics High-tech Co., Ltd, Tianjin, China) in a three-electrode system: a GCE or modified GCE as the working electrode, a platinum electrode as the counter electrode, and a saturated calomel electrode as the reference electrode. The initial parameters of the CV method in capsaicin solution were as follows: the potential range was between 0 and + 1.0 V, the scan rate was 100 mV/s, and the equilibrium time was 2 s. The initial parameters of CV in 5 mmol/L [Fe(CN) 6 ] 3−/4− solution were as follows: the potential range was between − 0.2 and + 0.6 V, and other parameters were consistent with those of in capsaicin solution. The initial parameters of differential pulse voltammetry (DPV) in capsaicin solution were that the potential range was between + 0.9 and + 0.2 V, impulse amplitude was 0.05 V, pulse width was 0.2 s, pulse period was 0.5 s, and equilibrium time was 2 s.

Statistical Analysis
The raw data were exported from the electrochemical workstation, then analyzed by SPSS 24.0. Imported processed data into OriginPro2017 to draw the graphics.

Micromorphology Analysis
The morphological analysis of the GCE, rGO/GCE, and β-CD/rGO/GCE is shown in Fig. 1. The surface of bare GCE was smooth, while the other two electrodes showed different morphological characteristics. For rGO/GCE, it can be clearly observed that the nanosheets of rGO were tightly stacked together to form a dense film with a wrinkled texture pattern. It has been reported that these wrinkles were due to capillary effects during drying, which can not only increase the surface roughness and specific surface area of rGO, but also speed up its electron transfer rate (Zhou et al. 2022). Interestingly, β-CD/rGO/ GCE presented the basic morphology of rGO, and there were many tubular structures embedded on the surface of the rGO, which may be because β-CD was electrically polymerized to form polycyclodextrins (Díaz et al. 2018). Previous studies have shown that electropolymerization of cyclodextrin can improve the ability of electrochemical sensor to recognize and detect capsaicin (Zhao et al. 2020). Therefore, we speculated that the emergence of these tubular structures provided a possibility for the synergistic effect of β-CD and rGO.

Analysis of Raman Spectra
The Raman spectra of rGO and β-CD/rGO are shown in Fig. 1D. Both curves had obvious characteristic peaks at 1346 cm −1 (D band) and 1558 cm −1 (G band), indicating that the functional group composition of rGO was retained after the addition of β-CD. Moreover, it has been reported that the D band was related to the second-order double resonance scattering process of graphite material defects, the G band represented the E 2g vibration mode of the sp 2 carbon atom in the two-dimensional hexagonal lattice, and the intensity ratio of the D and G band could reflect the structure disorder degree of carbon material (Zhao et al. 2020). Compared with rGO (the ratio was 1.38), the I D /I G of β-CD/rGO was markedly decreased (the ratio was 1.31), indicating that rGO was more dispersed on the electrode surface after the addition of β-CD (Ma et al. 2017). Our previous research has proved that the better the dispersion of carbon materials, the stronger the detection performance of capsaicin .

Electrode Transfer Capacity of Different Electrodes
The electrode transfer capacity of different electrodes was tested by CV scanning in 5 mmol/L [Fe(CN) 6 ] 3−/4− solution. It was observed from Fig. 2A that the peak currents of different electrodes were significantly different, and the peak current values in descending order were β-CD/rGO/GCE, rGO/GCE, and GCE. This result showed that the β-CD/rGO/ GCE had the optimum electron transfer capacity, which may be due to the addition of more electrochemical active sites after modification of the material.
Meanwhile, the effect of scan rate on the redox peak current of [Fe(CN) 6 ] 3−/4− solution is shown in Fig. 2B. It was found that the redox peak current value increased with the increase of scan rate, and showed a good linear relationship with the square root of scan rate, indicating that the electrochemical reaction process of Fe 2+ /Fe 3+ was controlled by diffusion (Lyu et al. 2019).
Electrochemical impedance spectroscopy (EIS) was used to evaluate the interface characteristics of modified electrodes. The Nyquist plots of bare GCE, rGO/GCE, and β-CD/rGO/GCE are shown in Fig. 3. The results were fitted into the R(C(RW)) equivalent circuit model, which was involved in the resistance of the electrolyte solution (R s ), the double layer capacitance (C dl ), charge transfer resistance of the redox probe (R ct ), and the Warburg impedance (Z w ). Compared to that of bare GCE (46.3 Ω), the R ct of rGO/GCE (23.8 Ω) decreased by half, which is attributed to the excellent electronic property of rGO. After adding β-CD, the semicircle decreased dramatically (5.4 Ω); this revealed that β-CD/rGO/GCE was more conductive.

Electrochemical Response of Capsaicin on Different Electrodes
The peak current of different electrodes in HCl solution containing 10 μg/mL capsaicin is shown in Fig. 4. It could be seen that there was a weak response current on the bare GCE of capsaicin with the anodic peak current (Ipa) and cathodic peak current (Ipc) of 0.84 μA and 0.89 μA, respectively. This could be because the charge transfer rate of bare GCE was slow. After modifying the rGO, the Ipa and Ipc were significantly increased to 40.5 μA and 40.0 μA, which were nearly 45 times with those of bare GCE. This could be due to that the electrochemical reduction process of GO can remove the oxygen-containing groups and form the wrinkled film, which significantly increased the electrochemical active surface area of rGO, promoting the electron transfer rate of electrochemical reaction (Kaleem et al. 2021;Li et al. 2021). Surprisingly, the peak current of capsaicin on β-CD/rGO/GCE was higher than that on rGO/GCE, and the Ipa and Ipc were 66.9 μA and 73.1 μA. There were two main reasons for the enhancement of electrochemical performance.
One was the addition of β-CD made rGO more dispersed, which increased the electrochemical active surface area of β-CD/rGO composite, thus further improving the charge transfer rate. The other might be that polycyclodextrins can selectively combine with capsaicin into their internal cavity to form stable host-guest inclusion complexes, which effectively improved the selectivity and catalytic ability of the sensor to capsaicin (Díaz et al. 2018;Huang et al. 2019).

CV Curves Analysis of Capsaicin
The electrochemical redox reaction of capsaicin on β-CD/rGO/ GCE was investigated by CV scanning. The voltammetric signal of capsaicin in 0.02 mol/L HCl solution was characterized by 4 consecutive forward scans and reverse scans with a scan rate of 200 mV/s (Fig. 5A). During the first forward scan, an irreversible oxidation peak appeared at 680 mV; when it came to reverse scan, a reduction peak appeared at 370 mV. However, during the second forward scan, the peak current of oxidation peak at 680 mV decreased obviously, and a new oxidation peak appeared at 440 mV. Based on our previous study, an electrochemical reaction pathway of capsaicin was proposed. Firstly, when the forward scan came to 680 mV, the phenolic hydroxyl group and methoxy group of capsaicin were oxidized and hydrolyzed respectively to form the o-benzoquinone structure (Lyu et al. 2019). Secondly, when the reserve scan came to 440 mV, the o-benzoquinone structure was reduced to the o-phenol structure. Interestingly, with the continuation of scanning, the current value of redox peaks at 370 and 440 mV gradually increased, and the oxidation peak current of 680 mV continued to decline, indicating that the mutual transformation of o-benzoquinone structure and o-phenol structure was mainly in the subsequent stage, and the reaction of capsaicin to o-benzoquinone gradually disappeared (Søpstad et al. 2018;Lyu et al. 2019).

Effect of Scan Rate
The β-CD/rGO/GCE was placed in capsaicin solution to study the relationship between scan rate (v) and peak current (Ip). As shown in Fig. 5B, with the increase in scan rate, the oxidation peak potential of capsaicin moved positively, and its peak current increased gradually. After linear fitting, the peak current featured a good linear relationship with both square root of scan rate (Fig. 5C) and scan rate (Fig. 5D), indicating that the electrochemical reaction of capsaicin on the surface of β-CD/rGO/GCE was controlled by diffusion and adsorption at the same time (Rezaei and Damiri 2008; CV curves of β-CD/rGO/GCE at different scan rates (B). The illustration is the correlation between the peak current and square root of scan rate Lyu et al. 2019). Simultaneously, the oxidation peak shifted positively and the reduction peak shifted negatively by changing the scan rate, indicating that there were electrons involved in the redox reaction of capsaicin. According to the Nernst equation and previous research, the number of electrons is about 2 (Supchocksoonthorn et al. 2020). Figure 6A displays the CV curves of capsaicin under different pH (from 1 to 8). Hydrochloric acid at 0.1 mol/L was used as the supporting electrolyte for pH = 1, and PBS was used as the supporting electrolyte for pH = 2-8. The potential of redox peak (Ep) shifted negatively with the increase of pH, and the peak current decreased gradually, reflecting that protons were involved in the redox reaction (Chen et al. 2018). Acidic conditions were more conductive to the detection of capsaicin, owing to the side effects of reducing the concentration of capsaicin under alkaline conditions, including deprotonation and dimerization (Kachoosangi et al. 2008;Yu et al. 2012). The calibration curve between Ep and pH was Ep (V) = − 0.05778 pH + 0.47197, R 2 = 0.9933 (Fig. 6B). In the equation, − 57.8 mV/pH was very close to the theoretical slope (− 59.1 mV/pH) for transferring an equal number of protons and electrons (Chen et al. 2018).

Optimization of Electrode Modification Conditions
According to the results of preliminary experiments, it was found that the performance of β-CD/rGO/GCE was mainly affected by the deposition parameters of β-CD/rGO, including the pH of the deposition solution, potential range, and sweep segment during the modification process. Thus, the  optimal parameters of the above influencing factors were explored by changing the experimental conditions.

Influence of pH Value of Deposition Solution
The response current of capsaicin obtained in different pH deposition solutions was significantly different. The response current increased rapidly when the pH of deposition solution increased from 3 to 5, but decreased significantly when the pH of deposition solution continued to rise. So the β-CD/rGO/ GCE had the highest peak current of capsaicin when the pH value of the deposition solution was 5. It has been reported that the pH values of both too low and too high were detrimental to the deposition of GO. The too low pH could cause GO to precipitate or gelation, promoting hydrogen evolution during the electrodeposition process, meanwhile too high pH could lead to rGO instability (Zhou et al. 2022). Therefore, PBS (pH = 5.0) was selected as the supporting deposition.

Influence of Potential Range
It has been experimentally found that when the upper limit of potential range was 0.4 V and the lower limit of potential range increased from − 1.6 V to − 1.2 V, the response current of capsaicin increased first and then decreased, and reached the maximum at − 1.4 V. This may be due to the fact that the lower limit was too high to carry out the electrochemical reduction reaction of GO normally, while the lower limit was too low, which could produce by-products affecting the electrochemical reaction (Wang et al. 2013;Zhou et al. 2022). However, when the lower limit of potential range was − 1.4 V and the upper limit of the potential increased from 0.4 to 0.8 V, the peak current of capsaicin fluctuated slightly, indicating that the effect of the upper limit on peak current of capsaicin was slight (Wang et al. 2013).

Influence of Sweep Segments
Additionally, it was observed that the peak current of capsaicin increased with the rise of sweep segments, and reached the maximum in the 25th segment, which may be because β-CD/rGO composite material deposited on the electrode increased with the increase of the number of sweep segments, thus increasing the active site of capsaicin (Zhou et al. 2010). However, the response current of capsaicin decreased as the number of sweep segments increased to 30, which may be because the increasing number of sweep segments led to excessive accumulation of β-CD/rGO, or because the electrochemical reaction time is too long, causing the occurrence of side reactions (Wang et al. 2013). Therefore, 25 segments were used as the optimal sweep segments.

Analytical Performance by DPV
The DPV response current of capsaicin with different concentrations on the surface of β-CD/rGO/GCE is shown in Fig. 7A. In the range of 0.1 ~ 40 μg/mL, the peak current value of capsaicin increased with the increase of concentration, and showed a good linear relationship. After linear fitting, the equation was Ip (μA) = 2.953 C (μg/mL) + 1.499 (n = 6), R 2 = 0.995, where Ip represents peak current value of capsaicin, and C represents the concentration of capsaicin. Based on the method supplied by the International Union of Pure and Applied Chemistry (IUPAC) , the limit of detection (LOD) of β-CD/rGO/GCE for capsaicin was calculated to be 0.05 μg/mL. The low detection limit of β-CD/rGO/GCE is attributed to the high electron transfer rate of β-CD/ rGO, and the recognition and adsorption of capsaicin by polycyclodextrins. The analytical properties of this method to determine capsaicin are compared to some of the reports in the literature (Table 1). The LOD of our proposed sensor was lower than that of Wang et al. (2016) and Gu et al. (2021), and the quantitative detection range was larger than that of Lyu et al. (2019) and Díaz et al. (2018), which meant that β-CD/rGO/ GCE had wider applications. Although the sensor reported by Supchocksoonthorn et al. (2020) had excellent performance, its preparation process is complex.

Reproducibility and Stability
In order to test the reproducibility of β-CD/rGO/GCE, capsaicin (30 μg/mL) was detected with the same electrode repeatedly modified 5 times. As a result, the response current values were 85.13, 83.89, 84.50, 86.81, and 84.97 μA respectively, and the relative standard deviation (RSD) was 1.28% (n = 5), indicating that β-CD/rGO/GCE had good reproducibility. To evaluate the long-term stability of β-CD/ rGO/GCE, we stored them at room temperature for 1 week and detected 30 μg/mL capsaicin solution. The results showed that the response current of capsaicin remained at 95% of its initial value, reflecting the good stability of the modified electrodes.

Anti-interference Analysis
The common additives and interfering ions in foods were added into capsaicin solution in order to test the antiinterference performance of β-CD/rGO/GCE. Ascorbic acid is common endogenous interference in plant samples (Lyu et al. 2019). Besides, sucrose and sodium glutamate are usually added to enhance the taste of food (Supchocksoonthorn et al. 2020). Sucrose (0.2 mg/mL), sodium glutamate (0.2 mg/mL), ascorbic acid (0.2 mg/mL), and inorganic salt ions (0.1 mg/mL of Na + , Cl − , K + , Ca 2+ , SO 4 2− , Mg 2+ ) were added into capsaicin solution, respectively. As shown in Fig. 7B, the peak current changes of capsaicin were not obviously under interfering substances, indicating that these interfering substances had little influence on the detection of capsaicin (P < 0.05).

Real Sample Analysis
In order to verify the accuracy and practicability of β-CD/ rGO/GCE, red pepper oil was used as the real sample ( Table 2). The content of capsaicin in red pepper oil was 105.714 mg/kg by the method proposed in our paper, which was close to the LC-MS test result of 107.615 mg/ kg. The recovery rates of capsaicin in red pepper oil were 94.83 ~ 115.75%, and the relative standard deviation (RSD) was less than 10.87%. Meanwhile, there was no significant difference between the results of our method and those of LC-MS through the paired t test (P < 0.05). These results indicated that the method proposed in our paper had strong application potential in red oil detection.

Conclusions
In this work, an electrochemical sensor for the rapid and accurate detection of capsaicin in red pepper oil was constructed based on rGO enhanced by β-CD. The excellent performance  of the sensor mainly is because the introduction of β-CD increased the electrochemical active surface area of β-CD/ rGO composite, and polycyclodextrin on the surface of rGO nanosheets provided the possibility of capsaicin enrichment on the surface of the sensor. The quantitative detection range and the detection limit of our sensor for capsaicin were 0.1 ~ 40 μg/ mL and 0.05 μg/mL, respectively. Meanwhile, the sensor also had good anti-interference and repeatability. When applied to red pepper oil samples, the sensor showed an acceptable practical application potential. The recovery rates of capsaicin in red pepper oil samples were 84.86 ~ 15.23%, and the RSD was less than 10.74%. In a word, our work is of great significance for the application of red pepper oil in the food industry.
Funding This work was financially supported by grants from the Major Science and Technology Projects of Anhui Province (202003a06020029) and the National Key R&D Program of China (2019YFC1605900).
Data Availability All data generated or analyzed during this study are included in this published article and its supplementary information files.

Declarations
Ethics Approval This article does not contain any studies with human participants performed by any of the authors.

Consent to Participate Not applicable.
Conflict of Interest Ning Yun declares that she has no conflict of interest. Chaoqun Lu declares that he has no conflict of interest. Tian Sun declares that she has no conflict of interest. Baocai Xu declares that he has no conflict of interest. Yunshen Song declares that he has no conflict of interest. Zibing Zong declares that he has no conflict of interest. Kangwen Chen declares that he has no conflict of interest. Ganhui Huang declares that he has no conflict of interest. Xingguang Chen declares that he has no conflict of interest. Qianhui Gu declares that he has no conflict of interest.