A novel molecular imprinted electrochemical biosensor for sensitive determination of Ganoderic acid A

Analytical approaches with the features of easy manipulation, cost-effectiveness, and miniature are needed for practice-oriented point-of-care diagnostics. This work prepared the molecularly imprinted polymers (MIPs) to selectively detect Ganoderic acid A (GAA). To determine the optimum composition, we examined diverse functional monomers through batch rebinding experiments. The new method uses nanomaterials-functionalized screen-printed carbon electrode (SPCE) to selectively measure GAA, the critical ingredient in Ganoderma lucidum (G. lucidum). Reduced graphene oxide (RGO) chitosan attached to SPCEs has offered the electrochemical sensor core to detect GAA. Cyclic voltammetry (CV) was performed on the SPCE to electropolymerize MIP using a solution containing GAA with o-phenylenediamine being the template. Thereafter, this sensor was characterized by scanning electron microscopy (SEM), and CV together with electrochemical impedance spectroscopy (EIS). GAA was analyzed through differential pulse voltammetry (DPV) within the 0.1-100 ng/mL linear range, and the limit of detection (LOD) was 0.03 ng/mL. The sensor’s simplicity, selectivity, and environmental friendliness make it ideal to detect GAA.


Introduction
The public has paid more and more attention to drug safety, and people tend to select natural medicinal products obtained from fungi and plants due to their distinct merits such as low side reactions and toxicity.Ganoderma lucidum (G.lucidum), a medicinal mushroom belonging to basidiomycetes, is extensively applied to promote health and longevity in Asian countries including China as the traditional medicine and food [1] .There are numerous studies conducted to analyze the bioactive ingredients in G. lucidum because of its diverse possible medicinal applications.According to relevant research results, most Ganoderma species possess hundreds of bioactive compounds such as triterpenoids, polysaccharides, sterols, and glycopeptides [2,3] .Of them, triterpenoids and polysaccharides may be the main bioactive ingredients, which display the pharmacological activities of immunoregulation, anticancer, anti-inflammation, hypolipidemia, and hypoglycemia [4,5] .Therefore, Ganoderma materials are always adopted for preventing and treating various diseases [6][7][8][9] .
In 1982, Kubota isolated GAA and Ganoderic acid B(GGB) in G. lucidum (FR.)KARST, since then, over 316 triterpenes have been extracted from spores, fruiting bodies, mycelia, and gills of numerous Ganoderma mushrooms [10] .In recent years, lax regulation has led to the chaotic healthcare product So far, diverse conventional approaches are proposed to determine GAA, like high-performance liquid chromatography (HPLC) [11] , thin-layer chromatography (TLC) [12] , immunochromatographic strip assay [13] , infrared spectroscopy (IR) [14] , and mass spectrometry (MS) [15] .But the above approaches are associated with drawbacks including costly equipment, complicated manipulation, poor real-time response, and low sensitivity.These traditional methods limit the development of Ganoderma lucidum triterpenoids due to the long time-consuming, high solvent consumption, and complexity of sample prepreparation and operation.Therefore, developing new analytical methods is particularly important.For drug regulatory authorities, an efficient and simple marker detection method is necessary.
In recent years, great attention has been paid to electrochemical sensors due to their rapid response, great sensitivity, easy manufacturing as well as real-time detection [16] .Besides, such sensors are costeffective, with higher field portability.Notably, tremendous interest has been shown in combining molecularly imprinted polymers (MIPs) and electrochemical sensors.MIPs can recognize templates due to those several binding sites generated between monomers and template molecules.In particular, MIPmodified electrochemical sensor is more sensitive and selective compared with conventional sensors during electrochemical analysis [17] .
In order to overcome the existing problems, a GAA molecular imprinting electrochemical detection method with high stability and strong specificity was designed by using MIP.The present work synthesized the novel electrochemical sensor on the basis of molecularly imprinted o-phenylenediamine (o-PD) on the reduced graphene oxide (RGO) nanocomposite-modified SPE surface for GAA determination.Besides, the electrochemical performances of this MIP sensor to GAA, including linearity, selectivity, stability, and reproducibility, were analyzed.

Apparatus
All voltammetric measurements were performed at room temperature using a CHI1030C potentiostat/galvanostat.The screen-printed carbon electrodes used in this study were purchased from Zensor R＆D CO. Ltd. (SPCEs, TE100-TH).Each of them consisted of a carbon working electrode, a graphite auxiliary electrode, and a silver pseudo-reference electrode.

Preparation of MIP/RGO-CS screen-printed electrodes (MIP/RGO-CS/SPCE)
The SPEs were rinsed with ultrapure water and dried naturally before use.50 mg RGO is added into 50 mL CS liquid followed by ultrasonic for 30 min.The SPE sensor was prepared by casting the suspension of RGO-CS suspension (5 μL) onto the working electrode area and dried with an infrared lamp.
PBS buffer solution (10 mmol• L-1, pH = 7.0) containing 5:1 o-phenylenediamine and GAA was prepared, and the modified electrode was placed in the buffer solution for electropolymerization.The electropolymerization reaction condition was that the scanning voltage range was between (-0.1⁓ 0.1 V) ⁓ (+0.7⁓+ 0.9V).The electrode was washed with ultra-pure water and dried to obtain a molecularly imprinted polymer film.The template molecule GAA was washed in ethanol-water eluent and rinsed with a pH of 7.0 PBS buffer to obtain the imprinting site.

Electrochemical measurements
All the solution was saturated with N 2 during electrochemical measurements.Cyclic voltammetry (CV) scans were conducted with 0.1 M phosphate buffer solution (PBS, pH 7.0, supplemented with 0.1 M KCl) to be the supporting electrolyte, and the scan potential region was -0.2 V~0.5 V at the 50 mV⋅s -1 scan rate apart from scan rate analysis part.In addition, we obtained differential pulse voltammetry (DPV) scans with 0.1 M PBS (pH 7.0, supplemented with 0.1 M KCl) to be the supporting electrolyte, and the scan potential region was -0.4 V~0.6 V at the 50 mV⋅s -1 scan rate apart from scan rate analysis part.

Morphological characterizations of the electrode surface
Figure 1 shows a scanning electron microscope (SEM) image of the sensitive membrane.The thickness of the sensitive film is 0.7-1.3nm, the diameter of the sensitive film reduced graphene oxide is 0.6-4.5 μm, and the single layer rate is 70-80%.The transverse size of the sensitive film is large, its surface is relatively smooth and slightly wrinkled, the edge of the layer folds and curls, and the number of layers is less.The functional membrane compounded by the sensitive membrane uses DPV to increase the peak value of the potassium ferricyanide electrolyte by 60-80 μ A. The sensitive film has good dispersion, stable structure, and excellent electrochemical performance.

Electrochemical behaviors
Figure 2 shows the CV test results of different modified electrodes in potassium ferricyanide and potassium chloride solutions.The electrode was placed in a phosphate probe solution of 0.1 mol• L-1 KCl and 5.0 mmol• L-1 K 3 [Fe (CN) 6 ] for CV scanning until a stable REDOX peak was obtained.CV scanning speed is 50 mV/s while scanning potential is -0.4 to + 0.6 V. Results as shown in curve a in Figure 2, it can be seen that naked SPCE has a good reversible REDOX reaction.As shown in Figure 2, Curve a represents the functional membrane without a modified sensitive membrane, and its peak value in potassium ferricyanide electrolyte is 40 μA.Curve b represents the functional membrane after modification of the sensitive membrane, and its peak value is 54 μA, indicating that the sensitive membrane has good electrocatalytic performance, and the peak value can be increased by 70μA in potassium ferricyanide electrolyte by using DPV.
Figure 2 shows the CV test results of different modified electrodes in potassium ferricyanide and potassium chloride solutions.The electrode was placed in a phosphate probe solution of 0.1 mol• L-1 KCl and 5.0 mmol• L-1 K 3 [Fe (CN) 6 ] for CV scanning until a stable REDOX peak was obtained.CV

(a) (b)
scanning speed is 50 mV/s while scanning potential is -0.4 to + 0.6 V. Results as shown in Curve a in Figure 2, it can be seen that naked SPCE has a good reversible REDOX reaction.As shown in Figure 2, Curve a represents the functional membrane without a modified sensitive membrane, and its peak value in potassium ferricyanide electrolyte is 40 μA.Curve b represents the functional membrane after modification of the sensitive membrane, and its peak value is 54 μA, indicating that the sensitive membrane has good electrocatalytic performance, and the peak value can be increased by 70 μA in potassium ferricyanide electrolyte by using DPV.

Effect of elution time.
The electropolymerized electrodes were placed into ethanol/water eluent for 2, 4, 6, 8, and 10 min, respectively, and the template molecules were washed away for DPV detection.
The results are in Figure 3(a).With the increase of elution time, ΔIp increases first and then basically stays the same, reaching the maximum value at 6 min, indicating that when the elution time is 6 min, the elution amount of template molecules is the largest, so the optimal elution equilibrium time is finally selected as 6 min.

Effect of adsorption time.
The electropolymerized electrodes were placed into 10 nmol/L GAA solution for adsorption for 5, 10, 15, 20, and 25min, and the template molecules were washed away for DPV detection.The results are shown in Figure 3(b).With the increase of adsorption time, the peak current value decreased first and then increased, and basically tended to remain unchanged after 15 min, indicating that the imprinted holes on MIP were basically saturated at 15 min, so the optimal adsorption time was finally selected as 15 min.

Number of electropolymerization cycles.
Under the conditions of an elution time of 6 min and an adsorption time of 15 min, only the number of scanning cycles of electropolymerization was changed, the peak current after electropolymerization and elution of each group of electrodes was measured, and the travel value was calculated.Figure 4(b) shows the results.Different electropolymerization cycle numbers will affect the thickness of the molecule-imprinted film on the electrode surface, and too thin or too thick will affect the subsequent experiments.According to experimental results, peak current difference first increases and then decreases as the electropolymerization cycle number increases, and reaches its maximum value at 10 cycles, so 10 cycles is chosen to be the best electropolymerization cycle number.

Determination of GAA
Through conducting CV at the 50 mV/s scan rate, we measured the responses of GAA at varying doses within 0.1 M PBS (pH 7.0) to evaluate the analysis performance of this sensing platform.Figure 4 shows the calibration plot of GAA sensing, in which Ipa was linearly related to concentration within the specific concentration range.The regression equation: ΔIp(μA) = 21.26LgC(ng• mL-1) +20.87 with R 2 =0.992.The linear relationship of current within the 0.1-100 ng• mL -1 concentration range is written in Figure 3. respectively.Additionally, such linearity was not observed after GAA was added at >100 ng• mL -1 , which was because active sites were saturated in this sensor at high concentrations.Additionally, Eq.LOD = 3σ/S was utilized to determine the LOD of this GAA sensor according to the standard deviation of the blank sample, LOD = 3σ/S, where the σ is the standard deviation for 11 blank samples and the S is the slope of the linearity.Finally, LOD was determined as 0.03 ng• mL -1 .Therefore, CV analysis can quantitatively and qualitatively measure the GAA level by using this sensor on the basis of RGO-CS/SPCE.This GAA sensor is advantageous compared with traditional sensors with regard to its low maintenance expenses, portability, simple synthesis, and on-site monitoring.We utilized SPE for preparing the sensing platform, and it is confirmed as a superb sensor, making it possible for in-situ GAA determination within real-world samples.Also, this is the first report about screen-printed electrochemical sensors for GAA.Therefore, this synthesized RGO-CS/SPCEbased BPSIP electrochemical sensor is comparable or superior to additional sensors reported in the literature with regard to the LOD, simplicity, accuracy, sensitivity, calibration range, cost-effectiveness, short-time analysis, and reliability in technological applications.For verifying the GAA sensitivity of this RGO-CS/SPCE sensor, we chose Ganoderic acids G, D, F, and C2 to be interferents.According to our observations (Figure 5), other Ganoderic acids did not interfere with GAA detection.Consequently, this RGO-CS/SPCE sensor is highly selective to GAA.

Conclusions
The present work manufactured RGO-CS and characterized it with suitable approaches.This RGO-CS was modified with commercial SPCE for designing RGO-CS/SPCE to electrochemically monitor GAA, the endocrine-disrupting factor.SPCE has not been applied in electrochemical GAA detection until the present study.This proposed sensor has shown a calibration range of 0.1-100 ng• mL -1 for GAA sensing, and its LOD was 0.03 ng• mL -1 .They were commonly seen as interferents like Ganoderic acid G, D, F, and C2, and did not interfere with GAA detection.According to Table 1, this RGO-CS/SPCE sensor achieved comparable or superior sensitivity, LOD, and linear range to sensors reported in the literature.Its high sensitivity, incomparable electrochemical response, low LOD, and reliability in in-situ monitoring have made it the potential next-generation sensing platform for economical production in bulk.Typically, the technique can be translated from the laboratory to the field, which can help the development of the Chinese medicine industry.Sensors can also be built to detect multiple ganoderic acids at the same time in the future.

Figure 4 .
Figure 4. (a) Linear relationship between peak current value and GAA concentration; (b) DPV curves of different GAA concentrations.
market, and the situation of infringing on the interests of consumers occurs from time to time, and also hinders the development of this field.The main reason lies in the lack of research on standard substances. 2