Comparative Studies on Carbon Paste Electrode Modified with Electroactive Polyamic Acid and Corresponding Polyimide without/with Attached Sulfonated Group for Electrochemical Sensing of Ascorbic Acid

In this study, electroactive poly (amic acid) (EPAA) and corresponding polyimide (EPI) without or with a sulfonated group (i.e., S-EPAA, and S-EPI) were prepared and applied in electrochemical sensing of ascorbic acid (AA). The electroactive polymers (EAPs) containing EPAA/EPI and S-EPAA/S-EPI were synthesized by using an amine-capped aniline trimer (ACAT) and sulfonated amine-capped aniline trimer (S-ACAT) as an electroactive segment that controlled the redox capability and influenced the degree of sensitivity of the EAPs towards AA. Characterization of the as-prepared EAPs was identified by FTIR spectra. The redox capability of the EAPs was investigated by electrochemical cyclic voltammetric studies. It should be noted that the redox capability of the EAPs was found to show the following trend: S-EPAA > S-EPI > EPAA > EPI. For the electrochemical sensing studies, a sensor constructed from an S-EPAA-modified carbon paste electrode (CPE) demonstrated 2-fold, 1.27-fold, and 1.35-fold higher electro-catalytic activity towards the oxidation of AA, compared to those constructed using a bare CPE, S-EPI-, and EPI/EPAA-modified CPE, respectively. The higher redox capability of S-EPAA-modified CPE exhibited a good electrochemical response towards AA at a low oxidative potential, with good stability and selectivity. Moreover, an electrochemical sensor constructed from S-EPAA-modified CPE was found to reveal better selectivity for a tertiary mixture of AA/DA/UA, as compared to that of EPI-modified, EPAA-modified and S-EPI-modified CPE, based on a series of differential pulse voltammograms.


Introduction
Vitamin C plays a vital role in the production of neurotransmitters and tissue repair [1]. It is necessary for the functions of several enzymes and the regulation of various biological functions, including functioning of the immune system, wound healing and collagen synthesis [2]. It is an essential nutrient for higher primates and a few other organisms. Vitamin C deficiency can cause scurvy [3,4]. The pharmacodynamic group of vitamin C is ascorbate ion. It is also of prime importance in some metabolic redox reactions in the human body. Therefore, ascorbic acid (AA) testing is of prime importance in the food, pharmaceutical and clinical industries. A variety of techniques for AA determination, including spectroscopy, chromatography, enzymatic and electroanalysis, have been reported [5][6][7][8]. Among them, because of its high sensitivity and simple operation, the electrochemical method is considered one of the best potential methods.
The sensitive materials currently used in electrochemical detection contain inorganic or organic materials. Studies have found that inorganic materials have excellent sensing capability and, therefore, affect the electrochemical sensing of AA. Hence, in this study, an electrochemical sensor was constructed by a carbon paste electrode (CPE) modified with EPAA, EPI, S-EPAA and S-EPI and applied in the detection of AA. The sensitivity, selectivity, and stability of CPE modified with four distinctive EPAs were investigated and compared systematically.

Preparation of Electroactive Polyamic Acid (EPAA/S-EPAA) and Polyimide (EPI/S-EPI)
Electroactive polyamic acid and the corresponding polyimides were prepared as reported in the literature [39] (Scheme 1). Briefly, EPAA and S-EPAA were synthesized from the polymerization of BPADA and ACAT/S-ACAT, respectively. The respective monomer was dissolved in DMSO at room temperature under nitrogen atmosphere, and then BSAA was added slowly into the solution. After that, the mixture was stirred for 6 h followed by precipitation, filtration and vacuum drying in oven at 60 • C for 3 h to obtain EPAA and S-EPAA.

Redox Property of the as-Prepared Materials
In this study, the synthesized materials (e.g., EPAA, S-EPAA, EPI and S-EPI) underwent CV measurements to determine the redox properties. Firstly, 1 wt% solution of the asprepared materials was prepared in DMAc, followed by spin coating on ITO electrodes (5 × 5 cm 2 ). Subsequently, the ITO electrodes were dried by heating at 120 • C for 1 h. CV measurements were recorded at room temperature, with a double-wall jacketed cell in 1.0 M H 2 SO 4 with platinum foil and Ag/AgCl (3 M NaCl solution) served as the auxiliary and the reference electrode, respectively.

Preparation of Electroactive Polyamic Acid (EPAA/S-EPAA) and Polyimide (EPI/S-EPI)
Electroactive polyamic acid and the corresponding polyimides were prepared as reported in the literature [39] (Scheme 1). Briefly, EPAA and S-EPAA were synthesized from the polymerization of BPADA and ACAT/S-ACAT, respectively. The respective monomer was dissolved in DMSO at room temperature under nitrogen atmosphere, and then BSAA was added slowly into the solution. After that, the mixture was stirred for 6 h followed by precipitation, filtration and vacuum drying in oven at 60 °C for 3 h to obtain EPAA and S-EPAA. Scheme 1. Preparation of EPAA/EPI and S-EPAA/S−EPI.

Preparation of Test Solutions
(1) PBS solution: 0.1 M PBS solution (pH 7.02) was prepared by dissolving 9.6 g of Dulbecco's PBS powder in 1 L of deionized water. (2) AA solution: 2 mM AA solution was prepared in PBS.
(3) CPE preparation: 30 mg carbon paste (weight ratio: graphite:paraffin oil, 3:1) and 20 mg of the given EAP were homogenously mixed and neatly filled in the groove at the front of the electrode to perform the electroactivity test. (4) AA sensing: A homogeneous mixture of the as-prepared EAP (i.e., EPAA, S-EPAA, EPI and S-EPI) and carbon paste (60:6 mg) was prepared by grounding it with a Platinum foil was used as the reference and Ag/AgCl (3 M NaCl solution) as the counter electrodes. The CPEs modified with EPAA, S-EPAA, EPI and S-EPI were used as the working electrodes at room temperature (25 • C).

Characterization
ACAT and S-ACAT were fully characterized as shown in Figures S1 and S2, respectively. EPAA/S-EPAA and EPI/S-EPI were characterized by FTIR, as shown in Figure 1. The main vibration peaks in the spectrum of EPAA in Figure 1a are the stretching vibration of the amide group (C=O) at 1605 cm −1 , carboxylic acid group (C=O) at 1717 cm −1 and the carboxylic acid group (O-H) at 2400-3400 cm −1 . After the ring closure of EPI, the spectrum in Figure 1a shows that the original peaks at 1605 and 1717 cm −1 became a unique stretching band of the imide ring (C=O) at 1724 cm −1 , and the vibration band of the carboxylic acid group (O-H) at 2400-3400 cm −1 also disappeared. In addition, the peak for imide ring (C-N-C) also appeared at 1367 cm −1 and the bending vibration of the imide ring (C-N-C) appeared at 740 cm −1 . Similarly, in the FTIR spectrum of S-EPAA in Figure 1b, the original vibration peaks of the amide group (C=O) and carboxylic acid group (C=O) were observed at 1600 and 1715 cm −1 , respectively. After the ring closure of the imidized material, the peak at 1720 cm −1 became remarkably enhanced in the FTIR spectrum of S-EPI, the stretching band of the imide ring (C-N-C) also appeared at 1370 cm −1 , and the bending vibration of the imide ring (C-N-C) also appeared at 740 cm −1 . The FTIR spectra confirmed that the four EAP materials, namely, EPAA, EPI, S-EPAA and S-EPI, were successfully prepared.

Polymer Electroactivity Measurement
The redox nature of ACAT/S-ACAT ( Figure S3) and the as-synthesized samples (i.e., EPAA, EPI, S-EPAA and S-EPI) was tested by CV in 1 M H 2 SO 4 . The observed oxidation current density of EPAA (39 µA/cm 2 ) was 1.6 times higher than that of EPI (24 µA/cm 2 ). The results suggest that the two carboxylic acid groups of EPAA can result in a greater redox capacity. Using the above measurement method, the electroactive polyamic acid with sulfonic acid and the electroactive polyimide with sulfonic acid were compared. The results showed that the redox abilities of S-EPAA and S-EPI were greater than those of EPAA and EPI, respectively. S-EPI showed a current density of 40 µA/cm 2 , whereas the highest value observed for S-EPAA was a current density of 49 µA/cm 2 . The CV test result in Figure 2 shows that the redox capabilities of the EAPs were in the following order: SEPAA > SEPI > EPAA > EPI. The CV results also showed that EPAA/S-EPAA and EPI/S-EPI have reversible redox abilities. The principle of electrochemical catalysis can be used to oxidize AA in the oxidized aniline segment, and the current can be detected by CV to achieve the AA detection.

Polymer Electroactivity Measurement
The redox nature of ACAT/S-ACAT ( Figure S3) and the as-synthesized samples (i.e., EPAA, EPI, S-EPAA and S-EPI) was tested by CV in 1 M H2SO4. The observed oxidation current density of EPAA (39 μA/cm 2 ) was 1.6 times higher than that of EPI (24 μA/cm 2 ). The results suggest that the two carboxylic acid groups of EPAA can result in a greater redox capacity. Using the above measurement method, the electroactive polyamic acid with sulfonic acid and the electroactive polyimide with sulfonic acid were compared. The results showed that the redox abilities of S-EPAA and S-EPI were greater than those of EPAA and EPI, respectively. S-EPI showed a current density of 40 μA/cm 2 , whereas the highest value observed for S-EPAA was a current density of 49 μA/cm 2 . The CV test result in Figure 2 shows that the redox capabilities of the EAPs were in the following order: SEPAA > SEPI > EPAA > EPI. The CV results also showed that EPAA/S-EPAA and EPI/S-EPI have reversible redox abilities. The principle of electrochemical catalysis can be used  Figure 3a shows the electroactivities of EPAA, EPI, S-EPAA and S-EPI via CV operating in 40 mL of 2 mM AA solution at 50 mV/s. The measured CV curves showed a clear oxidation peak, which indicates that AA was oxidized after electrochemical catalysis. The lower oxidation potential or the larger oxidation current, the more suitable the material is for the electrocatalytic oxidation of AA. to oxidize AA in the oxidized aniline segment, and the current can be detected by CV to achieve the AA detection.  Figure 3a shows the electroactivities of EPAA, EPI, S-EPAA and S-EPI via CV operating in 40 mL of 2 mM AA solution at 50 mV/s. The measured CV curves showed a clear oxidation peak, which indicates that AA was oxidized after electrochemical catalysis. The lower oxidation potential or the larger oxidation current, the more suitable the material is for the electrocatalytic oxidation of AA.
Oxidation currents were generated at the working electrodes of 2 mM AA in PBS solution. The electrode sensing ability of EPAA was better than that of EPI, and S-EPAA had better sensing ability than S-EPI. In general, the resonance effect of the sulfonate-enhanced aniline ring helps to enhance its oxidation ability, which results in better sensing ability for AA, and the best sensing ability was found in S-EPAA, which has sulfonate and carboxylic acid groups. Additionally, AA undergoes oxidation via the following mechanism, as shown in Scheme 2. [30] Scheme 2. Redox reaction that occurred between AA and S-EPAA. Furthermore, the repeatability of the constructed electrochemical sensor for AA detection was also investigated, as shown in Figure 3b. It should be noted that, by detection of 2 mM of AA, S-EPAA-modified CPE was found to reveal good repeatability at the stable current density of 122 A/cm 2 , with the minimum relative standard deviation of 0.43%, as shown in Figure 3b. However, the S-EPI-modified CPE was found to exhibit a stable current density of ~110 A/cm 2 , with the minimum relative standard deviation of 0.59%. After this testing period, the constructed electrochemical sensor was found to retain ~93-94% of its initial response, indicating that the four constructed electrochemical sensors all reveal excellent repeatability.

Amperometry
The cyclic voltammograms of the modified CPE electrodes performed at various concentrations of AA (0-10 mM) in PBS (pH 7.0) are shown in Figure S4. The detection was attributed to the reduction reaction of EPAA, EPI, S-EPAA and S-EPI and oxidation of AA. The increase in AA concentration increased the current value. Figure 4 shows the plot of the AA oxidation peak current density versus AA concentration, which indicates that the concentration of AA and oxidation peak current have a direct relationship and the slope values of 36.5, 27.3, 28.7 and 34.5 μA/mM were observed for EPAA, EPI, S-EPAA and S-EPI, respectively. These results indicate that the modified CPE electrodes effectively catalyzed the oxidation of AA. Oxidation currents were generated at the working electrodes of 2 mM AA in PBS solution. The electrode sensing ability of EPAA was better than that of EPI, and S-EPAA had better sensing ability than S-EPI. In general, the resonance effect of the sulfonate-enhanced aniline ring helps to enhance its oxidation ability, which results in better sensing ability for AA, and the best sensing ability was found in S-EPAA, which has sulfonate and carboxylic acid groups. Additionally, AA undergoes oxidation via the following mechanism, as shown in Scheme 2 [30].
Oxidation currents were generated at the working electrodes of 2 mM AA in PBS solution. The electrode sensing ability of EPAA was better than that of EPI, and S-EPAA had better sensing ability than S-EPI. In general, the resonance effect of the sulfonate-enhanced aniline ring helps to enhance its oxidation ability, which results in better sensing ability for AA, and the best sensing ability was found in S-EPAA, which has sulfonate and carboxylic acid groups. Additionally, AA undergoes oxidation via the following mechanism, as shown in Scheme 2. [30] Scheme 2. Redox reaction that occurred between AA and S-EPAA.

Scheme 2.
Redox reaction that occurred between AA and S-EPAA.
Furthermore, the repeatability of the constructed electrochemical sensor for AA detection was also investigated, as shown in Figure 3b. It should be noted that, by detection of 2 mM of AA, S-EPAA-modified CPE was found to reveal good repeatability at the stable current density of 122 µA/cm 2 , with the minimum relative standard deviation of 0.43%, as shown in Figure 3b. However, the S-EPI-modified CPE was found to exhibit a stable current density of~110 µA/cm 2 , with the minimum relative standard deviation of 0.59%. After this testing period, the constructed electrochemical sensor was found to retain~93-94% of its initial response, indicating that the four constructed electrochemical sensors all reveal excellent repeatability.  Figure S4. The detection was attributed to the reduction reaction of EPAA, EPI, S-EPAA and S-EPI and oxidation of AA. The increase in AA concentration increased the current value. Figure 4 shows the plot of the AA oxidation peak current density versus AA concentration, which indicates that the concentration of AA and oxidation peak current have a direct relationship and the slope values of 36.5, 27.3, 28.7 and 34.5 µA/mM were observed for EPAA, EPI, S-EPAA and S-EPI, respectively. These results indicate that the modified CPE electrodes effectively catalyzed the oxidation of AA.
Sensitivity is a very important reference value for materials used as sensors. The fixedampere method was used to test the current response ability and measure the difference in sensitivity between the CPEs and CPEs modified with EPAA, EPI, S-EPAA and S-EPI. The modified CPE was placed in a homogenized stirred solution of PBS (30 mL). The chronoamperometry program was set at 510 mV, with 5 s −1 scanning frequency. Figure 5a shows the amperometric responses of the bare CPEs and CPEs modified with EPAA, EPI, S-EPAA and S-EPI, with the continuous addition of AA in the PBS solution. The as-synthesized electrode increased the current successively with the successive addition of AA in 0.1 M PBS (potential 0.51 V). Each step showed a current increase with an increment of 15 mM AA. CPE can perform detection. Only a weak oxidation current response was generated by the successive addition of AA. All other electrodes showed a higher response compared with CPE.  Sensitivity is a very important reference value for materials used as sensors. The fixed-ampere method was used to test the current response ability and measure the difference in sensitivity between the CPEs and CPEs modified with EPAA, EPI, S-EPAA and S-EPI. The modified CPE was placed in a homogenized stirred solution of PBS (30 mL). The chronoamperometry program was set at 510 mV, with 5 s −1 scanning frequency. Figure 5a shows the amperometric responses of the bare CPEs and CPEs modified with EPAA, EPI, S-EPAA and S-EPI, with the continuous addition of AA in the PBS solution. The as-synthesized electrode increased the current successively with the successive addition of AA in 0.1 M PBS (potential 0.51 V). Each step showed a current increase with an increment of 15 mM AA. CPE can perform detection. Only a weak oxidation current response was generated by the successive addition of AA. All other electrodes showed a higher response compared with CPE.
Starting with EPAA with the -COOH group, the response was 1.76 times higher than that of CPE. Moreover, the corresponding EPI response was 1.57 times higher than that of CPE. This decrease in response may be associated with the absence of the -COOH group. Similarly, S-EPI showed higher sensitivity (1.86 times) than CPE. EPAA had a higher sensitivity compared with EPI (×1.12). The S-EPAA electrode showed the highest response among all with sensitivities, which were 2.45, 1.39, 1.55 and 1.27 times higher than CPE, EPAA, EPI and S-EPI, respectively. The reason is that the sulfonic group on the aniline ring can enhance the resonance effect and increase the attraction between AA and sulfonic acid molecules; this phenomenon enhances the electro-catalytic reaction on the electrode surface of S-EPAA, and the oxidation current response is relatively obvious. The sensitivity of the as-prepared electrodes towards electrochemical sensing of AA is in the order following: S-EPI < EPI < EPAA < S-EPAA.
The linear calibration curve in Figure 5b shows that the change in the peak current and AA concentration had a linear relationship. Table 1  Starting with EPAA with the -COOH group, the response was 1.76 times higher than that of CPE. Moreover, the corresponding EPI response was 1.57 times higher than that of CPE. This decrease in response may be associated with the absence of the -COOH group. Similarly, S-EPI showed higher sensitivity (1.86 times) than CPE. EPAA had a higher sensitivity compared with EPI (×1.12). The S-EPAA electrode showed the highest response among all with sensitivities, which were 2.45, 1.39, 1.55 and 1.27 times higher than CPE, EPAA, EPI and S-EPI, respectively. The reason is that the sulfonic group on the aniline ring can enhance the resonance effect and increase the attraction between AA and sulfonic acid molecules; this phenomenon enhances the electro-catalytic reaction on the electrode surface of S-EPAA, and the oxidation current response is relatively obvious. The sensitivity of the as-prepared electrodes towards electrochemical sensing of AA is in the order following: S-EPI < EPI < EPAA < S-EPAA.
The linear calibration curve in Figure 5b shows that the change in the peak current and AA concentration had a linear relationship. Table 1 shows the measuring limit of detection, linear dynamic range and sensitivity values for the EPAA, EPI, S-EPI and S-EPAA sensors. The table also summarizes the analytical performance of the other related sensors based on CP-modified electrodes in the present work. A better sensitivity performance was achieved in the present work, compared with the reported PANI systems.

Kinetic Parameter Study
Cyclic voltammograms of the CPEs modified with EPAA, EPI, S-EPAA and S-EPI in PBS towards the oxidation of 2 mM AA, with increasing scan rates between 10 and 500 mV/s, are shown in Figure S5.
The electrode process is diffusion-controlled, as suggested by the linear plots for Ip versus υ 1/2 in Figure 6a

Kinetic Parameter Study
Cyclic voltammograms of the CPEs modified with EPAA, EPI, S-EPAA and S-EPI in PBS towards the oxidation of 2 mM AA, with increasing scan rates between 10 and 500 mV/s, are shown in Figure S5.

Differential Pulse Voltammetry (DPV)
Generally, co-existing electroactive components, such as uric acid (UA) and dopamine (DA), show serious interference in the electrochemical detection of AA. Therefore, the amperometric responses of DA and UA are usually studied by DPV. A tertiary mixture of ascorbic acid (AA), dopamine (DA) and uric acid (UA) was used as an interfering species for the evaluation of selectivity by differential pulse voltammograms (DPV). Figure 7a showed the DPV recorded for bare and modified CPEs in PBS for a mixture of interferents. The concentration of each interferent in a mixture was 20 µM, with a potential window of −0.2 V to 0.6 V. Notably, for the tertiary mixture, bare CPE showed poor selectivity. On the other hand, the selectivity improved with the CPEs modified with EPAA, EPI, S-EPAA and S-EPI, respectively. The CPE modified with S-EPAA exhibited excellent selectivity, with three distinct voltammetric peaks at potentials of 0.022, 0.145 and 0.337 V, corresponding to the oxidation of AA, DA and UA, respectively. The oxidation of DA and UA does not influence the current response of AA, as shown in Figure 7b. This result convincingly indicated that the S-EPAA-modified CPE can be used as a sensor for the selective determination of AA in the presence of interfering components, such as DA and UA.
Generally, co-existing electroactive components, such as uric acid (UA) and dopamine (DA), show serious interference in the electrochemical detection of AA. Therefore, the amperometric responses of DA and UA are usually studied by DPV. A tertiary mixture of ascorbic acid (AA), dopamine (DA) and uric acid (UA) was used as an interfering species for the evaluation of selectivity by differential pulse voltammograms (DPV). Figure  7a showed the DPV recorded for bare and modified CPEs in PBS for a mixture of interferents. The concentration of each interferent in a mixture was 20 μM, with a potential window of −0.2 V to 0.6 V. Notably, for the tertiary mixture, bare CPE showed poor selectivity. On the other hand, the selectivity improved with the CPEs modified with EPAA, EPI, S-EPAA and S-EPI, respectively. The CPE modified with S-EPAA exhibited excellent selectivity, with three distinct voltammetric peaks at potentials of 0.022, 0.145 and 0.337 V, corresponding to the oxidation of AA, DA and UA, respectively. The oxidation of DA and UA does not influence the current response of AA, as shown in Figure 7b. This result convincingly indicated that the S-EPAA-modified CPE can be used as a sensor for the selective determination of AA in the presence of interfering components, such as DA and UA.

Conclusions
In this work, we reported the preparation of sulfonated and non-sulfonated electroactive monomers (ACAT and S-ACAT) and polymers (EPAA, EPI, S-EPAA, S-EPI), which were applied in electrochemical sensing of ascorbic acid. Based on the electrochemical CV studies, the redox capabilities of the as-synthesized materials were in the following order: S-EPAA > S-EPI > EPAA> EPI. In AA sensing, the CPE modified with S-EPAA had higher sensitivity (172 μM.mM − ) and relatively low LOD of 0.001 μM at the potential of 0.51 V among all electrodes. Moreover, the S-EPAA electrode exhibited excellent selectivity, with distinct voltammetric peaks. Thus, S-EPAA showed the most reliable results. Keeping in mind the higher sensitivity, good selectivity, low cost and simple preparation, the practical application value of this system will be evaluated in the future using real samples.

Conclusions
In this work, we reported the preparation of sulfonated and non-sulfonated electroactive monomers (ACAT and S-ACAT) and polymers (EPAA, EPI, S-EPAA, S-EPI), which were applied in electrochemical sensing of ascorbic acid. Based on the electrochemical CV studies, the redox capabilities of the as-synthesized materials were in the following order: S-EPAA > S-EPI > EPAA> EPI. In AA sensing, the CPE modified with S-EPAA had higher sensitivity (172 µM.mM − ) and relatively low LOD of 0.001 µM at the potential of 0.51 V among all electrodes. Moreover, the S-EPAA electrode exhibited excellent selectivity, with distinct voltammetric peaks. Thus, S-EPAA showed the most reliable results. Keeping in mind the higher sensitivity, good selectivity, low cost and simple preparation, the practical application value of this system will be evaluated in the future using real samples.