Pectinose electrochemical quantitative analysis method using functional metal materials

ABSTRACT As a soluble dietary fiber, pectin has the functions of lowering cholesterol, reducing weight and improving human immunity. However, nowadays, many studies have been reported on the extraction of pectin, but few on its quantitative analysis. In this paper, graphene modified copper foam and copper sheet were used as working electrodes, respectively. Pt electrode as counter electrode and Ag/AgCl (saturated KCl solution) electrode was used as reference electrode to build electrochemical detection system. The electrochemical responses to pectinose solution were measured by voltammetry (CV) and chronoamperometry (i-t). The detection data were analyzed to develop pectinose quantitative analysis model. Results indicated that the proposed method could determine pectinose concentration in solution, and graphene modified copper foam presented better responses. The sensitivity was 0.68851A cm−2 mmol/L, the detection limit was 0.24825 mol/L. This method presents some advantages including fast response, easy operation, and low cost. It provides a new method for pectinose quantitative detection.


Introduction
Nowadays, most of the food will contain sugar, and many people will unconsciously consume too much sugar, making the global rates of obesity, diabetes, atherosclerosis, and other diseases rise, so urgent need to find a kind of healthy diet, the quantitative detection of sugar, reduce the damage to the human body by the excessive consumption. Pectin is a complex mixture of polysaccharides, mainly composed of α-1, 4-glycosidic bonds linked to galacturonic acid. [1] It is a natural polysaccharide that can be used as thickening agent, suspension agent, emulsifier, etc. [2] It can also be used as soluble dietary fiber, which can reduce cholesterol levels, reduce glucose and lose weight, [3] improve human immunity and fight cancer. [4] It also has antioxidant properties. [5] It is widely found in the cell walls of higher plants, and in addition to the common pericarp, hemp pericarp also contains pectin. [6][7][8] In addition, some researchers have extracted pectin from blueberries, beets, citrus, coffee, and other foods and drinks by alcohol precipitation method, physical enzyme method, [9] water-based extraction method, [10] ultra-high pressure method, [11] etc., which has proved the widespread existence of pectin.
At present, there are many extraction methods for pectin, but the analysis of pectin is very few. The methods that can be used for carbohydrate analysis of pectin are mainly based on the determination of residual sugar composition of pectin obtained after chemical hydrolysis or enzymatic hydrolysis. [11] Carlo et al. used enhanced Raman spectroscopy to detect pectin in fruits and realized the quantitative detection of pectin. [12] Quemener et al. used hydrolysis method for quantitative analysis of pectin, but their method required extremely high-temperature control. [13] Manam et al. used high-performance anion exchange chromatography-pulsed amperometric method to determine the sugar content in pectin, [14] which has the characteristics of high accuracy, good selectivity, and high sensitivity. However, the chromatogram of the method is complicated, which may lead to the inaccurate determination of sugar. Voland-stevens LFRA analyzer was used to analyze the linear relationship between jelly fracture strength and pectin content to determine the content of pectin. [15] Archut Artwin et al. used the complex co-osmosis of pea protein and pectin to allow a quantitative assessment of the interaction using isothermal titration calorimetry complex. [16] Functional materials were also utilized in sugar quantitative analysis. [17][18][19][20] Based on the above analysis, pectinose quantitative analysis method based on functional sensing materials and electrochemical scanning was investigated. The copper foam sheet and graphenemodified copper foam were used as working electrodes, respectively. Pt electrode was used as counter electrode and Ag/AgCl (saturated KCl solution) electrode was used as reference electrode. The electrochemical responses of pectinose solutions were measured by CV and i-t methods. The measurement data were analyzed to develop the pectinose quantitative determination model. This method has advantages of quick reaction, simple operation, and low cost.

Reagent and instrument
NaOH was from Sinopharm Chemical Reagent Co., Ltd, Shanghai, China; D-psicose was from Juping Technology Co., Ltd, Shandong, China; NaCl was from Kelong Chemical Reagent Co., Ltd, Chengdu, China; D-pectinose was from Beilian Fine Chemicals Development Co., Ltd, Tianjin, China; CHI-614E electrochemical workstation was from Chenhua Instrument, Shanghai, China; Model 85-2A automatic stirrer from Xinrui Instrument Co., Ltd, Jiangsu; Pipetting gun from Dalong Xingchuang Experimental Instrument Co., Ltd, Beijing; Distilled water. The detecting system was displayed in Figure 1(a). Pt electrode was used as counter electrode. Ag/AgCl electrode was used as reference electrode. The working electrode was graphene-modified copper foam or copper sheet.

Copper foam preparation
Cu foam, with 70% porosity and 1 mm thickness, was directly formed on polyurethane foam substrates using electrodeposition strategy. First, the processes of degreasing, roughening, sensitization, activation, and peptization were performed to increase substrate surface functionalities and roughness and remove off surface contamination. Then electroless copper plating was carried out in a mixture solution containing 12.0g/L CuSO 4 · 5H 2 O, 42.0 g/L EDTA, 20.0 g/L Na 2 SO 4 , and 20.0ml/L HCHO. Then electrodeposition was performed on the prepared polyurethane foam in the electroplate solution containing 70 g/L CuSO 4 · 5H 2 O, 0.6 g/L NaCl, 0.03 g/L polyethylene glycol, 0.05 g/L sodium lauryl sulfate, and 25 mL/L H 2 SO 4 . During electrodeposition procedure, copper plate was used as anode, and polyurethane foam was used as cathode. Direct current was applied between anode and cathode to obtain stable copper layer. The obtained specimen was calcined at 600°C to remove polyurethane foam and then experienced hydrogen thermal reduction at 700°C for 30 min to obtain the uniform structure.
A new efficient and simple graphene modification method for engineering applications was explored. 30 mg graphene oxidated was added to a 50 mL beaker. Then 30 ml H 2 O was added. After ultrasonic treatment of 15 min, the copper foam was input. Keep heating until the water totally evaporates. Graphene is hydrophobic, and copper foam has specifically large surface area, which allows graphene to make full contact with metal foam and evenly adhere to its surface. After modification, the copper foam is kept at constant temperature at 30°C and sealed for further usage.

CV measurement
The copper electrode, copper plate, and graphene modified copper foam were used as working electrodes respectively. The electrochemical oxidation process was used to measure the signal of carbohydrate oxidation reduction reaction on the interface of the electrode. The basic solution is 20 ml NaOH solution with concentration of 0.1mol/L, and the detection solution is 100 µL pectin sugar solution. The scanning voltage range of the working electrode using copper sheet is [−0.8V, −0.2V], and the voltage range of graphene modified foam copper electrode is [−0.7V, −0.25 V]. The scanning rate is 100 mV/s, and the scanning round is 100.

I-t measurement
NaOH solution (20 ml) of 0.1 mol/L concentration was used as basic solution. A temperature adjustable magnetic mixer was used to stir the solution at a constant speed of 250 r/min. The contact area between the working electrode and the solution was 0.5 cm × 0.5 cm. In the first 200s of the testing, the electrical current reaches a stable state. After 200s, 0.05 mL pectinose solution was added into a basic solution every 50s. The experiment lasted to 1200s and 1.0 mL of pectinose solution was dipped into NaOH solution. The i-t responses were recorded for further analysis.

EIS
In this paper, EIS is used to test the impedance of electrodes. In the environment of 0.1mol/L NaOH solution, the initial voltage is open-circuit voltage value, the highest frequency is 10 6 HZ, the lowest frequency is 0.01 HZ, and the amplitude is 5 mV state. Figure 1(d) shows the Nyquist impedance spectroscopy of graphene copper foam and copper sheet. Graphene-modified copper foams had a large and stable ring. Its diffusion impedance was smaller than copper sheet. It had better electrochemical impedance characteristics.

CV measurement results
Load-free CV scanning results: Load-free CV scanning can characterize the stability and repeatability of the experimental electrodes. Graphene modified foam copper was used as the working electrode to conduct CV scanning in 0.1mol/L NaOH solution. The scanning range was [−0.75 V, −0.25 V], and the scanning speed was 100 mV/s. The scanning round is 100. Figure 2(a) displays the scanning results, indicating that the electrode had obvious oxidation peak and reduction peak. The oxidation peak increased with the increase of scanning rounds, demonstrating that the electrode had good stability and repeatability. The reason for this phenomenon lies in the oxidant film around the electrode. With the increase of scanning rounds, the oxidant film on the electrode surface could be gradually removed under the function of current. Finally, the activity of the electrode is greatly improved.
CV measurement results: Figure 2(b) and (c) shows the CV scanning results of different working electrodes in the mixture of 20 mL 0.1 mol/L NaOH with 100 µL 0.01 mol/L pectinose. The oxidation/ reduction peak value of graphene modified foam copper and copper sheet is much higher than that of copper sheet. The oxidation peaks of the two electrodes differ a lot. The peak values present little changes with the increase of scanning rounds, indicating that the electrodes have good stability and that the electrochemical window of each electrode fulfills the demand of pectinose detection. In general, the oxidation/reduction current of copper sheet is the higher, and the copper foam is the smaller. A possible explanation lies in the interface effect of electrode material in solution. The surface of copper sheet is relatively regular, and pectinose molecules are evenly distributed on the surface of copper sheet. Therefore, the current formed under the catalysis of copper atom is evenly distributed and the current is relatively large and stable. The graphene modified foam copper fully contacts with pectinose solution. The three-dimensional structure is beneficial to increase the contact area between copper and pectinose. However, the space structure is not enough to compensate for the contacting area induced by the fine copper branch due to the finer diameter of metal branch in copper foam network structure. So the current of copper foam electrode is not as high as the copper sheet electrode. Due to a thin layer of copper metal on PVC substrate, leading to the decrease of electrochemical signal.
CV scanning at different rates: Take 20 ml of 0.1 mol/L NaOH solution containing 100 μl of 0.01 mol/L pectin. The scanning speed is 0.01 V/s, 0.02 V/s, 0.03 V/s, 0.04 V/s, 0.05 V/s, 0.06 V/s, 0.07 V/s, 0.08 V/s, 0.09 V/s, 0.1 V/s, to judge the REDOX reaction. As shown in Figure 2(d), the working electrode graphene-modified copper foam has good repeatability and stability through CV results of different scanning rates. The larger the scanning speed, the more obvious the oxidation and reduction reaction.
To judge the nature of the electrochemical experiment, the root means square of the scanning speed and the current intensity of oxidation and reduction peaks were fitted. As shown in Figure 2(f), the fitting effect is good, and it can be judged that it is an electrochemical experiment with diffusion properties.
CV experiments with different scanning rates under 0.0 mol/L NaOH as the base solution and 0.01 mol/L petinose as the solution to be tested: Could be seen in Figure 2(d) and Figure 2(e). When 0.1 mol/L was used as the base solution, the response current ranged from −2 mA to 1.6 mA. When 0.01 mol/L was used as the base solution, the response current ranged from −1 mA to 1.5 mA. Therefore, when PH value becomes smaller, oxidation and reduction peak also becomes smaller and unstable.

I-T measurement results
I-t relationship analysis of the electrodes: Figure 3(a) and Figure 3(b) display the pectinose i-t scanning results using two kinds of working electrodes. Both i-t experiments were tested at a voltage of +0.5 V, and 0.1 mol/L NaOH was used as the base solution. After 200s, 50 μL of pectin solution was dropped every 50s, and the degree of current response changed. It can be seen from the figures that the response of the graphene modified foam copper presents the larger responses, and the responses of copper sheet are much smaller. The difference in responding current lies in the difference in the electrode interface chemical properties. Moreover, the modified foam copper is fully contacted with pectin solution with the assistance of graphene, which promotes pectinose oxidation in its three-dimensional structure.
In addition, it can be seen from Figure 3(c), the larger the PH value, the larger the current value of pectin solution oxidized. Graphene copper foam was used as the working electrode, 0.1 mol/L NaOH solution and 0.01 mol/L NaOH solution were used as the base solution, and 0.1 mol/L pectin solution was used as the test solution. Copper is not oxidized in nature, but when the electrolytic cell is energized, copper ions are formed to oxidize the pectin solution and generate current values. Pectinose quantitative analysis model development: Based on the above analysis, a quantitative pectin detection model was constructed using the responses of modified copper foam and copper electrode. In the procedure of i-t scanning, the pectinose solution was added 20 times in the last 1000s. The average value of the current response intensity within 50s after each drop of pectinose solution is used as the response current of this group of data. The testing is repeated multiply, and the average value of current data is taken as the ordinate. The pectinose concentration at the corresponding time is taken as the abscissa. Linear fitting regression between current data and pectinose concentration is conducted. Results are shown in Figure 4, and the fitting equation is displayed in the following.
Modified foam Cu electrode: Cu sheet electrode: The linear fitting results indicate good linear relationship between current responses and pectinose concentration. The slope of the graphene modified foam copper electrode is larger than that of the copper sheet, indicating that the electrode with the modified graphene foam copper has a better responding sensitivity than copper sheet electrode. Therefore, the graphene modified foam copper electrode is more suitable for pectinose quantitative detection than copper sheet electrode.

Negative control
NaOH solution is easy to react with CO 2 within the environment, forming disturbing substances (such as Na 2 CO 3 ). Here, Na 2 CO 3 is used to explore whether the interfering substances have impact on the detecting results. Pectinose i-t responding curves of were displayed in Figure 4 (c), and the i-t responses of pectinose with Na 2 CO 3 are shown in Figure 4 (d). Results indicate that the presence of Na 2 CO 3 does not have obvious influences on current responses, demonstrating that small amount of Na 2 CO 3 has no impact on pectinose detection. The reason for this phenomenon lies in that Na 2 CO 3 does not substantially participate in the oxidation/reduction reaction of pectinose molecules on the surface of the working electrode, so there is no corresponding interference current. The control experimental results demonstrate that the proposed method in this study has good anti-interference properties.

Conclusion
The quantitative detection method of pectin based on functional metal materials was studied in this paper. Graphene-modified copper foam and copper sheet were used as working electrodes. Pt electrode counter electrode, Ag/AgCl (containing saturated KCl solution) electrode reference electrode. The electrochemical detection system was developed and CV and I-T experiments were carried out. Firstly, CV scanning results showed that the working electrodes with different functional metal materials had good repeatability and stability. Graphene-modified foamed copper electrode and copper electrode had more obvious oxidation and reduction peaks. The scanning results showed that the graphene-modified foamed copper electrode had greater response current and higher sensitivity than the copper sheet electrode. Secondly, the quantitative detection model of pectin was established by linear regression of response current and pectin concentration, and the fitting effect was good Y ¼ 0:6669X þ 0:022 (R2 = 0.9995). Moreover, the sensitivity was 0:68851A � cm À 2 � mmol=L, the signal-tonoise ratio was 0:24825mol=L. Therefore, the graphene-modified copper foam electrode had the advantages of sensitivity, low detection limit and fast response speed. Thirdly, at different PH values, the higher the pH value, the higher the current value of pectin solution detection and the more obvious the oxidation and reduction peak. According to the Nyquist impedance spectrum, the graphenemodified copper foam has good electrochemical impedance characteristics. In addition, the results of negative control experiment showed that the interfering substances did no effect on the results of pectin analysis. The method studied in this paper has the advantages of high efficiency, good accuracy, and low cost.