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Amperometric Sulfite Sensor Using Electrodecorated Pt Particles onto Aminated Glassy Carbon Electrode Prepared by Stepwise Electrolysis.

A novel modified glassy carbon electrode with platinum (Pt)-electrodecorated and nitrogen-containing functional groups was prepared by stepwise electrolysis. The prepared electrode exhibited electrocatalytic activity towards sulfite oxidation that was better than that of a bare glassy carbon electrode. The electrocatalytic activity of sulfite oxidation has been applied to an amperometric sulfite sensor. A favorable linear relationship between the current response (ΔI) and the sulfite concentration up to 500 μM was exhibited. The detection limit was estimated to be 3 μM based on the criterion of a signal-to-noise (S/N) ratio of 3 under optimized conditions. In regards to the reproducibility, the RSD (n = 10) was 8.9% for 80 μM sulfite.


Introduction
Sulfite is an additive that is widely used in pharmaceutical products, beverages, and food so as to prevent oxidation, inhibit bacterial growth, and control enzymatic reactions during production and storage. 1,2 Despite these useful advantages, sulfite must be applied in extremely limited amounts due to its potential toxicity and harmful effects on human bodies. Exceeding the consumption limit may lead to headaches, nausea, diarrhea, asthma attacks, stomach irritation, nettle rash, and swelling. [3][4][5][6] The existence of accurate methods for determining sulfite is fundamental for the food industry to ensure product quality. At present, a number of analytical methods for detecting sulfite, such as iodimetric titration, 7 high-performance liquid chromatography, 8 fluorescence, 9 chemiluminescence, 10 capillary electrophoresis, 11 and electrochemical detection, 12,13 have been reported. Unlike these methods, electrochemical detection techniques realize simplicity, sensitivity, reliability, rapid detection, and are also inexpensive.
Most researchers have paid attention to the use of electrodes made of carbon material, such as glassy carbon (GC; type of 2 dimensional shapes) and carbon felt (CF; type of 3 dimensional shapes) electrodes. Two electrodes are useful in the fields of electrochemical sensing owing to their higher overpotential for oxygen reduction, 14,15 accessibility, inexpensive, chemical stability, wide potential window, 16 and large surface area 17 compared with metal electrodes, such as platinum and gold. Nowadays, chemical modification of the carbon surface has attracted great attention in various fields, such as electroanalytical chemistry. [18][19][20][21][22] Electrochemical sulfite detection may be based on direct sulfite reduction on glassy carbon 23 or after electrode modification. 24 Otherwise, direct sulfite oxidation may be obtained at platinum, 25 gold, 26 glassy carbon, 27 and platinummodified glassy carbon. 28 However, there are still challenges, such as problems correlated with fouling of the electrode and a high positive potential for oxidation of the sulfite. [25][26][27] As a consenquence, a number of substances may interfere with the detection and measurement of the analyte.
Due to the above challenges, our group has particularly focused on the development of electrochemical modifications on the carbon surface (GC electrode and CF electrode). The nitrogen-containing functional groups can form chains on the surface of the GC electrode by the electro-oxidation treatment process using ammonium carbamate in an aqueous medium solution at a high positive electrode potential, 29 we called it an aminated glassy carbon (AGC) electrode. In particular, it has been revealed by us that nitrogen atoms containing functional groups, such as aromatic amine groups, like aniline, can easily be introduced to the surfaces of the glassy carbon and carbon felt electrodes by the electrode oxidation of ammonium carbamate, 30 the electron transfer rates of many inorganic and organic compounds are accelerated to be able to measure excellent redox waves. 31,32 Our previous study reported, redox waves between hydrogen ions and hydrogen molecules (H2) at a highly positive potential range after a long-term electroreduction of the AGC electrode in a sulfuric acid electrolyte solution. During electrode reduction of the AGC electrode in sulfuric acid, platinum ion dissolved from a platinum wire counter electrode was electrodeposited onto the surface of nitrogen-containing functional groups to which a GC electrode was introduced. We named this electrode a Pt-NGC electrode, for which Pt particles are electrodeposited onto a GC electrode modified with nitrogen-containing functional groups. 33 In the present work, a simple and environment-friendly method was used for the preparation of Pt particles electrodeposited on the nitrogen-containing functional groups for a new-type modified electrode (Pt-NGC electrode) by stepwise electrolysis. The electrocatalytic oxidation of sulfite has been evaluated by cyclic voltammetry (CV) by comparing the Pt-NGC and various electrodes. The Pt-NGC electrode was also used for measurements of sulfite by CV and amperometry under optimized conditions. Moreover, we also evaluated the detection limit of sulfite, reproducibility, and the interference substance in an amperometric sulfite sensor.

Reagents and chemicals
All chemicals used for the study were of analytical reagent grades. Deionized water (Millipore Milli-Q System, Japan) was used to prepare the solutions used in this investigation. Sodium sulfite (Na2SO3), sulfuric acid (H2SO4), dipotassium hydrogen phosphate (K2HPO4), and potassium dihydrogen phosphate (KH2PO4) were purchased from Fujifilm Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Ammonium carbamate was purchased from Merck KGaA (Darmstadt, Germany). The glassy carbon (GC) electrode (diameter of 3 mm) was obtained from BAS Co., Ltd. (Tokyo, Japan).

Preparation of Pt-NGC electrode
In the present work, the GC electrode was polished with 1.0 μm polishing diamond and 0.05 μm polishing alumina on the polishing micro cloth, treated with an ultrasonic bath (1 min), and rinsed with deionized water.
A potential-controlled electrolysis process was performed with a potentiostat/galvanostat (HA-151B, Hokuto Denko Co., Ltd., Japan). A platinum spiral wire (diameter, 0.5 mm; length, ca. 70 mm) and an Ag/AgCl (3 mol/L NaCl electrolyte) were used as a counter electrode and a reference electrode, respectively. The two step processes for Pt-NGC electrode preparation are described below. First, the GC electrode was processed in an electrode oxidation process with a 0.1 mol/L ammonium carbamate aqueous solution at a constant potential of +1.1 V for 60 min. Next, the electro-oxidized GC electrode was electro-reduced in 1.0 mol/L sulfuric acid at -1.1 V (vs. Ag/AgCl) for 20 h. Throughout the electrode reduction of the electro-oxidized GC electrode in sulfuric acid, platinum ion dissolved from platinum wire counter electrode was electrodeposited onto the surface of nitrogen-containing functional groups to which a GC electrode was introduced. All experiments were performed at room temperature.

Electrochemical measurements
The voltammetric and amperometric measurements were performed with an automatic polarization system (HZ-3000, Hokuto Denko Co., Ltd., Japan) with a three-electrode cell consisting of a working electrode as a Pt-NGC electrode, a counter electrode as platinum wire, and an Ag/AgCl (3 mol/L NaCl electrolyte) as a reference electrode. During amperometric measurements, N2 gas was flowed into the electrolyte. The purpose was to block oxygen molecules so that the sulfite could not oxidize to sulfate. Additionally, the Pt-NGC electrode was covered with a dialysis membrane for reducing the diffusion of sulfite from bulk to the Pt-NGC surface. According to this treatment with the dialysis membrane, the background noise current is additionally reduced during amperometric measurements of sulfite due to reducing of the electrolyte diffusion. The supporting electrolyte was a 0.1 mol/L phosphate buffer solution (pH 7.0).

Results and Discussion
Electrochemical responses of 3 mM sulfite at various electrodes in a 0.1 M phosphate buffer solution (pH 7.0) were evaluated by cyclic voltammetry, as shown in Fig. 1. The electrocatalytic oxidation of sulfite on the AGC electrode ( Fig. 1(b)) and Pt-NGC electrode (Fig. 1(d)) was seen more clearly in the presence of the sulfite oxidation peak than in the sulfite oxidation performed on a bare GC electrode ( Fig. 1(a)). The nitrogencontaining functional groups, with the AGC electrode, introduced by electrode oxidation in an ammonium carbamate aqueous solution function as electron-transfer mediators for sulfite oxidation, which indicates that the AGC electrode had electrocatalytic activity and the oxidation peak potential moved to the negative direction of potential compared with the bare GC electrode. Next, a significant oxidation peak current of sulfite was observed by using the Pt-NGC electrode. In addition, the oxidation peak potential shifted to the increasingly negative direction of the potential compared with the AGC electrode ( Fig. 1(b)) and the Pt-electrodeposited glassy carbon (Pt-GC) electrode ( Fig. 1(c)). Hence, the Pt-NGC electrode easily promotes the electrode oxidation of sulfite. On the other hand, the above probably exhibits that the electro-deposited Pt in collaboration with the nitrogen-containing functional groups (such as the primary amine group and other N-containing functional groups), introduced by stepwise electrolysis in ammonium carbamate solution and sulfuric acid, act as active sites of the specific electrocatalytic current for sulfite. We previously exhibited that the electrocatalytic activity of the Pt-NGC electrode for the electrode oxidation of hydrogen molecule did not decrease after ultrasonication performed for 5 min, which indicates that platinum particles on nitorgen-containing functional groups are securely adsorbed onto the GC surface. 33 Based on the results in Fig. 1, it is understood that the Pt-NGC electrode is superior to the other electrodes in terms of sulfite oxidation. Figure 2 shows a comparison of the amperometric response of The relationship between the current response (ΔI) and the sulfite concentration is shown in Fig. 3. The oxidation current increased sharply up to 5000 μM in sulfite concentration, and achieved 95% of the maximum steady-state current within ca. 30 s. A favorable linear relationship (R 2 = 0.9979) was provided between the ΔI and the sulfite concentration up to 500 μM in Fig. 3 (inset). The detection limit was estimated to be 3 μM based on the criterion of a signal-to-noise (S/N) ratio of 3 under the optimized conditions. Based on the reproducibility of the 10 successive measurements with 80 μM of sulfite, the relative standard deviation (RSD) was calculated to be 8.9%. All of these results demonstrated that the current response was suitable with the increase of the sulfite concentration.
The selectivity of the Pt-NGC electrode has been investigated by recovery tests of sulfite. Figure 4 shows a typical amperometric response of 200 μM sulfite prepared by two different sample solutions, such as a test solution (0.1 M phosphate buffer) (a) and a red wine sample (b). Sulfite was added into various sample solutions. Inset is the recovery test results of sulfite from real sample, such as red wine. The results show that the favorable recovery of sulfite using each possible interfering substance in a real sample was obtained. In addition, we have also found that other substances, such as common metal cations and anions (Na + , K + , Cl -, NO3 -, HCO3 -, H2PO4 -, and HPO4 2-), glucose, fructose, sucrose, oxalate, citric acid, also did not interfere with the determination of sulfite (data not shown). Therefore, the amperometric sulfite sensor based on the Pt-NGC electrode that we propose is a useful analytical technique, since it enables simple and rapid determinations of the sulfite concentration to be performed easily.

Conclusions
In conclusions, a new-type modified glassy carbon electrode has been developed on the basis of Pt-electrodecorated on the nitrogen-containing functional groups by stepwise electrolysis. The Pt-NGC electrode exhibited a favorable electrocatalytic activity towards sulfite oxidation compared with the bare GC electrode and the AGC electrode. The electrocatalytic activity of sulfite oxidation has been applied to the amperometric sensor of sulfite under the optimized conditions. A favorable linear relationship was provided between the current response and the sulfite concentration up to 500 μM. The detection limit of sulfite was 3 μM (S/N = 3) under the optimal experimental conditions. As for the reproducibility, the RSD (n = 10) was 8.9% for 80 μM sulfite. The Pt-NGC electrode also showed high selectivity towards sulfite in the presence of common metal ions and other interfering substance. The good sulfite recovery test results using a possible real sample, such as red wine, were obtained. The Pt-NGC electrode is promising material for a wide range of applications related to sulfite measurements with high performance as a substitute for metal electrodes.