Optimization of Hydrogen Peroxide Detection for a Methyl Mercaptan Biosensor

Several kinds of modified carbon screen printed electrodes (CSPEs) for amperometric detection of hydrogen peroxide (H2O2) are presented in order to propose a methyl mercaptan (MM) biosensor. Unmodified, carbon nanotubes (CNTs), cobalt phthalocyanine (CoPC), Prussian blue (PB), and Os-wired HRP modified CSPE sensors were fabricated and tested to detect H2O2, applying a potential of +0.6 V, +0.6 V, +0.4 V, −0.2 V and −0.1 V (versus Ag/AgCl), respectively. The limits of detection of these electrodes for H2O2 were 3.1 μM, 1.3 μM, 71 nM, 1.3 μM, 13.7 nM, respectively. The results demonstrated that the Os-wired HRP modified CSPEs gives the lowest limit of detection (LOD) for H2O2 at a working potential as low as −0.1 V. Os-wired HRP is the optimum choice for establishment of a MM biosensor and gives a detection limit of 0.5 μM.


Introduction
Methyl mercaptan (MM) is one of the volatile sulfur compounds (VSCs), which are known to be involved in halitosis (bad breath) [1,2] and periodontal diseases [2], and the predominant causative factor of noticeable oral malodor [3]. MM is also present in several other cases such as the bottle storage of wines [4]; wood-pulp mills, sewage treatment plants and factories producing jet fuel, pesticides and plastics [5]; and even in the atmosphere and on the ocean surface [6]. Consequently, MM detection is important in the dental, medical, food, environment and atmosphere fields.
A low-cost, sensitive and specific sensor for detecting MM could be an interesting alternative to conventional MM monitoring methods such as the use of a halimeter, an expensive device, in the dental field [7,8]. Biosensors to monitor MM have been described by Mitsubayashi et al. [9][10][11][12]. In their work, monoamine oxidase A (MAO-A) or flavin-containing monooxygenase (FMO) was used to catalytically oxidize MM, and the oxygen consumption induced by this reaction was monitored. Coupled with this system, a substrate regeneration cycle with ascorbic acid was carried out. However, a sensor for the detection of O 2 depletion, which has the initially high current background of the oxygen electrode [13], is less sensitive than one for H 2 O 2 measurement. To solve this problem and seek sensitive detection methods, our objective was to develop a MM biosensor coupled with sensitive hydrogen peroxide detection. Alcohol oxidase (AOX) is known to catalytically oxidize MM with production of formaldehyde, sulfide and H 2 O 2 [14] according to the reaction: Hydrogen peroxide generated during enzyme-catalyzed reactions can be electrochemically detected on modified/unmodified carbon matrixes [15][16][17][18][19][20][21][22][23][24][25][26][27]. In this work, unmodified carbon nanotube (CNT), cobalt phthalocyanine (CoPC), Prussian blue (PB), and Os-wired HRP modified screen printed electrode (CSPE) sensors were fabricated and tested to detect H 2 O 2 . Our aim was to seek the most sensitive and optimal detection method of H 2 O 2 for a MM amperometric biosensor.

Instrument
CSPEs were produced in the laboratory using a semi-automatic DEK 248 screen-printing system (DEK, Weymounth, UK). The working electrode was a 4 mm diameter disk, the auxiliary electrode was a 16 mm × 1.5 mm curved wire and the Ag/AgCl reference electrode was a 5 mm × 1.5 mm straight wire (Figure 1). Cyclic voltammetry (CV) measurements, PB electrodeposition, PPD electropolymerisation and amperometric measurements were carried out on an AUTOLAB PGSTAT100 (Metrohm, Switzerland), using GPES v4.7 (Metrohm) as informatic interface. All potential values are reported versus Ag/AgCl. Amperometric measurements were performed in a 10 mL glass bath cell with magnetic stirring at room temperature.

Preparation of CNT Modified CSPEs
CNT modified CSPEs were prepared as described in the work of Silveira et al. [28]. Briefly, 10 µL of 0.3 mg/mL SWCNT water dispersion were successively deposited on the CSPEs working electrode, drying each layer one by one under vacuum. The electrodes were then washed with water.

Preparation of CoPC Modified CSPEs
Cobalt-phtalocyanine-modified paste was purchased from Gwent Electronic Materials, Ltd. (Gwent, UK) and modifications were performed on working electrode by the DEK screen-printing system.

Preparation of PB/PPD Modified CSPEs
The PB film was first deposited by covering the CSPEs with a solution containing 2.5 mM FeCl 3 , 2.5 mM K 3 Fe(CN) 6  Counter electrode of 50 mV·s −1 . After washing with distilled water, it was dried for 1 h at 100 °C in oven. To improve the stability and selectivity properties of the PB electrodes, the electropolymerisation of a poly-(o-phenylenediamine) (PPD) coat was formed. The PPD layer was deposited by electrochemically cycling the PB modified electrode with potential between −0.5 V and 0.7 V versus Ag/AgCl at a scan rate of 50 mV·s −1 in deaerated 0.1 M, pH 5.0 acetate buffer solution containing 0.5 mM o-phenylenediamine under a stream of nitrogen [29].

Preparation of Os Wired HRP (Os-HRP) Modified CSPEs
For the Os-HRP modified CSPEs, 10 μL 0.1 M phosphate buffer solution, pH 7.5, containing 10% (v/v) Os-HRP was deposited on the surface of CSPEs. It was allowed to dry at room temperature for 2 h. It was thoroughly washed with buffer before use.

H 2 O 2 Detection with Unmodified CSPEs
The cyclic voltammetry studies were performed in 0.1 M phosphate buffer solution, pH 7.5 to investigate the CSPEs' electrochemical behavior ( Figure 2). CSPEs showed no obvious peak in the absence of H 2 O 2 in the potential range from −0.2 V~0.8 V versus Ag/AgCl. In the presence of H 2 O 2 , CSPEs started to perform current response at potential around +0.3 V versus Ag/AgCl, indicating the onset potential of the H 2 O 2 electrooxidation.  For the investigation of H 2 O 2 limit detection, chronoamperometry experiments were carried out with several concentrations of H 2 O 2 injected into the stirred bath cell (Figure 3). For unmodified CSPEs, the H 2 O 2 detection limit was 3.1 μM (S/N = 3) applying a +0.6 V potential versus Ag/AgCl, and the current response slope of the calibration curve was 0.208 μA/mM. To investigate the reproducibility, three parallel measurements with 0.1 mM H 2 O 2 revealed a relative standard deviation (RSD) of 12.1%. The high RSD observed of unmodified CPSEs is likely related to the marked differences in the real active electrode area, which is difficult to handle and adjust.

H 2 O 2 Detection with CNT/CSPEs
Both CSPEs ( Figure 2) and CNT/CSPEs ( Figure 4) showed a H 2 O 2 oxidation peak in the cyclic voltammetry experiments for the studied potential range. The onset potential of the H 2 O 2 electrooxidation for CNT/CSPEs was around +0.2 V versus Ag/AgCl, the detection limit was 1.  Both unmodified CSPEs and CNT are intrinsically carbon. Compared to unmodified CSPEs, detections of H 2 O 2 for CNT/CSPEs need lower oxidation potential (CSPEs, +0.3 V; CNT/CSPEs, +0.2 V), and have lower detection limit (CSPEs, 3.1 μM; CNT/CSPEs, 1.3 μM) with higher current response (CSPEs, 0.208 μA/mM; CNT/CSPEs, 32.1 μA/mM). The increased current response may arise from the large electric active area and a thin, porous diffusion layer [30]; the reasons of lower onset oxidation potential and lower detection limit are still controversial [31], because CNT may contain metal impurities derived from the catalysts used for their growth [32,33]. In a sense, CNT/CSPEs could be more favorable than unmodified CSPEs for H 2 O 2 detection.

H 2 O 2 Detection with CoPC/CSPEs
To investigate the electrochemical behavior of CoPC/CSPEs in phosphate buffer solution, cyclic voltammetry experiments were carried out in the potential range of −0.2 V~0.8 V versus Ag/AgCl ( Figure 5). The presence of a well-defined oxidation current peak at around +0.3 V versus Ag/AgCl is consistent with the following reaction [21]: The reaction can be described by a chemical-electrochemical (CE) mechanism [21]: H 2 O 2 chemically reduces Co 2+ to Co + and its subsequent electrochemical re-oxidation is observed as an oxidation peak. Consequently, this peak is used for the quantification of H 2 O 2 . For CoPC/CSPEs, the H 2 O 2 detection limit was calculated as 71 nM (S/N = 3) applying a positive potential of +0.4 V versus Ag/AgCl in chronoamperometry experiments, and the slope of the calibration curve was 3.7 μA/mM by. Three parallel measurements with 10 μM H 2 O 2 reveal a RSD of 2.4%, indicating a good reproducibility for this sensor.

H 2 O 2 Detection with PPD/PB/CSPEs
PB is known to have a high solubility in neutral and basic solutions [34,35], consequently the PB-modified electrodes may have stability problems under our conditions. To improve the stability and selectivity of the PB electrodes a PPD coat is formed. In order to assess the effect of the PPD coat on the stability of the PB modified electrodes, cyclic voltammetry experiments were carried out in 0.1 M phosphate buffer pH 7.5 (not shown in this paper).
Without PPD layer coating, PB dissolved in solution, resulting in a CV current decrease in the scanning process. After 20 scan cycles, the PB/CSPEs CV's performance were similar to the unmodified CSPEs ( Figure 2). With PPD layer coating, the response of redox peaks of PB reduced slightly even after 50 scan cycles, indicating that the PPD layer stabilized the PB. To investigate the electrochemical performance of PPD/PB/CSPEs H 2 O 2 was added to the solution ( Figure 6).

H 2 O 2 Detection with Os-HRP/CSPEs
The Os-HRP/CSPEs show a couple of stable and well-defined redox peaks at around +30 mV and +70 mV at a scan rate of 20 mV·s −1 (Figure 7 curve a). In the presence of H 2 O 2 (Figure 7 curve b), the electrocatalytical reductive reaction process of Os-HRP towards to H 2 O 2 can be described by Scheme 1. The comparison of the amperometric analytical behavior to H 2 O 2 of the five kinds of electrodes developed in this study is summarized in Table 1. It shows that the Os-HRP/CSPEs are the most sensitive electrodes with a low reduction potential applied for H 2 O 2 detection. This is due to the specific, sensitive and rapid turnover of Os-HRP to H 2 O 2 . Interference of electro-active species [25,[37][38][39] is often encountered when using amperometric biosensors and applying a high potential in real samples. The decrease of the applied potential can be effective to avoid a lot of electrochemical interferences. With this consideration, PPD/PB/CSPEs and Os-HRP/CSPEs are used to combine with alcohol oxidase (AOX) in bovine serum albumin matrix to detect methyl mercaptan (MM) applying a low potential in the aqueous phase. The limit of detection of AOX/PPD/PB/CSPEs to MM is 10 μM; of AOX/Os-HRP/CSPEs to MM is 0.5 μM. For AOX/Os-HRP/CSPEs, the calibration curve of the response to MM is linear in the concentration range 0~15 μM with a good correlation with the classical analytical method. We are working on the stability of the biosensor which is the crucial point to improve its accuracy and reliability. Consequently, Os-HRP/CSPEs are combined with alcohol oxidase an optimum method which gives the best sensitivity in methyl mercaptan detection.

Conclusions
Five kinds of modified carbon screen printed electrodes applied for H 2 O 2 amperometric detection for MM biosensors were presented in this work. In comparison, and despite a worse reproducibility, CNT/CSPEs are a better choice than unmodified CPSEs in H 2 O 2 detection resulting from their lowest detection limit, lowest onset oxidation potential and highest current response of CNT/CSPEs. However, the applied potential of +0.6 V versus Ag/AgCl is too positive to avoid the interference of electro-active species. CoPC/CSPEs and PPD/PB/CSPEs are also a good choice in H 2 O 2 detection because of their low applied potential, low detection limit and good reproducibility. Os-HRP/CSPEs display the lowest detection limit and the best operational reproducibility towards H 2 O 2. With the cathode potential applied and the use of HRP, Os-HRP/CSPEs can avoid the interference of electro-active species and be specific for H 2 O 2 detection. The Os-wired HRP modified screen printed electrode is the optimum method we used to combine with alcohol oxidase in a methyl mercaptan biosensor, usable in both aqueous and gaseous phase detection.