Non-enzymatic Hydrogen Peroxide Sensors Based on Multi-wall Carbon Nanotube/Pt Nanoparticle Nanohybrids

A novel strategy to fabricate a hydrogen peroxide (H2O2) sensor was developed by using platinum (Pt) electrodes modified with multi-wall carbon nanotube-platinum nanoparticle nanohybrids (MWCNTs/Pt nanohybrids). The process to synthesize MWCNTs/Pt nanohybrids was simple and effective. Pt nanoparticles (Pt NPs) were generated in situ in a potassium chloroplatinate aqueous solution in the presence of multi-wall carbon nanotubes (MWCNTs), and readily attached to the MWCNTs convex surfaces without any additional reducing reagents or irradiation treatment. The MWCNT/Pt nanohybrids were characterized by transmission electron microscope (TEM), and the redox properties of MWCNTs/Pt nanohybrids-modified Pt electrode were studied by electrochemical measurements. The MWCNTs/Pt-modified electrodes exhibited a favorable catalytic ability in the reduction of H2O2. The modified electrodes can be used to detect H2O2 in the range of 0.01–2 mM with a lower detection limit of 0.3 μM at a signal-to-noise ratio of 3. The sensitivity of the electrode to H2O2 was calculated to be 205.80 μA mM−1 cm−2 at working potential of 0 mV. In addition, the electrodes exhibited an excellent reusability and long-term stability as well as negligible interference from ascorbic acid, uric acid, and acetaminophen.


Introduction
Hydrogen peroxide is a major messenger molecule in various redox-dependent cellular signaling transductions [1]. It is also known that H 2 O 2 is abnormally produced in the progress of inflammation by causing oxidative damage [2]. Therefore, sensitive detection of a trace level of H 2 O 2 is of great importance in health inspection and environment protection [3][4][5][6]. Various analytical methods including spectroscopy and electrochemistry have been used to detect H 2 O 2 [7][8][9]. In particular, electrochemical techniques based on enzyme-modified electrodes have attracted much interest [10,11]. However, enzyme-modified electrodes usually suffer from high cost, limited lifetime, inherent instability, and complicated immobilization procedure [12]. Consequently, it is imperative to develop non-enzymatic H 2 O 2 sensors with high sensitivity.
Recent studies indicated that Pt nanoparticles (Pt NPs) exhibited catalytic activity for the reduction of H 2 O 2 [13][14][15]. Meanwhile carbon nanotubes (CNTs) have attracted tremendous attention as a result of their ability to promote electron transfer reactions and high thermal stability [16][17][18]. Therefore, both CNTs [19,20] and Pt NPs [13][14][15] have been widely employed for detecting H 2 O 2 . In order to take full advantage of these nanomaterials, it is desirable to create novel CNT/Pt nanohybrids, so that the unique properties of each material can be integrated because the interactions between the two components may bring out novel properties.
To fabricate Pt NPs coated MWCNTs, electrochemical, chemical and physical methods have been employed so far [21,22]. However, most of the methods in the synthesis of MWCNTs/Pt NPs nanohybrids are time-consuming and complicated, requiring different kinds of chemicals and templates [23][24][25].
In this article, by combining the advantages of carbon nanotubes and Pt NPs, MWCNT/Pt NPs nanohybrids were designed and synthesized for the detection of H 2 O 2 . In our protocol, no additional reagent or irradiation was required because Pt NPs were reduced in situ onto the MWCNTs, in clear contrast to other reported procedures. The resulted nanomaterials were characterized by transmission electron microscopy (TEM), and the response of the modified electrode to H 2 O 2 was studied by amperometric measurements. The Pt NPs-modified electrodes showed a high activity in reduction of H 2 O 2 with a negligible interference from other electroactive molecules. The study can provide a promising platform for fabricating nonenzymatic electrodes and affinity matrix.

Microscopic Observations of MWCNTs-Pt NPs Nanohybrids
The morphology of nanohybrids was shown in Figure 1. Figure 1(a) shows TEM images of the MWCNTs modified with Pt NPs. Figure 1(c) shows that Pt NPs with a uniform size of about 1-2 nm were deposited onto the MWCNTs surface. In contrast, no such morphology of nanoparticles was observed on the surfaces of unmodified MWCNTs (Figure 1(b)). Figure 1(d) shows the selected area electron diffraction (SAED) image of the nanoparticles, indicating the phase structures of Pt single nanoparticles [26]. Although the spherical structure of Pt NPs decomposed to some degree, the main structure could still be observed on MWCNTs after stored at 4 °C for a month, showing a firm binding of PtNPs on the MWCNTs.  The numerous carboxylic acid groups on the MWCNTs could play the roles of catalyst, Pt "catcher", and supporter. In this case, Pt NPs were in situ generated from the K 2 PtCl 6 aqueous solution at room temperature and attached to the convex surfaces of MWCNTs, forming the MWCNTs/Pt NPs nanohybrids. We have tried to use also MWCNTs without carboxylic acid groups to prepare MWCNTs/Pt nanohybrids, but no Pt NPs could be obtained on the surface of the MWCNTs, indicating an essential role of carboxylic acid residues in the reduction of Pt on the surface of MWCNTs.

Electrochemical Response of MWCNTs/Pt Nanohybrids to H 2 O 2
Cyclic voltammetry (CV) is often used to estimate the true electroactive surface area of the modified electrode [27,28]. CVs of bare Pt electrode, MWCNTs/Pt electrode, and MWCNTs/Pt NPs/Pt electrode recorded in 0.1 M PBS were illustrated in Figure 3(a). There was no apparent redox process on the bare electrode. Diffusion current clearly increased for the MWCNTs-modified electrode owing to increased catalytically-active surface area. The diffusion current of the MWCNTs/Pt NPs nanohybrids-modified electrode further increased. An electroactive surface area of electrode can be estimated for a reversible and diffusion controlled process according to the Randles-Sevcik equation (Equation (1)) [29], where Ip relates to the redox peak current, A is the area of the electrode (cm 2 ), n represents the number of electron participating in the reaction which is equal to 1, D is the diffusion coefficient of the molecule in solution which is (6.70 ± 0.02) × 10 −6 cm 2 s −1 , C is the concentration of the probe molecule in the solution which is 10 mM and v is the scan rate (V s −1 ).
According to the above equation, we can obtain the surface area of the electrode. The calculated value of the electroactive surface area for the MWCNTs/Pt NPs nanohybrids-modified electrode was about 3.04-and 2.06-times higher than those of the bare Pt electrode and MWCNTs modified electrodes, respectively. In addition, the combination of the advantages of MWCNTs (large edge plane/basal plane ratio, enhanced conductivity, and rapid electrode kinetics) with well dispersive Pt NPs (high catalytic activity and large surface area) possesses higher electro-active surface areas, which facilitates the adsorption of detection molecules. CVs of the electrodes in the presence of H 2 O 2 are shown in Figure 3(b). For the MWCNTs-modified electrode, nearly no redox activity is observed for H 2 O 2 . In contrast, CV for the MWCNTs/Pt NPs-modified electrodes changed dramatically, in which the reduction (cathodic) current centered around 0 mV increased. This observation is a clear evidence for the electrocatalysis by Pt NPs. The highest current signal with lower reduction overvoltage are observed for the MWCNTs/Pt NPs modified electrode, which means that the MWCNTs/Pt NPs-modified electrode exhibits the best electrocatalytic activity towards H 2 O 2 among them. It is likely that the MWCNTs used here as a supporting matrix could well disperse Pt NPs, preventing them from aggregation and thus making them exhibit large active sites to easily contact H 2 O 2 for the electrocatalytic process. We can detect H 2 O 2 at 0 mV as working potential using the MWCNTs/Pt NPs-modified electrode, judging from the CV reported in Figure 3(b). The applied potential for the MWCNTs/Pt NPs-modified electrode is much lower than those for previously-reported H 2 O 2 sensors [30][31][32]. Therefore, the background current may be decreased and the response to interference materials can be minimized.
The amperometric response of the modified Pt electrode upon successive addition of H 2 O 2 in PBS (pH 7.0) was studied (Figure 4). When the H 2 O 2 was added to the PBS solution, the reduction current increased rapidly to reach a steady-state value within 5 s (achieving 95% of the steady-state current).
The inset of Figure 4 shows a calibration curve of the H 2 O 2 sensor. The response was proportional to the H 2 O 2 concentration in the range from 0.01 to 2.0 mM with a correlation coefficient of 0.997. The sensitivity was 205.80 μA mM −1 cm −2 , which was much higher than the previously reported values [33,34]. In addition, the lower detection limit was 0.3 μM at the signal-to-noise ratio of 3, which was lower than the reported values [32,35]   The analysis of real samples has also been carried out. Table 2 collects the results for the determination of H 2 O 2 in disinfected fetal bovine serum (FBS). The recoveries are between 99.8% and 103.4%, indicating that the proposed method can be applied in real sample analysis.

Interference Study
In real samples, some co-existing electroactive species such as ascorbic acid (AA), uric acid (UA), acetaminophen (AP) might affect the sensor response. The effects of the interference compounds to the H 2 O 2 sensor were studied by comparing the amperometric responses of the electroactive species (0.1 mM) and H 2 O 2 (1 mM) at the potential of 0 mV. Figure 5 shows that the successive addition of each interfering species brought out hardly discernible current response. The responses caused by AA, UA and AP could be negligible, and a well-defined H 2 O 2 response was obtained. The anti-interference ability is largely attributed to the low working potential of 0 mV employed in the determination of H 2 O 2 .

Reagents and Materials
Multi-wall carbon nanotubes (30-50 nm diameter and 0.5-1 μm length, with >95% purity, and carboxylic acid groups 0.73 wt%) were obtained from Institute of Organic Chemistry, Chinese Academy of Sciences (ChengDu, China). Hydrogen peroxide (30%, v/v aqueous solution) was obtained from Tianjin Eastern Chemical Reagent Co. Potassium chloroplatinate (K 2 PtCl 6 ) was obtained from Tianjin KRS Fine Chemcal Co. Ltd. (Tianjin, China). Uric acid, ascorbic acid and acetaminophen were obtained from Tianjin Damao Chemical Reagent Co. (Tianjin, China). All other reagents were of analytical grade and used without further purification. All aqueous solutions were prepared with Milli-Q deionized water. Phosphate-buffered saline (PBS) was prepared by mixing 25 mL 0.2 M KH 2 PO 4 aqueous solution with 29.54 mL 0.1 M NaOH and diluted to 100 mL with deionized water. All experiments were performed in PBS at room temperature, approximately 25 °C .

Apparatus and Electrochemical Measurements
The MWCNTs/Pt NPs nanohybrids were characterized with transmission electron microscopy (TEM, Philips T20 microscopy). Electrochemical measurements were carried out in a conventional three-electrode system. The MWCNTs/Pt NPs nanohybrids-modified Pt electrodes (3 mm diameter) were used as a working electrode, with a platinum spiral wire (1 mm diameter) as a counter electrode and an Ag/AgCl electrode (saturated with KCl) as a reference electrode. Amperometric measurements were performed by using Potentiostat-Galvanostat (EG&G PARC Model 283 with a software M270) (USA).

Modification of Multi-wall Carbon Nanotubes
Pristine MWCNTs were purified under strong sonication in a mixture of concentrated sulfuric and nitric acid 3/1 (v/v) for 4 h. The resultant black suspension was then diluted with water, through the high-speed centrifugal way to remove the supernatant, then dissolved in water to prepare a solution of 1 mg mL −1 .

Synthesis of MWCNTs/Pt NPs Nanohybrids
1 mL MWCNTs (1 mg mL −1 ) were put into a flask on a magnetic stirrer. While stirring at room temperature, 2 mL of K 2 PtCl 6 solution (0.01 M) was added dropwise into the flask. The reaction mixture was ultra-sonicated for 2 h and then stirred for 20 h at room temperature. The reaction product (MWCNTs/Pt NPs nanohybrids) was collected by centrifuging.

Electrode Modification with MWCNTs/Pt NPs Nanohybrids
Pt electrode was polished with a chamois leather containing 0.05 μm alumina powders, rinsed thoroughly with doubly distilled water, then immersed into HNO 3 :HCl:H 2 O (V:V:V = 1:3:4) solution for 3 min. The electrode was ultrasonically cleaned in ethanol and doubly distilled water, and dried at room temperature. MWCNTs/Pt NPs nanohybrids solutions (5 μL, 1 mg mL −1 ) were dropped on the surface of the Pt electrode. After drying, the modified electrode was washed and used as an amperometric sensor in phosphate buffer (pH 7.0).

Conclusions
The main feature of this work was to propose a new non-enzymatic H 2 O 2 sensor based on MWCNTs/Pt nanohybrids, which was prepared in a fast and simple procedure. In particular, experimental results demonstrated that Pt NPs were able to catalyze the electro-reduction of H 2 O 2 . The modified electrode exhibited an excellent selectivity to H 2 O 2 in the presence of possible interference compounds, which can be attributed to the relatively lower working potential at 0 mV. The present study may provide a feasible approach to develop new kinds of non-enzymatic amperometric sensors.