Facile and controllable synthesis of Prussian blue on chitosan-functionalized graphene nanosheets for the electrochemical detection of hydrogen peroxide

Electrochimica Acta 81 (2012) 37–43

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Facile and controllable synthesis of Prussian blue on chitosan-functionalized graphene nanosheets for the electrochemical detection of hydrogen peroxide Ji-Hoon Yang a , Noseung Myoung b , Hun-Gi Hong a,∗ a b

Department of Chemistry Education, Seoul National University, 599 Gwanangno, Gwanak-Gu, Seoul 151-748, Republic of Korea Department of Applied Chemistry, Konkuk University, Chungju Campus, Chungju, Chungbuk 380-701, Republic of Korea

a r t i c l e

i n f o

Article history: Received 1 November 2011 Received in revised form 31 May 2012 Accepted 12 July 2012 Available online 20 July 2012 Keywords: Graphene Chitosan (CS) Prussian blue (PB) Hydrogen peroxide (H2 O2 ) Amperometry

a b s t r a c t This work describes a new Prussian blue (PB)-based electrochemical sensor based on chitosan (CS)functionalized graphene nanosheets which exhibit good dispersibility and stability in aqueous solution. The morphology and composition of the graphene-CS/PB nanocomposite sheets were characterized by transmission electron microscopy (TEM), X-ray photoelectron spectroscopy (XPS), and X-ray diffraction (XRD). The electrochemical behavior of the resulting sensor was investigated using cyclic voltammetry and amperometry. The modified sensor showed good electrocatalytic activity in the reduction of hydrogen peroxide and was used as an amperometric sensor. The sensor exhibited a linear response for H2 O2 over concentrations ranging from 0.01 to 0.4 mM with a high sensitivity of 816.4 ␮A/(mM cm2 ) and a low detection limit (S/N = 3) of 0.213 ␮M. These parameters compare favorably with other PB-based electrodes. Furthermore, the new sensor exhibited freedom from interference from other co-existing electroactive species in human blood. This work describes a new type of graphene nanocomposite-modified electrode for amperometric biosensors. © 2012 Elsevier Ltd. All rights reserved.

1. Introduction Graphene, a honeycomb-shaped two-dimensional (2-D) lattice and one-atom-thick sheet of sp2 -bonded carbon atoms, has attracted much attention from both experimental and theoretical scientists in recent years [1,2]. The unique nanostructure of graphene, which has long-range ␲-conjugation, provides not only fascinating electronic and catalytic properties but also thermal and chemical stability that has shown great promise in applications in nanocomposites, batteries, supercapacitors, nanoelectronics, and chemical and biological sensors [3–9]. In particular, the electrochemical field is primed to explore the applications of graphene nanosheets. Several groups have demonstrated that graphene nanosheets exhibit very fast electron transfer kinetics and a large redox-active surface area, imbuing them with excellent electrocatalytic characteristics compared with other carbon-based materials [10–14]. For this reason, many graphene-based electrochemical sensors have been developed and used in areas such as the electrochemical determination of cadmium [15], dopamine [16], caffeine [17], hydroquinone [18], and other small molecules. More recently, to synergistically take advantage of the electrocatalytic activity, selectivity, and stability, various forms of

∗ Corresponding author. Tel.: +82 2 880 9115; fax: +82 2 889 0749. E-mail address: [email protected] (H.-G. Hong). 0013-4686/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.electacta.2012.07.038

graphene-based nanocomposite films have been developed. For example, Niu’s group reported new graphene nanocomposites with a polyethylenimine-functionalized ionic liquid used in the reduction of H2 O2 [8], and cationic polyelectrolyte-functionalized graphene nanocomposite sheets with gold nanoparticles were developed by Dong and co-workers for hydrogen peroxide sensing [20]. Hydrogen peroxide (H2 O2 ) is a very important analyte because it is not only an important byproduct from many peroxidasecatalyzed reactions but also an essential mediator in food, industrial, clinical, pharmaceutical, and environmental assays [21,22]. Prussian blue (PB), which is the prototypical metal hexacyanoferrate [23], has been extensively used to develop a new amperometric biosensor due to the characteristics of the reduced form of PB, Prussian white (PW). PW is known to catalyze the electrochemical reduction of H2 O2 at low overpotentials with remarkable selectivity and sensitivity, so that PB is often considered an “artificial peroxidase enzyme” [24,25]. Furthermore, PB has the advantages of lower cost and easier preparation compared with many other materials [26]. Recently, to improve the sensitivity and stability of the PB-based electrochemical sensors, many material supports have also been used to fabricate PB nanocomposite films, such as several forms of carbon materials, metal nanoparticles, and conducting polymers [27–31]. However, more recently, graphene nanosheets have been highlighted as a support for PB because of a large surface area that provides a large reactive space, a low cost

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and a convenient of the preparation compared with other materials [32]. For example, Jia and co-workers have developed graphene oxide/PB nanocomposite sheets used in the electrochemical detection of glucose [33], and graphene (reduced graphene oxide)/PB composite nanosheets have been developed by Wang’s group [34]. Nevertheless, it has still been necessary to develop a new strategy for the fabrication of graphene nanocomposites with PB, because the direct combination of graphene nanosheets, which are the reduced form of graphene oxide, and PB nanoparticles present two main difficulties. One challenge is that the chemical method of reducing graphene oxide tends to form irreversible agglomerates in aqueous solutions caused by the strong van der Waals interactions imposed by its large surface area [35]. The second challenge is that the residual negative charge of the reduced graphene oxide prevents the direct assembly of the negative charged PB nanoparticles through electrostatic repulsion. As a result, the uniformity of the PB deposited on graphene nanosheets is poor, and the morphology of the PB cannot be controlled precisely. It has been already demonstrated that the uniformity and morphology of PB nanoparticles are very important to the electrocatalytic performance in the reduction of H2 O2 [36]. Therefore, the development of new strategy that controls the size and shape of PB on graphene nanosheets precisely and yields high electrocatalytic activity and stability remains a challenge for the research community. In this paper, we demonstrate a new procedure using chitosan (CS)-functionalized graphene as a support material to improve the electrocatalytic function of PB nanoparticles. When directly combined with graphene nanosheets, chitosan could provide a stable cationic charge on the surface of reduced graphene oxide in neutral aqueous solutions. The cationic functionalization of the graphene nanosheets would lead to a high affinity between the nanosheets and the negative charged PB nanoparticles due to electrostatic attraction, such that the uniformity and morphology of the PB on the graphene nanosheets could be controlled and stable. By combining the respective strengths of graphene nanosheets, Prussian blue and chitosan, a novel PB-immobilized graphene nanocomposite biosensor has been developed, and its electrochemical characteristics for the reduction of H2 O2 have been studied. In particular, the size-dependent electrocatalytic activity of the PB on graphene nanocomposite sheets for the reduction of H2 O2 has been demonstrated. A high sensitivity, wide linear range and low detection limit were measured using amperometric analysis and other electrochemical methods. 2. Experimental 2.1. Chemicals and reagents Graphite powder (400 mesh), chitosan (low molecular weight), acetic acid (99.7 wt% in H2 O), ammonia (25 wt% in H2 O), potassium ferricyanide(III) (K3 Fe(CN)6 , 99+%), iron(III) chloride (FeCl3 , 97%), hydrogen peroxide (H2 O2 , 30 wt% in H2 O), and Nafion (5 wt%) were purchased from Sigma–Aldrich (USA) and used without further purification. Multi-walled carbon nanotube (MWCNT, 95% purity, diameter 10–15 nm, and length 10–50 ␮m) were purchased from Iljin (Korea). All other chemicals were of analytical grade, and all solutions were prepared with deionized water obtained from an Ultrapure water purification system (LabTech, Korea) with a resistivity of not less than 18.2 M cm. 2.2. Apparatus and measurements Cyclic voltammetry (CV) and amperometry were performed using a computer-controlled CHI 760B potentiostat with a conventional three-electrode system. A glass carbon (GC) working

electrode (3 mm diameter, GCE), purchased from Bioanalytical Systems (BAS), was used in the preparation of the modified electrode. A platinum (Pt) counter electrode and a silver/silver chloride (Ag/AgCl, 3 M KCl) reference electrode were used to complete the three-electrode system. Transmission electron microscopy (TEM) measurements were performed on a LIBRA 120 with an accelerating voltage of 120 kV, and the samples for TEM were prepared by placing a drop of the dispersion on a carbon-coated copper grid. Powder X-ray diffraction (XRD) analyses were performed on a M18XHF-SRA diffractometer, and X-ray photoelectron spectroscopy (XPS) was performed on an ESCALAB 250 spectrometer. 2.3. Preparation of the chitosan-functionalized graphene (rGO-CS) nanocomposite sheets Graphene oxide (GO) was prepared using a modified Hummers method [37], starting from graphite powder. Graphene-chitosan (rGO-CS) nanocomposite sheets were synthesized using Niu’s method [38]. First, 6 mg chitosan was added into 20 mL of an aqueous GO solution (0.5 mg/mL) at a pH of 3.5, adjusted with acetic acid. The obtained yellowish-brown GO–CS solution was then stirred at 60 ◦ C for 2 h. Subsequently, 7 ␮L hydrazine and the amount of ammonia (25 wt% in H2 O) necessary to adjust the pH to 8.5 were added. These additions were followed by continuous stirring at 90 ◦ C for 1 h. The final dispersion was filtered, centrifuged, washed with deionized water several times and dried in an oven at 40 ◦ C for 12 h. The material was finally redispersed in an acetic acid solution at pH 3.5. 2.4. Preparation of the graphene-chitosan/Prussian blue (rGO-CS/PB) nanocomposite sheets The above rGO-CS nanocomposite dispersion (0.8 mL at a concentration of 0.5 mg/mL) was added into 25 mL of a 0.1 M KCl aqueous solution (pH 2.7, adjusted with acetic acid) containing 1 mM K3 Fe(CN)6 under stirring for 5 min. 1 mM FeCl3 was then added and stirred at room temperature for 1 h. Afterwards, 2 ␮L of hydrogen peroxide (30 wt%) was added, causing the color of the mixed solution to gradually change from yellowish orange to dark cyan. After 3 h, the obtained dispersion was filtered through a membrane filter (with a pore size of 0.2 ␮m), rinsed with deionized water several times and finally redispersed in 10 mL of water. 2.5. Preparation of the PB, MWCNT/PB and rGO-CS/PB modified GCE A glassy carbon electrode was polished with aqueous slurries of successively finer alumina powder (particle size: 0.3 and 0.05 ␮m) on a polishing pad. The GC electrode was washed and then sonicated in deionized water and isopropyl alcohol for 10 min each. After the electrode was dry, 8 ␮L of the above rGO-CS/PB solution was dropped onto the purified GC surface, and then, 2 ␮L of Nafion (0.5 wt %) was also placed on the surface of the composite nanosheets. The system was then allowed to dry at room temperature in air. In order to compare the electrocatalytic behavior of rGO-CS/PB modified GCE for the detection of H2 O2 with those at PB and MWCNT/PB modified GCE, we also prepared nanosized PB and MWCNT/PB nanoparticles according to the literatures [24] and [28] previously reported, respectively. These nanoparticles were redispersed in deionized water by sonication, dropped onto the purified GC electrode, and then covered with Nafion solution as described above.

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Scheme 1. Synthesis of rGO-CS/Prussian blue.

3. Results and discussion 3.1. Characterization of the rGO-CS/PB nanocomposite sheets The synthesis of the rGO-CS/PB nanocomposite sheets is illustrated in Scheme 1, and snapshots of the outcomes at different stages of the process are shown in Scheme S1. In the first step, the GO nanosheets were synthesized by oxidizing graphite according to a modified Hummers method [37]. The nanosheets were subsequently redispersed in deionized water by ultrasonic treatment to obtain a yellowish brown dispersion (Scheme S1a). As shown in the snapshot in Scheme S1b, the rGO-CS nanocomposite sheets were aggregated because they had lost their charge in the alkaline solution (pH 8.5). However, after the precipitate was redispersed into an acidic solution (pH 2.7) to recover its cationic charge, a stable and homogeneous dispersion was obtained (shown in Scheme S1c). In the next step, the cationic rGO-CS nanocomposite sheets were mixed with an acidic solution (pH 2.7) containing 1 mM K3 Fe(CN)6 , and after 5 min, 1 mM FeCl3 was added and stirred for 1 h. Pre-adhered ferricyanide anions onto the cationic rGO-CS nanocomposite sheets allow selective growth of PB nanocubes (in contrast to free particle growth in solution). Due to the electrostatic attraction between the negatively charged iron hexacyanoferrate (PB) [39] and the cationic functionalized rGO nanosheets, the PB nanocubes strongly adhered to the surface of rGO-CS. A color change of the dispersion from black to dark cyan confirmed that the rGO-CS/PB assembly was successfully obtained (Scheme S1d). Typical TEM images of the as-prepared rGO-CS/PB nanocomposite sheets at different magnifications are shown in Fig. 1. Many PB nanocubes, with an average size of 25 nm, were spread out on the surface of the rGO-CS nanocomposite sheets (Fig. 1B and C) compared with the TEM image of the bulk graphene nanosheets (Fig. 1A). Moreover, because the PB nanocubes can be loaded at both sides of graphene, the PB nanocubes were found stacked as two layers [19]. Thus, the TEM images showed the high-loading and uniform distribution of the PB nanocubes on the rGO-CS nanocomposite sheets, demonstrating that this method can effectively produce the homogeneous high-loading of PB nanocubes on graphene hybrids. The structure and composition of the rGO-CS/PB nanocomposite sheets were characterized by XRD and XPS. Fig. 2A shows the XRD patterns of the rGO-CS and the rGO-CS/PB composite sheets for structural comparison. Compared to the XRD patterns of the rGO-CS nanocomposite sheets (Fig. 2A(a)), which showed a broad peak at 15–30◦ indicating the disordered stacking of graphene sheets after destroying the regular stack of graphite [40], it was definitively demonstrated that a pure face-centered-cubic phase of PB was

Fig. 1. (A) TEM image of rGO-CS/PB nanocomposite sheets; (B) and (C) at different magnifications.

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Fig. 2. (A) XRD patterns of (a) rGO-CS and (b) rGO-CS/PB nanocomposite sheets; (B) XPS C 1s spectrum of GO and (C) rGO-CS/PB.

synthesized on the graphene hybrid films (Fig. 2A(b)). The peaks at 2 = 17.3◦ , 24.8◦ , 35.3◦ , 39.6◦ , 50.7◦ , 54.1◦ , and 57.3◦ corresponded to the (2 0 0), (2 2 0), (4 0 0), (4 2 0), (4 4 0), (6 0 0), and (6 2 0) reflections, respectively [41]. The mean diameters of the PB nanocubes were calculated from the width of the strongest diffraction lines (the (2 0 0) diffraction line) using the Scherrer formula [42]: t=

0.9 B cos B

The linewidth B is usually measured in radians at the halfmaximum intensity.  B is the Bragg angle for the reflection, t is the crystal size, and  is the wavelength of the X-ray. The calculated mean diameter of the PB nanocubes was approximately 24.7 nm, which was similar to the diameter of the nanocubes determined from the TEM images. The chemical composition of the as-synthesized nanocomposite sheets was also characterized by XPS. The XPS survey spectra of the GO and the rGO-CS/PB are presented in Fig. 3a and b. Only C and O were present in the GO sample (Fig. S1A), whereas the rGO-CS/PB nanocomposite sheets contained C, O, N and Fe, with the latter two elements attributed to the PB (Fig. S1B). To further understand the electronic states of the elements, the high-resolution XPS spectra were examined. The peaks at 711.3 and 724.8 eV correspond to Fe 2p3/2 and Fe 2p1/2 and indicate the presence of Fe3+ [43], as shown in Fig. S1C. An additional peak was also observed at 708.2 eV, which can be assigned to Fe 2p3/2 originating from the Fe(CN)6 4− species [44]. According to the high-resolution Fe 2p spectra, the Fe3+ cations combine with the Fe(CN)6 4− anions to form PB on the graphene nanocomposite sheets. The C 1s spectrum of both the GO and the rGO-CS/PB samples could provide direct evidence of the reduction of GO. Fig. 2B shows the C 1s spectrum of the GO, which consists of four main components arising from C C (284.6 eV), C O (286.7 eV), C O (287.6 eV) and O C O (288.7 eV)

Fig. 3. CV of (a) GCE/PB, (b) GCE/MWCNT/PB and (c) GCE/rGO-CS/PB in the absence of H2 O2 from −0.3 to 0.6 V in 0.1 M KCl solution pH 2.7 at scan rate of 50 mV s−1 .

species. However, after the chemical reduction, the magnitudes of the peaks for the oxygen-containing species at C O (286.7 eV), C O (287.6 eV) and O C = O (288.7 eV) were decreased significantly (Fig. 2C), indicative of efficient deoxygenation. In addition, a component at 285.7 eV corresponds to C N bonds, which suggests the combination of the chitosan and graphene nanosheets.

3.2. Electrochemical performance of the rGO-CS/PB nanocomposite sheet-modified GCE To understand the synergistic effect of the rGO-CS/PB nanocomposite sheets, the electrochemical properties of the composites were characterized by cyclic voltammetry in a 0.1 M KCl (pH 2.7) aqueous solution at a scan rate of 50 mV s−1 . Fig. 3 represents the typical cyclic voltammograms (CVs) of the PB, MWCNT/PB and the rGO-CS/PB nanocomposite-modified GCE in the absence of H2 O2 . Each cyclic voltammogram shows a pair of well-developed redox waves corresponding to the inter-conversion between Prussian blue (PB) and Prussian white (PW). Among those CVs, the observed redox peak current is much larger at the rGO-CS/PB-modified GCE (Fig. 3c) than at the PB (Fig. 3a) and MWCNT/PB-modified GCE (Fig. 3b). It might be due to a number of PB nanoparticles adsorbed on large surface area and high electric conductivity of the reduced graphene oxide sheet [10–14]. When in the presence of H2 O2 , CVs of the PB, MWCNT/PB and the rGO-CS/PB nanocomposite-modified GCE (shown in Fig. 4) show much larger reduction current than the corresponding oxidation current in each redox wave, which are different from CVs observed in Fig. 3. This phenomenon is attributed to the existence of PB nanocrystals on the modified GCE. Prussian blue is well known for its ability to catalyze the electrochemical

Fig. 4. CV of (a) GCE/PB, (b) GCE/MWCNT/PB and (c) GCE/rGO-CS/PB in 5 mM H2 O2 from −0.3 to 0.6 V in 0.1 M KCl solution pH 2.7 at scan rate of 50 mV s−1 .

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reduction of H2 O2 at relatively low potentials and the electrocatalytic process can be described in two steps as follows [25]: KFe(III)[Fe(II)(CN)6 ](PB) + e− + K+ → K2 Fe(II)[Fe(II)(CN)6 ](PW)

(1)

2K2 Fe(II)[Fe(II)(CN)6 ](PW) + H2 O2 → 2KFe(III)[Fe(II)CN6 ](PB) + 2OH− + 2K+

(2)

Therefore, the larger reduction current is observed than the corresponding oxidation current because the electrocatalytic reduction of H2 O2 takes place at the redox potential of PB nanocrystals. The fact that the electrocatalytic reduction at rGOCS/PB-modified GCE (Fig. 4c) is much larger those that at PB (Fig. 4a) and MWCNT/PB-modified GCE (Fig. 4b) results from the existence of the reduced graphene oxide nanosheet providing large surface area. The effect of the scan rate on the electrocatalytic H2 O2 reduction current was investigated to evaluate the kinetics of the electrochemical performance of the as-synthesized graphene nanocomposite sheets, as shown in Fig. 5. The reduction current increased with increasing scan rates from 10 to 200 mV s−1 in proportion to the square root of the scan rate (inset in Fig. 5), indicating that the electrocatalytic reaction is a diffusion-limited process. The morphology and size of the PB nanoparticles on the rGOCS nanocomposite sheets are the important factors in determining the catalytic activity for the reduction of H2 O2 . To find the optimal condition for fabricating the PB nanocrystals on the graphene nanosheets, we controlled the concentration of the PB precursors (K3 Fe(CN)6 and FeCl3 ). TEM images of the rGO-CS/PB nanocomposite sheets prepared with different concentrations of the PB

Fig. 5. CV of the rGO-CS/PB nanocomposite sheets modified GCE in 5 mM H2 O2 and 0.1 M KCl aqueous solution (pH 2.7) at different scan rates of 10, 20, 50, 100, 200 mV s−1 . Insert: the calibration curve of reduction current on the square root of the scan rate.

precursors are shown in Fig. 6. When the concentration of the PB precursor was 0.5 mM, only small PB nanoparticles were observed, with a 19 nm crystal nucleus (Fig. 6A). At a concentration of 1 mM, the average size of the PB nanoparticles increased to 25 nm in diameter, and the morphology became cubic (Fig. 6B). When the concentration was further increased to 2 and 5 mM, the average size of the PB particles increased to approximately 42 nm and 63 nm, but the uniformity of the PB nanoparticles on the graphene nanosheets

Fig. 6. TEM image of rGO-CS/PB nanocomposite sheets with different concentration of PB precursor, K3 F2 (CN)6 and FeCl3 : 0.5 mM (A), 1 mM (B), 2 mM (C) and 5 mM (D).

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Table 1 Electroanalytical characteristics of various modified electrodes toward hydrogen peroxide. Linear range (mM) GCE/PBNPs/Nafion GCE/MWCNT/PB GCE/[PB@Pt/PCNTs]2 GCE/PB-SWNTs GCE/graphene/PB GCE/rGO-CS/PB

−3

2.1 × 10 –0.14 0.01–0.4 2.5 × 10−4 –1.5 0.5–27.5 0.02–0.2 0.01–0.4

Sensitivity (␮A/(mM cm2 ))

Limit of detection (␮M)

Potential (V)

References

138.6 153.7 850 – 196.6 816.4

1 0.567 0.15 0.01 1.9 0.213

−0.05 0 0.1 −0.1 −0.05 0.05

[45] [28] [30] [36] [32] This work

decreased, and the cubic morphology was lost in favor of spheres (Fig. 6C and D). Figs. S2 and S3 show the cyclic voltammograms with the catalytic reduction current for H2 O2 changing with the increasing size of the PB nanoparticles. Although the non-catalytic redox current exhibited by the PB increased with increasing size (Fig. S2), the highest ratio of electrocatalytic current (Ic ) to non-catalytic redox current (I0 ) was observed for the PB synthesized with a precursor concentration of 1 mM (Fig. S3). This is because as the size of PB nanoparticles grow larger than an optimum level, the active surface area for the reduction of H2 O2 decreased. In addition, these electrodes exhibited a redox reaction with a large peak-to-peak separation (Ep ) and poor reversibility upon increasing the particle size (Fig. S2), due to the relatively high resistance of the large PB nanoparticles. Fig. 7 displays a typical amperometric response of the rGO-CS/PB nanocomposite sheet-modified electrode upon the successive addition of H2 O2 in the 0.1 M KCl (pH 2.7) aqueous solution under stirring at 0.05 V. A remarkable increase of the reduction current was observed upon a subsequent addition of H2 O2 . Inset is the calibration curve of the reduction peak current vs. the concentration of H2 O2 . A linear response was observed in the range from 0.01 mM to 0.4 mM with a correlation coefficient of 0.996, and the sensitivity was estimated to be 816.4 ␮A/(mM cm2 ). The limit of detection was 0.213 ␮M with a signal-to-noise ratio of 3, and the time required to reach the 95% steady-state response was less than 5 s. The electroanalytical characteristics of some PB-based electrodes using different modification strategies to detect H2 O2 are summarized in Table 1. The sensitivity and detection limit of our new strategy improve upon those of the other reported modified electrodes. Furthermore, the as-synthesized sensor has good reproducibility. The relative standard deviation (RSD) of the response to 1 mM

Fig. 8. Amperometric response of the rGO-CS/PB nanocomposite sheets modified GCE to (A) 0.1 mM H2 O2 , (B) 0.1 mM cysteine, (C) 0.1 mM ascorbic acid and (D) citric acid in a stirring 0.1 M KCl (pH 2.8) solution. The working potential was +0.05 V.

H2 O2 for five sensors prepared under the same conditions was 7.5%. The stability of the rGO-CS/PB nanocomposite-modified electrode was investigated by measuring the response with a H2 O2 concentration of 2 mM. After 50 scanning cycles, only 2.2% of the signal was lost, and after 100 scanning cycles, the loss of signal reached only 4.7%. The life time of the as-synthesized sensor was also tested by measuring the current response to 2 mM H2 O2 , and it was found to decrease to 83.7% of its initial value after 30 days when exposed to air at ambient conditions, indicating good stability. One of the most important challenges in the detection of hydrogen peroxide in human blood is to decrease the response to other typical interfering electroactive species, which include ascorbic acid, cysteine, and citric acid. Fig. 8 shows a typical amperometric curve of the modified GCE according to the injection of identical concentrations of ascorbic acid, cysteine, citric acid, and hydrogen peroxide. The current responses generated by 0.1 mM ascorbic acid, cysteine, and citric acid were negligible compared with the current of 0.1 mM hydrogen peroxide, suggestive of high selectivity and catalytic activity of the new biosensor. 4. Conclusions

Fig. 7. Amperometric response of the rGO-CS/PB nanocomposite sheets modified electrode to successive injection of H2 O2 in 0.1 M KCl aqueous solution (pH 2.7) under stirring. Applied potential: 0.05 V. Insert: the calibration curve of the reduction peak current vs. concentration of H2 O2 .

This study has demonstrated that PB nanoparticles can be easily and strongly adsorbed on the surface of graphene nanosheets using chitosan, and that the modified electrode system can be used for the electrochemical detection of H2 O2 . We demonstrated that the electrocatalytic reduction of H2 O2 varies according to the size of the PB nanoparticles on the graphene nanocomposite sheets and determined the optimal concentration of the PB precursors for detecting H2 O2 . The as-synthesized electrochemical sensor exhibited not only a high sensitivity but also low limit of detection compared with other PB-based modified electrodes and can be used to detect H2 O2 without interference from other electroactive species in body.

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