Green synthesis of reduced graphene oxide/nanopolypyrrole composite: characterization and H₂O₂ determination in urine

Abstract

Here we report on a novel, simple and eco-friendly approach for the fabrication of a reduced Graphene Oxide/nanopolypyrrole (rGO/nPPy) composite material and its electrochemical performance for detection of hydrogen peroxide on a glassy carbon electrode. The characterization of the as-prepared rGO/nPPy composite was investigated by Fourier transform infrared spectroscopy, thermogravimetric analysis, ultraviolet-visible spectroscopy, scanning electron microscopy, contact angle measurement, cyclic voltammetry and electrochemical impedance spectroscopy. Cyclic voltammetry, differential pulse voltammetry and chronoamperometry techniques were used to investigate and optimize the performance of the developed electrochemical biosensor. The proposed biosensor showed excellent analytical response towards the quantification of H₂O₂ at pH 7.40. Under the optimized conditions, the biosensor shows a linear response range from 1.0 × 10⁻⁷ to 4.0 × 10⁻⁶ M concentrations of H₂O₂. The limit of detection was determined to be 34 nM. Reproducibility, sensitivity, stability and anti-interference capability of the fabricated biosensor for the detection of H₂O₂ were examined. The biological relevance of the developed electrochemical biosensor was further studied by the determination of H₂O₂ in urine samples. The real sample analysis of H₂O₂ was achieved before and after drinking coffee in urine samples. The successful and sensitive determination of H₂O₂ urine samples indicates that the proposed electrochemical biosensor can be applied to the quantification analysis of H₂O₂ in real samples.

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1. Introduction

Hydrogen peroxide is a strong oxidant used for medical and pharmaceutical sterilization, paper bleaching, industrial products and waste.^1,2 The design and development of new, rapid, accurate and reliable materials and methods for determination of H₂O₂ is of great significance due to the fact that its usage is continuously expanding in food and industrial processes.^3,4 The electrochemical quantification of H₂O₂ remains one of the predominant techniques used in analytical applications. However, the direct oxidation or reduction of H₂O₂ at bare electrode surfaces is not suited for analytical applications due to high over potentials and slow electron transfer kinetics on many electrode materials.⁵ For this reason, redox mediators, such as nanoparticles,^6,7 conducting polymers,⁸ and hybrid⁹ and composite¹⁰ materials have been widely used in order to decrease the over potential and enhance the electrode kinetics. Beside these materials, polymer based nanocomposite materials have recently attracted a lot of scientific interest due to their dramatic improvement in electron transfer rate, high conductivity, easy preparation procedures, good thermal and electrochemical stability, etc.^11–14

Graphene (G) has recently received significant research attention for different applications in sensor chemistry, such as enantioselective sensors,^15,16 biosensors,^17,18 gas sensors,^19–21 ion sensors,^22,23 due to its unique electronic, mechanical properties and large surface area. Graphene-based polymer composites (G-PCs), combination of polymer and graphene, which can be obtained through simple chemical procedures such as in situ chemical or electrochemical polymerization and non-covalent functionalization, have attracted much attention as electrode materials for sensors.²⁴ Polypyrrole (PPy) based G-PCs are one of the most widely used composite materials in sensor chemistry.²⁵ In comparison with other PCs, G-PPy has attracted great attention as a redox mediator in the design of electrochemical sensors due to its excellent conductivity, electroactivity and easy preparation.²⁶ The positively charged surface of the PPy can provide an interface for the interaction with graphene oxide. Moreover, the existence of amine group (–NH–) on the pyrrole ring may lead to increase for biomolecular sensing.²⁷

An alternative approach for polymer entrapment on graphene might be synthesis of polymers catalyzed by enzymatic reaction.²⁸ The enzyme-catalyzed reactions has received much attention because this simple process is one-step, eco-friendly and does not require strong acidic media.²⁹ Different oxidoreductases have been employed for the synthesis of conducting polymers.³⁰ While some studies for the enzymatic polymerization of pyrrole using soybean peroxidase,³¹ glucose oxidase,³² laccase,³³ horseradish peroxidase,³⁴ have been reported, to the best of our knowledge there is no study on incorporation of graphene with polypyrrole resulting G-PCs by enzymatic polymerization.

Here we report a simple, enzymatic, non-toxic and oxidative polymerization of pyrrole on graphene sheets using glucose oxidase in aqueous solution. The synthesized composite material was characterized with different methods. It was used for fabrication of electrochemical biosensor in order to determinate of hydrogen peroxide in aqueous solution and urine samples. Also, the determination of hydrogen peroxide was successfully achieved in urine before and after drinking coffee. The results ensure that proposed electrochemical sensor can be used for determination of H₂O₂ concentration in real samples at nanomolar level.

2. Experimental

2.1. Chemicals and equipment

Unless otherwise noted, all commercial chemicals were of analytical grade and purchased from global suppliers. Graphite powder (99.99%), glucose oxidase (GOx) from Aspergillus niger (E.C.1.1.3.4.) 295 U mg⁻¹, D-(+)-glucose were purchased from Sigma-Aldrich. All aqueous solutions were freshly prepared using ultra-pure water with a resistivity of 18.2 MΩ cm.

Electrode morphologies were investigated by scanning electron microscopy (SEM), performed on a ZEISS EVO LS 10. Before scanning process, all samples were coated with gold to enhance the electron conductivity. Fourier transformed infrared (FT-IR) spectra of the samples were recorded between 550 and 4000 cm⁻¹ using ATR FT-IR spectrometer (Perkin Elmer 100 FT-IR). Thermogravimetric analysis (TGA) of the samples (10–15 mg) was performed on Setaram thermal gravimetric analyzer (France) at temperature range of 25–1200 °C with 10 °C min⁻¹ heating ramp in argon atmosphere (gas flow rate: 20 mL min⁻¹). Contact angle measurements were carried out by a horizontal beam comparator (KSV CAM 200). The contact angle measurement was calculated as the mean value of 3 different points on each material. UV-vis absorption spectra were obtained on Shimadzu UV-1800 double beam spectrophotometer. Electrochemical measurements were performed with an IVIUM-CompactStat potentiostat (Ivium Technologies, Netherlands) combined with a BAS/C3 electrochemical cell stand using three electrode configuration cell. GCE, Ag/AgCl electrode and platinum electrode were used as working electrode, reference electrode and counter electrode, respectively. Electrochemical impedance spectroscopy (EIS) measurements were conducted in A-PBS solution pH 7.01 containing 1.0 mM [Fe(CN)₆]^3−/4− redox couples. The impedance measurements were performed in the frequency range from 10 Hz to 100 kHz with 5 mV signal amplitude. Cyclic voltammetry (CV) measurements were carried out in a mixed solution of 50 mM sodium acetate and 50 mM phosphate buffer (pH 7.40, as an optimum condition) with 100 mM KCl at ambient temperature (at 25 °C).

2.2. Green synthesis of reduced graphene oxide/nanopolypyrrole composite (rGO/nPPy)

The schematic diagram for producing of reduced graphene oxide/nanopolypyrrole composite (rGO/nPPy) was represented in Scheme 1. Graphene oxide (GO) was synthesized from graphite powder by the improved method,³⁵ which has significant advantages over Hummers' method,³⁶ as indicated in our previous paper.^15,16 In order to prepare rGO/nPPy, 20 mL of GO aqueous suspension (1 mg mL⁻¹) sonicated 30 min was added into GOx solution (1 mg mL⁻¹) in a round bottom flask and mixed for 30 min at 4 °C. Then, glucose (40 mM) and pyrrole (200 mM) were added into the solution and mixed at 4 °C for 72 h. Finally, the product was filtered with 0.2 micrometer, 25 mm cellulose membrane and washed with ultra-pure water (5 × 20 mL) and methanol (5 × 20 mL), and dried in vacuum oven at 50 °C.

Scheme 1 The schematic diagram for producing of reduced graphene oxide/nanopolypyrrole (rGO/nPPy) composite.

2.3. The preparation rGO/nPPy modified electrode

The fabrication of electrochemical biosensor for determination of H₂O₂ can be briefly explained as follows: prior to each experiment, in order to avoid contamination of oxidation products and to obtain a clean electrode, the surface of the GCE was pre-cleaned with acetone, ethanol and ultra-pure water. Then, GCE surface was hand-polished with 1.0, 0.3 and 0.05 μm alumina powder (PACE Technologies, USA) on a felt pad, and washed with a copious amount of ultra-pure water. The GCE was washed with HNO₃ and then was immersed in water and methanol for 15 minutes, respectively in an ultrasonic bath (Sonorex Super RK 106, Germany) in order to remove residual alumina particles by sonicating. The electrode was dried under N₂ atmosphere at room temperature before modification step. After drying, 5.0 μL suspension of rGO/nPPy (0.2 mg mL⁻¹) sonicated 30 min was dropped on the GCE surfaces as three times (as an optimum condition) in order to obtain rGO/nPPy/GCE. The electrode was then washed with ultra-pure water and used for electrochemical measurements after rinsing with ultra-pure water and drying under N₂ atmosphere.

3. Results and discussion

The occurring polymerization of pyrrole on graphene sheets is based on four major substrates; pyrrole: monomer for polymerization; GOx: enzyme for producing hydrogen peroxide; glucose: reducing substrate of graphene oxide (GO); dissolved oxygen: oxidizer for GOx. The reaction occurring with these compounds can be explained as follows; graphene oxide is reduced by glucose (eqn (1)).³⁷ At the same time, GOx starts to generate hydrogen peroxide and gluconolactone in the presence of glucose and dissolved oxygen (eqn (2)). It should be noted that pH gradient locally decreases while H₂O₂ gradient increases because of gluconic acid is formed in the solution (eqn (3)). Thus, the optimal conditions for the polymerization of pyrrole on rGO sheets (rGO/nPPy) might be created by the locally decreased pH and H₂O₂ (eqn (4)).^29,32

Gluconolactone + H₂O → gluconic acid (3)

3.1. Characterization of the rGO/nPPy composite and rGO/nPPy/GCE

The UV-vis absorption spectra of GO and rGO/nPPy are shown in Fig. 1A. GO dispersion displays an absorption maximum at 232 nm, which is due to the π–π* transition of aromatic C=C bonds and a shoulder at 304 nm, which corresponds to the n–π* transition of the C=O bond.³⁸ For rGO/nPPy composite, the color of the dispersion changed from pale-brown to black (the inset of Fig. 1A) and the absorption peak of the GO dispersion at 232 nm red-shifted to 269 nm suggesting that the conjugation within the rGO is formed due to the reduction by glucose.³⁷ rGO/nPPy also shows a shoulder at approximately 468 nm, which is characteristic absorption peak of PPy, attributed to the transition from the valence band to the anti-bonding polaron state.³⁹

Fig. 1 UV-vis spectra of GO and rGO/nPPy composite. The inset is the photographs of aqueous dispersion for the synthesized GO and rGO/nPPy composite (A) TGA curves of GO and rGO/nPPy composite (B) FT-IR spectroscopic analysis of GO and rGO/nPPy composite (C).

Changes in the thermal stability and the composition of GO and rGO/nPPy composite material were analyzed by TGA under argon atmosphere (Fig. 1B). The weight loss of GO below 100 °C is a result of the evaporation of the absorbed water and the major weight loss occurring around 200 °C can be attributed to the liable hydrophilic groups, yielding CO, CO₂ and steam, as similarly indicated in the earlier reports.^40,41 For rGO/nPPy composite, nearly a 4.3% weight loss occurred at 100 °C due to the de-intercalation of water from the gallery space of the GO framework.⁴² The weight loss near 200 °C for the rGO/nPPy composite presumably stemmed from the pyrolysis of the labile oxygen-containing functional groups. After 250 °C, the major weight loss (27.6%) occurred can be assigned to decomposition of the PPy from the composite.

FT-IR analysis of GO and rGO/nPPy composite was represented in Fig. 1C. The peaks at 2959 and 2882 cm⁻¹ in both spectra demonstrate the asymmetric stretching and symmetric vibrations of CH₂. In the FT-IR spectrum of GO, the characteristic absorption peaks were obtained. The strong and broad peak at 3273–3205 cm⁻¹ can be attributed to O–H groups stretching vibration, which is assigned to the adsorbed water molecules and the hydroxyl groups on the surface. The well-defined peak at 1731 cm⁻¹ is related to the C=O stretching vibrations in the carbonyl group. It shows a strong absorption band at 1632 cm⁻¹ due to the vibrations of the aromatic C=C group. The peaks at 1399–1221–1059 cm⁻¹ for GO were also assigned to the C–O (carboxyl), C–O (epoxy) and C–O (alkoxy) functional groups, respectively.^43,44 After the reduction, the peak intensity at 1731 cm⁻¹ described above greatly decreased for rGO/nPPy composite. Also, the presence of polypyrrole in the rGO/nPPy composite was confirmed by the appearance of characteristic peaks of C–N and C–C at 1506 and 1438 cm⁻¹, indicating asymmetric and symmetric ring-stretching of PPy, respectively.³⁹ The bands at 1399 and 1060 cm⁻¹ are attributed to the C–N stretching and C–H deformation vibrations in PPy ring, respectively. In addition, the presence of the doping state of nPPy can be explained with the presence of the peaks at 1221 and 931 cm⁻¹.^44,45

The morphology of the GO and rGO/nPPy composite sheet on GCE surface were characterized by scanning electron microscopy (SEM) and the images were shown in Fig. 2A and Fig. 2B, respectively. The GO/GCE reveals a characteristic flaky-like structure indicating that GO homogenously exfoliated on the electrode surface as represented in the inset of Fig 2A.^11,46 Fig. 2B shows the structure of rGO/nPPy composite sheet after the enzymatic polymerization of pyrrole on graphene oxide. Sphere-like nanoparticles wrapped on reduced graphene oxide are clearly observed, which are completely different from the sheet-like shape of GO.^27,47,48 The image of rGO/nPPy composite in the inset of Fig. 2B at the larger magnification also displays the rGO sheet coated with nPPy, which further confirms the growth of PPy along the sheet as nanoparticles.

Fig. 2 SEM images of GO sheet (A) and rGO/nPPy composite (B) on GCE surface; the insets display the larger magnification of the rGO/nPPy/GCE.

Contact angle measurements were performed on the surface of silicon wafer. The sessile drop method was used to measure the contact angle of the prepared materials.^49,50 A 4 μL droplet of distilled water was placed on the samples surface by a 0.10 mL syringe. A magnified image of the droplet was recorded by a digital camera. The static contact angles were determined from these images with calculation software (Fig. S1†). The water contact angles of the blank, GO and rGO/nPPy samples were measured as 57.57°, 60.10° and 60.23°, respectively. According to the results, it can be concluded that the rGO/nPPy surface has the highest hydrophobicity.

The characterization of the bare GCE and the rGO/nPPy/GCE was also investigated electrochemically by CV and EIS in A-PBS solution (pH 7.01) containing 1.0 mM [Fe(CN)₆]^3−/4− redox couple. The occurring changes at the surface of GCE after modification can be obviously seen by peak-to-peak potential difference (ΔE_p) and peak currents ratio (I_p,a/I_p,c) of the [Fe(CN)₆]^3−/4− redox couple as presented in Fig. 3A. The bare GCE showed a typical reversible electrochemical response for the [Fe(CN)₆]^3−/4− redox couple at 223 mV with I_p,a/I_p,c of 1.03 and ΔE_p of 114 mV,⁵¹ while the rGO/nPPy/GCE exhibited an increase at I_p,a/I_p,c of 1.06 and a remarkable amount of decrease for ΔE_p of 86 mV at the same redox potential. The peak currents ratios in both cases are very close unity. Considering of the EIS measurements given in Fig. 3B, the diameter of the semicircle indicates the charge transfer resistance that can be used to define the interfacial properties of the modified electrode. The semicircular part at higher frequency corresponds to the electron transfer limiting process, and the linear portion at a lower frequency corresponds to the diffusion process. The impedance values were fitted to standard Randle's equivalent circuit (the inset of Fig. 3B) comprising of the charge transfer resistance (R_ct), ohmic resistance of the electrolyte solution (R_s), Warburg impedance (Z_w) and surface double-layer capacitance (C_dl). In accordance with the CV results, the bare GCE generated an interfacial charge transfer resistance (R_ct) of 1.558 kΩ with a semicircle portion at high frequency region and a straight line at low frequency. By the modification of GCE surface with rGO/nPPy, the semicircle part on the impedance spectrum obviously decreased with R_ct of 0.460 kΩ. The obvious change in the diameter of the semicircle portion indicates that rGO/nPPy composite facilitates the electron transfer of [Fe(CN)₆]^3−/4−, because rGO generates excellent electrocatalytic activity and nPPy act as an electron transfer enhancing layer.

Fig. 3 Cyclic voltammograms (A) and Nyquist diagram (B) obtained at bare GCE and rGO/nPPy/GCE electrode in the presence of 1 mM [Fe(CN)₆]^3−/4− in A-PBS solution (pH 7.01). The inset is Randle's equivalent circuit used to model impedance data in the presence of the redox couples.

3.2. Electrochemical determination of H₂O₂ at rGO/nPPy/GCE

Fig. 4A shows the cyclic voltammograms obtained at rGO/nPPy/GCE in the absence and presence of H₂O₂ (10 μM) in 0.1 M A-PBS (pH 7.40) at the scan rate of 50 mV s⁻¹. Similar to the previous study in which stacked graphene nanofibers doped polypyrrole nanocomposite modified electrode was used,⁵² a well-defined irreversible anodic peak was observed at 0.83 V with rGO/nPPy/GCE in the presence of H₂O₂. No redox peak was observed at bare GCE and rGO/GCE surfaces under the same experimental conditions as indicated in the literature.^10,53 The irreversible anodic peak at rGO/nPPy/GCE can be explained by the oxidation of H₂O₂ with two electron transfer given by following equation.⁵⁴

H₂O₂ → O₂ + 2H⁺ + 2e⁻ (5)

Fig. 4 Cyclic voltammograms in the absence and presence of 10 μM H₂O₂ at GO/nPPy/GCE electrode (A) cyclic voltammograms in the anodic range for the different concentration of H₂O₂; the inset shows DPV measurements in the anodic range for the lower concentrations of H₂O₂ (B) cyclic voltammograms in the anodic range for the different scan rate (v); the inset is the linear graph between the anodic peak currents (I_pa) and the square root of scan rate (v^1/2) (C) the linear relationship between the oxidation peak potential (E_p) and the logarithm of scan rate (D) in 0.1 M A-PBS at pH 7.40.

Fig. 4B shows the additional CV measurements at anodic range carried out in order to investigate the relationship between the peak current and the concentration in the range of 0.50–14.0 μM of H₂O₂. The height of the oxidation peaks showed linear variation (R² = 0.9942) with concentration, indicating that the electron transfer is diffusion-controlled (data not shown). The inset in Fig. 4B shows the DPV measurements performed in order to gain an insight to the relationship between the peak current and of H₂O₂ amount at lower concentrations in the range 0.20 μM to 2.50 μM. In accordance with CV measurements, well-defined strong oxidation peaks were also observed for low concentration values of H₂O₂ which increased with increasing concentration of H₂O₂.

Fig. 4C shows anodic voltammograms for the oxidation process of H₂O₂ obtained at different scan rates. As shown in the inset of Fig. 4C, the plot of the oxidation peak current versus the square root of the scan rate (v^1/2) is a straight line at scan rate ranging 50–500 mV s⁻¹, as expected for a diffusion-limited electrochemical process. In addition, while the peak current was increased with an increase at the scan rate, the peak was shifted toward more positive potential values, indicating the electrochemical irreversible processes.⁵⁵ In order to obtain an insight on the rate determining step, Tafel slope, b, was determined using the following equation valid for a totally irreversible diffusion controlled process.^56,57

E_p = (b/2)log(v) + constant (6)

The slope of the linear plot is equal to b/2 = 0.059/αn V. Where α and n is the product of an electron transfer coefficient and number of electrons transferred in rate determining step, respectively. There is a linear correlation between the oxidation peak potential and the logarithm of scan rate, log(ν), as is illustrated in Fig. 4D. The slope is ∂E_p/∂log(ν), which was found to be 0.0953 V. So, b = 2 × 0.0953 V = 0.1906 V. Considering the number of electrons transferred in the rate-limiting step, n is equal to 2 (from eqn (5)), the anodic electron transfer coefficient (α) was estimated as 0.310.

3.3. Amperometric response and selectivity of the sensor

The amperometric response of the rGO/nPPy/GCE versus H₂O₂ was investigated by CA technique by successive addition of 0.10 μM–4.0 μM H₂O₂ to a continuously stirred 0.1 M A-PBS solution at 0.00 and 0.83 V vs. Ag/AgCl (3 M KCl) and results are shown in Fig. 5A. When an aliquot of H₂O₂ was dropped into the stirring A-PBS solution, the oxidation current value steeply rose to reach a stable value. The sensor could accomplish 99% of the steady state current within lower than 1 s, indicating that rGO/nPPy/GCE exhibits a very fast amperometric response behavior towards H₂O₂. As it can be clearly seen, rGO/nPPy/GCE exhibited an excellent amperometric response to the increasing concentration of H₂O₂ with ΔI_max of 186 μA. In accordance with the CV and DPV measurements, the amperometric results show that the oxidation peak current of H₂O₂ also linearly increases with increasing concentration of H₂O₂ in the range of 0.10 μM–4.0 μM as presented in the Fig. 5B. Considering the equation of the linear regression of calibration graph, the limit of detection (LOD) and the limit of quantitation (LOQ) were calculated as 34 nM and 102 nM, respectively, using the standard deviation of y-intercept and the slope of the regression line.

Fig. 5 The chronoamperograms for increasing concentration of H₂O₂ (0.1–4.0 μM) at rGO/nPPy/GCE in a continuously stirred 0.1 M A-PBS solution between 0.00 and 0.83 V vs. Ag/AgCl (3 M KCl) (A) the linear relationship between H₂O₂ concentrations and the oxidation peak currents (B) the amperometric responses of the fabricated sensor to H₂O₂ and different interfering substances at the applied potential of 0.83 V vs. Ag/AgCl in a continuously stirred A-PBS solution at pH 7.40 (C).

This high sensitive capability of rGO/nPPy/GCE can be attributed to (1) the electrocatalytic activity of rGO (due to the large surface area and high density of edge-plane-like defects, which might allow rapid heterogeneous electron transfer) significantly increased the catalytic activity⁵⁸ and (2) excellent conductivity of nPPy that acts as a mediator enhancing the electron transfer rate. These reasons indicate that very low charge transfer resistance of the rGO/nPPy/GCE, as demonstrated in the EIS test, may also allow faster electron transfer for the oxidation of H₂O₂ at the surfaces exposed between the rGO sheets.

The effects of three common interfering electro-active substances, ascorbic acid (AA), uric acid (UA) and glucose were investigated. Fig. 5C shows the responses to sequential addition of 0.10 μM H₂O₂, 1.0 mM AA, 1.0 mM UA, 1.0 mM glucose and 0.50 μM H₂O₂ measured at 0.83 V. As the optimum electrode potential 0.83 V was selected in order to avoid a possible interference of these electro active species. The results show that the potential interferents showed no significant effects on the response of the proposed biosensor which exhibits a high selectivity towards H₂O₂. The analytical performances of the proposed sensor were compared in Table 1 with other recently reported H₂O₂ biosensors in the literature. As it is clearly seen, the proposed sensor exhibits excellent performance in terms of good limit of detection value as well as good selectivity.

Table 1 Comparison of the performances of various H₂O₂ sensors^a

Sensor	Applied potential (V)	Linear range (M)	Limit of detection (M)	Ref.
a MCNs – mesoporous carbon nanospheres; CNT – carbon nanotube; PDDA – polydiallyldimethylammonium; G – graphene; f-G – functionalized-graphene; rGO – graphene oxide; nPPy – nanopolypyrrole.
Pd/MCNs	−0.3	7.5 × 10^−6–1 × 10^−2	1.0 × 10^−6	7
MnO2/OMC/GCE	0.45	0.5 × 10^−6–6.00 × 10^−4	7.0 × 10^−8	10
PDDA-G/Fe3O4	−0.4	2.0 × 10^−5–6.25 × 10^−3	25.0 × 10^−7	63
(HRP-Pd)/f-G	−0.1	2.5 × 10^−5–3.5 × 10^−3	5.0 × 10^−8	64
MnO2/G/CNT	0.4	1 × 10^−6–1.030 × 10^−3	1.0 × 10^−7	65
rGO/nPPy	0.83	1.0 × 10^−7–4.0 × 10^−6	34.0 × 10^−9	This work

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3.4. The stability, reproducibility and repeatability of the rGO/nPPy/GCE

The reproducibility of the rGO/nPPy/GCE was investigated by successive detection of 1.0 μM H₂O₂ with independently prepared five electrodes, and relative standard deviation (RSD) was calculated as 3.54%. The repeatability of modified electrodes was evaluated by analysis of chronoamperometric responses for five times using the same electrode and relative standard deviations (RSD) was calculated as 2.52%. The stability of the rGO/nPPy/GCE was investigated by CA in a constant H₂O₂ concentration (1.0 μM) over a period of 2 weeks. It was observed that during first week the current responses almost remained stable and showed a decrease in current density about 12.19% of its initial value after 14 days. The decrease of current response may be attributed to the degradation of rGO/nPPy composite from the GCE surface.

3.5. The real sample analysis performance capability of rGO/nPPy/GCE

As presented in literature,^59,60 a substantial amount of H₂O₂ is excreted in normal human urine, thus it might be a valuable biomarker of the extent of oxidative stress in vivo. For this purpose, the response capability of the proposed sensor for real sample was investigated in order to determine the spiked concentration of H₂O₂ in freshly collected human urine from healthy volunteers. The concentration of H₂O₂ was determined using the regression equation of calibration curve (Fig. 5B) and the obtained results for different urine samples were shown in Table 2.

Table 2 The determination of the spiked concentrations of H₂O₂ at the rGO/nPPy/GCE in urine samples

Sample	Spiked (μM)	Detected (μM)	Recovery (%)
1	0.00	0.98	—
	1.00	2.02	104
	2.00	2.97	99.5
2	0.00	0.87	—
	1.00	1.93	106
	2.00	3.07	110
3	0.00	0.42	—
	1.00	1.38	96
	2.00	2.47	102.5

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3.6. The determination of H₂O₂ in human urine stems from coffee

The amperometric response of the proposed sensor was tested before and after (60 min following the intake of coffee) drinking coffee as employed by Chatterjee and Chen.⁶¹ Fig. 6 shows the chronoamperograms indicating that coffee drinking leads to a detectable rise in urinary H₂O₂ levels. The rGO/nPPy/GCE exhibited good amperometric response for H₂O₂ in the presence of urine sample (diluted ten times) with ΔI of 3.02 μA before drinking coffee. After the intake of coffee, ΔI rose to 6.01 μA indicating that coffee drinking increased the H₂O₂ concentration with a difference of 2.99 μA in current. After that, the addition of 0.2 μM H₂O₂ gave rise to an increase in current with 2.85 μA. Considering the difference in current and the dilution of urine sample, the concentration of H₂O₂ in urine after drinking coffee was calculated as 1.98 μM from the calibration graph. The results can be explained by the presence of hydroxy hydroquinone component in coffee beans which generates hydrogen peroxide.⁶² The hydroxy hydroquinone in coffee is taken into the body, excreted into the urine and auto-oxidized to produce H₂O₂. Considering of the excellent amperometric response, it can be concluded that the fabricated biosensor can be used for detection of H₂O₂ in urine.

Fig. 6 The chronoamperometric response for H₂O₂ at the rGO/nPPy/GCE before and after 60 min of intake of coffee in urine samples.

4. Conclusion

In this paper, a novel H₂O₂ sensor based on rGO/nPPy composite enzymatically produced by green synthesis method was fabricated. The experimental results showed that the proposed sensor possesses an excellent electrocatalytic activity towards H₂O₂ in a 0.1 M A-PBS solution at pH 7.40. Also, The it should be noted that the developed electrochemical biosensor can be applied for the successful and sensitive determination of H₂O₂ urine. The produced H₂O₂ sensor excluded the interference of substances such as ascorbic acid, uric acid, and glucose typically present in the biological samples. Although the electrode was tested on hydrogen peroxide, the scope may be further extended for sensing of various other biological compounds. This method for production of rGO/nPPy composite allows for a high output of sensitive, portable and most importantly disposable electrodes for biosensing. Also, this simple, rapid and especially non-toxic enzymatic approach is expected to be a promising platform for incorporation of graphene sheets to the other polymers for the synthesis of novel graphene/polymer composites using different enzymes and polymerizable monomers.

Abbreviations

GO	Graphene oxide
PPy	Polypyrrole
rGO/nPPy	reduced Graphene Oxide/nanopolypyrrole
G-PCs	Graphene-based polymer composites
GCE	Glassy carbon electrode
rGO/nPPy/GCE	rGO/nPPy modified glassy carbon electrode
GOx	Glucose oxidase
FT-IR	Fourier transform infrared spectroscopy
TGA	Thermogravimetric analysis
UV-vis	Ultraviolet-visible spectroscopy
SEM	Scanning electron microscopy
CV	Cyclic voltammetry
EIS	Electrochemical impedance spectroscopy
DPV	Differential pulse voltammetry
CA	Chronoamperometry
LOD	Limit of detection
LOQ	Limit of quantitation
AA	Ascorbic acid
UA	Uric acid

Acknowledgements

This work was partially supported by Scientific Research Projects (121210004/122010005) of Necmettin Erbakan University.

References

1. E. Kurowska, A. Brzózka, M. Jarosz, G. D. Sulka and M. Jaskuła, Electrochim. Acta, 2013, 104, 439–447.
2. M. R. Guascito, E. Filippo, C. Malitesta, D. Manno, A. Serra and A. Turco, Biosens. Bioelectron., 2008, 24, 1057–1063.
3. S. Zhu, L. Fan, X. Liu, L. Shi, H. Li, S. Han and G. Xu, Electrochem. Commun., 2008, 10, 695–698.
4. E. M. I. Mala Ekanayake, D. M. G. Preethichandra and K. Kaneto, Sens. Actuators, B, 2008, 132, 166–171.
5. A. Salimia, R. Hallaj, S. Soltanian and H. Mamkhezri, Anal. Chim. Acta, 2007, 594, 24–31.
6. Z. Zhang, S. Gu, Y. Ding and J. Jin, Anal. Chim. Acta, 2012, 745, 112–117.
7. X. Bian, K. Guo, L. Liao, J. Xiao, J. Kong, C. Ji and B. Liu, Talanta, 2012, 99, 256–261.
8. F. Tian, B. Xu, L. Zhu and G. Zhu, Anal. Chim. Acta, 2001, 443, 9–16.
9. G. Bencsik, C. Janáky, B. Endrodi and C. Visy, Electrochim. Acta, 2012, 73, 53–58.
10. L. Luo, F. Li, L. Zhu, Z. Zhang, Y. Ding and D. Deng, Electrochim. Acta, 2012, 77, 179–183.
11. Y. Liu, H. Wang, J. Zhou, L. Bian, E. Zhu, J. Hai, J. Tang and W. Tang, Electrochim. Acta, 2013, 112, 44–52.
12. C. Xu, J. Sun and L. Gao, J. Mater. Chem., 2011, 21, 11253–11258.
13. R. Devi, S. Relhan and C. S. Pundir, Sens. Actuators, B, 2013, 186, 17–26.
14. J. Du and H.-M. Cheng, Macromol. Chem. Phys., 2012, 213, 1060–1077.
15. E. Zor, I. Hatay Patir, H. Bingol and M. Ersoz, Biosens. Bioelectron., 2013, 42, 321–325.
16. E. Zor, A. O. Saf, H. Bingol and M. Ersoz, Anal. Biochem., 2014, 449, 83–89.
17. K.-J. Huang, Y.-J. Liu, H.-B. Wang, T. Gan, Y.-M. Liu and L.-L. Wang, Sens. Actuators, B, 2014, 191, 828–836.
18. X. Han, X. Fang, A. Shi, J. Wang and Y. Zhang, Anal. Biochem., 2013, 443, 117–123.
19. F. Liu, X. Chu, Y. Dong, W. Zhang, W. Sun and L. Shen, Sens. Actuators, B, 2013, 188, 469–474.
20. R. K. Joshi, H. Gomez, F. Alvi and A. Kumar, J. Phys. Chem. C, 2010, 114, 6610–6613.
21. R. K. Paul, S. Badhulika, N. M. Saucedo and A. Mulchandani, Anal. Chem., 2012, 84, 8171–8178.
22. X.-R. Li, F.-Y. Kong, J. Liu, T.-M. Liang, J.-J. Xu and H.-Y. Chen, Adv. Funct. Mater., 2012, 22, 1981–1988.
23. Y. Wen, F. Y. Li, X. Dong, J. Zhang, Q. Xiong and P. Chen, Adv. Healthcare Mater., 2013, 2, 271–274.
24. H. J. Salavagione, G. Martínez and G. Ellis, Macromol. Rapid Commun., 2011, 32, 1771–1789.
25. Q. Xu, S.-X. Gu, L. Jin, Y. Zhou, Z. Yang, W. Wang and X. Hu, Sens. Actuators, B, 2014, 190, 562–569.
26. C. Bora and S. K. Dolui, Polymer, 2012, 53, 923–932.
27. T. Qian, S. Wu and J. Shen, Chem. Commun., 2013, 49, 4610–4612.
28. A. Ramanavicius, A. Kausaite and A. Ramanaviciene, Sens. Actuators, B, 2005, 111–112, 532–539.
29. A. Kausaite-Minkstimiene, V. Mazeiko, A. Ramanaviciene and A. Ramanavicius, Sens. Actuators, B, 2011, 158, 278–285.
30. A. Kausaite-Minkstimiene, V. Mazeiko, A. Ramanaviciene and A. Ramanavicius, Biosens. Bioelectron., 2010, 26, 790–797.
31. R. Bouldin, S. Ravichandran, A. Kokil, R. Garhwal, S. Nagarajan, J. Kumar, F. F. Bruno, L. A. Samuelson and R. Nagarajan, Synth. Met., 2011, 161, 1611–1617.
32. A. Ramanavicius, A. Kausaite, A. Ramanaviciene, J. Acaite and A. Malinauskas, Synth. Met., 2006, 156, 409–413.
33. H. K. Song and G. T. R. Palmore, J. Phys. Chem. B, 2005, 109, 19278–19287.
34. M. R. Nabid and A. A. Entezami, J. Appl. Polym. Sci., 2004, 94, 254–258.
35. D. C. Marcano, D. V. Kosynkin, J. M. Berlin, A. Sinitskii, Z. Sun, A. Slesarev, L. B. Alemany, W. Lu and J. M. Tour, ACS Nano, 2010, 4, 4806–4814.
36. W. S. Hummers and R. E. Offeman, J. Am. Chem. Soc., 1958, 80, 1339.
37. C. Zhu, S. Guo, Y. Fang and S. Dong, ACS Nano, 2010, 4, 2429–2437.
38. J.-L. Chen, X.-P. Yan, K. Meng and S.-F. Wang, Anal. Chem., 2011, 83, 8787–8793.
39. F.-H. Hsu and T.-M. Wu, Synth. Met., 2012, 162, 682–687.
40. I. K. Moon, J. Lee, R. S. Ruoff and H. Lee, Nat. Commun., 2010, 1(73), 1–6.
41. H. Feng, R. Cheng, X. Zhao, X. Duan and J. Li, Nat. Commun., 2013, 4, 1539–1546.
42. S. Bose, T. Kuila, M. E. Uddin, N. H. Kim, A. K. T. Lau and J. H. Lee, Polymer, 2010, 51, 5921–5928.
43. N. A. Kumar, H.-J. Choi, Y. R. Shin, D. W. Chang, L. Dai and J.-B. Baek, ACS Nano, 2012, 6, 1715–1723.
44. J. Zhang and X. S. Zhao, J. Phys. Chem. C, 2012, 116, 5420–5426.
45. V. Chandra and K. S. Kim, Chem. Commun., 2011, 47, 3942–3944.
46. H. He and C. Gao, ACS Appl. Mater. Interfaces, 2010, 2, 3201–3210.
47. H. Mudila, M. G. H. Zaidi, S. Rana, V. Joshi and S. Alam, Int. J. Chem. Anal. Sci., 2013, 4, 139–145,  DOI:10.1016/j.ijcas.2013.09.001.
48. F.-H. Hsu and T.-M. Wu, Synth. Met., 2012, 162, 682–687.
49. F. Garbassi, M. Morra and E. Occhiello, Polymer surfaces from physics to technology, Wiley, New York, 1994.
50. S.-T. Hwang and K. Kammermeyer, Membranes in Separations, Techniques of Chemistry, Wiley-Interscience, 1975, vol. VI.
51. F. C. Moraes, I. Cesarino, V. Cesarino, L. H. Mascaro and S. A. S. Machado, Electrochim. Acta, 2012, 85, 560–565.
52. C. L. Scott, G. Zhao and M. Pumera, Electrochem. Commun., 2010, 12, 1788–1791.
53. F. Jiang, R. Yue, Y. Du, J. Xu and P. Yang, Biosens. Bioelectron., 2013, 44, 127–131.
54. L. Giannoudi, E. V. Piletska and S. A. Piletsky, Development of biosensors for the detection of hydrogen peroxide, biotechnological applications of photosynthetic proteins: Biochips, Biosensors and Biodevices Biotechnology Intelligence Unit, Springer, Landes Bioscience, 2006, pp. 175–191.
55. A. J. Bard and L. R. Faulkner, Electrochemical Methods: Fundamentals and Applications, Wiley, New York, 2nd edn, 2001.
56. M. A. Kamyabi and F. Aghajanloo, J. Electroanal. Chem., 2008, 614, 157–165.
57. L. Fotouhi, N. Farzinnegad, M. M. Heravi and S. Khaleghi, Bull. Korean Chem. Soc., 2003, 24, 1751–1756.
58. J.-M. You, D. Kim, S. K. Kim, M.-S. Kim, H. S. Han and S. Jeon, Sens. Actuators, B, 2013, 178, 450–457.
59. N. Kuge, M. Kohzuki and T. Sato, Free Radical Res., 1999, 30, 119–123.
60. L. H. Long, P. J. Evans and B. Halliwell, Biochem. Biophys. Res. Commun., 1999, 262, 605–609.
61. S. Chatterjee and A. Chen, Biosens. Bioelectron., 2012, 35, 302–307.
62. B. Halliwell, L. H. Long, T. P. Yee, S. Lim and R. Kelly, Curr. Med. Chem., 2004, 11, 1085–1092.
63. X. Liu, H. Zhu and X. Yang, Talanta, 2011, 87, 243–248.
64. S. Nandini, S. Nalini, R. Manjunatha, S. Shanmugam, J. S. Melo and G. S. Suresh, J. Electroanal. Chem., 2013, 689, 233–242.
65. D. Ye, H. Li, G. Liang, J. Luo, X. Zhang, S. Zhang, H. Chen and J. Kong, Electrochim. Acta, 2013, 109, 195–200.

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Footnote

† Electronic supplementary information (ESI) available: Contact angle images. See DOI: 10.1039/c4ra00578c

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